This PDF file contains the front matter associated with SPIE Proceedings Volume 11046, including the Title Page, Copyright information, Table of Contents, Author and Conference Committee lists

To improve the localization accuracy and to reduce the overall photo-damage in Single Molecule Localization Microscopy (SMLM), we introduce a holography based Region-of-Interest (ROI) illumination with oblique angles. The ROI illumination is realized in two different application modes which offer different advantages. Both modes (A,B) are implemented within the same setup by modifying the illumination light using a phase-only spatial light modulator (SLM) twice. This allows to adaptively modify the size and the (excitation) angle of the required ROI illumination, resulting in reduced out-of focus signal and less overall phototoxicity. Mode A generates a nearly speckle free, circular ROI which can be obtained instantly (no iterative algorithm needed). Mode B allows to realize an arbitrarily shaped ROI but comes at the cost of a higher presence of speckle structures as well as in an increased calculation time of the holograms. In both cases illumination angles up to 60° with high NA objectives are realizable, enabling the effective selection of specific parts of a biological probe to be illuminated.

Super-resolution localization microscopy is a powerful tool to visualize molecular structures at a nanoscale resolution. High-density emitter localization combined with a large field of view and fast imaging frame rate is an effective strategy to achieve a high throughput. But the complex algorithms used to precisely localize the overlapping molecules in dense emitter scenarios limits their usage to mostly small image size. Here we present a computationally simple non-iterative method for high-density emitter localization to enable online image processing that remains robust even for low signals and heterogeneous background. Through numerical simulation and biological experiments, we demonstrate that our approach improves the computation speed by two orders of magnitude on CPU and three orders of magnitude upon GPU acceleration to realize online image processing, without compromising localization accuracy for various image characteristics.

Among super-resolved microscopy (SRM) methods, single molecule localisation microscopy techniques, such as photo-activated localisation microscopy (PALM) [1] and stochastic optical reconstruction microscopy (STORM) [2], enable imaging beyond the classical diffraction limit to gain new insights in subcellular biological processes with relatively simple instrumentation. This has led to a number of low-cost instruments, e.g. for STORM microscopy [3-6], which can benefit from an array of software tools for the single molecule localisation microscopy (SMLM) data analysis [7]. Our low-cost “easySTORM” approach [4] implements dSTORM [8] with multimode diode lasers and optical fibres to provide STORM images with fields of view up to ~125 μm diameter using μManager [9] to control the image data acquisition and ThunderSTORM [10] to analyse the SMLM data. We and others [11,12] are motivated to develop automated SMLM for high content analysis (HCA) that enable rapid imaging of sample arrays, allows statistical analysis of samples that may vary in terms of labelling and biological heterogeneity and enable moderate throughput screening applications.

In the paper recently developed microscopes operating in the soft X-rays and extreme ultraviolet (EUV) spectral regions are presented. Soft X-rays and EUV were generated using laser plasma light sources with a double-stream gas puff target. Compact Nd:YAG lasers, generating 4 ns pulses with energy up to 0.8 J at 10 Hz repetition rate, were used to irradiate the targets. Two full-field microscopes based on Fresnel optics have been developed with the use of these sources. The EUV microscope was operating at the wavelength of 13.8 nm and used for imaging of nanostructures with sub-100 nm spatial resolution. The soft X-ray microscope was operating at the wavelength of 2.8 nm and was used for imaging of dry and hydrated biological samples with spatial resolution below 100 nm. The third microscope is based on soft X-ray contact microscopy (SXCM) approach. The new microscopes and their application for imaging at the nanoscale are presented and discussed.

Large-scale microscopy approaches are transforming brain imaging, but currently lack efficient multicolor contrast modalities. We address this issue by introducing chromatic multiphoton serial (ChroMS) microscopy, a method combining multicolor multiphoton excitation through wavelength mixing and microtome-assisted serial block-face image acquisition. This approach delivers large-scale micrometric imaging of spectrally distinct fluorescent proteins with constant micrometer-scale resolution and sub-micron channel registration over the entire imaged volume. We achieve multicolor 3D imaging over several cubic millimeters and brain-wide serial 2D multicolor imaging. We illustrate the potential of this method for several novel types of measurements interesting for region-scale or whole brain studies: (i) color-based analysis of astrocyte morphology and spatial interactions in the mouse cerebral cortex, (ii) tracing of densely labeled neurons, and (iii) brain-wide mapping of axonal projections labeled with distinct tracers.

Here we present label-free imaging of retinal bipolar cell axons by second-harmonic generation microscopy. The nonlinear optical signal arises from uniformly polarized microtubules, providing a surrogate contrast for the functional integrity of axons. The unique properties of microtubule SHG from the inner plexiform layer were employed for testing an outstanding hypothesis in glaucoma, namely, the persistence of BC axons in the disease during a critical period when the synaptic connections to the retinal ganglion cells are lost.

In the last few years, optogenetic tools and optical functional indicators are increasingly used together to perform simultaneous manipulation and recording of neuronal activity. Nevertheless, this method has still some limitations mainly due to the spectral cross-talk between optogenetic actuators and functional sensors [1;2]. To address this issue, red variants of genetically encoded calcium indicators (red-GECIs) have been recently developed [3;4]. The main goal of this project is to develop a full-optical system that allows effective interrogation of brain circuits. To this aim, we combined a red-shifted calcium indicator (jRCaMP1a), with the most common blue-light activated opsin, Channelrhodopsin II (ChR2). The results presented here show: (I) extended expression of the full-optical system that covers all the motor areas, (II) functional correlation between the laser power and the evoked neuronal activity, (III) segregation of the cortical functional areas of two different forelimb evoked movements. The future perspective of this project concerns the study of the functional areas correlation during optogenetically-evoked forelimb complex movements.

Myelination in the brain has been typically associated with the white matter. Recently, however, cortical myelination has gained much interest not only as a possible lesion of multiple sclerosis (MS) but also for its significance in the plasticity of the central nervous system (CNS). It is urgent to understand the functional role of cortical myelination and its involvement in the MS pathogenesis. Here we demonstrate a novel nonlinear optical contrast, namely third-harmonic generation (THG), as a basis of label-free imaging of cortical myelin in living animals. Using the fixed brain slices of fluorescently labeled transgenic mice, we examined the distribution of myelin in the normal brain. We measured the spatial organization of cortical myelin. Finally, the capability of THG microscopy was tested for visualizing cortical myelin in living, unlabeled animals.

The three-dimensional reconstruction of large volumes of the human neural networks at cellular resolution is one of the biggest challenges of our days. Commonly, fine slices of samples marked with colorimetric techniques are individually imaged. This approach in addition to being time-consuming does not consider space cell organization, leading to loss of information. The aim of this work was to develop a methodology that allows analyzing the cytoarchitecture of the human brain in three dimensions at high resolution. In particular, we exploit the possibility of combining high-resolution 3D imaging techniques with clearing methodologies. We successfully integrate the SWITCH immunohistochemistry technique with the TDE clearing method to image a large volume of human brain tissue with two-photon fluorescence microscopy. In conclusion, this new approach enables to characterize large human brain specimens with high-resolution optical techniques, giving the possibility to expand the histological studies to the third dimension.

Deciphering brain architecture at a system level requires the ability to quantitatively map its structure with cellular and subcellular resolution. Besides posing significant challenges to current optical microscopy methods, this ambitious goal requires the development of a new generation of tools to make sense of the huge number of raw images generated, which can easily exceed several TeraBytes for a single sample. We present an integrated pipeline enabling transformation of the acquired dataset from a collection of voxel gray levels to a semantic representation of the sample. This pipeline starts with a software for image stitching that computes global optimal alignment of the 3D tiles. The fused volume is then accessed virtually by means of a dedicated API (Application Programming Interface). The virtually fused volume is then processed to extract meaningful information. We demonstrate two complementary approaches based on deep convolutional networks. In one case, a 3D conv-net is used to ‘semantically deconvolve’ the image, allowing accurate localization of neuronal bodies with standard clustering algorithms (e.g. mean shift). The scalability of this approach is demonstrated by mapping the spatial distribution of different neuronal populations in a whole mouse brain with singlecell resolution. To go beyond simple localization, we exploited a 2D conv-net estimating for each pixel the probability of being part of a neuron. The output of the net is then processed with a contour finding algorithm, obtaining reliable segmentation of cell morphology. This information can be used to classify neurons, expanding the potential of chemical labeling strategies.

In bone tissue, multiscale interfaces provide the structural basis of essential bone functions and contribute to its macroscopic mechanical properties. The lacuno-canalicular network (LCN) hosting the osteocytes in the bone matrix, in particular, represents a biological signature of the mechanotransduction activity in response to external biomechanical loading. We have demonstrated that label-free third-harmonic generation (THG) microscopy reveals the structure of the LCN in 3D with submicron precision over millimetric fields of view compatible with histology and can be coupled to second-harmonic generation (SHG) signals relating to the collagen organization in the bone matrix. Taking advantage of these label-free imaging methods, we investigate the impact of microgravity on the LCN structure in mice following a 1- month space flight. We show that our current lack of understanding of the extent of the LCN heterogeneity at the organ level hinders the interpretation of such investigations based on a limited number of samples and we discuss the implications for future biomedical studies.

Collagen is the most abundant protein in mammals and represents the main component of connective tissues, such as skin, cornea, artery or tendon. The three-dimensional multiscale organization of collagen is highly specific to every tissue and directly determines its physical and mechanical properties. This project aims at developing a new analytical method for in situ mapping of the fibrillar and denatured collagen multiscale structure in label-free biological tissues. To address this issue, infra-red nanospectroscopy (AFM-IR), which enables chemical mapping at nanometer scale, is combined to multiphoton microscopy based on Second Harmonic Generation (SHG) and 2PEF (two-photon excited fluorescence) signals, which probes collagen structure at micrometer scale. Optical signatures from multiphoton microscopy show that fibrillar collagen exhibits strong SHG signals and gelatin emits fluorescence signals. AFM-IR analysis shows that IR spectra exhibit amide I band and only in the case of gelatin an absorbing band around 1730 cm^-1. Correlation of both techniques before and after denaturation on the same samples confirms this optical and chemical signatures of gelatinization process. The correlative imaging of IR nanospectroscopy and multiphoton microscopy of fibrillar collagen and gelatin structural states provide a calibration of the multiphoton signals that can be further used for the assessment of degradation of the collagen within tissues such as cornea or skin due to injuries or diseases.

A developed temporal focusing-based multiphoton microscope (TFMPM) has a digital micromirror device (DMD) which is adopted not only as a blazed grating for light spatial dispersion but also for patterned illumination simultaneously. Herein, the TFMPM has been extended to implement spatially modulated illumination at structured frequency and orientation to increase the beam coverage at the back-focal aperture of the objective lens. The axial excitation confinement (AEC) of TFMPM can be condensed from 3.0 μm to 1.5 μm for a 50% improvement. Furthermore, a multiline scanning mechanism based on the DMD can push its AEC nearly equivalent to line scanningbased TFMPM to 1.5 μm from optimal 3.0 μm of the conventional TFMPM. By using the TFMPM with structured illumination and multiline scanning, reconstructed biotissue images according to the condensed AEC structured illumination are shown obviously superior in contrast and better scattering suppression.

In this study nonlinear microscopy techniques utilized to shed new light in breast cancer diagnosis. In particular, the nonlinear imaging modalities of multi-photon excitation fluorescence (MPEF) second and third harmonic generation (SHG, THG) used as non-destructive, label free diagnostic tools for the qualitative discrimination of breast cancer tissues. Nonlinear signals were collected from unstained thin histological sections of breast biopsies samples. In an attempt to discriminate the different types of tissues, quantitative analysis of the recorded THG signals on specific cells in tissue was also performed.

Endoscopy is a key technology for minimally-invasive optical access to deep tissues in humans and living animals. However, modern endoscopes, such as fiber bundles, still suffer from low spatial resolution. Multimode fiber is a very promising tool for high-resolution endo-microscopy. We use advanced wavefront shaping technique and experimentally demonstrate high-resolution fluorescent and label-free imaging through a multimode fiber. We also present an ultra-thin Raman imaging probe with an excellent ratio between field of view and probe diameter. However, state-of-the-art multimode fiber endo-microscopy still has several problems limiting its broad applications: slow speed, as well as requirements of complex wavefront shaping procedure and expensive spatial light modulators. Here we show the solution to all these problems. We propose and experimentally demonstrate a new method of high-resolution endoscopy: compressive multimode fiber imaging. The key idea is to integrate the compressive sensing technique with a multimode fiber probe, which produces a random basis of speckle patterns, collects the optical response and provides optical sectioning. This new approach allows high-speed diffraction-limited imaging at the full field of view of a probe and does not require complex elements, such as spatial light modulators or knowledge of the transfer matrix of the multimode fiber. We demonstrate high-resolution imaging through a fiber probe with the total number of measurements 20 times less than required for the standard raster scanning approach. Compressive multimode fiber imaging offers a unique tool for in vivo high-speed high-resolution endoscopy.

We demonstrated flat-field illumination (FFI) for multi-color wide-field fluorescence microscopy using a refractionbased beam shaping system. The non-homogeneous illumination of a Gaussian intensity profile makes quantitative analysis in laser-assisted wide-field fluorescence microscopy very difficult. As contrasted with other approaches, our method is applicable to TIRF illumination, which effectively rejects background fluorescence.

Our beam shaping device is extremely tolerant to variations in size of the incoming laser beam by accepting ± 10% variation, while being achromatic as well. This behavior originates from the well-balanced mapping of the incoming rays to the intended flattop beam profile in combination with a sophisticated material choice, which decreases the sensitivity to input beam diameter. The homogenous illumination profile of FFI will enable quantitative single-molecule analysis based on intensity information. This has powerful implications when combined with a pull-down assay, which can probe the oligomerization state of endogenous proteins. When combined with one-to-one fluorophore labeling, the stoichiometry of proteins related to neurodegenerative diseases could be readily determined by intensity distribution analysis, which is critical to not only diagnosing but also understanding the pathogenesis of these complex disorders that are particularly difficult to analyze.

An additional application of FFI is high quality super-resolution imaging with a uniform spatial resolution over a large FOV, where the full power of the excitation beam could be utilized. A new optical design approach based on refractive freeform surfaces generating a square-shaped beam instead of a round one will be presented, which would yield greater illumination efficiency.

Light-sheet microscopy has become one of the leading techniques for 3D in vivo imaging, thanks to its reduced photo-damaging effects on the specimen, its optical sectioning capability and its imaging speed. Various modifications of a Selective Plane Illumination Microscope (SPIM) have been proposed, with the aim to further increase 3D imaging speed, quality and resolution, and decrease photo-bleaching. We recently presented a modified SPIM, in which a Spatial Light Modulator (SLM) is used to move and modulate the light-sheet to perform different existing imaging techniques which improve image quality. We now discuss how such a system can be further exploited, and present two new light-sheet modulations specifically designed to help reduce the effect of photo-bleaching and, more in general, to improve the system's light-efficiency

Breast cancer and Glioblastoma brain cancer are aggressive malignancies with poor prognosis. In this study primary Glioblastoma and secondary breast cancer spheroids are formed and treated with the well-known Temozolomide and Doxorubicin chemotherapeutics, respectively. A custom multi-angle Light Sheet Fluorescence Microscope is employed for high resolution imaging of both cancer cell spheroids. Such a technique is successful in realizing pre-clinical drug screening, while enables the discrimination among physiologic tumor parameters. LSFM technique, parameters and method followed are also presented.

Capturing highly dynamic biological processes at cellular resolution is a recurring challenge in biology. Here we show that combining selective-volume illumination with simultaneous acquisition of orthogonal light-fields yields 3D images with high, isotropic spatial resolution and a significant reduction of reconstruction artefacts, thereby overcoming current limitations of light-field microscopy implementations. We demonstrate Medaka heart and blood flow imaging with single-cell resolution and free of motion artefacts at volume rates <200Hz.

One of the drawbacks of using Lloyd’s mirror interferometer for quantitative phase contrast microscopy is that it leads to overlapping of the object information which restricts the use of field of view. One way to overcome this limitation is to prepare the sample slide in such a way that the object information is contained in only one portion of the illuminating beam, while the other acts as a separate reference beam. This geometry has excellent temporal stability and is used to examine human erythrocytes.

For the example of digital holographic microscopy (DHM), a variant of quantitative phase imaging (QPI), we explored strategies for the analysis of confluent cell layers utilizing histogram based-evaluation of quantitative phase images. The applicability of the proposed numerical evaluation procedures is illustrated by the DHM-based quantification of drug induced cell morphology changes. Our results show that histogram-based evaluation of quantitative phase images enables a highly reliable quantitative observation of global cellular morphology changes in confluent cell layers.

In this work we demonstrate large scale high sensitivity optical diffraction tomography (ODT) of zebrafish. Compared to previous work the scale and sensitivity are enhanced by the following steps. First, we obtain a large field of view while still maintaining a high image resolution by using a high magnification over numerical aperture ratio ODT set-up. With the inclusion of phase shifting we demonstrate that we operate close to the optimum magnification over numerical aperture ratio. Second, we decrease the noise in the reconstructed images by implementing off-axis sample placement and numerical focus tracking in combination with the acquisition of a large number of projections. Although both techniques lead to an increase in sensitivity independently, we show that combining them is necessary in order to make optimal use of the potential gain offered by each respective method and obtain a refractive index (RI) sensitivity of 8•10^-5. In this way, high RI sensitivity can be achieved that is necessary for phase tomography of optically cleared tissue structures, which we can identify for features with RI down to 6•10^-4. Third, we optimize the optical clearing procedure to prevent scattering and refraction to deteriorate our large scale images. We demonstrate our technique by imaging a 3 day old zebrafish and an adult cryoinjured zebrafish heart in a large 5.5 x 5.5 x 4.1 mm³ volume with 4 micrometer resolution. Various tissue structures can be clearly identified. The volume of the cryoinjured heart is segmented and quantified based on the refractive index distribution.

Numerous biophysical questions require the quantification of short-range interactions between (functionalized) surfaces and synthetic or biological objects such as cells. Here, we present an original, custom built setup for reflection interference contrast microscopy that can assess distances between a substrate and a flowing object at high speed with nanometric accuracy. We demonstrate its use to decipher the complex biochemical and mechanical interplay regulating blood cell homing at the vessel wall in the microcirculation using an in vitro approach. We show that in the absence of specific biochemical interactions, flowing cells are repelled from the soft layer lining the vessel wall, contributing to red blood cell repulsion in vivo. In contrast, this so-called glycocalyx stabilizes rolling of cells under flow in the presence of a specific receptor naturally present on activated leucocytes and a number of cancer cell lines.

In this project it was found that Fourier ptychographic microscopy can be improved far beyond its conventional limits via waveguide-based optical systems. Extensive in silico studies showed that images obtained on high-refractive index material waveguide chips in conjunction with hyperspectral illumination light and finely designed waveguide geometries can be combined via a modified phase-retrieval algorithm to yield a resolution below 150 nm.

Lens-Free microscopy aims at recovering an observed object such as cell cultures from its diffraction measurements. Diffraction acquisitions are processed with an inverse problem approach to recover optical path difference (OPD) images of the object. Phase unwrapping issue is solved here by using a convolutional neural network (CNN) trained on simulations. The procedure was applied successfully on a neuron cells culture video acquisition.

One of the greatest challenges for early cancer detection is how to effectively manage patients who are at risk for developing invasive cancer. As most at-risk patients will not develop cancer, frequent and invasive surveillance of atrisk patients carries financial, physical, and emotional burdens. But clinicians lack tools to accurately predict which patients will likely progress into malignancy. With our increased understanding of molecular changes in cancer development, it is now established that disrupted epigenome that can alter nuclear architecture occurs in all stages of cancer development including in normal precursor cells. Therefore, assessment of nanoscale nuclear architecture represents a promising strategy for identifying pre-cancerous changes. Here we present the development of threedimensional nuclear architecture mapping (3D-nanoNAM) to assess the depth-resolved properties of phase objects with slowly varying refractive index without a strong interface, based on a variant form of Fourier-domain optical coherence tomography (FD-OCT). By computing the Fourier phase of the FD-OCT signal resulting from the light back-scattered by cell nuclei, 3D-nanoNAM quantifies, with nanoscale sensitivity, the depth-resolved alterations in mean nuclear optical density, and localized heterogeneity in optical density of the cell nuclei. We demonstrate that 3D-nanoNAM distinguishes high-risk patients with inflammatory bowel disease (IBD) colitis from those at low-risk via/through imaging tissue sections that appear histologically normal according to pathologists. As 3D-nanoNAM uses clinically prepared formalin-fixed, paraffin-embedded tissue sections, it can be integrated into the clinical workflow.

The histopathological diagnosis in malignances requests well trained specialists and multi-step operational procedures for sample preparation. Faster and more objective evaluation protocols should be implemented to give support to the pathologists. The Quantitative Phase Imaging based methods are biological-proved to be efficient in revealing important characteristics of the living structures without any labeling. These can be further exploited for an automatic evaluation of complex tissues. Using an off-axis Digital Holographic Microscopy setup, biopsies of two histological origins: cerebral (grade II glioma and grade IV glioblastoma) and colonic malignancies (dysplastic and malignant colonic adenomatous polyps), were investigated. Various parameters of quantitative phase shift maps (QPMs) were computed (mean, variance, median, kurtosis, skewness, energy, entropy). The possibility of automatic discrimination of tumor tissues having different structural complexity and presenting various malignancy grades was evaluated using supervised machine learning algorithms. The analysis of phase shift maps has successfully discriminated between levels of malignancy with high statistical confidence in the case of gliomas. Moreover an algorithm with the ability to classify the tissue biopsies in different malignant stages using parameters based on QPMs has been implemented on glioma tissues having a high level of homogeneity. In case of colonic polyps, the heterogeneity of the multilayered tissue demanded QPMs analysis to be performed on selected area of interest even though some statistical differences were obtained for global evaluation of phase shift distributions. In case of colonic polyps, for a good accuracy of classification algorithm a larger library of QPMs is under construction.

The application range of P-THG microscopy has been so far restricted to studies on molecular order and anisotropy of static specimen removed from their biological environment. Slow polarization commutation limits the investigation of highly dynamic systems because of motion artifacts. Here we have developed a new fast-P-THG microscope enabling efficient in vivo studies in dynamic biological samples. Our P-THG scheme benefits from a built-in EOM that switch polarization states at kilohertz between image lines to provide artefact-free P-THG images with micrometric resolution. Furthermore, we have developed a fast Fourier analysis enabling rapid P-THG processing to quantify lipid order and angular maps. We demonstrated that fast-P-THG is suitable in two major applications. Using first a linear polarization configuration, fast P-THG imaging revealed molecular order changes in MLVs undergoing phase-transition upon heating despite sample distortions. Anisotropy properties of small endogenous microparticles swimming in the otolith cavity embryos were also reported in early zebrafish embryos. A second configuration with linear-circular polarization commutation enabled efficient detection of birefringent media such as anisotropic vesicles in C-elegans gut cells.

Polarization resolved coherent Raman scattering (polar-CRS) provides information on molecular orientational organization, with the strong advantages of being a label free and chemically specific imaging method. Polarization tuning imposes however slow imaging acquisition rates. Here we demonstrate two strategies to fasten polarization resolved CRS and reach real time capabilities. First, fast polar-CRS imaging is obtained by a combined electro-optic polarization and acousto-optic amplitude modulations, applicable to both Stimulated Raman Scattering (SRS) and Coherent anti-Stokes Raman Scattering (CARS) point scanning imaging. The proposed scheme increases the speed of orientational imaging by two orders of magnitude as compared to polarization tuning approaches, and does not require post processing analyzes. Second, a combination of circular-based polarizations is used to directly filter the organization components of the probed samples, benefiting from symmetry matching properties between light and molecular assemblies. We demonstrate the potential of those methods for sub-second time scale imaging of lipid order packing and local lipid membrane deformations in artificial lipid multilayers, single cell ghosts, and myelin-rich tissues from spinal cords in live mice. Notably, these images permit to extract molecular scale signatures that bring for instance new information on the early stage of the development of neurodegenerative diseases.

Mueller transmission microscopy has been used for both theoretical and experimental studies of anisotropic scattering biological tissue. In our prior study, the linear dependence of retardance and quadratic dependence of depolarization on thickness was demonstrated for a dermal layer of skin model. During the primary analysis of polarimetric images of histological cuts both epidermal and dermal layers were delineated manually in order to calculate the spatially averaged values of retardance and depolarization parameters. Consequently, these average values contained the contribution of outliers (noise, not correctly identified pixels, etc.) which produces large standard deviation and biased mean values of the parameters mentioned above. For preventing the errors, the normalized maps of optical properties were calculated pixel-wise taking into account local optical density (e. i. logarithm of M11 element of Mueller matrix at each image pixel) to compensate varying tissue thickness across the cut area. Furthermore, the DBSCAN (Density-based spatial clustering of applications with noise) algorithm was applied for segmentation of microscopic images using the normalized values of retardance, depolarization, and intensity. From the results of image segmentation, we could discriminate the regions of dermal and epidermal layers in Muller microscopic images of skin cuts more accurately and obtain more reliable values of tissue’s optical properties.

Mueller microscopes enable imaging of the optical anisotropic properties of biological or non-biological samples, in phase and amplitude, at sub-micrometer scale. However, the development of Mueller microscopes faces instrumental challenges: whilst adjusting the microscope, the operator needs a polarimetric image as guidance and the production of polarimetric parameters must be sufficiently quick to ensure fast imaging. To mitigate this issue, in this paper, a full Mueller scanning microscope based on spectral encoding of polarization is presented. The spectrum collected every 10 ms for each position of the optical beam on the specimen, incorporates all the information needed to produce the full Mueller matrix, which allows simultaneous images of all the polarimetric parameters at the unequalled rate of 1.5 Hz (for an image of 256×256 pixels). The design of the optical blocks allows for the real-time display of linear birefringent images which serve as guidance for the operator. In addition, the instrument has the capability to easily switch its functionality from a Mueller to a Second Harmonic Generation (SHG) microscope, providing a pixel-to-pixel matching of the images produced by the two modalities. The device performance is illustrated by imaging various unstained biological specimens.

Quantitative measurement of protein-protein interaction is important for many biological processes, including cell growth, intercellular communication, gene expression and apoptosis. Förster Resonance Energy Transfer (FRET) provides a molecular level ruler to measure the distance, within a few nanometers, between two proteins. FRET can be measured by changes in fluorescence lifetime of the fluorophors by Fluorescence lifetime imaging microscopy (FLIM) in live cells. Combined with confocal imaging, FLIM can achieve 3-dimensional optical sectional capability and resolve sub-cellular structures. The change in lifetime is inversely proportional to the ratio of bound to non-bound proteins. Time-resolved conventional confocal scanning microscopy is inherently slow and not suitable for rapid imaging applications. We present a 32×32 multiplexing confocal microscope, equipped with a 64×32 time-gated single-photon avalanche photodiode (SPAD) sparse array detector. The multiplexing setup allows the use of the sparse array with high frame rate and sub-nanosecond time-gating to achieve high throughput FLIM acquisition. We used this multiplexing confocal FLIM system to measure Bcl-2 family proteins interactions in live cells and are able to capture a 240×240 μm FOV multi-channel confocal FLIM images in less than 1.5s. Protein binding affinities are estimated by measuring the changes in FRET as function of acceptor to donor ratio.

In this research work, we present a novel indirect immunolabeling method for labeling up to three different antigens using just two primary and fluorophore tagged secondary antibodies. We propose a viable solution to overcome the limitations imposed by limited variability in primary and secondary antibody type by leveraging cross-labeling phenomenon. Cross-labeling among two fluorophore conjugated secondary antibodies leads to FRET effects, resulting in changed spectral and fluorescence lifetime properties of donor molecule. To detect and quantify these changes in photophysical properties of immunolabeled species, we developed an eight-channel spectrally resolved fluorescence lifetime imaging (sFLIM) system. We demonstrate the capabilities of our approach in case of multi-targeted immunostaining in A549 cells. Efficient excitation of samples is achieved using two pulsed laser of wavelengths 485 nm and 561 nm operating in alternating/interleaved manner. Acquired multi-dimensional sFLIM data was pre-processed and analysed using state-of-the-art pattern-matching algorithm¹ which takes into account the information of fluorescence emission spectra as well as lifetime. The sFLIM detection system together with pattern-matching analysis enables separation of cross-linked labels from single labeled species.

We propose a disruptive point-of-care (PoC) imaging platform based on lens-free interference phase-contrast imaging for rapid detection of biomarker such as for sepsis and potentially other diseases (e.g. cancer). It enables simultaneous analysis of potentially up to 10,000 functionalized microarray spots with different biomarkers with fast time-to-results (few minutes) and by consuming a small sample volume (~10 μL). The high sensitivity allows direct measurements of the biomarker binding without the use of fluorescent labels (e.g. ELISA) or microbial culture methods. In addition, adhoc plasmonic nano-structuring is utilized to significantly improve the sensitivity for biomarker detection (optical path difference ~Å) to concentration levels relevant for disease diagnosis.

The proposed technology incorporates a portable and low-cost lens-free imaging reader made of consumer electronic components, plasmonic microarrays with distinct functionalization, and user-friendly software based on a novel phaseshifting interferometry method for topography and refractive index analysis. Due to its compactness and cost-efficiency, we foresee a great potential for PoC applications, especially for the rapid detection of infectious diseases or lifethreatening conditions, e.g. sepsis, but also for clinical trials of drugs and food control.

Trichomoniasis is one of the most common non-viral sexually transmitted infection caused by a parasite called Trichomonas vaginalis. Currently, there is no timely, cost-efficient diagnostic test exist for Trichomoniasis. We present a lensless optofluidic imaging technique for label-free point-of-care detection of motile parasites in bodily fluids. With this setup we are able to achieve a FOV of ~2 mm² at a frame rate of 30 fps and can see ~200 μl of sample in one image. Trichomonas vaginalis is identifiable through simple morphological features. Contrary to common practice of minimizing the distance between the sample and the sensor to maximize the resolution, we demonstrated that a greater distance is more advantageous by using the parasites natural ability to focus light as a contrast mechanism. This technique uses a low-cost high frame rate CMOS detector, which enables high throughput operation with requires minimal sample preparation, making it a promising solution to the rapid diagnosis of Trichomoniasis.

Microplastics are small plastic particles with a size of less than 5 millimeters from cosmetics or results of abrasion and decomposition of plastic waste. The tremendous marine pollution by plastic particles and fibers and their increasing presence in the human environment from drinking water reservoirs to waste water demands for an environmental management and effective detection methods. The uptake of microplastics by living organisms may cause injuries of the gastrointestinal tract, trigger inflammation or cause cell toxicity by intrinsic particle properties or adsorbed pollutants. Thus, there is an urgent need for methods to identify microplastics in the environment as well as its sources and associated risks. The German joint research project MicroPlastiCarrier focusses on the development of new technologies for the optical detection and identification of microplastic particles in wastewater. In order to monitor particle uptake minimally-invasively in living organisms and cellular specimens in a label-free manner, we applied high resolution optical coherence tomography (OCT) and phase tomographic approaches. Moreover, multi-spectral digital holographic microscopy (DHM) is combined with innovative microfluidics to quantify morphological particle properties and their refractive index signatures at different wavelengths. Our results demonstrate that label-free optical metrology, as provided by OCT, DHM and tomographic phase microscopy (TPM), forms a promising powerful multi-functional toolbox for quantitative imaging to identify and determine the influence and risks of microplastics in the environment.

In view of a pharmacological test system with living cells we examined Förster Resonance Energy Transfer (FRET) between fluorescent proteins upon Total Internal Reflection (TIR) of a laser beam. We measured in particular HeLa cells expressing membrane associated EGFR-CFP and Grb2-YFP prior to and subsequent to stimulation with the epidermal growth factor EGF. In addition, HeLa cells expressing a membrane associated Green Fluorescent Protein (HeLa hFR-GPI-GFP) were incubated with the energy acceptor Nile Red for further validation. Measurements range from fluorescence microscopy to Fluorescence Lifetime Imaging (FLIM) of a larger number of samples in a TIR reader.

Fibre-bundle endomicroscopy is an emerging medical imaging tool. Inter-core coupling within coherent fibre bundles limits the technology's imaging capabilities. We introduce a novel approach for quantifying and modelling cross coupling, optimising image reconstruction.

We present a two-dimensional piezoelectric fiber actuator and a novel actuation scheme to establish three different scan patterns with single device. The flexibility in choosing scan patterns offers multiple options for the operator to choose between improved uniformity, high frame-rate to image abrupt biological applications, or circular field-of- view to better image naturally cylindrical cavities (gastrointestinal tract, esophagus, etc.). The presented device is capable of achieving different scan patterns with a robust actuation scheme that demonstrated through first presenting its mechanical frequency behavior, then generating 256 pixel width or diameter raster, spiral, and Lissajous patterns at ≥20 frames-per-second.The presented device, along with the actuation strategy is a promising candidate for integration with miniaturized laser scanning devices towards clinical use.

Rapid and label-free cell assay presents a challenging and significant problem that have wide applications in life science and clinics. We report here a method that combines polarization diffraction imaging flow cytometry (p-DIFC) with deep convolutional neural network (CNN) based image analysis for solving the above problem. Cross-polarized diffraction image (p-DI) pairs were acquired from 6185 cells in 5 types to investigate their uses for accurate classification. Different CNN architects have been studied to develop a compact architect named DINet which has relatively small set of network parameter for fast training and test. The averaged accuracy among the 5 groups of p-DI data ranges from 98.7% to 99.2%. With the DINet, the strong potentials of the p-DIFC method for morphology based and label-free cell assay have been demonstrated.

Type 1 Collagen is the most abundant member of the family of collagens, which are the dominant proteins constituting the extracellular matrix (ECM) of multicellular organisms. Within tissues, Type 1 collagen exhibits a fibrillar geometry that serves as a mechanical scaffold for cells. The latter remodel the collagen through the secretion of proteoglycans (proteins with long chains of sulfated glycosaminoglycans (GAGs)), both within physiological and pathological contexts. The dermatan sulfate proteoglycans (DS-PGs) are abundantly present within the developing organs and are known to be dysregulated in diseases such as cancer. How DS alters the fibrillar architecture of collagen is however, not well known. Herein, we have used second harmonic generation (SHG) microscopy to dissect the effects of DS GAGs on Type1 Collagen polymerization. We observe that the presence of DS during polymerization enhances the width and number of the fibers, the surface occupancy (which we define as the ability of the collagen matrix to fill a given volume) and the mean SHG signals. We then image polymerizing collagen matrices at temporal intervals: at very early time points (<6 h), the SHG signals in both control and DS-treated polymerizing Type-1 collagen are low and do not show any difference. However, there is a sudden increase in SHG signals 6 h onwards, with a sharper and significantly increased enhancement in the presence of DS. Our results suggest the presence of DS kinetically alters the collagen polymerization leading to significant changes in its eventual architecture.

Graded index (GRIN) lens-based microendoscopes are widely used to perform two-photon fluorescence microscopy in deep (> 1 mm) regions of highly scattering biological tissue, such as the mammalian brain. However, GRIN microendoscopes are limited by intrinsic aberrations which severely restrict the usable field-of-view (FOV). The effect of aberrations is particularly relevant in ultrathin (diameter < 500 μm) microendoscopes which allow a less invasive insertion of the optical probe into the brain tissue but which are characterized by relatively small imaging FOV. Currently, there are limited commercially available solutions to correct aberrations in these ultrathin microendoscopes because of the difficulty in fabricating corrective optics at the small spatial scale corresponding to the microendoscope diameter. Here, we report the development and application of a new approach to correct aberrations in GRIN microendoscopes using microfabricated polymeric lenses. Corrective optical elements were first designed using optical simulation software, then fabricated by two-photon lithography, and finally coupled with the GRIN lens to generate aberration-corrected microendoscopic probes. The method that we developed was applied to several types of GRIN lenses that differed in length and diameter, and corrected microendoscopes had up to 9 folds larger FOV compared to uncorrected probes. We put corrected microendoscopes to the test by performing high-resolution functional imaging of hundreds of hippocampal or thalamic cells expressing genetically encoded fluorescent indicators in the mouse brain in vivo.

Current microscopy systems for the imaging of micro-organisms are expensive because of their optimized design for resolution maximization and aberration correction. In situations where such an optimization is not needed, for instance to merely detect the presence of pathogens in liquids, a potential approach is to use highly-refractive spheres in combination with low-magnification objectives to increase the resolution and the sensitivity of the optical sensing system in a cost-effective fashion. Here, we report a study on highly-refractive dielectric mm-size spheres that are partially-embedded in polymeric membranes of mismatched refractive index. We computed the transformation that the sphere mediates on the light originating from the sample towards the optical detector, and show its enhanced-detection potential. We then propose a method to easily fabricate microfluidic chips with custom designs and precise location of dielectric spheres for enhanced imaging of micro-organisms. Our technique is based on the creation of polymeric membranes with partially-embedded spheres by spin-coating of polydimethylsiloxane pre-polymer on micro-structured Si wafers, followed by manual positioning of the dielectric spheres and polymer cross-linking. We applied this concept to the detection of living fluorescent bacteria in a water flow by creating a microfluidic chip with suitable design to confine the bacteria in the imaging region of the sphere. Based on the fluorescence-image analysis, we quantified the net contrast gain provided by the sphere for short exposure time, showing its potential for fast imaging. This fabrication method combines the flexibility of microfluidic handling with the optical enhancement of low-magnification systems and has potential for use in flow cytometry applications.

Colorectal cancer is one of the most commonly diagnosed and poorly responding to chemotherapy types of cancer, which emphasizes the importance of personalized approach to treatment selection. Short-term primary cell cultures established from patients’ tumors represent a valuable model for testing drug response. In this study, we developed protocols for generation of the short-term primary cell cultures from colorectal cancer tissue and assessment their chemosensitivity using MTT test. Additionally, we showed the possibility of metabolic analysis of patient-derived cancer cells using fluorescence lifetime imaging (FLIM) of autofluorescent cofactor NAD(P)H. Since FLIM of NAD(P)H demonstrates the potential to detect early responses to cancer treatment, we assume that this method, alone or in combination with MTT assay, can be used for choosing the optimal chemotherapy for patients.

Phase contrast images of stromal region of different stages of cervical pre-cancer were captured from tissue sections. A wavelet leader based multifractal analysis was performed on the phase contrast images to estimate multifractal spectrum for each image. Wavelet leaders were calculated through discrete wavelet transform using bi-orthogonal mother wavelets. The derived multifractal parameters, namely, width of singularity spectrum shows good discrimination between different grades of cervical pre-cancer.

In the present study, observations of temperature-mediated phase transitions of the components of adipose tissue were done. Pieces of pork abdominal fat were used as samples of adipose tissue. These samples were heated using a temperature-controlled table and observed using a MPTflex two-photon microscope (JenLab GmbH) with fluorescence lifetime imaging microscopy (FLIM) mode. The analysis of FLIM data was done with a software package “Becker&Hickl”. The developed approach has a great potential for obtaining accurate information on the phase-transition processes occurring during metabolism alterations.

We propose a fast surface profiling measurement method using a color confocal microscope based on time-encoded spectroscopy. The chromatic confocal microscopy can acquire depth information at high speed because it does not require depth scanning. On the other hand, in chromatic confocal microscopy, depth information is obtained through the wavelength of the reflected light, which is difficult for wide field imaging. By applying time encoded spectroscopy technology, depth information can be obtained at high speed through time information of reflected light. As a result, we could obtain the 3D surface shape without scanning by measuring the reflected light through the CCD over time.

The optical parameters of temporal-focusing multiphoton excitation microscopy (TFMPEM), which is capable of achieving varying wavelength excitation for multiple fluorophore measurement, was systematically examined to have better excitation performance. For this purpose, the approaches were adopted to quantitatively evaluate the grating groove density, focal length of the collimating lens and objective, and different excitation wavelengths. A grating with a groove density of 830 lines/mm enables the TFMPEM system to achieve a wavelength range of 700-1000 nm by adjusting the incident angle of the ultrafast laser on the grating; a diffraction efficiency of 81 ± 3 % was obtained at this wavelength range. By using the 830 lines/mm grating, a collimating lens with a 500 mm focal length, and a 60× water immersion objective, we achieved a large excitation area and a better filling effect of the spectrum band of the pulse on the back focal plane of the objective; these parameters ensure high optical sectioning and small variation in the illumination power density within this wavelength range. The variation ranges of the excitation area, optical sectioning, and illumination power density of 4272 ± 798 μm2, 2.6 ± 0.3 μm, and 71 ± 29 %, respectively, were obtained in two photon excitation fluorescence imaging at 700-1000 nm excitation wavelengths.

In the last years, there has been an increasing interest to the elaboration of new biocompatible and biodegradable medical polymers meant to contact the living body milieu. Amine-based biopolymer films with intrinsic biological activity have a significant potential for the synthesis of contemporaneous materials intended for surgery and tissue engineering. Our investigation is aimed to perform a non-invasive assessment of the structural characteristics and biological properties of biodegradable polymeric composites with anti-inflammatory activity, by means of ultra-high resolution laser interferometric microscopy.

Various samples of biodegradable polymers were studied with a phase-modulation laser interferometric microscope MIM-340 (Yekaterinburg, Russia) at a wavelength of 532 nm and magnification of x 20, with superficial plane resolution of up to 15 nm, vertical resolution of 0,1 nm and possibility to control the relief depth of up to 600 nm.

We have performed an in vitro non-invasive assessment of the impact of the structure, composition and modification conditions of the obtained biopolymer composites on the viability, adhesive properties and functional activity of the living blood cells (neutrophils, lymphocytes, and platelets). We propose a number of densitometry criteria to identify the most promising biopolymer samples for the development of medical products with characteristics maximally resembling the physiological ones.

A non-invasive optical measurement system based on a broadband light source and color filters has been developed for determining pulse rate and arterial blood oxygen saturation (SaO₂). In contrast to classical pulse oximetry using red and infrared LEDs to measure the peripheral capillary oxygen saturation (SpO₂), we use color filters in our system. Spectral analysis of human tissue can be easily achieved by combining a tiny color filter matrix and a commercial CMOS/CCD image sensor. During system operation, white LED light illuminates our tissue (e.g., a finger), while a CCD sensor covered by filters detects the light transmitted through that tissue. The CCD sensor is controlled by a Field Programmable Gate Array (FPGA) and a microcontroller. The detected photoplethysmographic (PPG) signal is transferred to a host computer and analyzed with MATLAB. After sensor system calibration, pulse rate and SpO₂ can be simply extracted from the PPG signal. The heart rate and SpO2 of different volunteers are then measured simultaneously by commercial pulse oximetry and the proposed sensor system, in which results from both devices show good agreement. To integrate more functions into system, nanostructured color filter matrix containing 15 filters for different wavelengths is designed and fabricated. This filter can be designed to provide transmission peaks over the visible and near-infrared range (i.e., the human tissue optical transparent window) and has a high potential to be fabricated directly on top of pixels of an image sensor.

We propose a new diagnostic tool for anemias identification based on quantitative phase imaging. We introduce a panel of label-free optical markers to identify red blood cell (RBC) phenotypes, demonstrating that an optical fingerprint of RBC is related to erythrocyte disease through modeling RBC as biological lens.

The complete cells characterization in microfluidic flow can be achieved by using the quantitative phase imaging by digital holography as imaging tool. In fact, by assuring the complete 3D rotation of flowing cells, it is possible to recover their 3D refractive index mapping by using the tomographic phase-contrast reconstruction. In this paper, we investigate all steps need to obtain the tomographic reconstruction of flowing cells. In particular, we employ a holographic 3D tracking algorithm to follow each cells that moves in the field of view, along with a suitable tracking angle method for the cell’s tumbling. Moreover, a fluid modeling is used to characterize the cell rotation effect. We test the proposed processing pipeline for circulating tumor cells.

In this work we have explored the live-cell friendly nanoscopy method Multiple Signal Classification Algorithm (MUSICAL) for multi-colour imaging of various organelles and sub-cellular structures in the cardiomyoblast cell line H2c9. We have tested MUSICAL for fast (up to 230Hz), multi-colour time-lapse sequences of various sub-cellular structures (mitochondria, endoplasmic reticulum, microtubules, endosomes and nuclei) in living cells using low excitation-light dose. Challenges and opportunities with applying MUSICAL for studying rapid sub-cellular dynamics are discussed.

We present a comparative analysis of photodynamic-treatment induced changes in optical parameters of cancer cells obtained from individual patients with three solid tumor localizations. Accumulation of photosensitizer inside living cells was validated using far-field fluorescence microscopy. Measurements of their optical characteristics were performed by means of digital holographic microscopy. The quantitative analysis of cell death dynamics performed by digital holographic microscopy was shown to be promising for investigation of cells resistivity to treatment. It was shown that both the photosensitizer accumulation and post-treatment dynamics of average phase shift may differ significantly in cell cultures obtained from different tumor localizations and different patients. Some of the cell cultures demonstrated very low or even no response to treatment.

The concept of the development of a microscope to provide research in the aquatic environment is proposed in conditions where the study of living and inanimate, static or moving microscopic objects in the environment of their natural location is required. Underwater microscope uses methods and technologies, similar conventional, based on the theory of diffraction. Achievable resolution is up to 0.3-0.5 micrometers (μm). The optical system of the underwater microscope should allow the study of microscopic objects in the water column (at different depths). In the case of an underwater microscope, the only way to get images is to use a digital receiver. Objective of microscope should be work in water immersion. The optical and the mechanical design of the objective for an underwater microscope must be adapted to the conditions of immersion in the water column, including to a considerable depth.

We integrated two-photon microscopy (TPM), second harmonic imaging microscopy (SHIM), near-infrared fluorescence microscopy (NIRF), confocal laser scanning microscopy (CLSM), and fluorescence lifetime imaging microscopy (FLIM) in one microscope system. Using our microscope system, five different images were obtained simultaneously.

We present all-optical imaging system for studying cerebral blood flow regulation. To investigate changes in blood flow in the brain, it is necessary to visualize the vascular microstructure between neurons, astrocytes, and vascular cells and monitor changes in vessel diameter and blood flow velocity. Optogenetics is an excellent technology that can be applied to cerebral blood flow regulation studies that require superior spatial and temporal resolution and individual control of cellular activity. The developed optical system is a new integrated optical imaging system that can apply all of the techniques mentioned above for cerebral blood flow regulation research. The optical system includes a dual-color fluorescence imaging system and a laser stimulation system. The system successfully performed dual-color fluorescence imaging with a 50 μm grid pattern in the two wavelength ranges of 515-545 nm and 608-631 nm. It also performed laser stimulation with a minimum output laser pattern size of 2 x 2 μm² and a maximum intensity of up to 120 mW/mm². In addition, the optical system measured the fast flow velocity of fluorescent microbeads up to 1.9 μm/ms. Experimental results show that the system can be a promising tool for cerebral blood flow regulation studies that require optogenetic stimulation and dual-fluorescence imaging while measuring blood flow velocity. In the future, the all-optical imaging system will be applied to fiber bundle-based endoscopic systems developed to study cerebral blood flow regulation using optogenetics in living animal brains.

We studied polarized fluorescence in biological coenzyme NADH in water-methanol solutions upon two-photon excitation with femtosecond laser pulses at 720 nm. The polarized fluorescence decay was recorded by a time correlated single photon counting (TCSPC) system. Fluorescence decay times, rotational diffusion time, fluorescence anisotropy, and the ratio of two pre-exponential factors have been determined and studied as a function of methanol concentration. The results obtained were interpreted on the basis of a model of NADH denaturation processes in solutions and can be used for modeling of NADH binding with various dehydrogenases in living cells.