Biophotonics as a research area provides and employs novel photonic technologies and tools for medical research, diagnosis and therapy. Biophotonics research aims for a deeper understanding of the processes within living cells, which is, on the other hand, a prerequisite for the improvement of early recognition and targeted treatment of diseases. The corresponding technological solutions and methods for diagnosis and therapy will be employed for an efficient and affordable health care which will help to deal with the challenges of aging societies and exploding health care costs. Resultant Biophotonics systems will support not only the preservation, but even the increase of the impressive double-digit annual growth rates of the related industries. The development of such systems will require highly interdisciplinary collaboration based on an intensive dialogue between scientists from the various disciplines in order to streamline common efforts. An important part of Biophotonic systems is based on spectroscopic, microscopic and imaging methods, not only for diagnostic purposes but also for surgical instruments, pathology applications and therapy control. Multimodal systems, based on combinations of different, not necessarily only photonic techniques will help to overcome the limits of the individual methods in combination with powerful deep learning and artificial intelligence based image analysis and chemometric methods.

Contributed papers are solicited concerning, but not limited to, the following areas:

  • OCT, Raman- and IR-based methods, optoacoustics
  • multiphoton methods (SHG, CARS, FlIM, FRET)
  • fast and ultrafast imaging methods
  • super-resolution imaging/adaptive optics
  • optical fibers for Imaging applications
  • analysis of molecules, cells/bacteria, tissues
  • understanding cell processes
  • image analysis, chemometrics, artificial intelligence-based methods.
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    In progress – view active session
    Conference 12144

    Biomedical Spectroscopy, Microscopy, and Imaging II

    4 - 7 April 2022
    View Session ∨
    • 1: Neurophotonics I
    • 2: Neurophotonics II
    • 3: Raman Spectroscopy and Imaging I
    • 4: Raman Spectroscopy and Imaging II
    • 5: Advanced Microscopy and Imaging I
    • 6: Advanced Microscopy and Imaging II
    • 7: Advanced Microscopy and Imaging III
    • 8: Multiphoton Microscopy I
    • 9: Optical Coherence Tomography
    • Posters-Wednesday
    • 10: Multiphoton Microscopy II
    Session 1: Neurophotonics I
    4 April 2022 • 1:30 PM - 3:10 PM CEST
    Author(s): Walther Akemann, Sébastien Wolf, Ecole Normale Supérieure (France); Vincent Villette, CNRS (France); Benjamin Mathieu, Astou Tangara, INSERM (France); Jozsua Fodor, YMETRY (France); Cathie Ventalon, Jean-François Léger, CNRS (France); Stéphane Dieudonné, INSERM (France); Laurent Bourdieu, CNRS (France)
    4 April 2022 • 1:30 PM - 1:50 PM CEST
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    Custom-access serial holography (CASH) is a new method for optical recording of neuronal activity in 3D at high speed in-vivo. Our implementation allows random access of 20 cells at 1 kHz up to 200 cells at 0.1 kHz in head-fixed behaving mice across a cortical space of 500 x 500 x 500 m3 size. Using fast acousto-optic spatial light modulation, every single laser pulse of a 40 kHz regenerative amplifier is individually patterned to serially access a selection of target cells with a square 5x5 spot excitation volume covering the cell body and, for the prevention of recording artefacts, the surrounding space in anticipation of the cell displacements during animal behavior. The recorded activity is corrected for neuropil signaling by weighted subtraction of a neuropil reference signal obtained by interleaved sampling of neuropil activity close to each cell. We performed 3D-CASH recordings of GCaMP6f expressing neurons in layer 2/3 and 5 of mouse primary visual cortex in response to moving contrast gratings and applied deconvolution to estimate spikes. Thanks to the fast recording permit by 3D-CASH, the cortical laminar structure is revealed in the temporal organization of the activity: pairwise correlation was higher between intralaminar vs. interlaminar neuron pairs; principal component analysis of the correlation matrix revealed a component assigning weights of opposite sign to neurons in different layers; closest follower spikes occurred with higher probability in a neuron of the same layer. 3D-CASH allows also following the response to the temporal periodicity of the stimulus, which features a phasic (R1) and a non-phasic component (R0). R1/R0 values are broadly distributed with weak bimodality resembling the transition between pure non-phasic response (complex receptive field) to phasic response (simple receptive field). Our data validate thus 3D-CASH as a method for assessing neuronal activity in 3D-distributed cortical circuits at high sampling rate.
    Author(s): Baptiste Grimaud, École normale supérieure Paris-Saclay (France); Maxence Frétaud, INRAE (France); Feriel Terras, École normale supérieure Paris-Saclay (France); Karine Duroure, Institut de la Vision (France); Valérie Bercier, KU Leuven (Belgium); Gaëlle Allard, École normale supérieure Paris-Saclay (France); Elodie Chaudan, Thierry Gacoin, Ecole Polytechnique (France); Filippo Del Bene, Institut de la Vision (France); François Marquier, École normale supérieure Paris-Saclay (France); Christelle Langevin, INRAE (France); François Treussart, École normale supérieure Paris-Saclay (France)
    4 April 2022 • 1:50 PM - 2:10 PM CEST
    Author(s): Nicolò Accanto, François Blot, Valeria Zampini, Florence Bui, Antonio Lorca Camara, Christophe Tourain, Institut de la Vision (France); Noam Badt, Ori Katz, The Hebrew Univ. of Jerusalem (Israel); Valentina Emiliani, Institut de la Vision (France)
    4 April 2022 • 2:10 PM - 2:30 PM CEST
    Author(s): Francesco A. Resta, LENS - Lab. Europeo di Spettroscopie Non-Lineari (Italy), Univ. degli Studi di Firenze (Italy); Giacomo Mazzamuto, Univ. degli Studi di Firenze (Italy), Istituto Nazionale di Ottica, Consiglio Nazionale delle Ricerche (Italy), LENS - Lab. Europeo di Spettroscopie Non-Lineari (Italy); Emilia Conti, Anna Letizia Allegra Mascaro, LENS - Lab. Europeo di Spettroscopie Non-Lineari (Italy), Istituto di Neuroscienze, Consiglio Nazionale delle Ricerche (Italy); Francesco Saverio S. Pavone, LENS - Lab. Europeo di Spettroscopie Non-Lineari (Italy), Istituto Nazionale di Ottica, Consiglio Nazionale delle Ricerche (Italy)
    4 April 2022 • 2:30 PM - 2:50 PM CEST
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    Brain responsiveness and its activation complexity are linked to the level of consciousness (Tononi et al., 2004). However, how these features change across brain states is still not clear. The combination of Transcranial Magnetic Stimulation and hd-EEG recordings represents the standard method to address this issue in humans. A preclinical analogous in lab animals would provide novel mechanistic insights on the brain-state-dependent complexity of the brain. A powerful technique to study mesoscale cortical connectivity in mice exploits wide-field fluorescence microscopy. This approach provides simultaneous information of neuronal ensemble activity from distributed cortical areas, while optogenetic has been demonstrated to be a powerful tool to activate cortical neuronal clusters. However, all-optical systems that combine these techniques critically suffer from crosstalk between imaging and photostimulation (Emiliani et al. 2015). Brain responsiveness and its activation complexity are linked to the level of consciousness (Tononi et al., 2004). However, how these features change across brain states is still not clear. The combination of Transcranial Magnetic Stimulation and hd-EEG recordings represents the standard method to address this issue in humans. A preclinical analogous in lab animals would provide novel mechanistic insights on the brain-state-dependent complexity of the brain. A powerful technique to study mesoscale cortical connectivity in mice exploits wide-field fluorescence microscopy. This approach provides simultaneous information of neuronal ensemble activity from distributed cortical areas, while optogenetic has been demonstrated to be a powerful tool to activate cortical neuronal clusters. However, all-optical systems that combine these techniques critically suffer for crosstalk between imaging and photostimulation (Emiliani et al. 2015). Here we established an all-optical method combining wide-field fluorescence imaging of the red-shifted calcium indicator jRCaMP1b and transcranial optogenetic stimulation of Channelrhodopsin-2 (ChR2). To achieve a cortex-wide expression of the calcium indicator, an adeno-associated virus (AAV.PHP.eb) carrying jRCaMP1b under the control of the synapsin promoter was injected in the retro-orbital sinus of anesthetized mice. This led to a uniform expression of the functional indicator in the whole cortex, giving the possibility to visualize the neuronal activity propagation in all the cortical areas. Due to the high opsins expression required for effective optogenetic stimulation, AAV9-ChR2 was locally injected in the somatosensory cortex (S1). Results show that in awake mice, optogenetic stimulations at increasing laser power evoke a distributed cortical response in several areas in the two cortical hemispheres, whereas, during anesthesia, stimulation led to a localized reponse limited in space and time. These results suggest that response complexity decrease with the levels of consciousness, as observed in pathological patients affected by disorders of consciousness (Massimini et al., 2009).
    Author(s): Pietro Ricci, LENS - Lab. Europeo di Spettroscopie Non-Lineari (Italy); Marco Marchetti, Light4Tech (Italy); Michele Sorelli, Lapo Turrini, Francesco A. Resta, Vladislav Gavryusev, Giuseppe de Vito, Giuseppe Sancataldo, Francesco Vanzi, Ludovico Silvestri, Francesco Saverio S. Pavone, LENS - Lab. Europeo di Spettroscopie Non-Lineari (Italy)
    4 April 2022 • 2:50 PM - 3:10 PM CEST
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    To understand the brain computation paradigms and the causal interactions in complex neuronal networks, we need methods and technologies to record and perturb neuronal distributions over large fields of view. In this application, two-photon (2P) imaging has become a cornerstone microscopy technique, widely used for deep optical access in biological samples and selective light targeting with submicrometric resolution. In parallel to structural and functional imaging, 2P optogenetics has represented a game-changer, allowing targeted stimulation of specific neural circuits. However, the long commutation times and refresh rates of traditional scanning methods substantially hinder near-simultaneous multi-site 3D stimulation. Acousto-optic deflectors (AODs), owing to their fastest scanning and refresh rates, can fulfil the temporal requirements for concurrent activation of sparsely distributed neurons. Nevertheless, their applicability to 2P optogenetics in large volumes has been limited so far by the massive efficiency drop along the optical axis during their use in axial scanning. To counteract this drawback, a compensation software module is frequently employed to flatten the power distribution throughout the volume. However, the power threshold is reduced to the minimum intensity value addressable, lowering the peak intensity released in the centre of the axial scan. Here, we propose a unique approach for overcoming this drawback which provided lifted axial power distribution while maintaining a uniform lateral illumination range. We tested this method by the 2P photoactivation of optogenetic actuators in 3D in zebrafish larvae, showing how the probability of evoking an electrophysiological response and the relative neuronal activity amplitude improved by carefully optimizing the light targeting time on different axial planes. In conclusion, fast and uniform axial light addressing with AODs enables unprecedented 3D 2P optostimulation, formerly not feasible. Furthermore, this approach can be adopted as an upgrade for existing microscopes designed for volumetric imaging, providing 3D multi-site imaging and random-access illumination.
    Session 2: Neurophotonics II
    4 April 2022 • 3:40 PM - 5:20 PM CEST
    Author(s): Karen Caicedo, Antony Lee, Pierre Bon, Laurent Cognet, Lab. Photonique, Numérique et Nanosciences (France)
    4 April 2022 • 3:40 PM - 4:00 PM CEST
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    Fluorescence microscopy has succeeded in attaining super-resolution localization of single emitters in cellular biology. However, 3D localization deep inside tissue is still challenging. A few years ago, we developed SELFI: self-interference 3D super-resolution microscopy, a framework for 3D single-molecule localization within multicellular specimens and tissues. Here, we extend the capability of SELFI to the near-infrared (NIR) region where carbon nanotubes (CNTs) are strong emitters. The aim of this work is to develop NIR SELFI for single-particle tracking applications of CNTs in live brain tissues or NIR quantum dots. SELFI uses a diffraction grating placed on the optical path of the sample image, generating an interference pattern within diffraction limited images of point emitters. A single image obtained with NIR SELFI contains two independent variables: the intensity distribution to extract the intensity centroid to determine the lateral localization, and the wavefront curvature (provided by the interfringes) to get the axial super-localization. SELFI was first developed to localize red emitting dyes and quantum dots. The performance of the system is examined by means of the standard deviation and root mean square error of the localizations. The experiments performed show that the 3D-precision and accuracy achieved with NIR SELFI are both below 100 nm for emission around 1000 nm and high photon budget. Therefore, we can now achieve 3D localization in the NIR, permitting 3D single-particle tracking of CNTs at video rate in complex environments.
    Author(s): Florian Semmer, Marie-Charlotte Chandeclerc, François Treussart, Karen perronet, François Marquier, LuMIn (France)
    4 April 2022 • 4:00 PM - 4:20 PM CEST
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    Neurodegenerative diseases such as Alzheimer’s disease present abnormalities in intraneuronal transport, suggesting the relevance of measuring this key biological process. In 2017, a sensitive method to measure changes in intraneuronal endosomal transport has been reported in 2D cultures of neurons using fluorescent nanodiamonds (fNDs) [1]. The high brightness, photostability and absence of cytotoxicity allow fNDs to be tracked with 50 nm spatial and 50 ms time resolutions. This nanoparticle tracking based-approach applies also to multiphoton imaging, opening the possibility of transport measurement in vivo. We use nanocrystals possessing a large nonlinear second order optical response. First results indicate that the intraneuronal transport measurement can be inferred from nonlinear microscopy data, opening applications to thicker samples owing to the low background of multiphoton imaging. In order to get a high spatio-temporal resolution (around 10 nanometers at 1 ms), we are developing a two-photon microscope, based on a digital holography method [2]. A Digital Micromirror Device (DMD) is used as a spatial light modulator, allowing a fast 3D motion of the excitation volume. We aim at reaching a time resolution below the millisecond and super-localization regime in the tens nanometer range using orbital tracking. References : [1] S. Haziza, et al. Nat. Nanotechnol. 12 (2017), 322. [2] Geng, Q., Gu, C., Cheng, J. & Chen, S. Digital micromirror device-based two-photon microscopy for three-dimensional and random-access imaging. Optica 4, 674 (2017)
    Author(s): Clara Manesco, Joshua de Lizaraga, Lab. Charles Coulomb, Univ. de Montpellier, CNRS (France); Bela Varga, Lab. Charles Coulomb, Univ. de Montpellier, CNRS (France); Thierry Cloitre, Lab. Charles Coulomb, Univ. de Montpellier, CNRS (France); Marta Martin, Lab. Charles Coulomb, Univ. de Montpellier, CNRS (France); Yannick Gerber, MMDN, Univ. de Montpellier, INSERM (France); Florence Perrin, MMDN, Univ. de Montpellier, INSERM (France); Csilla Gergely, Lab. Charles Coulomb, Univ. de Montpellier, CNRS (France)
    4 April 2022 • 4:20 PM - 4:40 PM CEST
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    Spinal cord injuries (SCI) affect between 2.5 and 4 million patients worldwide, with no current curative treatment. To understand the mechanisms underlying the absence of spontaneous regeneration following injury, we are combining the non-linear multiphoton microscopy (MPM) technique with force measurements via atomic force microscopy (AFM), in a mouse model, to monitor the glial scar, a scar that inhibits the axonal regeneration by forming a physical and chemical barrier composed mainly of astrocytes and microglia. We recorded 2-photon excited fluorescence (2PEF) and second harmonic generation (SHG) signals of excised mice SC injured tissues in MPM at 72h, 1week and 6 weeks post-lesion, and further performed polarization dependent measurements of the SHG signal to assess the preferential orientation of the collagen bundles. Our MPM images revealed a strong SHG signal at 1 week post injury, due to the formation of fibrillary collagen fibers (collagen type I) by the injury site. The SHG signal was increased at 6 weeks after injury, and associated with (1) a higher fiber density (2) a shorter fiber length and less fibers oriented in the same direction. AFM based force spectroscopy measurements, performed at the same post-lesion time-points to map the elastic properties of the spared grey and white matters and injured (lesion) parts of the tissue, suggested an increase of the lesion area stiffness over time. These results together indicate the presence of a fibrotic process seven days after injury, that is further increased at later time points. We similarly started to investigate the effect of a treatment (pharmacological transient depletion of microglia/macrophage proliferation) in mice that underwent SCI. Our preliminary results suggested an increase in fibers length in treated tissues, as well as a reduction of the collagen extension around the injury site.
    Author(s): Jelena Petrovic, Fred Lange, Dennis Hohlfeld, Univ. Rostock (Germany)
    4 April 2022 • 4:40 PM - 5:00 PM CEST
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    A miniaturized fiber-based optrode, microfabricated to perform a simultaneous light delivery and electrophysiological recording with a minimal tissue damage is presented. The device consists of a tapered fiber tip and three separate metal electrodes deposited on the cylindrical surface of an optical fiber. Tapered fiber tip of a good optical quality is created using our custom made mechanical grinding setup which gives the possibility of achieving different cone angles. Using our custom precision fiber lateral and angular alignment setup as a step before sputtering through a shadow mask, allows us to fabricate a novel fiber based optrode with three separate equidistant metal electrodes along the fiber as well as on the fiber tip.
    Author(s): Liang-Wei Chen, Feng-Chun Hsu, Chun-Yu Lin, National Yang Ming Chiao Tung Univ. (Taiwan); Yvonne Yuling Y. Hu, National Cheng Kung Univ. (Taiwan); Shean-Jen Chen, National Yang Ming Chiao Tung Univ. (Taiwan), Taiwan Instrument Research Institute, National Applied Research Labs. (Taiwan)
    4 April 2022 • 5:00 PM - 5:20 PM CEST
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    Optogenetics contributes toward a clearer understanding of neural activity by manipulating the light to precisely and rapidly control cellular activity. Multiphoton fluorescence excitation has the advantage of lower photobleaching effect, less tissue scattering, and better penetration depth in biological sample. Herein, a femtosecond laser with the wavelength of 1250 nm has been utilized to stimulate drosophila neurons with two-photon excitation. With computer-generated holography (CGH), customized 3D excitation micropatterns can be offered. Traditionally, the digital holograms are generated by iterative optimization algorithms. However, the iterative algorithms cannot produce high-resolution holograms in a very short periods of time. Therefore, we apply deep-learning based CGH for generating the holograms to synthesize customized 3D micropatterns. However, the patterns would suffer from low axial resolution as using CGH to generate 3D micropatterns. This drawback is the main reason that we cannot stimulate the drosophila neurons in single-cell resolution. To solve this problem, we integrated the temporal focusing (TF) technique to improve the axial resolution. Due to the expanded frequencies of the incident laser beam under TF scheme, the deep-learning based CGH algorithm is modified into a multi-wavelength deep-learning based CGH algorithm. We use a grating to generate TF effect for better axial confinement and multiplex the stimulation pattern to arbitrary 3D positions in the volume of interest by apply the hologram that is generated by the deep-learning based CGH algorithm on the SLM. Through this progress, 3D neural activity and connection could be observed. Therefore, the developed deep learning-based CGH with TF axial confinement can provide 3D micropatterned multiphoton stimulation at the few-micron level. The approach not only improves the axial accuracy of the stimulation pattern, but also speeds up the computation time to tens of milliseconds. As orders of magnitude faster computation time, we can achieve real-time and precise stimulation on the target sample.
    Session 3: Raman Spectroscopy and Imaging I
    5 April 2022 • 8:30 AM - 10:30 AM CEST
    Author(s): Suraj Kumar Singh, Taru Verma, Dipankar Nandi, Indian Institute of Science, Bengaluru (India); Siva Umapathy, Indian Institute of Science Education and Research, Bhopal (India)
    5 April 2022 • 8:30 AM - 9:00 AM CEST
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    Antibiotics have transformed medicine in many aspects and their rampant and reckless use has resulted in the rapid emergence of antibiotic resistance, giving rise to bacteria that are resistant to a multitude of antibiotics. There are multiple mechanisms by which bacteria become antibiotic resistant. Apart from genetic mechanisms, certain compounds present in the environment like disinfectants, commonly prescribed anti-inflammatory drugs, phenolics, tobacco smoke, herbicides etc can also contribute towards the development of resistant strains. This is known as phenotypic antibiotic resistance. These compounds reduce the influx of antibiotics and enhance drug efflux from the bacteria, resulting in an increase in the MIC of several antibiotics like cephalosporins, carbapenems, tetracyclines, fluroquinolones etc in multiple bacterial species. Sodium Salicylate (NaSal) and Sodium Benzoate have been shown to potent inducers of antibiotic resistance. Also, herbicides such as 2,4 d and glyphosate have been shown to induce changes in the response of E. coli and S. Typhimurium to antibiotics. The combination of high use of both antibiotics and herbicides in proximity to farm animals, poultry and important insects, such as honeybees, might also compromise their therapeutic effects and drive greater use of antibiotics. In this Raman spectroscopic study, we have used E. coli as our model system and treated it with sub-lethal concentrations of bacteriostatic (tetracycline and rifampicin) and bactericidal (ciprofloxacin, ceftriaxone and ampicillin) antibiotics with distinct mechanism of action. Tetracycline belongs to the class of tetracyclines which arrest the bacterial growth by interrupting protein synthesis whereas rifampicin is a member of rifamycin class and blocks transcription. On the other hand, ciprofloxacin is a member of fluroquinolones and kills bacteria by binding to DNA gyrase, causing extensive DNA damage. Ceftriaxone belongs to the class of cephalosporins while ampicillin is a penicillin derivative. Both these antibiotics inhibit cell wall synthesis to bring about bacterial cell death. We identified various biomolecular changes occurring as a function of antibiotic concentration using Raman spectroscopy and partial least square discriminant analysis (PLS-DA). The ability of Raman spectroscopy in differentiating resistant and sensitive bacteria was also established by employing two genetic mutants of E. coli, ∆lon and ∆acrB, which display differential susceptibilities towards antibiotics. Also, we induced resistance using 2,4 d and Glyphosate and demonstrated that Raman spectroscopy could efficiently identify the emergence of resistance in bacterial strains that were previously susceptible to antibiotics.
    Author(s): Tamiki Komatsuzaki, Koji Tabata, Hokkaido Univ. (Japan); Hiroyuki Kawagoe, Osaka Univ. (Japan); James Nicholas Taylor, Hokkaido Univ. (Japan); Kentaro Mochizuki, Kyoto Prefectural Univ. of Medicine (Japan); Toshiki Kubo, Osaka Univ. (Japan); Jean-Emmanuel Clement, Hokkaido Univ. (Japan); Yasuaki Kumamoto, Osaka Univ. (Japan); Yoshinori Harada, Kyoto Prefectural Univ. of Medicine (Japan); Atsuyoshi Nakamura, Hokkaido Univ. (Japan); Katsumasa Fujita, Osaka Univ. (Japan)
    5 April 2022 • 9:00 AM - 9:30 AM CEST
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    We present our recent study combined multi-armed Bandits algorithm in reinforcement learning with spontaneous Raman microscope for the acceleration of the measurements by designing and generating optimal illumination pattern “on the fly” during the measurements while keeping the accuracy of diagnosis. We present our simulation and experimental studies using Raman images in the diagnosis of follicular thyroid carcinoma and non-alcoholic fatty liver disease, and show that this protocol can accelerate more than a few tens times in speedy and accurate diagnoses faster than line-scanning Raman microscope that requires the full detailed scanning over all pixels. The on-the-fly Raman image microscopy designs to accelerate measurements by combining one of reinforcement machine learning techniques, bandit algorithm utilized in the Monte Carlo tree search in alpha-GO, and a programmable illumination system. Given a descriptor based on Raman signals to quantify the likelihood of the predefined quantity to be evaluated, e.g., the degree of cancers, the on-the-fly Raman image microscopy evaluates the upper and lower confidence bounds in addition to the sample average of that quantity based on finite point/line illuminations, and then the bandit algorithm feedbacks the desired illumination pattern to accelerate the detection of the anomaly, during the measurement to the microscope. Most conventional bandit algorithms assume that infinite number of measurements or samples provides us with 100% accuracy. However, in Raman measurements we should develop both a Raman descriptor to quantify the degree of anomaly, and a new algorithm to take into account the finite accuracy lower than 100%. This microscope can also be applied to other problems, besides detection of cancer cells, such as anomaly or defects of materials. The algorithm itself is also beneficial and transferrable to the other microscopes such as infrared image microscope.
    Author(s): Subitcha Jayasankar, Sujatha N., Indian Institute of Technology Madras (India)
    5 April 2022 • 9:30 AM - 9:50 AM CEST
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    Raman spectroscopy is a powerful optical tool in cancer diagnosis. In addition to diagnosis during intraoperative procedures, insight on tumor location and geometry is required. This work utilizes Spatially offset Raman spectroscopy (SORS) to estimate the varying size of an ellipsoid tumor located at various subsurface depths and tumor thicknesses. The prediction is based on the incremental movement of the probe on the surface to analyze the relative tumor contribution change calculated from SORS signal. Regression on Monte Carlo simulated SORS signals predicted the dimension with a coefficient of determination of 0.93 and root mean square error less than 2%.
    Author(s): Sarika Hinge, Jyotsana P. Dixit, Gauri R. Kulkarni, Pandit B. Vidyasagar, Savitribai Phule Pune Univ. (India)
    5 April 2022 • 9:50 AM - 10:10 AM CEST
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    Deformability of red blood cell is an important aspect in hemodynamic and related diseases. There has been an increasing interest to understand the biochemical and structural changes in Red blood cell (RBC) due to microgravity conditions. In the present work, isolated human red blood cells are exposed to simulated microgravity condition using 2D clinostat. Comparative analysis of normal RBC and RBC in simulated microgravity condition is done using UV-Visible spectroscopy, FTIR Spectroscopy, Raman spectroscopy and single beam Optical Tweezer. The change in position of Soret band absorption peak is observed after the exposure of simulated microgravity. However, shift in biochemical functional groups of RBC is studied using FTIR spectroscopy with change in % Transmittance. Conformational changes in RBC membrane protein is examined through Raman spectroscopy. To understand, how microgravity affects the mechanobiology of red blood cell; Optical Tweezer is used as a probe to measure mechanical properties of single red blood cell. Significant changes are reported in measurement of trapping force by Optical Tweezer. Microgravity induces stress which are likely to change morphology and rigidity of red blood cell. Therefore, trapping force measurement of RBC can be correlated with the elasticity and flexibility of RBC membrane which are important properties for blood circulation during Space mission.
    Author(s): Artem Tabarov, Vladimir V. Vitkin, ITMO Univ. (Russian Federation); Daria Danilenko, Smorodintsev Research Institute of Influenza (Russian Federation); Evgeniy Popov, Alexander Dobroslavin, Olga Andreeva, Arina Shemanaeva, Valeriia Kurikova, Olga Kuznetsova, ITMO Univ. (Russian Federation)
    5 April 2022 • 10:10 AM - 10:30 AM CEST
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    According to the World Health Organization (WHO), respiratory diseases are one of the five leading causes of death worldwide. Respiratory viral infections place an enormous burden on the health care system and lead to both a decrease in labor productivity and an increase in medical expenses. The global COVID-19 pandemic has demonstrated the danger from zoonotic viral infections and pointed out the problem of modern methods imperfection at the disease diagnosis. The classical methods of polymerase chain reaction (PCR) and enzyme immunoassay (ELISA) do not have sufficient throughput capability to be used during infection outbreaks. The express tests available today still give a large percentage of false-positive results and lack the ability to detect several distinct pathogens. Thus, the problem of rapid and accurate diagnosis of various respiratory diseases remains relevant. An important scientific problem solved in this work is providing the possibility of detecting a pathogen in a transport medium by surface-enhanced Raman spectroscopy without the additional use of antibodies or specific proteins. This study provides and systematizes information on the peak positions in the surface-enhanced Raman spectra of influenza A and B viruses, which allows for judging a pathogen's presence in a sample. The classification of obtained spectral patterns, the subsequent creation of a spectral database, and its use as a basis for machine learning will provide an opportunity for rapid identification of viruses for high-precision differential diagnosis of infectious diseases. Viruses' spectral data were obtained by adsorbing them onto a SERS substrate. The differences in the spectra are explained by the different structure and amino acid composition of the influenza A and B viruses' surface proteins. Machine learning technologies were used to analyze and visualize the spectral differences between influenza A and B viruses. This approach to the obtained data processing makes it possible to create a technology for rapidly diagnosing various viral diseases.
    Session 4: Raman Spectroscopy and Imaging II
    5 April 2022 • 11:00 AM - 1:00 PM CEST
    Author(s): Kotaro Hiramatsu, The Univ. of Tokyo (Japan), PRESTO, Japan Science and Technology Agency (Japan); Keisuke Goda, The Univ. of Tokyo (Japan), Univ. of California, Los Angeles (United States), Wuhan Univ. (China)
    5 April 2022 • 11:00 AM - 11:30 AM CEST
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    High-speed Raman spectroscopy has enabled label-free characterization of molecules in cells and materials in a space- and time-resolved manner. Among these, time-domain Raman spectroscopy (TDRS) techniques, such as Fourier-transform coherent anti-Stokes Raman scattering (FT-CARS) and impulsive stimulated Raman scattering (ISRS) spectroscopies, have unique capabilities such as high spectral acquisition rates, broadband spectral sensitivity in the fingerprint region, and nonresonant-background-free spectral acquisition. With a few exceptions, most TDRS studies have focused only on the fingerprint region (200 – 1800 cm-1) because the ultrashort pulses typically used for ultrabroadband (200 – 3200 cm-1) spectral acquisitions are difficult to generate and handle. For example, detecting Raman peaks above 3000 cm-1 necessitates a pulse duration of < 10 fs, which demands an expensive laser source and careful dispersion control. Furthermore, with sub-10-fs pulses, Raman detection sensitivity in the fingerprint is compromised because the spectral power density is diluted in the spectrally broad ultrashort pulse. The present research demonstrates FT-CARS spectroscopy covering both the fingerprint and CH-stretching regions by employing synchronized mode-locked Ti:Sapphire and Yb-doped fiber lasers as the light source. With this method, we show that ultra-broadband FT-CARS spectra can be obtained without using sub-10-fs pulses, which significantly mitigates experimental complexity. More importantly, ultra-broadband Raman detection can be achieved in this scheme without compromising the sensitivity in the fingerprint region, unlike previous ultrashort-pulse approaches. The present method will significantly broaden the application range of TDRS for biomedical and material science research.
    Author(s): Hannah U. Holtkamp, Claude Aguergaray, Michel K. Nieuwoudt, Gus Grey, The Univ. of Auckland (New Zealand); Federico Marini, Sapienza Univ. di Roma (Italy); Cather M. Simpson, The Univ. of Auckland (New Zealand); Paul Jarrett, Middlemore Hospital (New Zealand), The Univ. of Auckland (New Zealand)
    5 April 2022 • 11:30 AM - 12:00 PM CEST
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    Discoid Lupus Erythematosus is chronic autoimmune disease that disproportionately affects parts of the population. Presently, the biochemical events involved in the formation of the disease and elements of the pathophysiology are poorly understood, demonstrating a need for improved analysis. We present the results of our multimodal imaging combining Raman spectroscopy and mass spectrometry and their chemometric analysis and models. We distinguish physiological features and how they differ between healthy and DLE. We show that by fusing the data we are able to build a classification model that can differentiate the two with higher accuracy than either technique alone. The findings from this study can serve as a basis for improved biomedical diagnostics and better informed potential treatment options.
    Author(s): Elisa Grassi, Siarhei P. Laptenok, Luca Genchi, Carlo Liberale, King Abdullah Univ. of Science and Technology (Saudi Arabia)
    5 April 2022 • 12:00 PM - 12:20 PM CEST
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    Stimulated Raman Scattering (SRS) microscopy is a Coherent Raman technique that emerged in recent years as a powerful tool for biomedical imaging due to its high specificity, high speed, and label-free capability. Despite its advantages, SRS microscopy can be affected by nonlinear competing phenomena, namely two-photon absorption (TPA), cross-phase modulation (XPM), and thermal lensing (TL), which generate a background signal that reduces the achievable specificity and sensitivity. These competing processes are quasi-instantaneous and spatially non-uniform in heterogeneous samples and require customized setups to be canceled in SRS acquisitions. A robust approach for background-free SRS measurements is the frequency-modulation (FM) SRS, which is based on the broad spectral dependence of the parasitic effects (typically tens of nanometers) compared to the narrower band of the SRS effect (~ 1 nm). Performing a differential measurement at two different wavenumbers, respectively on- and off- Raman resonance, it is possible to selectively detect the SRS process. Different solutions for background cancellation via FM have been reported in the literature, but they present various drawbacks, such as the limited applicability over certain ranges of the vibrational spectrum or the necessity to modify the optical setup when performing measurements at different Raman shifts. We propose an FM-SRS configuration realized for the first time with an acousto-optic tunable filter, able to perform measurements from the fingerprint to the CH-stretch region of the spectrum without any modification of the optical setup. We determined its efficiency in canceling the background signal due to different types of competing effects on various samples: polymer beads, human hair, and human cells. These results underline the importance of an effective cancellation of background signals of diverse nature when collecting SRS images. Our FM-SRS setup demonstrated critical advantages compared to other FM configurations.
    Author(s): Bryce Manifold, Dan Fu, Univ. of Washington (United States)
    5 April 2022 • 12:20 PM - 12:40 PM CEST
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    T cells are an integral part of the adaptive immune system. The various types of T cells and their respective functions are an important point of study in immune response, vaccine development, and cancer treatment. Current methods for studying T cells typically rely on extraction, fluorescent antibody staining, and separation via flow cytometry. While these methods can readily identify and collect the varying T cell differentiations, they are not applicable to in situ study of T cell function and response. Microscopy based studies of T cells are possible, but they often involve fluorescent labeling which limits the observation specificity and duration. We present hyperspectral stimulated Raman scattering (SRS) microscopy as a potential label-free alternative to directly observing and characterizing T cells. SRS microscopy targets the endogenous vibrational chemical differences of T cells at high spatial resolution without fluorescent labeling. We show that a deep learning model can be trained to identify and classify T cell differentiations from hyperspectral SRS images with 96% accuracy. SRS microscopy augmented with deep learning shows strong promise towards label-free in situ observation of T Cells.
    Author(s): Maximilian Brinkmann, Sven Dobner, Tim Hellwig, Refined Laser Systems GmbH (Germany)
    5 April 2022 • 12:40 PM - 1:00 PM CEST
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    We present our recent developments in utilizing a compact and portable light source for high-speed multicolor stimulated Raman scattering imaging (SRS) in biomedical and medical environments. The source combines a rapid and wide tunability for accessing Raman bands between 700 and 3300 cm-1 with high stability in terms of power (deviation < 0.3 %) and wavelength (deviation < 0.5 pm) over more than 100 h. We highlight applications in metabolic cell imaging and the identification of pharmaceuticals in complex environments such as skin by harvesting contrast from several Raman bands.
    Session 5: Advanced Microscopy and Imaging I
    5 April 2022 • 2:10 PM - 4:10 PM CEST
    Author(s): Lorry Mazzella, Guillaume Giroussens, Benoît Rogez, Aix-Marseille Univ. (France), CNRS (France); Jérome Idier, Lab. des Sciences du Numérique de Nantes, CNRS (France); Marc Allain, Anne Sentenac, Loïc Le Goff, Aix-Marseille Univ. (France), CNRS (France)
    5 April 2022 • 2:10 PM - 2:30 PM CEST
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    Structured light microscopy relies on multiple images captures under different illuminations to retrieve a super-resolved image. In the context of volume imaging, the total number of required frames can severely slow down imaging. We applied extended depth of field (EDF) to random illumination microscopy (RIM, Mangeat 2021) to speed up super-resolved imaging of sparse 3D biological objects. We demonstrate the gain in contrast and resolution of EDF-RIM compared to conventional EDF on target samples with simple geometries and developing Drosophila tissues.
    Author(s): Kevin Affannoukoué, Guillaume Maire, Institut Fresnel (France); Thomas Mangeat, Ctr. de Biologie Intégrative, Univ. de Toulouse (France); Simon Labouesse, Institut de Biologie du Développement de Marseille (France); Claire Estibal, Ctr. de Biologie Intégrative (France); Benoît Rogez, Guillaume Giroussens, Loic Legoff, Julien Savatier, Laurent Gallais, Marc Allain, Institut Fresnel (France); Jérome Idier, Lab. des Sciences du Numérique de Nantes (France); Anne Sentenac, Institut Fresnel (France)
    5 April 2022 • 2:30 PM - 2:50 PM CEST
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    Total Internal Reflection Fluorescence Microscopy (TIRFM) exploits an evanescent field induced at the boundary between high and low refractive index media to selectively excite the sample inside a very thin region (from 100 to 300 nm depending on the illumination angle) above the coverslip surface. The minimum exposure of the sample to light above the excitation slice reduces significantly the out-of-focus fluorescence and phototoxicity which are major issues in live-cell imaging. It has become an indispensable tool in biology, in particular to study the molecular traffic at the cell plasma membranes. However, in many applications, the lateral resolution of TIRF, which is diffraction limited to about 300 nm, is not sufficient. In addition, the optical sectioning of the evanescent illumination of TIRF is seldom perfect. Propagative waves stemming from imperfections in the optical train of the instrument and/or light scattering by the sample itself are able to excite the fluorescence in the volume of the sample. When the latter is densely marked, these leaks result in out-of-focus fluorescence which deteriorates the signal to noise ratio. To improve simultaneously the lateral resolution and the image contrast, and to address the difficulties related to the control of the illumination patterns, we propose to adapt the recently developed Random Illumination Microscope (RIM) to the TIR configuration. We show that this approach yields a two-fold resolution gain and ameliorates the image contrast without compromising the ease of use of standard TIRFM. We apply TIRF-RIM to calibrated targets and to fixed and live biological samples with a sub-100nm resolution.
    Author(s): Mathias Mercier, Sophia Imperato, Ecole Supérieure de Physique et de Chimie Industrielles de la Ville de Paris (France); Cynthia Veilly, Fabrice Harms, Imagine Optic SA (France); Alexandra Fragola, Ecole Supérieure de Physique et de Chimie Industrielles de la Ville de Paris (France)
    5 April 2022 • 2:50 PM - 3:10 PM CEST
    Author(s): Jorge Tordera Mora, Liang Gao, UCLA Samueli School of Engineering (United States)
    5 April 2022 • 3:10 PM - 3:30 PM CEST
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    Cameras with extreme speeds are enabling technologies in both fundamental and applied sciences. However, existing ultrafast cameras are incapable of coping with extended three-dimensional (3D) scenes. To address this unmet need, we developed a new category of computational ultrafast imaging technique, light field tomography (LIFT), which can perform 3D snapshot transient (time-resolved) imaging at an unprecedented frame rate with full-fledged light field imaging capabilities including depth retrieval, post-capture refocusing, and extended depth of field. As a niche application, we demonstrated real-time non-line-of-sight imaging of fast- moving hidden objects, which is previously impossible without the presented technique. Moreover, we showcased 3D imaging of fiber-guided light propagation along a twisted path and the capability of resolving extended 3D objects. The advantage of such recordings is that even visually simple systems can be scientifically interesting when they are captured at such a high speed and in 3D. The ability to film the propagation of light through a curved optical path, for example, could inform the design of invisibility cloaks and other optical metamaterials
    Author(s): Chenting Lai, Bernhard Messerschmidt, Sven Flämig, Karl Reichwald, Grintech GmbH (Germany); Hyeonsoo Bae, Tobias Meyer, Leibniz-Institut für Photonische Technologien e.V. (Germany), Institutes für Physikalische Chemie, Friedrich-Schiller-Univ. Jena (Germany); Michael Schmitt, Institutes für Physikalische Chemie, Friedrich-Schiller-Univ. Jena (Germany); Herbert Gross, Institut für Angewandte Physik, Friedrich-Schiller-Univ. Jena (Germany); Orlando Guntinas-Lichius, Universitätsklinikum Jena (Germany); Jürgen Popp, Institutes für Physikalische Chemie, Friedrich-Schiller-Univ. Jena (Germany), Leibniz-Institut für Photonische Technologien e.V. (Germany)
    5 April 2022 • 3:30 PM - 3:50 PM CEST
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    Here, we report a new handheld endoscopic system for nonlinear multimodal imaging of the head and neck region. It has a long rigid endomicroscopic probe with two versions (0° and 45° bended tip), connecting with a compact scan head of approx. 10×12×6 cm3 size. The rigid probe is 6 mm in diameter and 24 cm long, and allows at least 430 µm field of view with a sub-micron resolution for multiphoton imaging. The signal of Coherent anti-Stokes Raman Scattering (CARS), second harmonic generation (SHG), and two-photon excited fluorescence (TPEF) are collected by a non-descanned detection path in the scan head, and the indocyanine green (ICG) can be detected by a confocal descanned configuration. Furthermore, this system is capable of guiding a high-power laser pulse for tissue ablation without the risk of damaging the glass components.
    Author(s): Elias Scharf, Robert Kuschmierz, TU Dresden (Germany); Ronja Stephan, Michael Steinke, Leibniz Univ. Hannover (Germany); Jürgen W. Czarske, TU Dresden (Germany)
    5 April 2022 • 3:50 PM - 4:10 PM CEST
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    Endoscopes with 3D imaging have been known for some time. Additional information about the depth allows further statements during the examination and better visualisation. However, the measuring heads of such endoscopes are bulky, since they must accommodate optics that enable axial scanning. Hence, the field of application remains very limited. We present an endoscope without optics on the distal end and a significantly smaller measuring head in the sub-millimetre range. This enables endoscope technology to be used in new areas of surgery, such as in brain or cochlea. Conventional endoscopes are too large for these regions. A static phase correction has been demonstrated to be sufficient to maintain phase information. Hence, programmable optics like spatial light modulators are no longer needed. Therefore, we applied 3D printed phase masks using 2-photon polymerisation. This allows a robust and cost-efficient system to be realised. In addition to the process of printing phase correction DOEs, we also present a new setup which allows the sample in front of the endoscope head to be imaged through the fibre bundle directly to a camera sensor. No raster scan is required like in past approaches. Hence, an image can be generated in a single shot without further computational reconstruction.
    Session 6: Advanced Microscopy and Imaging II
    6 April 2022 • 8:30 AM - 10:30 AM CEST
    Author(s): Shane Carney, Ting C. Khoo, Anna V. Sharikova, Univ. at Albany (United States); Margarida Barroso, Albany Medical College (United States); Supriya D. Mahajan, Univ. at Buffalo (United States); Jonathan C. Petruccelli, Alexander Khmaladze, Univ. at Albany (United States)
    6 April 2022 • 8:30 AM - 9:00 AM CEST
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    Non-invasive methods of tracking morphological cell changes are based on measurements of phase, which is proportional to the cell thickness and can be extended to measure the optical path length, and, therefore, cell volume. Additionally, Raman micro-spectroscopy is widely used for the mapping of chemical composition within live biological samples, such as cells, organoids, and tissues. It permits non-invasive and non-destructive measurements that do not require special sample preparation processes, such as dye labelling or staining. We have previously reported that we used Raman spectroscopy and Digital Holography Microscopy (DHM) to study cells undergoing apoptosis. Another form of programmed cell death, ferroptosis, is the main cause of tissue damage driven by iron overload and lipid peroxidation. Currently, only invasive cell biological assays are used to monitor the expression level and subcellular location of proteins that are known to bind iron or be involved in ferroptosis. Our group has previously reported a Raman spectroscopic method to visualize and quantify the distribution of multiple iron-binding proteins in intact cells and tissues. Here, we have replaced DHM with another method that is capable of real-time high resolution phase reconstruction. Assembling or altering a system to make the measurements required to solve the Transport-of-Intensity Equation (TIE) is cheaper and easier than implementing the associated DHM setup and measurements. For the full phase retrieval TIE requires only the data collected in the focal plane and in two symmetrically separated planes about the focus. Furthermore, TIE is robust to reduced spatial and temporal coherence, and therefore can potentially achieve a higher resolution than DHM. Since TIE can utilize incoherent sources of illumination, we implemented the TIE setup within an existing Raman microscope, which provided near simultaneous chemical composition and morphological data about the cells.
    Author(s): Jürgen W. Czarske, Nektarios Koukourakis, Stefan Rothe, Felix Wagner, Mike O. Karl, TU Dresden (Germany)
    6 April 2022 • 9:00 AM - 9:30 AM CEST
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    The retina is an epithelium composed of different cell layers with unique optical properties and detects light by photoreceptor neurons for visual function. The quest for suitable measurement methods to detect the health status of retinal tissues is ongoing. We study the capability of the optical transmission matrix, which fully describes the transition of a light field propagating through a scattering sample. Despite its rich information content, the transmission matrix is commonly just used for light delivery through scattering media. Digital holography is employed to measure the transmitted light. We demonstrate that singular value decomposition of the transmission matrix allows to discriminate phantom tissues with varying scattering coefficient. We apply these findings to retinal organoid tissues. Application of an inducer of retinal damage in animals, caused cell death and structural changes in human retinal organoids, which resulted in distinct changes in the transmission matrix. Our data indicate that the analysis of the transmis-sion matrix can distinguish pathologic changes of the retina towards the development of imag-ing-based biomarkers. Laser microscopy of retinal organoid samples from human induced plu-ripotent stem cells is a disruptive technology that promises paradigm shifts for biomedicine.
    Author(s): Irina V. Semenova, Ioffe Institute (Russian Federation); Daria A. Gorbenko, ITMO Univ. (Russian Federation); Anna A. Zhikhoreva, Andrey V. Belashov, Ioffe Institute (Russian Federation); Ilya Litvinov, Tatyana N. Belyaeva, Elena S. Kornilova, Institute of Cytology (Russian Federation); Oleg S. Vasyutinskii, Ioffe Institute (Russian Federation)
    6 April 2022 • 9:30 AM - 9:50 AM CEST
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    In this report we present our analysis of HeLa cells response to photodynamic treatment with 5-ALA-induced endogenously generated photosensitizer protoporphyrin IX (PpIX). Accumulation of PpIX in cells was analyzed by confocal fluorescent microscopy. The cell death dynamics resulting from photodynamic treatment at different doses was monitored by holographic and holotomographic microscopy in two regimes: in cell cultures in vitro during 3 hours after irradiation and on fixed samples obtained in 24 hours after treatment. Changes in cellular parameters: thickness, volume, projected area, dry mass and 3D distribution of refractive index, were monitored and analyzed. The analysis performed allowed for distinguishing between cell death pathways and dynamics, namely for differentiating between necrotic and apoptotic death pathways and for identifying the prevailing pathways at different doses. It was shown that cells response to treatment with PpIX was drastically different in samples cultivated with 5-ALA in serum-containing and serum-free culture media. At the same irradiation doses most of cells in serum-containing samples were characterized by sharp increase in the average cell height and significant decrease in the projected area and volume. This behavior of cellular morphology indicates apoptotic cell death pathway, which is accompanied by cell shrinkage and rounding, nuclear condensation and formation of apoptotic bodies. Whereas in serum-free medium the projected area of cells increased dramatically and their height and volume decreased substantially, that is associated with rupture of cellular membranes and unregulated efflux of intracellular content to extracellular medium, that is characteristic for necrotic pathway. The standard fluorescent analysis performed using acridine orange and ethidium bromide test assay (AO/EB) with further observations in the confocal fluorescence microscope confirmed the results obtained by holographic methods. The AO/EB test on cell membrane integrity confirmed that in general cells in FBS-free samples were more sensitive to photodynamic treatment.
    Author(s): Ilya Balmages, Riga Technical Univ. (Latvia); Janis Liepins, Ernests Tomass Auzins, Anitra Zile, Institute of Microbiology and Biotechnology, Univ. of Latvia (Latvia); Dmitrijs Bliznuks, Riga Technical Univ. (Latvia); Ilze Lihacova, Alexey Lihachev, Institute of Atomic Physics and Spectroscopy, Univ. of Latvia (Latvia)
    6 April 2022 • 9:50 AM - 10:10 AM CEST
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    Our previous studies have shown that it is possible to determine the growth activity of a microbial colony by laser speckle imaging techniques. A subpixel correlation method was proposed to detect small changes in the sequence of laser speckle images. In this study, we compared the laser speckle imaging method to the reference method - image time series under white light to detect the colony growth parameters (growth rate, critical detection time, maximum activity time).
    Author(s): Samer Alhaddad, Houda Bey, Institut Langevin Ondes et Images, Ecole Supérieure de Physique et de Chimie Industrielles de la Ville de Paris, CNRS (France), Univ. PSL (France); Pascale Boulanger, Institut de Biologie Integrative de la Cellule, Univ. Paris-Saclay, CEA, CNRS (France); Claude Boccara, Institut Langevin Ondes et Images, Ecole Supérieure de Physique et de Chimie Industrielles de la Ville de Paris, CNRS (France), Univ. PSL (France); Martine Boccara, Institut Langevin Ondes et Images, Ecole Supérieure de Physique et de Chimie Industrielles de la Ville de Paris, CNRS (France), Univ. PSL (France), Institut de biologie de l'Ecole Normale Supérieure (France); Ignacio Izeddin, Institut Langevin Ondes et Images, Ecole Supérieure de Physique et de Chimie Industrielles de la Ville de Paris, CNRS (France), Univ. PSL (France)
    6 April 2022 • 10:10 AM - 10:30 AM CEST
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    Interferometric microscopy techniques have been recently developed to detect small particles at the nanoscale without the need for specific labeling. Here, we introduce a highly sensitive, label-free approach on a common path interferometric set-up working on a transmission that amplifies weak scattering signals coming from small particles. Using single-particle tracking analysis, we can track and differentiate viruses and other biotic particles in an aquatic environment. Furthermore, we have developed a fast assay based on antibody recognition of targeted virus in solution. We associate changes in diffusion and in the interferometric signal of the detected particles with the immune reaction between antibodies and surface proteins of the virus.
    Session 7: Advanced Microscopy and Imaging III
    6 April 2022 • 11:00 AM - 1:00 PM CEST
    Author(s): Volodymyr N. Borovytsky, National Technical Univ. of Ukraine “Igor Sikorsky Kyiv Polytechnic Institute” (Ukraine)
    6 April 2022 • 11:00 AM - 11:30 AM CEST
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    The mathematical technique for calculation of the three-dimensional spatial bandwidth of optical systems, in particular – in optical microscopes, is presented and discussed. This technique is extension of Abbe theory from two-dimensional to three-dimensional space. An optical system that forms an image of a grating in an object space is studied. There are introduced various inclinations of this grating relatively the optical axis round the axis in the object plane that is parallel to its slits. In this case, the minimum resolvable period and the corresponded spatial cut off frequency of the optical system may be calculated using the analytical functions of the angle of grating inclination, the numerical apertures and the wavelength using the idea of Abbe theory. This function for calculation of the spatial cut off frequencies describes a surface in the three-dimensional space of spatial frequencies that covers the three-dimensional spatial bandwidth. The spatial harmonics with spatial frequencies inside this spatial bandwidth can pass through an optical system and they participate in formation of three-dimensional images. The other spatial harmonics cannot pass through it. This approach makes understanding and explanation of formation of three-dimensional images by optical systems clearly and logically completed.
    Author(s): Boris S. Gurevich, Kirill V. Zaitchenko, Institute for Analytical Instrumentation (Russian Federation)
    6 April 2022 • 11:30 AM - 11:50 AM CEST
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    The resent development of the modern optical-electronic, especially spectroscopic technologies opened the possibilities of multispectral processing image processing for the analysis of the state of different biological objects. The human skin is among such objects. Fast foundation and diagnostics the skin lesions are the very actual problem of the modern medicine. The applied nowadays spectroscopic methods of the skin cancer diagnostics have some deficiencies. In order to eliminate the mentioned above deficiencies, we have proposed to realize the skin lesion sub-images multispectral processing method. Our approach means the conversion of the initial polychromic image into the series of the monochromic sub-images, each of which represents the light intensity distribution at one of the selected wavelengths. Hence, the bigger number of the wavelengths we use, the larger amount of the spectral information can be obtained regarding the studied bio-object. The functional circuit of the installation is represented in our talk as well as the results of the experiments carried out at it. For example, the experiments of the hand skin areas images multispectral processing at different persons in the light spectral range of 450…815 nm. The experiments results confirmed the operation ability of the installation as well as possibility to form the necessary set of the studied skin areas sub-images. The results showed that the considered method can reliably define the spectral composition of light reflected from the human skin with the lesion. It allows to suppose that it is possible due to the obtained redundant spectral information to recognize the malignant lesion character at the early stage of its development.
    Author(s): Haider Al-Juboori, Institute of Technology Carlow (Ireland); Tom D. McCormack, Univ. College Dublin (Ireland)
    6 April 2022 • 11:50 AM - 12:10 PM CEST
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    A systematic investigation of the expansion dynamics of plasma plumes generated by two Q-switched Nd:YAG lasers at 1064 nm wavelength operating on homogenous and heterogeneous targets was undertaken using a technique involving fast-gated intensified charge-coupled device imaging. Our experiments present the results on the temporal, spatial and semi-spectrally imaging of colliding plasmas of aluminium and silicon targets. The aim of the work presented here is to further advance and study colliding plasma techniques, as well as other methods to realize and control species density and expansion, with a view to a deep understanding of these complex mechanisms and optimize emission in the visible wavelength range. The analysis is focused on describing the velocity of the expanding plasma front for the interaction zone where the present results found the expansion velocity of the station layer increases with time additionally the fact that the laser energy reduces the velocity. All investigations focus on studies of CLPP characterizations formed on wedge-shaped targets where the angle of the wedge varies from 180o to 80o. The target materials were Aluminum and Silicon in both homogenous and heterogeneous mixtures. Time-resolved emission imaging was employed to track the size, shape and velocity of the stagnation layer which might act as a signature of hard versus soft stagnation. Moreover, this work investigated the effect on the homogeneity of the stagnation layer with the target angle. The analysis suggests that there is significant collisional reheating of the stagnation layer followed by radiative recombination as well as this study provides a considerable amount of detailed data related to expansion velocity of the interaction zone which extends the understanding of the behaviour of particular species within colliding laser-produced plasmas.
    Author(s): Claire Lefort, Erwan Ferrandon, XLIM (France); Cecile McLaughlin, Univ. de Limoges (France), Ctr. de Recherches Sémiotiques (France); Sophie Alain, Univ. de Limoges (France), RESINFIT UMR-S 1092, INSERM (France), Ctr. Hospitalier Univ. de Limoges (France)
    6 April 2022 • 12:10 PM - 12:40 PM CEST
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    We are presenting the application of an optical and computational pipeline FAMOUS for revealing the presence of free viral particles named “virions” [1]. The idea of such a protocol is to give rise to images of virions in their environment with a soft solution for recording the native image, contrary to the standard solution of imaging virions with electron microscopy (EM) for visualizing viral particles. Indeed, EM is a highly destructive method due to the vacuum required, associated with electron beam. For this first demonstration, the chosen free virions are cytomegalovirus (CMV), a virus from the herpesvirus family also named “Human HerpesVirus 5”, with two kinds of cultures: a fluorescent culture (BAD) and a label-free one (VHLE), both being collected from infected cell culture. These virions are interesting for our demonstration thanks to their dimensions: between 150 and 300 nm of diameter, at the limit of the standard optical resolution [2]. On the one hand, VHLE virions were observed after double immunostaining and concentrated with magnetic nanoparticles. Then, VHLE emission signals are recorded without labelling with a multiphoton optical process: two-photon fluorescence [3). The optical protocol rests on a standard solution of multiphoton microscopy combined with a computational strategy based on the point-spread-function (PSF) recording, its mathematical estimation and the restauration of the image resting on the PSF evaluation. The resulting images are then compared to images recorded from EM. The visualization of objects aggregates obtained in both situations confirm the relevance of the pipeline FAMOUS for imaging free virions. [1] C. Lefort, M. Chalvidal, A. Parenté, V. Blanquet, H. Massias, L. Magnol, E. Chouzenoux, “FAMOUS: a fast instrumental and computational pipeline for multiphoton microscopy applied to 3D imaging of muscle ultrastructure”, J. Phys. D : Appl. Phys, 54 (2021) 274005 (2021) [2] M.K. Gandhi and R. Khanna, “Human cytomegalovirus: clinical aspects, immune regulation, and emerging treatments”, The Lancet, 4, 725-738 (2004) [3] A. M. Larson, “Multiphoton microscopy”, Nature Photonics, 5, 1 (2011) [4] B. Zhang, J. Zerubia, J.-C. Olivo-Marin, “Gaussian approximations of fluorescence microscope point-spread function models”, Applied Optics, 46 (10), 1819-1829 (2007)
    Author(s): Laura Sironi, Univ. degli Studi di Milano-Bicocca (Italy); Claudio Conci, Lorenzo Gentili, Politecnico di Milano (Italy); Mario Marini, Univ. degli Studi di Milano-Bicocca (Italy); Margaux Bouzin, Univ. degli Studi di Milano Bicocca (Italy); Rebeca Martínez Vázquez, CNR-Istituto di Fotonica e Nanotecnologie (Italy); Emanuela Jacchetti, Manuela Teresa Raimondi, Giulio Cerullo, Politecnico di Milano (Italy); Roberto Osellame, CNR-Istituto di Fotonica e Nanotecnologie (Italy), Politecnico di Milano (Italy); Maria Farsari, Elmina Kabouraki, Foundation for Research and Technology-Hellas (Greece); Anthi Ranella, Institute of Electronic Structure and Laser, Foundation for Research and Technology-Hellas (Greece); Laura D'Alfonso, Univ. degli Studi di Milano-Bicocca (Italy); Nikos Kehagias, Nanotypos (Greece); Maddalena Collini, Giuseppe Chirico, Univ. degli Studi di Milano-Bicocca (Italy)
    6 April 2022 • 12:40 PM - 1:00 PM CEST
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    The current protocols for biocompatibility assessment of biomaterials, based on histopathology, require the sacrifice of a huge number of laboratory animals with an unsustainable ethical burden and remarkable cost. Intravital microscopy techniques can be used to study implantation outcomes in real time though with limited capabilities of quantification in longitudinal studies, mainly restricted by the light penetration and the spatial resolution in deep tissues. We present the outline and first tests of a novel chip which aims to enable longitudinal studies of the reaction to the biomaterial implant. The chip, which we call In2Sight, is composed of a regular reference microstructure and a set of microlenses, both fabricated via two-photon polymerization in the SZ2080 resist. The geometrical design and the planar raster spacing largely determine the mechanical and spectroscopic features of the microstructures. The development, in-vitro characterization and in vivo validation of the In2Sight chip is performed in living chicken embryos by fluorescence microscopy 3 and 4 days after the implant; the quantification of cell infiltration inside the In2Sight chip demonstrates its potential as novel scaffold for tissue regeneration. Arrays of two-photon polymerized microlenses were first characterized for the optical point spread function under one and two-photon excitation and then tested for the effective focal length in ex-vivo tissue in dependence of the laser writing parameters and the surface shape and roughness.
    Session 8: Multiphoton Microscopy I
    6 April 2022 • 2:10 PM - 3:10 PM CEST
    Author(s): YuHao Tseng, National Yang Ming Chiao Tung Univ. (Taiwan); Yvonne Yuling Y. Hu, National Cheng Kung Univ. (Taiwan); Chia-Wei Hsu, Chun-Yu Lin, National Yang Ming Chiao Tung Univ. (Taiwan); Hsueh-Cheng Chiang, National Cheng Kung Univ. (Taiwan); Shean-Jen Chen, National Yang Ming Chiao Tung Univ. (Taiwan), National Applied Research Labs., Taiwan Instrument Research Institute (Taiwan)
    6 April 2022 • 2:10 PM - 2:30 PM CEST
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    Temporal focusing multiphoton excitation microscopy (TFMPEM) with a diffraction grating to disperse the laser pulse and a 4-f system composed of a high numerical aperture objective lens to recombine the pulse to achieve widefield imaging with optical sectioning at the focal plane, which is capable of fast 3D imaging while maintaining both lateral and axial resolutions compared to the traditional point-scanning method. However, the plane illumination manner suffers from the sever scattering of biotissue and signal crosstalk that blurs the image. Nevertheless, the high acquisition rate decreases the effective excited fluorescent, which reduces the signal-to-noise (SNR) ratio of the image. Moreover, the intrinsic noise of the electronic device under high bit rate, which is often underestimated. In order to solve the scattering and low SNR issues, the deep learning method is proposed. A multi-stage U-Net architecture consists of two 3D U-Nets were utilized. Unlike a cascade scheme, the multi-stage method works parallelly by sharing the feature map in the different stages. Briefly, the images with higher quality acquired via point-scanning (PS) manner was used as the ground truth in the network training, while inputs were the highly scattered images acquired via TFMPEM in 100 frames per second (fps). Before the training, registration must be done since the two utilized schemes were not sharing the same optical system. A 3D image registration network was utilized to find the global deformation vector field to match the image shift between the two modalities. This deep-learning based methodology works more flexible than traditional methods, such as scale invariant feature transform, and provides faster registration with higher precision. The peak signal-to-noise ratio (PSNR) is used to evaluate the quality of the restored images by comparing with the ground truth PSNR images. The PSNR of the original TFMPEM images are less than 10 dB, while the PSNR of the restored TFMPEM images can be enhanced to 30~40 dB, which show a significant improvement using the multi-stage 3D U-Net image registration. Hence, the proposed method shows the feasibility of TFMPEM image restoration with enhanced image quality while keeping the high frame rate advantage of TFMPEM system.
    Author(s): Viktoras Mažeika, Mykolas Maciulis, Laser Research Ctr., Vilnius Univ. (Lithuania); Lukas Kontenis, Laser Research Ctr., Vilnius Univ. (Lithuania), Light Conversion Ltd. (Lithuania); Edvardas Zurauskas, Vilnius Univ. (Lithuania); Martynas Riauka, Mehdi Alizadeh, Laser Research Ctr., Vilnius Univ. (Lithuania); Kamdin Mirsanaye, Univ. of Toronto (Canada); Virginijus Barzda, Laser Research Ctr., Vilnius Univ. (Lithuania), Univ. of Toronto (Canada)
    6 April 2022 • 2:30 PM - 2:50 PM CEST
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    Extracellular matrix (ECM) has important functions in cell proliferation, differentiation, and migration, which influence the development and progression of cancer. ECM in tumor microenvironment experiences changes in composition and structure that can appear early in tumor development and could serve as a biomarker for cancer diagnostics. In addition, some changes in ECM may correlate with the rate of tumor progression or its tendency to form metastases and would allow to predict future tumor development [1]. Collagen is an important structural protein found in ECM. It has a non-centrosymmetric structure, and, thus, can be easily visualized using second harmonic generation (SHG) microscopy. SHG microscopy employs certain polarimetric techniques to gain detailed information about the organization of collagen in various tissues [2]. In this work, polarimetric SHG microscopy is used to acquire collagen images from normal and cancerous regions of human colon and pancreas histological samples. Texture analysis is performed on SHG intensity and polarization images to characterize the distribution of ultrastructure parameters in the tissue. Significant differences are observed in collagen ultrastructure between normal and tumor areas. Further, collagen structures of colon and pancreas tumor microenvironments are compared to investigate relative differences in ECM organization between the tissues. Finally, a machine learning classifier is used to group the acquired images in tumor and normal groups. The results show potential for development of novel cancer diagnostic technique using polarimetric second harmonic generation microscopy and texture analysis. [1] Winkler, J. et al., “Concepts of extracellular matrix remodelling in tumour progression and metastasis”, Nat Commun 11, 5120 (2020). [2] Golaraei, A. et al., “Polarimetric second-harmonic generation microscopy of the hierarchical structure of collagen in stage I-III non-small cell lung carcinoma,” Biomed. Opt. Express 11, 1851-1863 (2020).
    Author(s): Martynas Riauka, Viktoras Mazeika, Mykolas Maciulis, Vilnius Univ. (Lithuania); Lukas Kontenis, Light Conversion Ltd. (Lithuania); Edvardas Zurauskas, Mehdi Alizadeh, Vilnius Univ. (Lithuania); Kamdin Mirsanaye, Virginijus Barzda, Univ. of Toronto (Canada)
    6 April 2022 • 2:50 PM - 3:10 PM CEST
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    Development and metastasis of cancer are known to change the structure of extracellular matrix (ECM), which affect the tumor's further growth and spread. A substantial part of ECM is comprised of collagen, which is a noncentrosymmetric structure. As a result, it generates second harmonic signals, dependent on the polarization of incoming light. This property of collagen led to the applications of polarization-resolved second-harmonic generation (P-SHG) microscopy in investigating collagen ultrastructure changes in different cancers. In this work, multiphoton absorption fluorescence (MPF), third-harmonic generation (THG) and polarimetric second-harmonic generation (P-SHG) measurements were performed on various types and staging of human melanoma histological sections. Reduced polarimetry techniques, employing linear and circular polarization states, were used to obtain polarimetric SHG parameters of collagen in both normal and cancerous tissues. These parameters provide important information about the structural properties of collagen. The parameter distributions were analyzed using a grey-level co-occurrence matrix (GLCM), which allows to obtain statistical parameters, such as correlation, contrast, entropy, angular second moment and inverse difference moment. Statistical tests were performed on polarimetric and texture analysis data in order to determine whether parameter distribution differences in normal and cancerous tissues are statistically significant. Furthermore, a machine learning classifier algorithm was trained to distinguish normal tissues from cancerous using aforementioned polarimetric and texture parameters as predictors. Firstly, separate training and testing datasets were formed from each sample and classification was carried out for each of them individually and afterwards, a common training dataset was used for all samples. The results suggest that normal and cancerous skin tissues can be distinguished from each other with the help of multimodal nonlinear polarimetric microscopy. Also, depending on the type and stage of melanoma, the differences in some polarimetric and texture parameters are more pronounced, suggesting its possible application in melanoma diagnostics and differentiation.
    Session 9: Optical Coherence Tomography
    6 April 2022 • 3:40 PM - 5:50 PM CEST
    Author(s): Yan Yan, Jose Galaz, Maryam Basij, Nardhy Gomez-Lopez, Mohammad Mehrmohammadi, Wayne State Univ. (United States)
    6 April 2022 • 3:40 PM - 4:10 PM CEST
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    A sonographic short cervix is a major risk factor for spontaneous preterm birth (PTB). However, the cervical length is a suboptimal means to assess cervical status due to the lack of functional and molecular information. Spectroscopic photoacoustic (sPA) imaging is a non-invasive ultrasound-based technology for assessing cervical tissue compositions, such as collagen-to-water ratio (CWR), which are the major molecular changes during cervical ripening. A longitudinal CWR measurement by sPA was performed in murine cervices (n=3 per group) through the gestational ages from non-pregnant, 13.5 to 19.5, 6 to 12 hours, and 69 to 94 hours postpartum. The sPA data acquisition was performed in a range of wavelengths covering the peak absorption of collagen and water (1100 to 1650 nm) with an amplified sPA wavelength unmixing method (sPA-CWR). Our imaging results indicated that the sPA-CWR method is capable of accurately quantifying cervical tissue composition changes during the process of cervical remodeling. The sPA-CWR measurements reveal that non-pregnant murine cervical samples have significantly higher CWR than any other tissue group. Furthermore, a decrement in CWR at larger gestational ages was detected, which follows the cervical ripening process. In addition, the repair process was detected through increased CWR in tissue samples collected 6-12 hours postpartum and completing their recovering process at about 69 to 94 hours postpartum. Finally, the imaging results were validated by quantitative histological analysis. The imaged tissue samples were stained by Hematoxylin and eosin (H&E) and Picrosirius Red stains (PSR) to compare the collagen content by calculating the averaged collagen fiber area and total optical density. These histological results confirm that the sPA-CWR measurements have a high correlation to the process of collagen reorganizing.
    Author(s): Ralph-Alexandru Erdelyi, Univ. Politehnica Timisoara (Romania); Virgil-Florin Duma, Univ. "Aurel Vlaicu" din Arad (Romania); Cosmin Sinescu, Univ. de Medicina si Farmacie "Victor Babes" din Timisoara (Romania); George M. Dobre, Adrian Bradu, Adrian G. H. Podoleanu, Univ. of Kent (United Kingdom)
    6 April 2022 • 4:10 PM - 4:30 PM CEST
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    Dental imaging combines several methods that provide specific images of hard and soft tissue. Each method and corresponding system should be improved to achieve high-quality dental images in order to help the medical doctor to diagnose or to assess a patient’s treatment. Commonly, X-ray imaging such as intraoral radiography, panoramic radiography of three-dimensional cone-beam computed tomography (3D CBCT) are utilized. Other possibilities include smart devices that spots cavities (i.e., Diagnocam or Diagnopen), micro-CT, or optical coherence tomography (OCT). The aim of this paper is to present the protocol we developed to improve the quality of X-ray imaging using OCT. This optimization comprises the adjustment of X-ray unit settings (i.e., radiation dose, current intensity, and voltage) by focusing on certain dental details visible only with the superior resolution technique, OCT. A trade-off must be reached, as according to the ALARA protocol, X-ray radiation must be as low as possible while preserving an as high as possible image quality. Therefore, there is a limitation of X-ray settings in terms of patient safety. The optimization protocol was carried out ex vivo, and then the results were used in vivo, for each type of dental investigation. To make sure the procedure is accurate, we compared radiographs before and after optimization. Also, a comparison between X-ray units with potential resolution of 200 to 75 µm was possible using OCT images with 10 µm axial resolution. In conclusion, OCT can be applied to improve the quality of each type of dental imaging investigation. Also, in certain situations when X-ray imaging cannot provide relevant images for dental diagnose or treatment assessment, OCT proves capable to obtain such images.
    Author(s): Thomas B. Rockett, Nada A. Adham, Matthew Carr, John P. R. David, Robert D. Richards, The Univ. of Sheffield (United Kingdom)
    6 April 2022 • 4:30 PM - 4:50 PM CEST
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    GaAsBi has attracted research for near-infrared (NIR) optoelectronics because bismuth incorporation causes a far greater band gap reduction per unit strain than indium incorporation. The bismuth atoms induce the formation of many localised states near the valence band maximum, which can take part in radiative transitions and result in a large broadening of the luminescence spectrum. The large linewidths observed in GaAsBi are typically seen as a disadvantage of the material and researchers have focussed on reducing the density of localised states. Superluminescent light emitting diodes with peak emission centred around 1050 nm are useful for ophthalmology applications such as OCT since these wavelengths are less strongly absorbed by ocular media. In this case, a large LED spectral linewidth leads to an improved axial resolution in OCT, enabling better imaging and subsequent analysis by doctors. Commercial LED based OCT light sources operating at 1050nm rely on emission from both ground and excited states in InGaAs quantum wells and have a linewidth around 70nm. State-of-the-art OCT light sources based on multiple layers of InAs self-assembled quantum dots have achieved linewidths of 160nm. Existing unoptimised GaAsBi single quantum well structures grown in our group by molecular beam epitaxy with a peak wavelength of 1050nm have a spectral linewidth of around 67 nm, which nearly matches the commercial LEDs used for OCT. This is despite our devices only containing emission from the ground state in the quantum wells. With careful control of the bismuth content and well thickness in future devices, the linewidth of GaAsBi based devices could match or exceed the state-of-the-art for NIR broadband light sources. In this work we study the applicability of GaAsBi quantum well LEDs as a competitor to InGaAs quantum well and InAs quantum dot LEDs for broadband NIR light sources. We show simulations of LED structures to find the optimum LED design parameters that will give the broadest linewidth centred on 1050nm while retaining an approximately Gaussian emission shape. The growth challenges associated with growing the structures are also discussed.
    Author(s): Valentin V. Demidov, Geisel School of Medicine, Dartmouth College (United States); Megan Clark, Petr Bruza, Thayer School of Engineering at Dartmouth (United States); I. Leah Gitajn, Dartmouth-Hitchcock Medical Ctr. (United States); Jonathan T. Elliott, Geisel School of Medicine, Dartmouth College (United States)
    6 April 2022 • 4:50 PM - 5:10 PM CEST
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    High-energy orthopedic injuries causing fractures of lower limbs, especially tibias, are prone to infection and healing complications, thus making their management challenging and increasing the patient’s risk of adverse outcomes. Further studies involving clinically translatable animal models of high-energy tibia trauma are needed to evaluate treatment options to improve the outcomes. As most of such models utilize a blunt guillotine principle, they can accurately break the bone but cause minimal soft-tissue injury with minimal complication rates. Taking the next step, we propose a novel approach to controlled tibia injury with severely damaged soft tissue around it. For this, we utilize the principle of compressed air-driven membrane rupture as the blast wave source. We developed a miniaturized model of a MacMillan blast device consisting of a cylindrical tube, separated into compression and expansion chambers by Mylar membrane. The compression chamber is continuously filled with air until the Mylar membrane spontaneously ruptures to create a shock wave propagating through the expansion chamber to hit the fixed animal leg at the open end. After blast tube calibration and tests on cadavers, anesthetized rats were used for in-vivo experiments. Various peak overpressure blasts were tested to determine what pressures caused the desired severity of injury and could be salvageable such that the animal could be repaired surgically and recovered in a humane way with the use of a multidrug analgesia plan. For this, fractured and surrounding bones were 3D-rendered with micro-CT and soft-tissue and bone perfusion were evaluated with fluorescence-guided imaging and optical coherence tomography. The optimal peak overpressure of 175kPa was found to create an open injury with complete tibia bone break and severe but manageable surrounding tissues injury. This carefully designed reproducible high-energy trauma model opens new, exciting ways to the incorporation of survival and infection models, as well as various treatment evaluations.
    Author(s): Yogeshwari S. Ambekar, Manmohan Singh, Alexander W. Schill, Univ. of Houston (United States); Jitao Zhang, Univ. of Maryland (United States); Christian Zevallos Delgado, Behzad Khajavi, Salavat R. Aglyamov, Univ. of Houston (United States); Giuliano Scarcelli, Univ. of Maryland, College Park (United States); Kirill V. Larin, Univ. of Houston (United States)
    6 April 2022 • 5:10 PM - 5:30 PM CEST
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    A multimodal, co-aligned imaging system combining optical coherence tomography (OCT) and Brillouin microscopy was developed. The main purpose of this study was to simultaneously image structure as well as the biomechanical properties of embryonic neurulation process in a murine model. Many complex processes are involved during embryogenesis, such as neurulation, which is the formation the neural tube. Mechanical forces play a major role during neurulation, and any disturbance can lead to severe birth defects such as spina bifida, which often results in lifelong disabilities. Thus, it is very important to study the interplay between forces and tissue stiffness during development. OCT and Brillouin microscopy are noninvasive high-resolution optical imaging modalities, where OCT provides structural information and Brillouin microscopy is capable of mapping tissue stiffness. Brillouin microscopy lacks the ability to provide structural images which limits its capabilities for imaging dynamic processes such as neurulation. To overcome the individual limitations, we have combined OCT with Brillouin microscopy in one synchronized and co-aligned instrument. We developed a custom instrumentation software that utilizes the OCT structural image to guide Brillouin imaging. 2D structural and mechanical maps of mouse embryos at gestational day (GD) 8.5 and 9.5 were acquired with the multimodal system. Brillouin microscopy showed the cell-dense layer of neural plate derived from the ectoderm at GD 8.5, which could not differentiate with OCT alone. At GD 9.5, the neuroepithelium could be clearly seen by Brillouin microscopy with a greater stiffness than the surrounding tissue and had slightly greater scattering surrounding tissue seen in the OCT image. The multimodal OCT and Brillouin system performed co-registered structural and function imaging of neural tube of mouse embryos.
    Author(s): Abdulrahman Aloraynan, Univ. of Waterloo (Canada), Umm Al-Qura Univ. (Saudi Arabia); Shazzad Rassel, Chao Xu, Dayan Ban, Univ. of Waterloo (Canada)
    6 April 2022 • 5:30 PM - 5:50 PM CEST
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    Despite the recent outstanding development, machine learning (ML) has not been utilized in MIR and PA spectroscopy for noninvasive glucose detection. ML models can assist in improving the detection sensitivity to meet FDA requirements. Furthermore, the employment of ML can help to solve the complexity of detecting glucose in the presence of different blood components or at various environmental conditions. In noninvasive optical spectroscopy, ML models can be developed to distinguish glucose signals despite the variations in human skin properties for \textit{in vivo} measurements. Different ML classification algorithms have been developed and employed to detect glucose levels using MIR-infrared photoacoustic spectroscopy. The photoacoustic system has been developed using a single wavelength quantum cascade laser, lasing at a glucose fingerprint of 1080 $cm^{-1}$ for noninvasive glucose monitoring. Artificial skin phantoms have been prepared as test models for the system with different glucose concentrations, covering the normal and hyperglycemia blood glucose ranges. Support vector machine, narrow neural network, and medium neural network algorithms have achieved high prediction accuracy to classify glucose levels.
    6 April 2022 • 5:40 PM - 7:30 PM CEST
    Conference attendees are invited to attend the Photonics Europe poster session on Wednesday evening. Come view the posters, enjoy light refreshments, ask questions, and network with colleagues in your field.

    Poster Setup: Wednesday 10:00 AM – 5:00 PM
    View poster presentation guidelines and set-up instructions at
    Author(s): Loredana Buzura, Univ. Tehnica din Cluj Napoca (Romania); Horea Demea, OftaReview Ctr. de Investigatii (Romania); Monica Loredana Budileanu, Radu Papara, Ramona M. Galatus, Univ. Tehnica din Cluj Napoca (Romania)
    6 April 2022 • 5:40 PM - 7:30 PM CEST
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    Age-related macular degeneration (AMD) is the leading cause of permanent vision loss and visual impairment in people over 60. Early detection of the disease is essential to prevent the evolution of the disease into an advance stage. An eye care specialist has to perform dilated eye exam, fundoscopy, a visual acuity test and fundus photography to determine if a patient has macular degeneration and the stage of the disease. Most of equipment used nowadays in eye care clinics are equipped in one system with both fundus camera and OCT technology that provides more comprehensive clinical evaluations. In most countries the healthcare system suffers from a low doctor to patients ratio; do to it, diagnosis can become time consuming and error-prone. To minimize this downfall, a computer aided diagnosis (CAD) strategy is proposed using machine learning techniques to predict the presence of age-related macular degeneration using both OCT and fundus images. The computer aided diagnosis (CAD) is using a portable device, Jetson TX2 board, a powerful AI computer device, to predict the presence of an abnormality in the retina. A dataset composed of four categories: normal retina, dry AMD, wet AMD and drusen from both OCT and fundus images has been used to evaluate the performance of different neuronal networks. Cost reduction and system portability is implemented with the proposed system for point of care in ophthalmology applications.
    Author(s): Daria A. Gorbenko, ITMO Univ. (Russian Federation); Andrey V. Belashov, Irina V. Semenova, Ioffe Institute (Russian Federation); Iliya K. Litvinov, Institute of Cytology (Russian Federation); Oleg S. Vasyutinskii, Ioffe Institute (Russian Federation); Tatyana N. Belyaeva, Anna V. Salova, Institute of Cytology (Russian Federation)
    6 April 2022 • 5:40 PM - 7:30 PM CEST
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    We present research on photodynamic treatment HeLa cells with 5-ALA-induced PpIX. Samples of HeLa cells were incubated for 3 hours with 5-ALA in the absence of FBS in the culture medium. The intracellular localization of the generated PpIX was determined, and combinations of the experimental parameters triggering cell death were identified. The dynamics of cell death was investigated by digital holographic tomography. The observation was carried out continuously for 24 hours after irradiation. It was shown that in the absence of FBS in the cell culture medium during incubation with 5-ALA, cells demonstrate signs of necrotic death at the earliest stages. Notable inappropriate protein aggregation was observed in the perinuclear space at practically every time/dose combination. We suppose that the observed morphological deformation of cell nuclei was due to an ER stress. The density of aggregated proteins was estimated from the data obtained by the analysis of segmented phase images.
    Author(s): Anna A. Zhikhoreva, Andrey V. Belashov, Ioffe Institute (Russian Federation); Iliya Litvinov, Anna V. Salova, Tatyana N. Belyaeva, Elena S. Kornilova, Institute of Cytology (Russian Federation); Irina V. Semenova, Oleg S. Vasyutinskii, Ioffe Institute (Russian Federation)
    6 April 2022 • 5:40 PM - 7:30 PM CEST
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    The paper presents investigation of accumulation, localization, photobleaching times, and fluorescence lifetimes of Radachlorin photosensitizer (PS) in HeLa cells. Experiments were performed at different incubation times in the Radachlorin-containing culture medium. Intracellular localization of the photosensitizer was studied by its colocalization with standard fluorescence tracers of lysosomes and mitochondria and the analysis by means of confocal fluorescence microscopy. The predominant accumulation of the PS in lysosomes was demonstrated. Lifetimes of Radachlorin fluorescence in different cell areas were analyzed by the FLIM System. Fluorescence lifetimes and photobleaching times were analyzed depending upon the PS localization and incubation duration.
    Author(s): Irina Matveeva, Oleg Myakinin, Yulia A. Khristoforova, Lyudmila A. Bratchenko, Ivan A. Bratchenko, Samara Univ. (Russian Federation); Alexander A. Moryatov, Sergey V. Kozlov, Samara State Medical Univ. (Russian Federation); Valery P. Zakharov, Samara Univ. (Russian Federation)
    6 April 2022 • 5:40 PM - 7:30 PM CEST
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    Cancer is a leading cause of death worldwide, and one of the most common in 2020 (in terms of new cases of cancer) was skin cancer. Raman spectroscopy has been increasingly used to diagnose skin neoplasms. The main purpose of the research is to study the possibilities of applying the multivariate curve resolution–alternating least squares (MCR-ALS) algorithm for the analysis of Raman spectra. In the research, we used in vivo Raman spectra of normal skin, basal cell carcinoma (BCC), malignant melanoma (MM) and pigmented nevus (PN). The Raman spectra was recorded using a portable spectroscopic setup which includes an excitation laser source with 785 ± 0.1 nm central wavelength. After pre-processing, which includes baseline removal, smoothing by the Savitzky‐Golay method and data normalization, Raman spectra was decomposed into several pure component Raman spectra and concentration profiles for each of the components. As a result of applying the MCR-ALS algorithm to in vivo Raman spectra, it was possible to reconstruct a number of spectra of individual components corresponding to the components of human skin tissue, as well as components associated with fluorescence and the optical system. The results of the study can be used in the field of medical diagnostics to analyze the Raman spectra of skin tissue. In addition, the ability to distinguish the components associated with the optical system and the fluorescence signal makes it possible to use this algorithm for preprocessing experimental Raman spectra.
    Author(s): Lyudmila A. Bratchenko, Ivan A. Bratchenko, Yulia A. Khristoforova, Samara Univ. (Russian Federation); Daria Y. Konovalova, Alexander A. Moryatov, Sergey V. Kozlov, Peter A. Lebedev, Samara State Medical Univ. (Russian Federation); Valery P. Zakharov, Samara Univ. (Russian Federation)
    6 April 2022 • 5:40 PM - 7:30 PM CEST
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    Various physiological and pathological processes occurring in the human body affect the component composition of body tissues. Therefore, the study of the metabolic profile of the human body tissues can provide information about the state of the body and possible pathological-associated changes in it. The aim of this work is in vivo study of skin spectral-characteristics at various physiological conditions by conventional Raman spectroscopy in near infrared region. The study included an analysis of metabolic changes in the skin in kidney failure, in skin cancer and during pregnancy. Comparative multivariate analysis of experimental data was carried out on the basis of discriminant analysis with the projection on latent structures (PLS-DA, basic solution without involving deep learning) and on the basis of one-dimensional convolutional neural networks (CNN, solution using deep learning). Application of Raman spectroscopy to investigate the forearm skin in 85 adult patients with kidney failure (90 spectra) and 40 healthy adult volunteers (80 spectra) has yielded the accuracy of 0.96, sensitivity of 0.94 and specificity of 0.99 in terms of identifying the target subjects with kidney failure. When classifying subjects by the presence of kidney failure using the PLS-DA method, the most informative Raman spectral bands are 1315-1330 cm-1, 1450-1460 cm-1, 1700-1800 cm-1. When analyzing the skin in the presence of oncopalotology, the obtained results for different classification tasks demonstrates that CNN significantly (p<0.01) outperforms PLS-DA. The demonstrated performance of CNN classification of skin tumors based on low SNR Raman spectra analysis is higher or comparable to the accuracy of trained dermatologists. The proposed approach may be combined with other optical techniques of skin lesion analysis, such as dermoscopy- and spectroscopy-based computer-assisted diagnosis systems to increase accuracy of neoplasms classification.
    Author(s): Anastasia A. Shatskaya, Yulia А. Frolova, Dmitry N. Artemyev, Samara Univ. (Russian Federation)
    6 April 2022 • 5:40 PM - 7:30 PM CEST
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    We propose the development design of a miniature fiber optic probe for the implementation of the Raman spectroscopy method in endoscopic applications. Optical model of a fiber optic probe was designed using special software. Fiber probe construction was adapted for endoscopic applications; optimization was performed to minimize the signal power losses of the main units of the optical system; tolerance analysis of the placing of optical elements and fibers was carried out. Also, analysis of the spectral deformations of Raman radiation was made during transportation through the optical scheme of the probe. We experimentally tested the developed design of the fiber optic probe. The results of this work can serve as a guideline for optimizing the schemes of fiber-optic Raman spectroscopy devices for applications in biomedicine.
    Author(s): Evgeniy Popov, Anton Polishchuk, Artem Tabarov, Valeriia Kurikova, Anton V. Kovalev, Vladimir V. Vitkin, ITMO Univ. (Russian Federation)
    6 April 2022 • 5:40 PM - 7:30 PM CEST
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    We demonstrate the possibility of detecting the presence of the bacterium Helicobacter pylori in the patient by measuring the change in the concentration of 13СО2 in the exhaled breath using Raman spectroscopy. The concentration of gas mixtures of known composition was measured and the spectrum of air exhaled by a person was measured. The measurement was carried out using a Raman spectrometer consisting of a continuous 532 nm laser, with 5 W output power; a spectrometer made according to the Czerny-Turner scheme and a CCD camera cooled down to -40°C. Gas mixtures with a concentration of 12СО2 - 107,3 ppm, 13СО2 - 104,5 ppm in first mixture and concentration of 12СО2 - 5,11 %, 13СО2 - 4,9 % in second mixture were measured. Gas pressure in a cuvette was 10 atmospheres. A calibration function was determined, and the uncertainty of 13CO2 concentration measurement required to detect the influence of the Helicobacter pylori bacteria in a sample was calculated – 1.1 ppm. The results showed that the intensity distribution over all measurements corresponds to a normal distribution, and is described by a Gaussian function. For each component of the mixture, the documentation contains an expanded measurement uncertainty, which is 0.6 ppm for 12СО2 and 0.7 ppm for 13СО2 in first mixture and, 0.03 % for 12СО2 and 0.03 % for 13СО2 in second mixture. This implies the probability density distribution for the concentration also in accordance with the normal distribution law. Thus, it is possible to construct a bivariable distribution describing the relationship between measurement intensity and concentration, which can be used to determine concentration. We measured the air exhaled by a person, the measurement uncertainty was calculated. The main factors influencing the measurement uncertainty are determined. It is shown that with a given measurement uncertainty, it is possible to determine the presence of Helicobacter pylori in a patient.
    Author(s): Yulia A. Khristoforova, Ivan A. Bratchenko, Lyudmila A. Bratchenko, Samara Univ. (Russian Federation); Alexandr A. Moryatov, Sergey V. Kozlov, Samara State Medical Univ. (Russian Federation); Valery P. Zakharov, Samara Univ. (Russian Federation)
    6 April 2022 • 5:40 PM - 7:30 PM CEST
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    The annual upward trend in the incidence of melanoma is observed worldwide. The growth of cancer can be caused by the different risk factors in especially genetics factors, patient behavior, environmental factors. We aimed to test the dependence of the optical biopsy performance on the individual patient factors and cancer growth risk factors. In our study, we performed in vivo clinical study of 617 skin cancer in Samara Regional Clinical Oncology Dispensary. The 204 malignant tumors and 413 benign tumors were enrolled in this study. At the appointment oncologist interviewed each patient and collected potential risk factors of skin cancer growth. Were analyzed the 8 individual patient factors and risk factors: Gender, Age, Location, Family history, Personal history, Burns, Size, and Occupational Hazards. All risk factors were digitized. The optical biopsy based on Raman and autofluorescence spectroscopy was performed to noninvasively preliminary diagnose of skin tumors. The Raman and autofluorescence skin spectra were registered using 785 nm wavelength and portable spectroscopic setup. For each tumor the spectroscopic parameters, digitized individual factors, and risk factors were combined in regression models and all parameters were analyzed to classify different skin cancer types using PLS-DA analysis. The ROC AUC was used to quantify the performed classification. The classification of the malignant versus benign neoplasms were performed with 0.61 ROC AUCs based on the spectral analysis and with 0.781 ROC AUCs incorporating with spectral and 8 individual and risk factors. This improvement was statistically significant (p<0.05). Combination of the spectral and 8 risk factors in melanoma versus benign pigmented tumors classification allows one to improve ROC AUCs from 0.789 to 0.849 but this improvement was insignificant (p=0.13). Finally, analysis of the spectral data with 8 factors insignificantly improves ROC AUCs from 0.814 to 0.820 to classify melanoma versus seborrheic keratosis. Therefore, individual and cancer growth risk factors in different way effect on the skin cancer diagnostics.
    Author(s): Rajesh Kumar, Indian Institute of Technology Ropar (India); Saurav Bharadwaj, Agriculture & Water Technology Development Hub, Indian Institute of Technology Ropar (India)
    6 April 2022 • 5:40 PM - 7:30 PM CEST
    Author(s): Imane Lboukili, Georgios N. Stamatas, Johnson & Johnson Santé Beauté France SAS (France); Xavier Descombes, Institut National de Recherche en Informatique et en Automatique, Univ. Côte d'Azur, CNRS (France), Lab. d'Informatique, Signaux et Systèmes de Sophia Antipolis (France)
    6 April 2022 • 5:40 PM - 7:30 PM CEST
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    Reflectance confocal microscopy (RCM) allows real-time in vivo visualization of the skin epidermis at cellular level. The study of RCM images provides information on the topological and geometrical properties of the epidermis. They change in each layer of the epidermis, with age and with certain skin conditions. Studying RCM images requires manual identification of cells to derive these properties which is time-consuming and subject to human error, highlighting the need for an automated cell identification method. We propose an automated pipeline to analyze the structure of the skin in reflectance confocal microscopy images. The first step is to identify the region of interest (ROI) containing the skin epidermis cells. We start by differentiating between the tissue area and the dark background using the Morphological Geodesic Active Contour method. We further refine the ROI by removing bright areas sometimes present in RCM images by applying an alternate sequential filter on the binarized blurred image. Other spurious areas are removed using a Support Vector Machine algorithm trained on four features from the Grey level Co-occurrence Matrix. The second step is to identify individual cells in the segmented tissue area. To perform this task six methods are compared: A) Gabor filtering, B) Sato filtering, C) Gabor filtering followed by Sato filtering, D) Sato filtering followed by Gabor filtering, E) Frangi filtering and F) application of a modified Frangi filter to detect blobs. The outputs of the six methods are then locally normalized, binarized, cleaned and skeletonized. The obtained skeletons (representing cell membranes) are cleaned by pruning and removing spurious branches. We further apply a Gaussian template matching to split detected areas that contain several cells. We then use the detected cell centres to apply a marker-controlled watershed algorithm. The frontiers between flooded areas define the cell borders. The results are evaluated both on simulated data and manually annotated real RCM data. This study shows that automatically identifying cells can be achieved, with accuracy (precision and recall) value that match inter-person variability of cell detection by experts.
    Author(s): Marina Scardigli, Univ. degli Studi di Firenze (Italy), LENS - Lab. Europeo di Spettroscopie Non-Lineari (Italy); Irene Costantini, Univ. degli Studi di Firenze (Italy), LENS - Lab. Europeo di Spettroscopie Non-Lineari (Italy), Istituto Nazionale di Ottica, Consiglio Nazionale delle Ricerche (Italy); Niamh Brady, Mohamed Baghdad, LENS - Lab. Europeo di Spettroscopie Non-Lineari (Italy); Josephine Ramazzotti, Giacomo Mazzamuto, Istituto Nazionale di Ottica, Consiglio Nazionale delle Ricerche (Italy), LENS - Lab. Europeo di Spettroscopie Non-Lineari (Italy); Filippo Maria Castelli, LENS - Lab. Europeo di Spettroscopie Non-Lineari (Italy); Curzio Checcucci, Univ. degli Studi di Firenze (Italy), LENS - Lab. Europeo di Spettroscopie Non-Lineari (Italy); Ludovico Silvestri, Istituto Nazionale di Ottica, Consiglio Nazionale delle Ricerche (Italy), LENS - Lab. Europeo di Spettroscopie Non-Lineari (Italy), Univ. degli Studi di Firenze (Italy); Paolo Frasconi, Univ. degli Studi di Firenze (Italy); Francesco Saverio S. Pavone, Univ. degli Studi di Firenze (Italy), LENS - Lab. Europeo di Spettroscopie Non-Lineari (Italy), Istituto Nazionale di Ottica, Consiglio Nazionale delle Ricerche (Italy)
    6 April 2022 • 5:40 PM - 7:30 PM CEST
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    A deep understanding of the human brain’s structural and functional organization is one of the current outstanding challenges in neuroscience. However, we still lack a detailed map of the anatomical disposition of neurons, obtained by volumetric imaging. Here, we report an innovative pipeline that enables high-resolution 3D mapping of neurons in large human brain samples using Light-Sheet Fluorescence Microscopy (LSFM). Exploiting the optimized SHORT clearing protocol, we cleared a whole human Broca's Area sectioned in 49 slabs each of 450μm in thickness. In order to map the cellular distribution and discriminate the excitatory and the inhibitory sub-population of neurons, each slab was stained using four different markers (targeting nuclei, NeuN, Calretinin, and Somatostatin). Then, we perform fast 3D imaging with subcellular resolution over the cm-sized samples using a custom-made inverted light-sheet fluorescence microscope capable of acquiring up to four channels, of which two simultaneously. Finally, we implemented a data management system to deal with the big data (≈14TB/slab) produced during the acquisition and a machine learning technique to automatically count the neurons in 3D with high precision and sensitivity. Using our approach, we reconstructed and analyzed a whole human Broca's Area of 4x4x2 cm3 with sub-cellular resolution. In conclusion, the proposed combination of a specialized tissue preparation protocol, an advanced LSFM, and big data analysis tools allows the human brain architecture to be mapped at the cellular level, paving the way for routinary analyses of 3D reconstructions of different tissue blocks up to the entire organ.
    Author(s): Elina K. Nepomnyashchaya, Maksim A. Baranov, Elena N. Velichko, Peter the Great Saint-Petersburg Polytechnic Univ. (Russian Federation)
    6 April 2022 • 5:40 PM - 7:30 PM CEST
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    For the development of semiconductor electronics, it is necessary to introduce new materials, including biomolecular materials. Both single protein molecules and molecular aggregates in the form of thin films can become potential materials for constructing the functional properties of biomolecular electronics. The paper describes conducted research in the field of biomolecular electronics aimed at revealing the fundamental regularities of the excitation of biological molecules by external influences such as an electromagnetic field in optical diapason. Optical diapason is used due to special interactions of molecules with field, providing changes in the energy of molecular oscillations. We investigated the energy and dipole moment of such molecules. The instantaneous time-dependent dipole vector moment and molecular system energy were calculated in the VMD program. The conducted studies can be used for further development of hybrid electronics devices, for instance, non-linear converters of electrical and optical signals, biological sensors.
    Session 10: Multiphoton Microscopy II
    7 April 2022 • 11:00 AM - 1:00 PM CEST
    Author(s): Chia Wei Hsu, Chi-Yu Wang, Chun-Yu Lin, National Yang Ming Chiao Tung Univ. (Taiwan); Yvonne Yuling Hu, Chun-Yuan Wu, Hsueh-Cheng Chiang, National Cheng Kung Univ. (Taiwan); Shean-Jen Chen, National Yang Ming Chiao Tung Univ. (Taiwan), National Applied Research Labs., Taiwan Instrument Research Institute (Taiwan)
    7 April 2022 • 11:00 AM - 11:20 AM CEST
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    Multiphoton microscopy is a very important and powerful technique in the application of in-vivo drosophila brain imaging. However, the photon budget, which is compromising between imaging speed, spatial resolution and accumulating time, remains a huge challenge in biological compatibility. Rapid dual-resonant volumetric multiphoton microscopy combines a tunable acoustic gradient (TAG) lens and a resonant mirror, which can achieve up to 8 kHz frame rate and hundreds of hertz volumes per second temporal resolution. Adaptively sampling each laser pulse by an embedded field programmable gate array (FPGA) with up to 80 MHz pixel rate enables efficient but restricted signal accumulation. This study has developed a generative neural network to restore images from degradation of low signal-to-noise ratio (SNR), missing pixels and pattern residual. A series of training data by adding noise and Lissajous scanning path into the known fluorescent bead images were used to pretrain the model for the rapid dual resonant volumetric multiphoton microscopy system. Experimental results verify that the axial distortion and the image resolution of noisy fluorescent bead images can be effectively restored with the deep-restoration neural network. The mushroom body (MB) of drosophila brain which contains thousands of Kenyon cells in a 200 × 200 × 100 µm3 volume is utilized to demonstrate the strategy. The deep-restoration rapid dual resonant volumetric multiphoton microscopy image not only maintains 256 × 256 × 128 voxels and ~30 volumes per second, but also significantly improves image quality which is compatible to the ground truth. However, in-vivo imaging is time-varying. Base on in-vitro image datasets, in-vivo images via transfer learning is utilized to enables fast and improved image quality efficiently. Despite of one pulse per pixel, in-vivo drosophila brain imaging with deep-restoration not only keeps the advantage of temporal resolution, but also obtains well image quality.
    Author(s): Sophia Imperato, Lab. de Physique et d’Etude des Matériaux, Ecole Supérieure de Physique et de Chimie Industrielles de la Ville de Paris, CNRS (France), Univ. PSL (France); Fabrice Harms, Cynthia Veilly, Imagine Optic SA (France); Mathias Mercier, Lab. de Physique et d’Etude des Matériaux, Ecole Supérieure de Physique et de Chimie Industrielles de la Ville de Paris, CNRS (France), Univ. PSL (France); Laurent Bourdieu, Institut de biologie de l'Ecole Normale Supérieure, Univ. PSL, INSERM, CNRS (France); Alexandra Fragola, Lab. de Physique et d’Etude des Matériaux, Ecole Supérieure de Physique et de Chimie Industrielles de la Ville de Paris, CNRS (France), Univ. PSL (France)
    7 April 2022 • 11:20 AM - 11:40 AM CEST
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    Optical microscopy allows to perform structural and functional imaging within large volume of tissues with subcellular resolution. Non-linear microscopy allows the interrogation of neuronal activity in mammalian brains but remains limited because of scattering and optical aberrations. To overcome these issues, Adaptive Optics (AO) strategies have been implemented to retrieve the microscope imaging quality while addressing important imaging depths. A first AO strategy implemented in non-linear microscopy relies on a sensorless configuration, but is a time-consuming iterative process hardly compatible with photobleaching issues. A second approach is based on direct wavefront sensing using Shack-Hartmann wavefront sensors and has proved its efficiency on in vivo experiments. However, this method fails at large depths because of the strong scattering of the emitted fluorescence. A method for direct wavefront sensing more resilient to scattering of the fluorescence emission would therefore facilitate the use of AO in optical microscopy. This work proposes an alternative method of direct wavefront measurement, which relies on the cross-correlation of images of an extended source obtained through a microlens array. This extended-source Shack-Hartmann wavefront sensor (ESSH) requires to be coupled to an optical sectioning method. Its efficiency has been proven when coupled to Light Sheet Fluorescence Microscopy in the adult drosophila brain in weekly scattering conditions. Here, we show that it allows quantitative aberration measurements through highly scattering fixed brain slices, up to four times the scattering length of the tissue. We demonstrate that it is more resilient to scattering compared to the current centroid-based approach. Taking advantage of its geometry, this new wavefront sensor also provides scattering coefficient measurements of biological tissues. Finally, we present its implementation on a two-photon microscope within a closed–loop configuration for in depth neuroimaging in mouse brain and compare its performances in scattering media to the classical centroid approach.
    Author(s): Anupama Nair, National Yang Ming Chiao Tung Univ. (Taiwan); Shu-Chun Chuang, Yi-Shan Lin, Chung-Hwan Chen, Kaohsiung Medical Univ. (Taiwan); Chi-Hsiang Lien, National United Univ. (Taiwan); Chun-Yu Lin, Shean-Jen Chen, National Yang Ming Chiao Tung Univ. (Taiwan)
    7 April 2022 • 11:40 AM - 12:00 PM CEST
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    Discriminating type I and type II collagen is important owing to its dominating presence in cartilage and connective tissues where an alteration of collagen matrix is observed in several diseases including Osteogenesis Imperfecta and Osteoarthritis. Unlike soft tissues, bone fracture healing is a complex process where an initial synthesis of cartilage or callus formation rich in type II collagen is followed by the type I collagen-rich endochondral ossification. For non-destructive investigation of the molecular level properties of collagen, a non-invasive Dual-liquid crystal based polarization-resolved second harmonic (SHG) microscopy is utilized to facilitate the quantitative characterization of collagen types I and II in fracture healing tissues. In this study, we extend an existing approach allowing the quick generation of any desired linear polarization states without any mechanical parts to quantify the characteristics of collagen types using pitch angle and anisotropy parameter. Furthermore, data reliability is ensured by using right and left-hand circular polarization imaging centered circular dichroism analysis. Our findings indicate that the effective pitch angle for the collagen at fracture healing tissue is 48.4° and 49.9° at two weeks and four weeks of repair respectively where type II collagen dominates in the former and type I in the latter. Similarly, a difference of 0.6 is observed for the anisotropy parameter of both collagen types. The mean SHG-CD response of the articular cartilage is 0.271 and 0.183 at the rich zone of collagen types II and I, respectively. These findings are correlated to the values obtained from the non-fractured control bone tissue. The measurements obtained reflect the different types of collagen in the molecular fibril assembly. Therefore, these methods demonstrate a powerful tool to provide new insights on understanding the role of collagen in ECM structure and on the development of cartilage repair.
    Author(s): Irina V. Larina, Baylor College of Medicine (United States)
    7 April 2022 • 12:00 PM - 12:20 PM CEST
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    Trough intravital functional optical coherence tomography (OCT) in mouse models, this study investigates physiological processes within the fallopian tube in vivo. The transport of oocytes and embryos through the oviduct (fallopian tube) is a fundamental reproductive processes of clinical importance. However, because mammalian fertilization and embryogenesis take place deep within the female body, these processes are hidden from direct observation. Therefore, much of what we know about the innerworkings of the female reproductive tract is extrapolated from in vitro and ex vivo experimental settings and does not necessarily represent the native state, limiting success in management of reproductive disorders. This study presents first in vivo volumetric dynamic imaging of oocytes and embryos as they are transported through the mouse oviduct. By implementation of new functional OCT methods, we established methods for tracking oviductal ciliary function and individual sperm movements. Supported by dynamic volumetric visualizations, the study reveals a variety of intriguing never-before-seen dynamic behaviors and suggest new regulatory mechanisms driving reproductive processes.
    Author(s): Mehdi Alizadeh, Viktoras Mažeika, Mykolas Maciulis, Martynas Riauka, Vilnius Univ. (Lithuania); Virginijus Barzda, Univ. of Toronto Mississauga (Canada), Vilnius Univ. (Lithuania), Univ. of Toronto (Canada)
    7 April 2022 • 12:20 PM - 12:40 PM CEST
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    Polarimetric second harmonic generation (SHG) microscopy techniques are powerful tools to reveal sub-molecular information from biological specimens. Among biological samples collagen with a noncentrosymmetric structure and efficient SHG conversion has been the focus of many studies. Since collagen remodeling takes place due to cancer progression, it is important to develop tools to detect and understand the ultrastructural changes in collagen assembly using polarimetric nonlinear microscopy. Several polarimetric techniques have been developed to probe susceptibility ratios, in-plane orientation, and out of the image plane orientation of collagen. Polarization-In Polarization-Out (PIPO) and SHG circular dichroism (SHG-CD) techniques have been used to calculate the out of the image plane orientation and chirality of collagen. In this work, we study the correlation between SHG-CD and the chiral susceptibility ratio (C) in order to reveal the collagen chirality, and the collagen fiber tilt out of image plane. A numerical modeling is used to understand the relation between aforementioned parameters and the chirality and out of the image plane orientation of collagen. The results of numerical modeling show similar behaviors for SHG-CD and the chiral susceptibility ratio (C) calculated from PIPO measurements. The results obtained from rat tail tendon collagen confirms that the sign of both SHG-CD and C ratio changes by flipping the sample as it is predicted by the numerical modeling. The results also show that both SHG-CD and C ratio may become miscalculated when antiparallel chiral fibers are present in the focal volume of the microscope. The results of this study confirm that polarimetric SHG microscopy techniques are able to reveal 3D structure of biological samples and therefore they are beneficial to the diagnosis of collagen related diseases.
    Author(s): Jyothsna Konkada Manattayil, A.S. Lal Krishna, Indian Institute of Science, Bengaluru (India); Hyunmin Kim, Daegu Gyeongbuk Institute of Science & Technology (Korea, Republic of); Varun Raghunathan, Indian Institute of Science, Bengaluru (India)
    7 April 2022 • 12:40 PM - 1:00 PM CEST
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    In this paper we present experimental demonstration of focal-field engineering in infrared-sensitive third-order sum frequency generation (TSFG) microscopy by utilizing beam-shaping technique. Two photons of the input mid-infrared (mid-IR) beam at 3000 nm are upconverted to 615 nm in the presence of a single photon at 1040 nm through the TSFG process. The focal-field engineering scheme studied here improves optical resolution and contrast of the TSFG imaging. Diffraction-limited resolution of the TSFG microscope when using reflective objective of numerical aperture 0.65 to focus incident beams is measured to be ~603 nm. The use of Toraldo-pupil functions with 0-π annular phase step mask at 1040 excitation beam (generated using a reflective spatial light modulator placed at the conjugate plane to the back-aperture) results in narrowing of the central lobe and increase in energy of the side-lobes for the TSFG focal field profile. We observed best improvement of ~15% in the central-lobe full-width half diameter (~522 nm) and increase in side-lobe strength to ~50% of that of the central-lobe with the use of optimum phase-mask of 0.41 times the diameter of the first mirror of the reflective objective and using isolated amorphous silicon (a-Si) nanodisks as the imaging sample. The TSFG process is also quantified by estimating the image contrast obtained by scanning one-dimensional a-Si gratings of varying pitch. The far-field TSFG signal as a function of varying grating positions is also calculated using Green’s function integral to model the radiation of the TSFG nonlinear dipoles from the sample. We compare the contrast enhancement between the experiments and simulations as a function of varying grating pitch and find good overall agreement between the two. In addition to annular phase masks, we also demonstrate edge contrast enhancement by imaging gratings with higher-order Hermite-Gaussian beams profile generated using horizontally partitioned 0-π phase profile.
    Conference Chair
    Leibniz-Institut für Photonische Technologien e.V. (Germany)
    Conference Chair
    Lab. Charles Coulomb (France)
    Conference Co-Chair
    Francesco Saverio S. Pavone
    LENS - Lab. Europeo di Spettroscopie Non-Lineari (Italy)
    Conference Co-Chair
    Univ. de Bordeaux (France)
    Program Committee
    Technical Univ. of Denmark (Denmark)
    Program Committee
    James M. Brewer
    Univ. of Glasgow (United Kingdom)
    Program Committee
    National Yang-Ming Univ. (Taiwan)
    Program Committee
    TU Dresden (Germany)
    Program Committee
    Vrije Univ. Amsterdam (Netherlands)
    Program Committee
    Univ. of St. Andrews (United Kingdom)
    Program Committee
    Bar-Ilan Univ. (Israel)
    Program Committee
    Univ. de Strasbourg (France)
    Program Committee
    Univ. of Houston (United States)
    Program Committee
    Hainan Univ. (China)
    Program Committee
    Thomas G. Mayerhöfer
    Leibniz-Institut für Photonische Technologien e.V. (Germany)
    Program Committee
    Helmholtz Zentrum München GmbH (Germany)
    Program Committee
    The Univ. of Western Australia (Australia)
    Program Committee
    Johann Wolfgang Goethe-Univ. Frankfurt am Main (Germany)
    Program Committee
    Vrije Univ. Brussel (Belgium)
    Program Committee
    Siva Umapathy
    Indian Institute of Science (India)
    Program Committee
    Ontario Cancer Institute (Canada)
    Program Committee
    Ctr. for Biomedical Optics and Photonics (Germany)
    Program Committee
    Princess Margaret Hospital (Canada)
    Additional Information