This PDF file contains the front matter associated with SPIE Proceedings Volume 10881, including the Title Page, Copyright information, Table of Contents, Author and Conference Committee lists.

The vascular endothelium is a complex single layered network of cells which signal via the release of Ca2+ ions; the study of endothelial cell function and interactions in response to stimuli provides useful information for medical research into, for example, hypertension, diabetes and heart failure. A side viewing GRIN imaging system has previously been used to view calcium signalling in the endothelium [1] utilising a low numerical aperture GRIN rod and microscope objective to increase the imaging depth and image a large number of cells over the curved inner artery surface. This allows cells to be imaged in near-physiological conditions, as opposed to imaging of flattened arteries; however, the use of GRIN lenses introduces optical aberrations. Resolution is also limited from using a low numerical aperture system. In this work we investigate this important imaging challenge further with a view to compensating for both the cylindrically-curved geometry of the arteries and field-aberrations present in the optical system. The field aberrations in the imaging optics and resulting from the curved surface/planar sensor mismatch are quantified to allow for corrections to be made through introducing field curvature and aberration correction into the imaging path. This new instrumentation opens up the potential to image calcium signalling within large numbers of cells to try and understand the complex patterns which are produced in response to a range of stimuli.

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.

Frequency-division-multiplexed laser-scanning fluorescence microscopy is a powerful imaging method for biological tissues that enables an imaging speed of >10,000 frames/s. Despite its unprecedented high speed, its large-scale implementation that includes a bulky and unstable Mach-Zehnder interferometer has hampered its practical applications, especially in biomedical studies. Here we present a compact implementation of frequency-division-multiplexed microscopy to overcome this issue. The compactness is enabled by introducing an inline interferometer for generating an excitation beam array. In this setup, the laser beam is separated and recombined with small beam separation angles (<2°) by optical components such as acousto-optic deflectors or Wollaston prisms, thus implementing an interferometer with a relay lens system and drastically downsizing the setup. Compared with our previous setup with a Mach-Zehnder interferometer, the footprint of the optical setup for the excitation beam generation was downsized from ~20 cm x 70 cm to ~130 cm x 2.54 cm (defined by one-inch optical components used in the setup). Furthermore, our design concept allows for an ultra-compact implementation (~10 cm x 1 cm) by using custom optical components and omitting the relay lens systems. As a proof-of-concept demonstration, we obtained two-color (fluorescence and brightfield) images of Euglena gracilis cells (autofluorescent) and MCF-7 cells (fluorescence from nuclei stained by SYTO16) at a scanning speed of 0.84 m/s, which corresponds to a frame rate of 15,300 frames/s at a 55-μm field of view in the scanning direction. By virtue of the wide modulation bandwidth of the excitation beam (200 MHz), it is also possible to measure fluorescence lifetimes of target fluorophores, leading to potential applications for fluorescence lifetime imaging (FLIM).

Understanding the energy transfer mechanisms of amino acids in real time can provide insight into the strain induced during modification of proteins. Protein phosphorylation, one of the most prevalent signal transduction mechanisms within biological cells, involves structure conformation and energy transfer using a phosphate group. In eukaryotic cells, specifically, there are three phosphorylatable amino acids: L-serine, L-threonine and L-tyrosine. These amino acids are particularly important because they are responsible for biochemical reactions and cellular functions such as metabolic regulation, muscular growth, cell differentiation and metastatic behavior. In an effort to study these energy transfer mechanisms in real time, this work focuses on noninvasive measurements, such as confocal Raman spectroscopy, using custom-designed microreactors and the flow of in-solution L-serine, L-threonine and L-tyrosine biomolecules during a wide range of measurement parameters. This study accounts for optical scattering, absorption, and reflection mechanisms of phosphorylatable amino acids in an effort to better understand the optical absorbance properties so that molecular fingerprinting before and after the chemical reaction can be precisely measured. The L-serine and L-threonine particles were elongated in shape while the L-tyrosine was a fine white powder. The confocal Raman spectroscopy tool produced a one-micron diameter laser spot, and spectra for each amino acid was collected and analyzed to account for optical energy scattered. Spectral data reveals relative resilience among the amino acid crystals. The absorbance characteristics at 532 nm Raman wavelength also reveal dependencies on optical power density and an attenuation spread approaching 12% among the amino acid group under study.

Imaging natural fluorescence of the endogenous molecules is of clinical importance. Amino acids like Phenylalanine, Tyrosine and Tryptophan, structural proteins like collagen and elastin, and coenzymes directly involved in cellular metabolism like Nicotinamide Adenine Dinucleotide Phosphate (NAD(P)H) and Flavin Adenine Dinucleotide (FAD). Each endogenous molecule absorbs and emits at different wavelengths and has been used to detect various cancer including prostate cancer (PCa), based on fluorescence intensity. NAD(P)H and FAD provide detectable natural autofluorescence signals to monitor metabolic flux. Among listed amino acids, tryptophan (Trp) provides the strongest fluorescence signals. It has been shown by biochemical methods that degradation or reduction in the abundance of the Trp indicates the presence of cancer in biological systems. The one-photon absorption of Trp is 260 nm which is phototoxic to the cells. The emission of Trp is 300-400 nm. Most of the optical microscopy system transmits maximally starting from 400 nm. To meet the Trp requirement, we configured the Zeiss 780 to provide maximum emission signal about 80% at 340 nm at the ND output port for three-photon excitation. The spectral sensitivity of the detector used for Trp imaging is 300-650 nm which is integrated with the Zeiss780 system. A ROI-based segmented cell FLIRR assays was developed to investigate the interaction between NAD(P)H and Trp.

Changes in morphology and swimming dynamics of plankton by exposure to toxic chemicals are studied using a novel a new paradigm of image acquisition and computer vision system. Single cell ciliate Stentor coeruleus enclosed in a drop of water provide a means to automatically deposit many individual samples on a at surface. Chemicals of interest are automatically added to each drop while the dynamical and morphological changes are captured with an optical microscope. With computer vision techniques, we analyze the motion trajectory of each plankton sample, along with its shape information, quantifying the sub-lethal impact of chemicals on plankton health. The system enables large screening of hundreds of chemicals of environmental interest which may make their way into water habitats.

Design of portable microfluidic-cytometry devices for measurements in the field (e.g. initial medical diagnostics and recommended actions for first responders and search and rescue teams) requires careful design in terms of power requirements and weight to allow for true portability. True portability with high-throughput microfluidic systems also requires sampling systems with minimal need for sheath hydrodynamic focusing both to avoid the need for large amounts of sheath fluid and to enable higher volumes of actual sample, rather than conventional sheath/sample combinations. Weight/power requirements dictate use of super-bright LEDs as light sources and very small silicon photomultiplier sensors, with tightly integrated electronics that can both be powered by small batteries or regenerative power sources such as solar. GPS-based positioning, and telecommunications (including possible satellite–based, if cellphone towers are not nearby) to export data to other medical facilities. Microfluidic-cytometry also requires judicious use of small sample volumes and appropriate statistical sampling to permit real-time (typically in less than 10-15 minutes) initial medical decisions, not just raw data, for first responders in the field who may need results which include on-board expert medical systems. The portable system should be robust for extreme environments and should be modular and flexible to allow for multiple applications and for plug-in repairs if subsystems should become damaged. For example, one or two drops of blood obtained by pin-prick should be able to provide statistically meaningful results for use in making real-time medical decisions without the need for blood fractionation, which is not realistic in the field.

Second order susceptibility microscopy was used to image and characterize chondrogenesis in cultured human mesesnchymal stem cells (hMSCs). Second order susceptibility analysis shows that the second order susceptibility tensor ratios can be used to characterize type I and II collagens in living tissues and that both collagen types are produce at the onset of chondrogenesis. Time-lapse analysis shows that relative to type I collagen, type II collagen increases with time. Eventually, type II collagen content stabilizes at the composition of 70% of total collagen content. This approach can be used to continuously and non-invasively monitor the production of collagens I/II and developed into an effective quality control tool for monitoring extracellular matrix production in engineered tissues.

Multiphoton microscopy uses ultrafast nonlinear light-matter interactions to generate signal contrast from biological samples. The imaging of tissue from various organs plays an important role for a better understanding of cellular processes within their microenvironment and helps to reveal mechanisms of cellular changes in tissues during disease processes. Most tissue imaging studies by the pharmaceutical industry or by pathologists have typically been performed using harvested and sectioned tissue from organs to investigate drug toxicity or disease-related changes. However, immediately following biopsy, tissues begin to degrade due to cell necrosis and apoptosis, and substantial information is lost during the process. We demonstrate tissue degradation monitoring at different time points after tissue excision by using our label-free multimodal multiphoton imaging system which integrates SHG, TPEF, FLIM, and CARS in one platform. We examined whole organs and tissues harvested from mice, including kidney, liver, pancreas, and brain, and immersed each in several different media including saline, Euro-Collins solution, UW solution, HTK, and formalin. We collected time-lapse images from each sample and compared rates of cell degradation, tissue structure changes, and variations in optical properties including the intensities of NADH and FAD, the metabolic redox ratio, and FLIM of free/bound NADH. As a result, we quantified rates of degradation and metabolic changes associated with the preservation methods based on these label-free optical properties. Therefore, these results can be used as reference values for most ex vivo tissue research that relies on tissue and cell viability.

We are engineering cartilage from autologous nasal chondrocytes and a collagen scaffold in chondrogenic conditions to treat knee cartilage defects in an ongoing phase II clinical trial. To comply with regulatory requirements, we are developing quality controls to characterize and ensure the safety and quality of the engineered cartilage products. Our preliminary results show that we can measure the Raman spectra of engineered cartilage. Here we propose a standardized procedure for collecting and preprocessing the Raman spectral data. We currently have experienced, trained technicians manufacturing the engineered cartilage, but in the future, these grafts will be made by various labs, therefore ensuring the standardization of the manufacturing process is a challenge that could be addressed with Raman spectroscopy-based quality controls. In this manuscript we discuss how Raman spectroscopy-based quality controls could be incorporated into the Good Manufacturing Practice (GMP) compliant process for our engineered cartilage.

Fluorescent nano-diamonds (fND) containing negatively charged Nitrogen Vacancy (NV) centers have remarkable applications in biology such as bio-labelling and nanoscale sensing of cellular processes. The NV centers also act as atomic scale probes that are highly sensitive to the magnitude, direction and fluctuation of local magnetic fields. The work presented here reports on the development of a wide-field microscope using fNDs as biologically targeted quantum probes to monitor live cell dynamics associated with fluctuations in local magnetic and electric fields. fNDs were functionalized using antibodies to enable site specific targeting. Strategies were also implemented to overcome the formation of endosomes around fNDs once inside the cytoplasma, which not only inhibits targeting but also the resultant sensitivity to the cellular environment. In addition to fluorescent mapping, the exchange of magnetization between the magnetically active NV defects in the diamond lattice and paramagnetic species in the local environment was studied via acquisition of optically detected magnetic resonance spectra. The results of this work demonstrate the utility of fNDs as probes to monitor subcellular dynamics. The wide-field configuration of the microscope enabled fast acquisition of images essential to characterize transient events in live cells. Looking to the future, fNDs have tremendous potential to augment existing fluorescent probes and to enable magnetic resonance measurements on a nanoscale in live cell cultures.

Plankton is at the bottom of the food chain. Microscopic phytoplankton account for about 50% of all photosynthesis on Earth, corresponding to 50 billion tons of carbon each year, or about 125 billion tonnes of sugar[1]. Plankton is also the food for most species of fish, and therefore it represents the backbone of the aquatic environment. Thus, monitoring plankton is paramount to infer potential dangerous changes to the ecosystem. In this work we use a collection of plankton species extracted from a large dataset of images from the Woods Hole Oceanographic Institute (WHOI), to establish a basic set of morphological features for supporting the use of plankton as a biosensor. Using a perturbation detection approach, we show that it is possible to detect deviation from the average space of features for each species of plankton microorganisms, that we propose could be related to environmental threat or perturbations. Such an approach, can open the way for the development of an automatic Artificial Intelligence (AI) based system for using plankton as biosensor.

With the development of fluorescence molecular tomography (FMT), it has become an important tool for life science research. Many methods were developed to improve the reconstruction performance of FMT. However, these methods only focus on the accuracy and speed of the reconstruction, while ignoring the morphological information of the tumor. In this paper, the modified L_2,1-norm method were adopted to reconstruct the morphology of the mouse orthotopic glioma. To verify the L_2,1-norm method, we carried out the simulation experiment and the in vivo orthotopic glioma mouse experiment. The iterated shrinkage (IS) method and the Tikhonov-based method were implemented for comparison. The results show that the modified L_2,1-norm method can obtain more accurate reconstruction in both tumor location and morphology reconstruction. We believe that the morphological reconstruction of FMT will provide more potential for preclinical research.

The current clinical standard for mass screening of prostate cancer are prostate-specific antigen (PSA) biomarker assays. Unfortunately, the low specificity of PSA’s bioassays to prostate cancer leads to high false-positive rates, as such there is an urgent need for the development of a more specific detection system independent of PSA levels. In our previous research, we have successfully demonstrated, with the use of our Photonic-Crystal based biosensor in a Total-Internal- Reflection (PC-TIR) configuration, detection of prostate cancer (PC-3) cells against benign prostate hyperplasia (BPH-1) cells. The PC-TIR biosensor achieved detection of individual prostate cancer cells utilizing cellular refractive index (RI) as the only contrast parameter. To further study this methodology in vitro, we report a comprehensive study of the cellular RI’s of various prostate cancer and noncancerous cell lines (i.e. RWPE-1, BPH-1, PC-3, DU-145, and LNCaP) via reflectance spectroscopy and single-cell RI imaging utilizing the PC-TIR biosensor. Our study shows promising clinical potential in utilizing the PC-TIR biosensor system for the detection of prostate cancer against noncancerous prostate epithelial cells.

Sucrose is important to be measured in food industry and Raman spectroscopy is preferred method for being fast, accurate, and non-destructive. Different concentration of sucrose was measured using Horiba’s modular probe-based Raman system. The signature peak of 824cm-1 caused by CH out-of-plane deformation was studied as a conventional fingerprint. There is a correlation between the intensity of each Raman peak and the final sample concentration. As one drops the other will follow linearly. Minimum concentration of 0.5% was recorded for short integration time of 0.5s. This finding can be revolutionary for food industry due to the high sensitivity and speed.

Lens-based imaging approaches are faced with a trade-off between resolution and field-of-view (FOV). Generally, the greater the resolvable detail in a sample, the smaller the FOV we can observe. In our lab, we study the behaviour of microorganisms within confined spaces using microfluidic devices. In order to capture the full scope of their behaviour, we need to be able to discern individual microorganisms as well as observe the full microfluidic device area in real time. As such, visualizing such systems can be challenging, since we require an imaging system that can provide a resolution as high as 1 um, with a FOV large enough to fit our region of interest. To that end, we used the Nokia Lumia 1020 mobile phone, which has a 41.3 megapixel (MP) image sensor with a pixel size of 1.14 um, with an external lens attached to the camera for better focus, and we characterized the imaging system to have a spatial resolution of 1.2 um, with a FOV of 3.6 x 2.7 mm, and a working distance of 0.6 mm. Moreover, we used the screen of a Retina display Apple device as a versatile illumination source for this system. The screen is used to project various illumination patterns onto the specimen being imaged, each corresponding to a different illumination mode, with the Nokia phone capturing the resulting image. We tested our system by using it to image microorganisms such as Escherichia coli and Euglena gracilis within our microfluidic devices.

Many hardware approaches have been developed for implementing hyperspectral imaging on fluorescence microscope systems; each with tradeoffs in spectral sensitivity and spectral, spatial, and temporal sampling. For example, tunable filter-based systems typically have limited wavelength switching speeds and sensitivities that preclude high-speed spectral imaging. Here, we present a novel approach combining multiple illumination wavelengths using solid state LEDs in a 2-mirror configuration similar to a Cassegrain reflector assembly. This approach provides spectral discrimination by scanning a range of fluorescence excitation wavelengths, which we have previously shown can improve spectral image acquisition time compared to traditional fluorescence emission-scanning hyperspectral imaging. In this work, the geometry of the LED and other optical components was optimized. A model of the spectral illuminator was designed using TracePro ray tracing software (Lambda Research Corp.) that included an emitter, lens, Spherical mirror, flat mirror, and liquid light guide input. A parametric sensitivity study was performed to optimize the optical throughput varying the LED viewing angle, properties of the Spherical reflectors, the lens configuration, focal length, and position. The following factors significantly affected the optical throughput: LED viewing angle, lens position, and lens focal length. Several types of configurations were evaluated, and an optimized lens and LED position were determined. Initial optimization results indicate that a 10% optical transmission can be achieved for either a 16 or 32 wavelength system. Future work will include continuing to optimize the ray trace model, prototyping, and experimental testing of the optimized configuration.

Hyperspectral imaging has numerous applications in a range of fields for target detection. While its original applications were in remote sensing, new uses include analyzing food quality, agriculture and medicine, Hyperspectral imaging has shown utility in fluorescence microscopy for detecting signatures from many fluorescent molecules, but acquisition speeds have been slow due to the need to acquire many spectral bands and the light losses associated with spectral filtering. Therefore, a novel confocal microscope, the 5- Dimensional Rapid Hyperspectral Imaging Platform (RHIP-5D) was designed and is undergoing testing to overcome acquisition speed and sensitivity limitations. The current design utilizes light-emitting diodes (LEDs) and a multifaceted mirror array to combine light sources into a liquid light guide. Initial tests demonstrated feasibility and we are now working on determining the ideal location of the liquid light guide, LEDs, lenses and mirror array to optimize optical transmission. A computational model was constructed using Monte Carlo optical ray tracing in TracePro software (Lambda Research Corp.). LED sources were simulated by importing irradiance properties from the manufacturers’ specifications. Optical properties of lenses were modeled using lens files available from the manufacturer. Analysis of the model includes geometry and parametric optimization, assessing lens power, mirror angles and location of optical elements. Initial results show an increase of transmission is possible by up to 20%. Future work will involve evaluating the position of the liquid light guide as well as analyzing lens configurations to further increase optical transmission.

Most existing methods using hyperspectral imaging (HSI) to estimate skin chromophore concentrations fail to give an account of scattering properties crucial to many medical applications. To address this limitation, we propose to combine HSI with spatial frequency domain imaging (SFDI). Total acquisition time is around five seconds, making the process suitable for in vivo application. Skin absorption and scattering analysis is performed from these images by successive optimizations on the absorption and scattering properties. The problem of shadows occurring on complex shapes such as the face is addressed by an original approach that make results robust to irradiance drift.

Glioblastomas are brain cancers with very poor patient prognosis. We have developed a Glioblastoma U87 MR model, using 4-dimensional imaging in multi-day tracking experiments. The cells have a tendency to form long-term cellular associations, and quantifying their motility by standard approaches is difficult. We cultured the cells in a structured environment (wound healing template), separated the X and Y information to define cumulative directionality plots providing a metric of the overall cell population movement analyzed by holographic imaging cytometry. With cellular tomography, we obtained 3D time lapse tomographs of cells at 0.2 um resolution, enabling sub-cellular analysis at levels not previously possible. Even in label-free cultures, sub-cellular components can be distinguished and color-coded based on differences of their refractive index values. We discovered that there are numerous mitochondria present, both single and also actively undergoing fission and fusion processes. Many thin mitochondrial networks are present within the cytoplasm, and also extending away from the cell in tunneling nanotubes. There is fusion of these networks to form larger structures that form connections between cells. Substances can be seen moving bi-directionally between cells. After several days of culture, the cells form large multicellular and highly connected spheroids. This is evident in widefield stitched images of the spheroids. While the tendency of U87 cells to form spheroids was previously known, the combined results from our multi-modality quantitative imaging platforms provide new insights into the cellular dynamics of glioblastoma cells, and the networks that they form. This knowledge is being applied to the development anti-glioblastoma treatments.

Biomarker detection and quantification in body fluids is widely utilized in medical diagnosis as it provides useful information for developing treatment strategies. Once attached to target biomarker molecules, fluorescence can be used to detect and quantify the target biomarkers. However, expression of target biomarkers in body fluids is generally weak, and therefore direct utilization of fluorescence for detection with high sensitivity and selectivity is challenging. To address this issue, fluorescence enhancement of target biomarkers has been investigated by several research groups. These studies utilized light-metal-fluorophore interactions and have reported about few thousand-fold fluorescence enhancement. Fluorescence enhancement of few thousand folds enables the detection of molarities up to about nanomolar. However, medical diagnosis, especially early diagnosis, requires detection limit of attomolar. To extend the capabilities of fluorescence enhancement to be applicable in the full range of medical diagnosis, we have used low frequency electric fields (<20 MHz) to further enhance the light-metal-fluorophore interactions. This paper presents the results from our simulation work performed to show how electric fields could modulate the fluorescence enhancement. Moreover, we have found that external electric fields can be used to place the fluorophore molecules outside the quenching regions, align fluorophore dipoles with plasmonic axis of metal and place the fluorophore in the high electric field region of the scattered light. These capabilities could lead to fluorescence enhancement up to about billion-fold that enables attomolar detection. In addition to enhancing the fluorescence, we have also studied the effect of electric fields on localized surface plasmon resonance (LSPR).

In nonlinear optical imaging of biological samples, epi-illumination collection of fluorescent signals results in half of the signal loss. We enhanced the collected luminescent signal by using a multilayer coating of tantalum pentoxide (Ta₂O₅) and cerium oxide (SiO₂). Our coatings are biocompatible, allowing visual inspection of samples and optimizing the collection of luminescent signals. This method was confirmed on a number of samples, including sulforhodamine solutions, fluorescent microspheres and labeled 3T3 cells. In all cases, the coated coverslips were used to enhance the signal by approximately 2 times. Image analysis also shows that signal enhancement does not result in additional photobleaching. Our results indicate that the designed coated coverslip enhances multi-photon microscopy detection signals.

Demands of multi-well plate readers has been on the rise for drug discovery and cell line development applications, as it can obtain fluorescence, absorbance, and morphology information from cell cultures grown in tens to hundreds of conditions. Existing systems typically only house one camera, requiring slow mechanical actuation to cover a large area on the multi-well plate, or to sacrifice speed for area, forgoing the precious spatial information. We mitigate the time-resolution trade off with the Fourier ptychographic microscopy (FPM) technology by simultaneously capturing 96 high-resolution phase images (>20,000 cells per plate) with CMOS-based cameras with custom-designed microscope objectives. By illuminating the samples with a permutation of lighting conditions, we achieve synthetic numerical aperture (NA) of 0.3 at an extended depth-of-field of 20 micrometer for at most 96 conditions at one time. Unlike our previous invention of the 6-well plate reader (EmSight), the same illumination condition can be shared among adjacent cameras. Therefore, image acquisition and data transfer can be performed in a massively parallel manner. Along with computational acceleration with graphical processing units (GPUs), all these approaches reduces the plate-to-image turnover from hours to minutes – an eight-fold reduction in time over existing mechanical-scanning plate readers. In addition to providing phase imaging, the system is also capable of fluorescence imaging at the native resolution of the objectives. We anticipate that our high-throughout 96-camera imaging system will help advance the high content analysis of cell cultures beyond hundreds of test conditions, thus facilitates more in-depth characterization of biological screens and drug testing.

Histological examination has been the primary imaging modality in the diagnosis of eases such as cancer. However, since cells are three-dimensional in nature, the use of traditional nucleus to cytoplasm ratio (N/C) in two dimensions does not represent their three-dimensional structures. In this study, we used two-photon microscopy to acquired threedimensional images of normal human lung cell line Beas2B, human lung adenocarcinoma CL1-0 and CL1-5 cell lines. We determined N/C ratios in two- and three-dimensions and found that 2D N/C-ratio is more precise than 3D N/C-ratio in discriminating normal and cancer cells.

Successful cancer treatment continues to elude modern medicine and its arsenal of therapeutic strategies. Therapy resistance is driven by significant tumor heterogeneity, complex interactions between malignant, microenvironmental and immune cells and cross talk between signaling pathways. Advances in molecular characterization technologies such as next generation sequencing have helped unravel this network of interactions and have vastly affected how cancer is diagnosed and treated. However, the translation of complex genomic analyses to pathological diagnosis remains challenging using conventional immunofluorescence (IF) staining, which is typically limited to 2-5 antigens. Numerous strategies to increase distinct antigen detection on a single sample have been investigated, but all have deleterious effects on the tissue limiting the maximum number of biomarkers that can be imaged on a single sample and none can be seamlessly integrated into routine clinical workflows. To facilitate ready integration into clinical histopathology, we have developed a novel cyclic IF (cycIF) technology based on antibody conjugated oligonucleotides (Ab-oligos). In situ hybridization of complementary oligonucleotides (oligos) facilitates biomarker labeling for imaging on any conventional fluorescent microscope. We have validated a variety of oligo configurations and their respective signal removal strategies capable of diminishing fluorescent signal to levels of autofluorescence before subsequent staining cycles. Robust signal removal is performed without the employment of harsh conditions or reagents, maintaining tissue integrity and antigenicity for higher dimensionality immunostaining of a single sample. Our platform Ab-oligo cycIF technology uses conventional fluorophores and microscopes, allowing for dissemination to a broad audience and congruent integration into clinical histopathology workflows.

A rapid and easy method for determining the ratio of live to dead bacteria in a sample is valuable in many fields of microbiological and pharmacodynamics studies, and for the monitoring of food safety and public health. Efficient, culture-independent detection of live and dead bacteria can be achieved using differentially staining fluorescent dyes SYTO 9 and propidium iodide (PI). Fluorescence microscopy and flow cytometry have been used extensively for detection of these live/dead cell fluorescence signals, however, both these methods require bulky equipment and are relatively expensive to implement. We are developing a cost-effective and portable fibre-based fluorometer for the accurate measurement of fluorescence signals to determine the proportions of live and dead bacteria present. Escherichia coli suspensions with differing ratios (from 0 to 100%) of live to dead cells were stained with SYTO 9 and PI. Fluorescence spectra were collected from samples using the optrode, which consists of a 473 nm solid state laser, DAQ controlled shutter, photodiode for monitoring the laser power and CCD spectrometer. Three regression techniques (simple SYTO 9/PI intensity ratio, adjusted SYTO 9/PI intensity ratio, and support vector regression (SVR)) were used to analyse the spectra to predict the percentage of live bacteria present in each sample. To date, SVR achieved the best correlation (R^2>0.99, for samples containing 108 bacteria/mL). Preliminary results are promising and show that this method may be extended to a wider concentration range or modified for the calibration of other bacterial species of interest.

Data from single-cell mRNA sequencing, made available by leading-edge experimental methods, demand proper representation and understanding. Multivariate statistics and graph theoretic methods represent cells in a suitable feature space, assign to each cell a time label known as “pseudo-time” and display “trajectories” (in fact orbits) in such space. Orbits shall describe a process by which progenitors differentiate into one or more types of adult cells: broncho-alveolar progenitors are e.g., found to evolve into two distinct pneumocyte types. This work aims at applying the qualitative theory of dynamical systems to describe the differentiation process. Some notions of qualitative theory are presented (§ 2). The main stages of single-cell data analysis are outlined (§ 3). Next, a two-dimensional continuous time, autonomous dynamical system of polynomial type is looked for, the orbits of which may interpret some sequences of data points in feature (⌘ state) space. Section 4 defines an energy function F of two variables, 1,!2}, and the autonomous dynamical system obtained from rF, which thus generates a gradient flow. Both F and the gradient flow give rise to a phase portrait with two attractors, A and B, a saddle point, O, and a separatrix. These properties are suggested by data from single cell sequencing. Initial states of the system correspond to progenitors. Attractors A and B correspond to the two cell types yielded by progenitor differentiation. The separatrix and the saddle point make sure an orbit asymptotically reaches either A or B. Why and how a gradient flow model shall be applied to data from single-cell sequencing is discussed in § 5. The application of dynamical system theory presented herewith relies on a heuristic basis, as all population dynamics models do. Nonetheless, placing a given cell on an orbit of its own enables time ordering and compliance with causality, unlike pseudo-time assignment induced by a minimum spanning tree. An earlier (2009) application in a much simpler context, the evolving morphology of cytoskeletal tubulines, is finally recalled: from cyto-toxicity experiments, epifluorescence images of tubulin filaments were obtained, then analysed and assigned to morphology classes; class centroids formed a sequence in feature (⌘ state) space describing loss of cytoskeletal structure followed by its recovery.

Successful development of a multimodal imaging system for the detection of live single cells is critical for enhancing diagnosis and treatment of diseases at an early stage. Recently, photoacoustic microscopy (PAM) and optical coherence tomography (OCT) have been investigated as high resolution, non-invasive imaging techniques for the detection of single cells. However, most cancerous cells lack sufficient endogenous contrast. The aim of the current study is to develop a multimodal imaging system that combines PAM, OCT, and fluorescence microscopy (FM) for the detection of single cells with the assistance of gold nanoparticles as contrast agents. Three different type of ultra-pure colloidal cluster gold nanoparticles (GNPs) were fabricated and functionalized with ligands. The synthesized GNPs, average particle size of 10, 20 nm, and 60 nm (cluster GNPs), were evaluated. Cluster nanoparticles were achieved by combining an average of 3 to4 colloidal GNPs. The peak absorption of GNPs is 532 nm (bare) and 650 nm (cluster). The functionalized GNPs were delivered into different cells (HeLa, OZ3, bovine retinal endothelial, and bovine brain endothelial cells) at various concentrations (0, 12.5, 25 and 50 µg/mL). The treated cells with GNPs were imaged using the custom-built multimodal PAM, OCT, and FM system. The PAM contrast was enhanced linearly with the increasing in GNP concentration. The OCT contrast was also increased up to 2 fold in comparison with control. FM results showed the position of accumulated GNPs co-registered with the dark field images. The proposed multimodal imaging system may provide a potential tool for the detection and imaging of live single cells.

Sensing photoacoustic waves brings a lot of loss since the detector distance is in the order of millimeters which also leads to noise in the measured signal. To solve this problem, we used an optically trapped silica particle as a transducer in this study. We used two laser sources, one for optical tweezers (976 nm, CW) and a fiber laser for photoacoustic imaging (SHG output: 532 nm, pulsed). The fiber laser was produced in our laboratory whose pulse duration is 8 ns, pulse energy is 10 µJ, and pulse repetition frequency is 65 kHz. The separation between them in the sample plane is 8 µm. The green laser excited several absorbing mediums such as trypan blue, horse hair, black ink and gold thin film. We tracked the position of trapped silica particle (5µm diameter) when the green laser is on and off. We observed dramatic difference between two states. We have validated that this effect is fully photoacoustic by changing the frequency of the green laser with a chopper which led to the exact same frequency when we calculated the Fourier transform of the position distribution of the trapped silica particle. Also, when we change the power of the green laser, the amplitude of the Fourier transformation of the position distribution of the trapped silica particle changes in the same way.

Otitis media (OM) is a common disease associated with high antibiotics prescription, high recurrence, and developmental issues in children. Its early detection is crucial to prevent sequelae. However, the diagnosis of OM is commonly based on subjective conclusions taken from observation of an eardrum through a conventional otoscope. Therefore, this often leads to misdiagnosis and moreover culminates in erroneous antibiotics prescription, occasioning the appearance of resistant bacteria. A smartphone-based imaging system allows an untrained user to acquire and transmit data to a specialist for remote diagnosis. We thus developed a smartphone-based multispectral imaging otoscope capable of offering quantitative information on the physiological state of an eardrum with the benefits from the portability and connectivity of the smartphone. The system consists of an Android application for the control of the hardware and also the display of classification results, a circuit board for the interface of an LED multiplexer with the smartphone, and a custom-made otoscope probe for uniform illumination onto the interior of an ear canal. The probe includes a set of lenses and eight optical fibers attached to the LED multiplexer. The multiplexer is composed of a white LED and eight LEDs with consecutive/sequential wavelengths. We examined a normal ear and an ear with OM with effusion using our developed system. The results showed that the smartphone-based multispectral imaging otoscope could quantitatively distinguish between a healthy ear and an ear with OM with effusion, suggesting its potential as a mobile healthcare tool for diagnosis and management of middle ear pathologies.

Performance of multi-spectral imaging critically depends on image acquisition time and working spectral bandwidths. Ultimate performance can be achieved if a set of monochromatic (single-wavelength) spectral images is obtained by a single snapshot - a technique provisionally called “snapshot multi-spectral-line imaging” or SMSLI. The SMSLI principle and the developed prototype devices for 3, 4 and 5 spectral line snapshot imaging are described. Two potential practical applications of SMSLI are discussed – for fast mapping of the main in-vivo skin chromophores and for detection of counterfeit banknotes and documents.

Aggregates of misfolded α-Synuclein in the brain represent a hallmark of Parkinson’s disease (PD). In patients and animal models, phosphorylated α-Synuclein was detected in the gut, hence, raising the hypothesis that early-stage PD could be diagnosed based on colon tissues. Marker-independent technologies represent an ideal method to monitor disease progression and potentially detect early-stage aggregated α-Synuclein in vivo. Here, formalin-fixed, paraffinembedded colon tissues of a transgenic rat model were analyzed using Raman imaging. Detailed spectral and imagebased analysis was performed indicating the major spectral shifts that alter in PD rat tissues in the amide I region. Peak fitting and multivariate analysis specified an increase of β-sheet proteins in transgenic rat colon compared with wild-type colon. In summary, Raman imaging is capable to detect α-Synuclein aggregates in colon tissues of a PD rat model, indicating that it could be a useful tool to support diagnosis in PD pathology.

Brightfield microscopy is a standard method for the identification and enumeration of different micro-organisms, specifically for analyzing different types of algae and planktonic organisms in water samples. Typically, bright- field microscopy is performed in a broadband visible spectrum configuration; however, important distinguishing features in various micro-organisms are much better captured using a narrow-band multispectral configuration. One challenge with leveraging multispectral microscopy, particularly in low-cost field-portable instrument setups, is the presence of significant chromatic aberrations. Therefore, we introduce a multispectral Bayesian-based computational microscopy method for enhancing image quality by jointly correcting for chromatic aberrations, illumination inhomogeneities and noise across multiple spectral wavelengths within a probabilistic framework. To test the efficacy of this method, calibration parameters associated with a field-portable multi-spectral mi- croscopy instrument are measured by characterizing the point spread functions at different spectral wavelengths ranging from 465 nm - 655 nm with a pinhole target. We demonstrate the effective optical resolution improvements of the microscopy instrument augmented with the proposed method using the 1951 USAF resolution test chart. Finally, we evaluate the qualitative performance of this instrument by imaging Anabaena flos-aqua, a toxin-producing cyanobacteria, as well as Ankistrodesmus falcatus, a type of green algae. The efficacy of this proposed framework shows the potential of having an in-situ instrument to observe biological organisms at mul- tiple narrow-band wavelengths, providing both additional spectral information and the ability for continuous detection and monitoring of micro-organisms.

The first commercial confocal microscope was released in 1987. Since then, laser scanning confocal microscopy has been widely used in tissue, cell, developmental and molecular biology, and even pathology. Currently, there is a growing need to image multiple fluorescent (and autofluorescent) markers, simultaneously and cost effectively, to detect, discriminate and quantitate various components and their interactions (e.g. by fluorescence resonance energy transfer) in biological materials, with enhanced axial resolution. In addition, with high enough spectral resolution, even cell nuclear size estimations are possible, using Mie scattering. Current multispectral/hyperspectral scanning laser confocal microscopes are extremely expensive and there is huge need for a cost-effective and efficient add-on system to upgrade conventional microscopes to spectral laser scanning confocal microscopes. We developed an affordable ultra-resolution spectral confocal add-on system and tested it on research-grade microscopes. Our system includes supercontinuum laser as a broadband light source, a confocal scanning system, and spectral selection using custom spectral tunable cavities (STCs) offering ultra-spectral (sub-nanometer) resolution. The STCs use picoliter volume Fabry–Perot-type optical cavities filled with liquid crystal for tuning and can be incorporated in excitation or emission optical paths as a single cell or an array format. The spectral selection is done with no moving parts and just applying voltage to the STC filter. We will present various fabrication methods of STCs with different geometrical and material selections (glass versus polymer substrates) to improve resolution, throughput and manufacturability. We present system design, validated spectral and spatial resolution and testing the confocal system on histopathology slides.

Adipose tissue derived stem cells (ASCs) has applications in soft tissue replacement-based tissue engineering. ASCs can potentially reduce many of the disadvantages of autologous fat transplantation such as donor-site morbidity and immune system rejection. Although, ASCs hold clinical relevance as a potential cell therapy candidate, widespread use of them is hampered due to inadequate data on the fate of stem cells after transplant. Hence a method to facilitate long term tracking of the cells will enable better understanding of stem cell fate in stem cell-based therapeutics. Here, we employ biocompatible surface functionalized nanorods for tracking the adipogenesis and osteogenesis differentiation of ASCs. Anisotropic plasmonic nanostructures based on silver (Ag) and gold (Au) have received much attention owing to their tunable size and shape dependent localized surface plasmon resonance (LSPR) with multiple applications such as biological contrast agents, photothermal conversion, plasmon-enhanced spectroscopies, optical sensors and in catalysis. Hyperspectral microscopy combining both nanoscale imaging and spectral characteristics from plasmonic nanostructures provides a powerful tool for their identification and quantitative spectral analysis of plasmonic nanostructures with unprecedented level of details. Here, we present the analysis of single particle spectroscopy of gold nanorods and their orientation dependent scattering properties using hyperspectral microscopy and validated with correlated high-resolution electron microscopy. Fairly monodisperse gold nanorods with bright longitudinal SPR centered at about 663 nm were synthesized using bromide-free surfactant mixture consisting of cetyltrimethylammonium chloride and sodium oleate. The nanorods were successfully characterized by UV-Visible spectroscopy, DLS, XPS, and TEM results. Dark-field hyperspectral and second harmonic generation (SHG) microscopy were performed on individual gold nanorods and their optical scattering spectra were analyzed for imaging orientation of single nanorods. The initial results revealed scattering spectra from individual gold nanorods displayed measurable spectral-shifts from their collective LSPR spectrum from bulk measurements performed using UV-Visible spectroscopy. The analysis and utility of gold nanorods for labeling stem cells and the orientation dependent spectral features of nanorods inside the cells will be characterized and discussed in detail. The cell viability, differentiation capacity, gene expression, potential cytotoxicity due to nanorods such as inflammatory molecule and reactive oxygen species production, adipogenic and osteogenic potential will be evaluated using histochemical staining and quantitative polymerase chain reaction (qPCR). The study has implications towards tracking individual nanorods in complex biological systems and beyond.

Abnormally shaped red blood cells (poikilocytes) can cause serious health problems such as anemia and increase the risk of death. At present, the biochemical abnormalities in poikilocytes is not well understood, especially at the single cell level. In this study, confocal Raman spectroscopy revealed biochemical differences between single normal red blood cells (RBCs) and poikilocytes. Intragastric administration of nanoparticulate titanium dioxide (TiO₂) was used to produce poikilocytes. Adult rats were administered by gavage 200mg/kg body weight TiO₂ every other day for 20 days (low-dose, N=5) or 250mg/kg every day for 60 days (high-dose, N=5). Low and high-dose controls (N=5 each) were administered distilled water for equal durations. Raman spectroscopy was performed on individual RBCs of low-dose subjects using 514nm excitation and a confocal setup. Whole blood from high-dose subjects underwent a Complete Blood Count (CBC) and inductively coupled plasma mass spectrometry (ICP-MS). Acanthocytes and echinocytes, two types of poikilocytes, were observed from TiO₂ subjects. RBCs were grouped into four types: normal RBCs from controls and normal-looking RBCs, Acanthocytes, and Echinocytes from TiO₂ subjects. The intensities of Raman bands at 1637, 1585, 1559, 1372 and 1228cm^-1 are larger in acanthocytes than normal and normal-looking RBCs. The 1342cm^-1 band is larger in normal RBCs, acanthocytes and echinocytes than in normal-looking RBCs. Also, the 975cm^-1 band is larger in acanthocytes than normal-looking RBCs. These bands are associated with oxygenated RBCs. Overall, poikilocytes, especially acanthocytes, carry more oxygen and haemoglobin and this is corroborated by CBC and ICPMS.

A major benefit of fluorescence microscopy is the now plentiful selection of fluorescent markers. These labels can be chosen to serve complementary functions, such as tracking labeled subcellular molecules near demarcated organelles. However, with the standard 3 or 4 emission channels, multiple label detection is restricted to segregated regions of the electromagnetic spectrum, as in RGB coloring. Hyperspectral imaging allows the user to discern many fluorescence labels by their unique spectral properties, provided there is significant differentiation of their emission spectra. The cost of this technique is often an increase in gain or exposure time to accommodate the signal reduction from separating the signal into many discrete excitation or emission channels. Recent advances in hyperspectral imaging have allowed the acquisition of more signal in a shorter time period by scanning the excitation spectra of fluorophores. Here, we explore the selection of optimal channels for both significant signal separation and sufficient signal detection using excitation-scanning hyperspectral imaging. Excitation spectra were obtained using a custom inverted microscope (TE-2000, Nikon Instruments) with a Xe arc lamp and thin film tunable filter array (VersaChrome, Semrock, Inc.) Tunable filters had bandwidths between 13 and 17 nm. Scans utilized excitation wavelengths between 340 nm and 550 nm. Hyperspectral image stacks were generated and analyzed using ENVI and custom MATLAB scripts. Among channel consideration criteria were: number of channels, spectral range of scan, spacing of center wavelengths, and acquisition time.

Microplastics are small plastic particles the 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 the 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. The urgent need for methods to identify microplastics in the environment, its sources of input and the risk of microplastic particles is the objective of the research project MicroPlastiCarrier. The project develops new tools for the optical detection and identification of microplastic particles from wastewater by a multiwavelength approach. The multiple labelfree optical toolbox is based on digital holographic microscopy using wavelengths from the visible to mid infrared. 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 multi-spectral digital holographic microscopy (DHM). In combination with microfluidics technologies as flow cytometry the project plans to identify particles based on size and their absorption and refraction index properties at several wavelengths. The technology should overcome the limitations of state of the art FT-IR.

In this paper, we introduce the improvement of the conventional skin color separation method using two input skin images. The conventional method needs to satisfy the severe limitations that input skin image has no shading component and has plenty variation of the pigments density distribution simultaneously. In the two input images at the proposed method, the one has no shading component and the other has plenty variation of the pigments density distribution. To verify the effect of our method, we taken four subjects and separate these images to pigments components and compare the independent evaluation value (IEV) of separated signals extracted by using our method and the conventional method. The result shows that we obtain the separated signals which have lower IEV by using our method than the conventional method.

Fluorescence Imaging (FI) has become a major diagnostic tool in the surgical field where Indocyanine Green (ICG) is mainly used as contrast agents, as it is currently the most widely compatible, FDA-approved contrast agent. ICG stained particles emit fluorescent light when irritated by pulses of laser. For in vivo surgical imaging, however, ICG is used in either different concentrations or mixed with various viscous solutions to avoid dispersion to unnecessary areas. Depending on the mixture and the concentration, absorption levels will fluctuate and wavelengths may even shift from what is determined to be the norm. Similarly, ICG has limited capabilities in detection under certain depths. This can lead to inconsistent decisions among surgeons, further leading to reoccurring problems both during and post-surgery as detection may become faint or inaccurate. Due to its high contrast, high sensitivity and affordability, much research has been done on the properties of ICG but there currently is a lack of sufficient data on the varying shifts and absorption levels caused by different conditions. By determining and solidifying a spectrum of wavelengths that different ICG solutions emit depending on its concentration, mixture and depth, ICG detection can greatly be enhanced through better calibration. Ultimately, it will increase the effectiveness of non-invasive imaging-guided treatments and diagnostics.

Ca²⁺ and cAMP are ubiquitous second messengers known to differentially regulate a variety of cellular functions over a wide range of timescales. Studies from a variety of groups support the hypothesis that these signals can be localized to discrete locations within cells, and that this subcellular localization is a critical component of signaling specificity. However, to date, it has been difficult to track second messenger signals at multiple locations within a single cell. This difficulty is largely due to the inability to measure multiplexed florescence signals in real time. To overcome this limitation, we have utilized both emission scan- and excitation scan-based hyperspectral imaging approaches to track second messenger signals as well as labeled cellular structures and/or proteins in the same cell. We have previously reported that hyperspectral imaging techniques improve the signal-to-noise ratios of both fluorescence and FRET measurements, and are thus well suited for the measurement of localized second messenger signals. Using these approaches, we have measured near plasma membrane and near nuclear membrane cAMP signals, as well as distributed signals within the cytosol, in several cell types including airway smooth muscle, pulmonary endothelial, and HEK-293 cells. We have also measured cAMP and Ca²⁺ signals near autofluorescent structures that appear to be golgi. Our data demonstrate that hyperspectral imaging approaches provide unique insight into the spatial and kinetic distributions of cAMP and Ca²⁺ signals in single cells.

Disruption of cell respiration and metabolic changes accompanies many tissue disorders including neurodegenerative diseases and cancers. Fluorescence lifetime imaging microscopy (FLIM), a non-invasive and information-rich technique, can be invoked to reveal these changes. The method allows for monitoring of fluorescent intrinsic metabolic coenzymes, first of all, NADH (nicotinamide adenine dinucleotide), on the level of single cells and can be applied to living tissue. The ratio between protein-bound and free NADH gives information about the balance between oxidative phosphorylation and glycolysis in cells. There is a correlation between cellular metabolic activity, redox ratio and fluorescence lifetime of NADH. In combination with laser scanning microscopy, the time-correlated single photon counting (TCSPC) technology enables FLIM NADH mapping of the biomedical samples. On the other hand, TCSPC provides quantification of the phosphorescence lifetime of oxygen-sensing molecules. Accordingly, the oxygendependent quenching of phosphorescence of compounds such as transition metal complexes can be employed for evaluation of oxygen partial pressure (pO2) by PLIM (phosphorescence lifetime imaging microscopy). We demonstrate correlated FLIM/PLIM imaging, which provides simultaneous mapping of NADH and oxygen in living cells. Continuous FLIM/PLIM imaging enables to monitor changes in oxygen levels and cell metabolic status dynamically during PDT and provides new opportunities in theranostics.

In this study, the spatial resolution of the existing 1.5 T magnetic resonance imaging (MRI) was attempted to be improved from 0.7 mm to 50 μm by accurately analyzing the MRI signal using high-precision signal analysis. Non-harmonic analysis (NHA) accurately estimates the Fourier coefficient based on the least mean squares method, and exhibits a higher frequency resolution that is than the fast Fourier transform. A numerical experiment based on measuring parameters of 1.5 T MRI demonstrates the potential use of NHA in visualizing finer structures.

Thyroid nodules are very common, and their incidence increase with age. The majority of thyroid nodules are benign, but approximately 5-15% are thyroid cancer. The cornerstone to evaluation of most thyroid nodules is a neck ultrasound followed by fine-needle aspiration (FNA) to evaluate for malignancy. Unfortunately, approximately 15-30% of FNAs are considered “indeterminate”. In these cases, cytopathologist cannot determine if the thyroid nodule is benign or malignant based on FNA alone. Although the majority of these “indeterminate” nodules are ultimately benign, majority of these patients require thyroidectomies to rule out cancer. This puts the patient at unnecessary risk of surgical complications and increases health care costs. A better method is needed to help physicians determine the risk of malignancy in patients with indeterminate thyroid nodules. In recent years there has been much interest in the use of optical diagnostic in cancer detection. Recent investigations potentially suggest that Raman spectroscopy (RS) can be used as a clinical tool that could confer great patient advantage with minimally invasive, non-destructive, rapid and accurate diagnosis. In this study, we investigate the use of line-scan RS and imaging in combination with multivariate statistical analysis of the spectral data for objective identification and classification of single cells isolated from frozen samples of different types of human thyroid nodules. Preliminary results indicate a high sensitivity and specificity for identifying different cell types.

The development of Bioluminescence Tomography (BLT) has allowed the quantitative three dimension (3D) whole body imaging and non-invasive study of biological behavior of cancer. However, the ill-posed problem of BLT reconstruction limits the quality of reconstruction result. In this work, we proposed a Bilateral Weight Laplace (BWL) method that utilizes a non-local Laplace regularization to improve the imaging quality of bioluminescence tomography reconstruction. The non-local Laplace regularization was constructed by spatial weight and range weight to penalize the neighborhood-variance of reconstructed source density in both spatial and range domain. To evaluate the performance of BWL method, both dual-source BLT reconstruction experiment in simulation data and in vivo BLT reconstruction in orthotopic glioma mouse model were designed. Furthermore, fast iterative shrinkage/threshold (FIST) method and Laplace method were utilized to compare with BWL. Both dual-source experiment and in vivo experiment demonstrate that the construction results of BWL method provide more accurate tumor position (BCE = 0.37mm in dual-source experiment) and better tumor morphological information.

Fluorescence molecular tomography (FMT) is a promising imaging technique in applications of preclinical research. However, the complexity of radiative transfer equation (RTE) and the ill-poseness of the inverse problem limit the effectiveness of FMT reconstruction. In this research, we proposed a novel Deep Convolutional Neural Network (DCNN), Gated Recurrent Unit (GRU) and Multiple Layer Perception (MLP) based method (DGMM) for FMT reconstruction. Instead of establishing the photon transmission models and solving the inverse problem, the proposed method directly fits the nonlinear relationship between fluorescence intensity at the boundary and fluorescent source in biological tissue. For details, DGMM consists of three stages: In the first stage, the measured optical intensity was encoded into a feature vector by transferring the VGG16 model; In the second stage, we fused all encoded feature vectors into one feature vector by using GRU based network; In the last stage, the fused feature vector was used to reconstruct the fluorescent sources by MLP model. To evaluate the performance of our proposed method, a 3D digital mouse was utilized to generate FMT Monte Carlo simulation samples. In quantitative analysis, the results demonstrated that DGMM method has comparable performance with conventional method in tumor position locating. To the best of our knowledge, this is the first study that employed DCNN based methods for FMT reconstruction, which holds a great potential of improving the reconstruction quality of FMT.

Bone healing is a complex process involving molecular changes. Bone matrix consists of collagen proteins that serve as the framework and minerals, calcium and phosphate, are deposited into the matrix accordingly. Raman spectroscopy is a promising technique to study bone mineral and matrix environments simultaneously. We studied the bone composition using 785 nm excitation during healing of subcritical calvarial defects without disrupting the fracture. Calvarial defects (in vivo) were created using 1 mm burr drill on the parietal bones of Sprague-Dawley rats (n=8). After 7 days, subjects were sacrificed and an additional defect (control) was created. Principal component analysis was utilized for the analysis of Raman spectra and helped in classifying normal and healing bone. Principal component 1 (PC1) shows that the major variation between in vivo and control defects and normal bone surface is at 958 cm^-1 (ʋ1 phosphate band). PC2 shows a major variation at 1448 cm^-1 (CH₂ deformation). PC2 score distinguishes in vivo defects from normal surface and control defects. The decrease in crystallinity and mineral to matrix ratio at the healing site as revealed by Raman confirms the new bone formation. Scanning electron and optical microscopy show the formation of newly generated matrix by means of bony bridges of collagens. The surface roughness increases by 23% from control to in vivo defects, as revealed by optical profiler. Overall, the new collagen formation shows the scaffolding of the bone is growing during healing.

Colloids and suspensions are part of our daily routines. Even the blood is considered a “naturally” occurring colloid. However, the majority of colloids are complex and composed by a diversity of nano to microparticles. The characterization of both synthetic and physiological fluids in terms of particulate types, size and surface characteristics plays a vital role in products formulation, and in the early diagnosis through the identification of abnormal scatterers in physiological fluids, respectively. Several methods have been proposed for characterizing suspensions, including imaging, electrical sensing counters, hydrodynamic or field flow fractionation. However, the Dynamic Light Scattering (DLS) has evolved as the most convenient method from these. Based also on the scattering signal, we propose a novel, simple and fast method able to determine the number of different scatterers type present in a suspension, without any previous information about its composition (in terms of particle classes). This is achieved by collecting features from a 980 nm laser back-scattered signal acquired through a polymeric lensed optical fiber tip dipped into the solution. Unlike DLS, this technique allows the trapping of particles whose diameter ≥ 1 μm. For smaller particles, despite not guaranteeing their immobilization, it is also able to determine the number of different nanoparticles classes in an ensemble. The number of particle types was correctly determined for suspensions of synthetic particles and yeasts; different bacteria; and 100 nm nanoparticles types, using both Principal Component Analysis and K-means algorithms. This method could be a valuable alternative to complex and time-consuming methods for particles separation, such as field flow fractionation.

The standard practice employed by dermatologists for examining skin lesions is dermoscopy, where an epiluminesence microscope (ELM) is used to examine the skin chrominance and micro-structural characteristics for anomalies. Conventional ELM instruments are being replaced by digital ELM instruments that enable der- matologists and other health care practitioners to digitally capture, archive, and analyze skin lesions using computer-aided diagnosis (CAD) software. One of the limiting factors of digital ELM is the fundamental trade- off between spatial resolution and field-of-view (FOV), where a larger FOV (which is needed to allow for larger skin lesions to be examined in their entirety) can be achieved by reducing magnification at the cost of spatial resolution (leading to a loss of fine details that can be indicative of malignancy and disease). Here, we introduce deep computational optics (DCO) for the purpose of resolution-enhanced digital ELM to improve the balance between spatial resolution and FOV. More specifically, the multitude of parameters of a deep computational model for numerically magnifying digital ELM images are learned through a wealth of low-resolution and high- resolution digital ELM image pairs. The proposed DCO approach was experimentally validated, demonstrating improvements in the spatial resolution of the resolution-enhanced digital ELM by two-fold while maintaining FOV.

In recent years, hyperpolarized noble gas MRI attracts attention. Spin-exchange optical pumping (SEOP) is often used for hyperpolarizing nuclear spin of noble gas. Although there are many reports on rare gas hyperpolarization devices using SEOP, most of them use high power CW lasers exceeding 10 W. Large output laser has high risk of exposure, high cost and high energy consumption. However, in SEOP, only a very small amount coincident with D1 line works and almost all other power is abandoned. Therefore, in this study, we development that Xe hyperpolarizing system using narrow linewidth Ti:Sapphire laser and demonstrated that this system can pump with lower pawer by tuning wavelength to D1 line. As a result, we show that system using narrow linewidth laser can get equally or lager signal augment ratio by 1/45 times lower irradiation power than conventional system.

Radiogenic dental damage is a common and crucial problem in patients receiving radiotherapy for malignancies in the head and neck region. Unfortunately, little is known about the development of complications after radiation therapy on the microstructure profiles of the human tooth. Therefore, we propose a novel method in which the primary focus is to investigate, in vitro, the direct influences of di↵erent radiation doses on elastic properties of enamel and dentin of human tooth by Scanning Acoustic Microscopy (SAM) at the microscale. We obtain two-dimensional (2D) acoustic impedance images from twenty-five sound human third molars each of which is cut into a 1 mm thick cross-sectional slices. Acoustic impedance by SAM operating at 320 MHz are recorded from the sections comprising enamel and dentin before and after every irradiation dose to a cumulative dose of 60 Gy. The findings of our study reveal that radiation therapy changes the micro-elastic features of enamel and dentin accompanied by the decreased acoustic impedance. We establish a relationship between cumulative irradiation doses and the measured acoustic impedance. The quantified acoustic impedance values for the different irradiation doses might be helpful in in vitro assays for the determination of the safe dose limits to prevent severe tooth damage in the treatment plan of the individuals having head and neck cancer.

Prostate cancer (PCA) is the most common cancer and the third most common cause of cancer death in men. Targeted nanoparticles (NPs) that deliver effective doses of chemotherapeutic drugs specifically to PCA could improve chemotherapy efficacy without the toxicities. In the relevant mouse models, the direct visualization of such drug nanoparticles along with the vasculature and fibrillar collagen matrix at submicron resolution are critically important for the accurate measurements of the drug distribution in the tissue matrix. Multiphoton microscopy, which uses ultra-short IR laser pulses as the excitation source, produces multiphoton excitation fluorescence (MPEF) signals from exogenous or endogenous fluorescent proteins and induces specific second harmonic generation (SHG) signals from non-centrosymmetric proteins such as fibrillar collagens. The objective here is to visualize and quantify the 3D distribution of an aptamer conjugated calcium phosphosilicate based drug nanoparticle carriers along with vasculature and tissue matrix in ex vivo thick mouse prostate tumor tissue with submicron resolution. Human prostate tumor xenografts were established in athymic mice by injecting prostate cell line derived from human (PC-3 cells) and were grown for 4 weeks. Near-infrared imaging agent indocyanine green (ICG) loaded calcium phosphosilicate nanoparticles (CPSNPs) including targeted CPSNPs bioconjugated with DNA Aptamer, empty non-ICG containing CPSNPs (Ghost) and Dil (for blood vessel painting) were injected into the tail vein. The spectral unmixing was performed to extract Dil signal from ICG signal using measured emission spectra. The 3D reconstructions and subsequent quantitation showed accumulation of ICG in blood capillaries versus tissue matrix. We here conclude that this multiphoton based multimodal imaging approach can provide spatially resolved 3D images with spectral specificities that are sensitive enough to identity and quantify the distributions of drug nanoparticle carriers in conjunction with vasculature and tissue matrix in prostate tumor with structural precision.

Some preliminary results and experimental details of Near-field Scanning Optical Microscopy (NSOM) operation in liquids have been reported by us earlier. Here we present the first use of custom made polystyrene/poly (methyl methacrylate) (PMMA) optical fibers to assemble new NSOM probe/sensor for operation in liquids. Assembled NSOM probe has quite large quality factor Q ranging 2000-6000 in air, and 300-900 when immersed 0.2-0.3 mm deep into the water. Such montage demonstrates high mechanical durability permitting to scan different samples during many hours or even days, and overall low cost in comparison with NSOM probes based on glass optical fibers. A specially prepared optical fiber with 125 μm diameter (from Paradigm Optics Company, USA) (polystyrene core diameter is 0.85 μm, ncore=1.59 and PMMA cladding, n_core=1.49) was chemically etched using a 9:1 mixture of dichloromethane and ethyl acetate. As result of the etching, a smooth and sharp tip is formed with a typical radius of the curvature equal to 50 - 170 nm. For completeness, earlier unpublished images of living Picocyanobacteria bacteria obtained using glass fiber- made NSOM probes are also presented.

By short-term irradiation of a cell with focused laser pulses, the cell membrane can be made transiently permeable. This "optoporation" allows for foreign macro molecules to pass this otherwise impenetrable barrier and enter the cell interior enabling a highly efficient and “hands-off” laser-assisted transfection of cells. We intend to combine a precise flow control of cells with the laser-assisted-transfection technique by employing microfluidics to control the cell flow through the laser-cell interaction region. The precise flow control will allow for a high throughput of cells and combine microfluid cell control with the advantages of the highly efficient laser-assisted transfection scheme. The development of the setup is still in progress. Here, the background and key features are outlined.