This PDF file contains the front matter associated with SPIE Proceedings Volume 11243, including the Title Page, Copyright information, Table of Contents, Author and Conference Committee lists.

Previous studies in this area of research have reported that merchantability of meat drops when the meat color. The changes in meat color are caused by met-myoglobin concentration changes. Despite, a few methods are presented to measure met-myoglobin concentration, those methods have a number of problem in use. In general, met-myoglobin concentrations increase inside the meat and spread to the surface. The main purpose of this study is the measurement of met-myoglobin proportion inside the meat by using diffuse optical spectroscopy (DOS) to predict meat color changes. To conduct the experiments, the DOS system consists of a spectrometer and the broadband light source. And 30 beef samples were taken on the day that the cattle were slaughtered. In order to measure met-myoglobin changes over time, Data were collected every day. The results show us increase and decrease of met-myoglobin during the storage. This study will help us to predict meat color changes and to qualify merchantability

We report a single-shot computational polarized-light microscopy (SCPLM) method for identifying pathological crystals in bodily-fluids. We utilize the four-directional polarizers integrated on the pixels of a CMOS image sensor to reconstruct the transmittance, retardance, and slow-axis orientation maps of the objects with a single image exposure. Using SCPLM, we imaged birefringent crystals found in synovial fluid, e.g., monosodium urate, calcium pyrophosphate dehydrate, and triamcinolone acetonide. The quantitative birefringence images created by our method are pseudo-colored and digitally-integrated with bright-field images to highlight the birefringent crystals within the background. We believe this single-shot, quantitative, and easy-to-operate method will significantly benefit rheumatology.

Measurement of melanin concentration, blood concentration and oxygen saturation in skin tissue is highly important technology in medical and cosmetic fields. Most previous studies neglect depth inhomogeneity of blood concentration distribution in skin tissue. However, skin tissue has a complex layered structure, and it is known that distribution of blood concentration differs greatly depending on the depth. Although a method has been proposed considering the inhomogeneity of blood distribution in depth direction, it is impossible to measure oxygen saturation. In addition, there is a limitation that shading influences the estimation as noise in any of the methods. Therefore, in this study, we propose a method to measure skin components concentration, which are melanin concentration, blood concentration, and oxygen saturation, in layers with different depths of skin tissue without influence of shading. First, to construct a dataset of absorbance corresponding to the skin components concentration we construct an optical multi-layered model of skin tissue and simulate light propagation and attenuation by light scattering and absorption with Monte Carlo simulation method. Each absorbance value is converted to a relative value based on an averaged absorbance value of all wavelengths to remove influence of shading. Next, we create a model describing an absorbance value corresponding to the skin components concentration with high dimensional multivariate regression based on the created dataset. This model enables to estimate the skin components concentration from spectral information without influence of shading. The proposed method is evaluated by simulation and shown to have state-of-the-art performance.

We investigate the process of induced pluripotent stem cell (iPSC) passaging. Subcultures are created by transferring cells from iPSC cultures to new growth mediums. We found that standard protocols for iPSC passaging primarily have researchers use their eyesight to determine cultures' confluencies and cell counts. With the consequences of inaccurate estimates going as far as cell death due to passaging at the suboptimal confluency, we sought to circumvent human error and develop a culture analyzing algorithm (CAA) that calculates both confluency and cell count primarily through Otsu's method. We incorporate multi-image machine learning into our CAA, improving its ability to recognize colonies as it is fed more images. In comparing our algorithm to standard protocols, we found that there was a significant percent difference between both methods when measuring the confluency and cell count of iPSC cultures. Through further refinement, we hope to streamline our CAA for large-scale use.

Drosophila Melanogaster is a sample of high biological interest that is being widely used as biological model, due to the relatively short life cycle, short genome and ease in culturing. In this work we present a microscope on chip capable of processing Drosophila embryos to obtain three dimensional fluorescent images at high throughput. This device, based on light sheet microscopy, uses a plane of light intercepting the sample channel to optically and noninvasively section the embryos while flowing. This permits to automatically acquire for each sample the stack of images necessary for the subsequent 3D reconstruction with no need of any manual sample positioning and alignment. The whole chip is fabricated in a glass substrate by femtosecond laser micromachining. The device has been optimized for the specific morphology of the sample. Indeed, the highly elliptical shape of the embryos (about 100 x 500 μm²) might affect the image quality degrading both the vertical and the axial resolution of the system. To overcome this issue, we have first optimized the layout of the fluidic channel to precisely control the sample orientation by means of hydrodynamic forces. Thereafter, we have optimized the properties of the optical circuit, to realize two opposite light sheets impinging on the sample, perfectly overlapped, with a high signal to noise ratio. With these actions, we have been able to obtain high quality Drosophila reconstruction.

Fluorescence imaging microscopy has traditionally been used because of the high specificity that is achievable through fluorescence labeling techniques and optical filtering. When combined with spectral imaging technologies, fluorescence microscopy can allow for quantitative identification of multiple fluorescent labels. We are working to develop a new approach for spectral imaging that samples the fluorescence excitation spectrum and may provide increased signal strength. The enhanced signal strength may be used to provide increased spectral sensitivity and spectral, spatial, and temporal sampling capabilities. A proof of concept excitation scanning system has shown over 10-fold increase in signal to noise ratio compared to emission scanning hyperspectral imaging. Traditional hyperspectral imaging fluorescence microscopy methods often require minutes of acquisition time. We are developing a new configuration that utilizes solid state LEDs to combine multiple illumination wavelengths in a 2-mirror assembly to overcome the temporal limitations of traditional hyperspectral imaging. We have previously reported on the theoretical performance of some of the aspects of this system by using optical ray trace modeling. Here, we present results from prototyping and benchtop testing of the system, including assembly, optical characterization, and data collection. This work required the assembly and characterization of a novel excitation scanning hyperspectral microscopy system, containing 12 LEDs ranging from 365- 425 nm, 12 lenses, a spherical mirror, and a flat mirror. This unique approach may reduce the long image acquisition times seen in traditional hyperspectral imaging while maintaining high specificity and sensitivity for multilabel identification and autofluorescence imaging in real time.

Most cancers originate from epithelium, the top layer of mucosa. We report single-snapshot Spatial Frequency Domain Imaging (SFDI) of mucosa with visible modulated light. A novel two-layer mucosa model enables the mapping of the total hemoglobin concentration, the oxygen saturation, the scattering characteristics of the top and bottom layers, and the thickness of the top layer from a single-snapshot. After validating with phantom studies, we demonstrate its applicability to the characterization of mucosa by imaging human mouth lips of healthy subjects. The proposed approach may find important applications in screening mucosa for early detection of cancer and other diseases.

A widefield system for multidimensional fluorescence imaging capable of resolving space, time and wavelength is developed and validated on a synthetic fluorescence sample. The system enables structured illumination and compressing detection. A compression strategy based on an a-priori information obtained by a camera is validated and proved to be effective at compression ratio of about 90%.

We designed a simple, compact and robust microscope for phase and fluorescent imaging. No mechanical movement of neither sample nor objective or any other part of the system is needed to change between the phase-contrast and fluorescence modality. We can observe a thousand cells in parallel in a single field of view with resolution down to 2 µm. We demonstrate the system on a FUCCI marked HeLa cell culture observed over several days directly in the standard incubator. We compare the obtained statistics to the flow cytometer data and show that we can produce a statistically relevant time-resolved measurement

Capillaroscopy is a simple microscopy technique able to measure important clinical biomarkers non-invasively. For example, optical absorption gaps between red blood cells in capillary vessels of the nailfold have been shown to correlate with severity of neutropenia. The direct visualization of individual white blood cells with capillaroscopic techniques is elusive because it is challenging to generate epiillumination phase contrast in thick turbid media. Here, we evaluate white blood cell visibility with graded-field capillaroscopy in a flow phantom. We fabricate capillary phantoms with soft photolithography using PDMS doped with TiO₂ and India ink to emulate skin optical properties. These glass-free phantoms feature channels embedded in scattering media at controlled depths (70-470 μm), as narrow as 15 x 15 μm, and permit blood flow up to 6 mm/s. We optimize the contrast of the graded-field capillaroscope in these tissue-realistic phantoms and demonstrate high speed imaging (200 Hz) of blood cells flowing through scattering media.

Time-domain microfluidic fluorescence lifetime flow cytometry enables observation of fluorescence decay of particles or cells over time using time-correlated single photon counting (TCSPC). This method requires the fluorescence lifetime measured from a limited number of photons and in a short amount of time. In current implementations of the technique, the low throughput of state of the art detectors and lack of real-time statistical analysis of the current technology, the timedomain approaches are usually coupled with off-line analysis which impedes its use in flow cell sorting, tracking and capturing. In this work, we apply a 32×32 CMOS SPAD array (MegaFrame camera) for real-time imaging flow cytometry analysis. This technology is integrated into a 1024-beam multifocal fluorescence microscope and incorporating a microfluidic chip at the sample plane enables imaging of cell flow and identification. Furthermore, the 1.5% native pixel fill-factor of the MegaFrame camera is overcome using beamlet reprojection with <10 μW laser power at 490 nm for each beam. Novel hardware algorithms incorporating the center-of-mass method (CMM) with real-time background subtraction and division are implemented within the firmware, allowing lossless recording of TCSPC events at a 500 kHz frame rate with 1024 histogram bins at 52 ps time resolution. Live calculation of background compensated CMM-based fluorescence lifetime is realized at a user-defined frame rate (typically 0.001 ~ 27 kHz) for each SPAD pixel. The work in this paper considers the application of the SPAD array to confocal fluorescence lifetime imaging of multiple coincident particles flowing within a microfluidic channel. Compared to previous flow systems based on single-point detectors, the multi-beam flow system enables visualization, detection and categorization of multiple groups of cells or particles according to their fluorescence lifetime.

Computer vision and deep learning are integral tools in the improvement of high-throughput analysis of cellular images. Specifically, optimization of algorithms for object detection and instance segmentation tasks are important in cellular image analysis to segment and classify multi-object, multi-class images. In this work, we employ an instance segmentation pipeline with Mask RCNN, using a ResNet-101 and Feature Pyramid Network convolutional backbone to segment and classify T cells and antigen presenting cells (APCs) in multi-channel fluorescence confocal images of lupus nephritis biopsies. This task was first performed on a dataset of fresh frozen biopsies stained for T cells (CD3 and CD4) and two APC populations: 1) myeloid dendritic cells (BDCA1 and CD11c), and 2) plasmacytoid dendritic cells (BDCA2 and CD123). The network achieved an average sensitivity of 0.82, specificity of 0.91, and Jaccard index of 0.79 across all cell types. However, relative to fresh frozen tissue, samples prepared through formalin fixation and paraffin embedding (FFPE) provide larger potential datasets for investigating immune activity. Training this same network architecture on an FFPE database of lupus nephritis tissue stained with the same antibody panel, the network achieved an average sensitivity of 0.82, specificity of 0.92, and Jaccard index of 0.77 across all cell types. In addition to working with FFPE tissue, it would also be beneficial to identify APCs with a single stain and image more cell types with a single staining panel. We have trained this network on a single-stained APC panel FFPE dataset to achieve an average sensitivity of 0.79, specificity of 0.86, and Jaccard index of 0.63 across all cell types. These three trained networks were used to assess differences in cell shape features between fixation and staining protocols.

Flow cytometer is a powerful tool for the quantitative analysis of a large population of cells. A key factor determining the measurement accuracy and system stability is the illumination beam. The conventional beam shaper is composed of multiple geometrical optical elements, which should be precisely calibrated to produce elliptical Gaussian beams. It is difficult to control the sizes and positions of the focal spots formed by different wavelengths. A new beam shaping method based on diffractive optical element (DOE) is developed for rectangular flattop spots with multi-wavelength illumination. Its benefits include high simplicity, design flexibility, uniform illumination, and multifunctionality.

Coherent anti-Stokes Raman scattering (CARS) microscopy was used to disclose dynamic information from lipid droplets (LDs) in living cancer cells. Statistical analyses of LD dynamics show different pools of LDs associated with LD synthesis and degradation processes. We found that each LD population satisfies a log-normal distribution, thus allowing quantitative comparison through fitting of population histograms. Two-dimensional analyses of LD displacements and speeds offer dynamic signatures of cells to monitor responses to stimuli and drugs. We revealed hypothermia-induced cellular metabolic changes and a two-step metabolic response during apoptosis. We also explored dynamic signatures of LDs during starvation and various drug exposures.

Time-gated fluorescence imaging of near-infrared emitting ZnCuInSe/ZnS quantum dots (QDs) with fluorescence lifetimes in the range of 150−300 ns enables the efficient rejection of fast autofluorescence photons and the selection of QD fluorescence photons. This leads to complete elimination of autofluorescence background and a significant increase of imaging sensitivity. We demonstrate efficient detection and imaging of individual QD-labeled lymphoma cells circulating at mm/s velocities in blood vessels. In a second application, these QDs were used as a sensing platform to detect enzymatic activity using a ratiometric time-gated sensing scheme.

We propose a high-throughput 3D imaging cytometer for fast quantification of DNA double strand break (DSB) frequency in cells for DNA damage study. With structured illumination enabled depth contrast and a fast focus tunable lens enabled scanning, this system generates a three-dimensional stack of clustered nuclei γH2AX foci with submicron resolution at a speed of 800 cells/second. Moreover, we unify the stack construction with the deep neural network, which largely improve quantification accuracy as well as the processing speed. Compared to previous 2D imaging approach, the addition of z-resolution in our 3D method provides an extra dimension of contrast and thus allows for more accurate DNA DSB quantification.

Förster resonance energy transfer (FRET) is a valuable tool for measuring molecular distances and the effects of biological processes such as cyclic nucleotide messenger signaling and protein localization. Most FRET techniques require two fluorescent proteins with overlapping excitation/emission spectral pairing to maximize detection sensitivity and FRET efficiency. FRET microscopy often utilizes differing peak intensities of the selected fluorophores measured through different optical filter sets to estimate the FRET index or efficiency. Microscopy platforms used to make these measurements include wide-field, laser scanning confocal, and fluorescence lifetime imaging. Each platform has associated advantages and disadvantages, such as speed, sensitivity, specificity, out-of-focus fluorescence, and Zresolution. In this study, we report comparisons among multiple microscopy and spectral filtering platforms such as standard 2-filter FRET, emission-scanning hyperspectral imaging, and excitation-scanning hyperspectral imaging. Samples of human embryonic kidney (HEK293) cells were grown on laminin-coated 28 mm round gridded glass coverslips (10816, Ibidi, Fitchburg, Wisconsin) and transfected with adenovirus encoding a cAMP-sensing FRET probe composed of a FRET donor (Turquoise) and acceptor (Venus). Additionally, 3 FRET “controls” with fixed linker lengths between Turquoise and Venus proteins were used for inter-platform validation. Grid locations were logged, recorded with light micrographs, and used to ensure that whole-cell FRET was compared on a cell-by-cell basis among the different microscopy platforms. FRET efficiencies were also calculated and compared for each method. Preliminary results indicate that hyperspectral methods increase the signal-to-noise ratio compared to a standard 2-filter approach.

Light sheet fluorescence microscopy (LSFM) enables fast 3D imaging of live cells, however the traditional LSFM geometry is not compatible with conventional multiwell plates used in high content microscopy. We have developed an automated LSFM platereader based on an oblique plane microscope (OPM) that is compatible with multiwell plates. The system enables automated studies of cells cultured in a collagen matrix. Cells across 30 different conditions are imaged every 5 minutes for 12 hours. A custom 3D segmentation and tracking pipeline analyses cell morphological dynamics, allowing the study of a range of treatment conditions on the ability of cancer cells to change shape and invade.

Recent biomedical developments towards compact, mobile, decentralized and system-integrated diagnostic platforms require automated and miniaturized microscopy technologies. Reducing system dimensions by downsizing classical single aperture optics limits either the field of view (FOV) or resolution. However, arranging multiple miniaturized objectives in parallel allows overcoming this restraint by decoupling the system’s FOV from the axial system length. Based on this principle, we propose a thin multi-aperture microscope concept to image a large FOV with μm resolution. Due to a total optical system length of only 10 mm, the imaging optics can be integrated into conventional camera housings. The approach’s potential is demonstrated by introducing a first prototype specified by life science requirements. The final system enables for bright field and fluorescence imaging of (1) multiple separated object fields simultaneously (e.g. parallel monitoring in microfluidics applications) or (2) extended continuous object areas via sample scanning. Hence, the micro-objective array approach provides a microscopy solution for biomedical applications with tight space requirements like point-of-care diagnostic devices, cell incubator microscopes and organ/lab-on-chip long term monitoring.

Endogenous and exogenous fluorescence imaging have shown great values for monitoring and studying all kind of biological processes including vascular tissue regeneration and disease progression. In this study, we present a fiber-based optical imaging instrument that is able to simultaneously acquire endogenous and exogenous fluorescence images from tissue samples using a reflective optical chopper wheel to temporally interleave the two modalities. The functionality of the system was demonstrated by imaging native tissue constructs seeded with cells labeled with different dyes. The cellular regions were clearly resolved in both modalities, providing an exogenous and in-situ validation for endogenous fluorescence lifetime images.

Microplastic particles are a major environmental pollution problem. Challenges in microplastics detection are the broad size range, chemical heterogeneity and low concentrations in liquids. Label free optical imaging methods for the detection and particle type identification were tested and compared. Quantitative Phase Imaging and holographic tomography were used to study cell response and cell uptake of microplastic particles. Optical Coherence Tomography could analyze larger organisms as Daphnia magna for microplastics uptake. Imaging methods combining particle localization with a chemical identification as RAMAN and mid IR spectroscopy could clearly identify different particle types and even detect specific microplastic chemistries inside cells.

Correctly diagnosing and staging prostate cancer continues to be a significant clinical challenge. Currently, the standard of care consists of a pathologist’s visual assessment of hematoxylin-and-eosin-stained (HE) histological sections, and designation of a Gleason score based on the top two most common patterns. However, this process is subjective and thus prone to error. Further, lack of standard protocols for staining, makes quantitative analysis of stained tissues difficult. Therefore, there is a significant need to develop new quantitative methods that can provide robust, objective, and accurate information of the aggressiveness and stage of prostate cancer. In this work, we seek to address this challenge using multi-spectral deep-UV microscopy of unstained tissue sections. This method yields valuable insight into the aggressiveness and stage of the disease due to its subcellular spatial resolution and high sensitivity to many endogenous biomolecules, including nucleic acid and proteins. In our approach we use a simple and cost effective wide-field imaging configuration with sequential illumination at multiple wavelengths ranging from 220 nm to 450 nm. Spectral signatures are analyzed in conjunction with the morphology using a geometrical representation of principal component analysis and principles of mathematical morphology. Our results reveal distinct morphological and molecular alterations in the tissue as cancer becomes more aggressive. In this presentation we will detail the design of the multispectral, deep UV microscope; describe our quantitative image analysis; and show preliminary results.

Genipin cross-linked engineered tissues are 10000 times less toxic than glutaraldehyde cross-linked tissues. Hence, genipin is a better fixative to support the recellularization of tissue-engineered constructs such as vascular grafts. Here, we demonstrate the ability of fiber-based Fluorescence Lifetime Imaging (FLIm) guided Raman spectroscopy to monitor the quality of genipin cross-linked vascular grafts with high speed and specificity. Current results indicate that the fluorescence lifetime of AR-BP shortens upon GE cross-linking. Raman spectroscopy reveals secondary structural changes occurring in the extracellular matrix of pericardia that correspond to Amide I, Amide III and C-C stretch vibrations. We conclude that FLIm guided Raman imaging can detect cross-linking signatures with biochemical specificity and that this imaging modality provides a non-destructive and label-free method to assess the quality of vascular grafts

Met-myoglobin is a major component related to meat discoloration, and it gradually accumulates over time after the meat is slaughtered. Recently, studies have been conducted to observe the changes in the composition of met-myoglobin in the meat along with its storage time using Diffuse Reflectance Spectroscopy(DRS). DRS is an optical technique that is simple and can estimate the composition of chromophores without damaging the sample. However, since DRS requires high resolution and complicated fitting process, it is difficult to apply DRS to the mobile environment. Therefore, the purpose of our study is to classify the freshness of meat by extracting features from low spectral resolution diffuse reflectance spectrum by using the deep learning model. To improve the generality of the model, a data augmentation was used. To consider the applicability at low-resolution spectrometer, the diffuse reflectance spectrum was down-sampled 5, 10, 30 and 50 times.

The differentiation of endothelial cells from human iPSC has incontestable advantages in diseases research and therapeutic applications. However, the safe use of iPSC derivatives in regenerative medicine requires an enhanced understanding and control of factors that optimize in vitro reprogramming and differentiation protocols. Shifts in cellular metabolism associated with intracellular pH changes affect the enzymes that control epigenetic configuration, which impact chromatin reorganization and gene expression changes during reprogramming and differentiation. FLIM-based metabolic imaging of NADH and FAD is a powerful tool for measuring mitochondrial metabolic state and widely used diagnostic method for identification of neoplastic diseases, skin diseases, ocular pathologies and stem cells differentiation. Therefore, in this study, we used the potential of FLIM-based metabolic imaging and fluorescence microscopy of NADH and FAD to study the metabolic changes during iPSC differentiation in endothelial cells. The evaluation of the intracellular pH was carried out with the fluorescent pH-sensor SypHer-2 and fluorescence microscopy to obtain complete information about metabolic status of iPSC and their endothelial derivatives. Based on the FAD/NAD(P)H optical redox ratios increase and the contributions rise of the NAD(P)H fluorescence lifetime in iPSC during endothelial differentiation, we demonstrated an contribution increase of OXPHOS to cellular metabolism. Based on the shift toward more acidic intracellular pH in endothelial cell derived from iPSCs we verified their oxidative state.

Chemical space for small molecule therapeutics discovery is greatly under-explored due to difficulties in animal testing, the first bottleneck compounds encounter in going from formula to human use. We developed and validated an assay that combines 3D tissue biofabrication with high-throughput imaging biomarkers. This may impact more diseases than just skin cancer, where we have recently shown promising preliminary findings. Our skin constructs have normal epidermis, with populations of human keratinocytes, dermis with human fibroblasts and tumor spheroids containing populations of human squamous cell carcinoma cells. We present imaging biomarkers that show the cellular chemotherapeutic treatment. This constitutes a novel chemotherapeutic assay that may enable a paradigm-shifting drug discovery pipeline. Such a pipeline could enable tissue-relevant assay on a high throughput scale and be both more robust than monolayer cell culture and easier than animal models.

Antibiotic resistance has become a great concern in the last decades. In the U.S., at least 2 million people suffer from antibiotic resistance-related illnesses. Otitis Media (OM) is the number one cause of antibiotic prescriptions for children in the US. However, not every case of OM requires antibiotic therapy and accurate diagnosis is crucial for the appropriate treatment. We investigate whether a multimode smartphone-based otoscope, which is capable of fluorescence and spectral imaging, allows us to discriminate between OM types with better accuracy. Experimental results demonstrate that the multimode smartphone-based otoscope may become a potential tool for the accurate discrimination between OM cases, preventing misprescriptions of antibiotic agents with the addition of remote diagnostics.

In a partial cornea transplant surgery, a procedure known as “Big Bubble” is used and it requires precise needle detection and tracking. To accomplish this goal, we used traditional image segmentation methods and trained a Convolutional Neural network (CNN) model to track the needle during the cornea transplant surgery guided by OCT B-scan imaging. The dataset was generated from the laboratory OCT system and we classified them to three categories. The network architecture is based on U-Net and modified to avoid overfitting. We are able to track the needle and detect the distance between the needle tip and cornea bottom layer based on these results.

Notch signaling is an evolutionarily conserved mechanoresponsive pathway that decides cell fate during developmental process. However, it is not clearly known whether cardiac contraction mediates Notch signaling during the development of the heart. In this study, we have developed a method of segmenting cardiomyocyte nuclei as markers to quantify the contractility of the heart on the basis of an improved Watershed segmentation technique using the Hessian Blob Algorithm. This method provides robust performance for a 4-D (3-D + time) region of interest (ROI) that we are interested in analyzing. Furthermore, we have overcome poor contrast and lack of detail due to refractive index mismatch between different media present in the light propagation path while taking images from LSFM. After image acquisition, we performed pre-processing using a dehazed PSF for deconvolution as the dehazing operation restores the light intensity of corrected PSF. To make the entire process automated, we used a pre-defined feature matching algorithm, whereby the user simply has to crop out a reference ROI, after which the algorithm will automatically segment the ROI from successive frames based on the reference frame. By the aforementioned processes, we have imaged tg(cmlc2:gfp-nuc) zebrafish to visualize and track nuclei of individual cardiomyocytes to quantify the contractility of the heart. To understand the contractility-mediated Notch signaling in cardiac development, tg(tp-1:gfp) has been used with pharmacologic treatments of isoproterenol and metoprolol to increase and decrease cardiac contractility respectively. Our image processing pipeline provided accurate quantification of understanding the heart development between Notch signaling and contractility.

The aim of this research was to visualize and measure the human gingival sulcus in vivo using the swept-source optical coherence tomography system based on 1310 nm wavelength source with the developed classification algorithm of gingival sulcus. Apart from the algorithm based examination procedure, the OCT cross-sectional images were involved in A-scan depth profile analysis to illustrate the intensity fluctuation of teeth and the periodontal tissue structures to clarify the end point of gingival sulcus. The quantitative measurement was assessed with 1.10 ± 0.26 mm. Thus, the swept-source optical coherence tomography system could be used to perform the gingival sulcus imaging.

Raman spectroscopy for the analysis of exosomes

Sentinel lymph node involvement is recognized as a prognostic factor in breast cancer staging and is essential to guide optimal treatment. The possibility of missed micrometastases by using conventional methods was estimated around 20-60% cases has created a demand for the development of more accurate approaches. A paired-agent imaging approach is presented by employing a control imaging agent to allow rapid, quantitative mapping of microscopic populations of tumor cells in lymph nodes to guide pathology sectioning. To test the feasibility of this approach to identify micrometastases, lymph node micrometastases biological tissue model was developed and were stained with targeted and control imaging agent solution to evaluate the binding potential of the agents of intact nodes. ABY-029, an EGFR specific affibody was labeled with IRDye-800CW(LICOR) as targeted agent and IRDye-700DX was hydrolyzed as control agent. Lymph nodes phantoms were stained for 60 min, followed by 60 min rinsing, and the fluorescence of whole lymph node phantoms were recorded to evaluate the spatial distribution of both agents in the entire phantom. Measured binding potential of targeted agent between micrometastases and control regions were 0.652 ± 0.130 and -0.008 ± 0.042 respectively (p < 0.0001). The results demonstrate the potential to enhance the sensitivity of lymph node pathology using paired-agent imaging in a whole human lymph node.

Living cell culture provides convenient and standard biotechnology chosen option in the laboratory. However, current imaging methods could not present real 3D models. Therefore, we have developed a compact, high-speed spectral-domain optical coherence microscopy (SD-OCM) system to observe the interaction of the tumor cell spheroid with gold nanoparticles. Volumetric OCM images of the cell spheroid were acquired using an in-house C++ interface and used a low-cost microcontroller for triggering to synchronize the galvanometer mirror to the detector array. We designed a hermetic chamber on the microscope stage to control temperature, humidity, CO2 concentration in the experiment.

Posterior tibial tendon dysfunction is one of the most common causes of acquired flat foot deformity in adults, and results in significant morbidity due to the pain and development of secondary osteoarthritis. The exact etiology of this condition is still unknown. Tibial tendons are predominately made up of fibrillar collagens and we hypothesis that their structural properties such as the collagen fiber density, orientation properties and cross-linking density essentially control the biomechanical properties of posterior tibial tendons. In this study, our aim is to visualize and quantitate fibrillar collagen distributions, their organization, crosslink densities and the fiber orientation (using Fourier analysis) in three distinct regions of posterior tibial tendon human samples namely proximal, middle and distal regions. The experiments conducted here are based on tendon specimens donated for research by persons that have their diseased tendon removed prior to surgery. Multiphoton microscopy provides a powerful imaging method for evaluation of remodeling of fibrillar collagen structures deep within tissues. Ultra-short IR laser pulses served as an excitation source to produce multiphoton excitation fluorescence (MPEF) from endogenously fluorescent macromolecular systems and to induce highly specific second harmonic generation (SHG) signals from fibrillar collagens. We systematically examined the nature of fibrillar collagen remodelling in relatively thick posterior tibial tendon tissues. Computed Orientation Index values obtained from Fourier analysis show statistically significant differences particularly between proximal and middle regions (p<0.0001). The crosslinking densities determined from the ratio of auto-fluorescence (MPEF) to BSHG again show differences particularly between proximal and middle regions (p<0.01). We have successfully demonstrated that the multiphoton and harmonic generation microscopy can be a powerful high resolution imaging method requiring minimal sample preparation that can provide structural information about spatially and spectrally resolved fibrillar collagens in three different posterior tibial tendon tendon regions.

Pharmacokinetic diffuse fluorescence tomography (DFT) can provide helpful diagnostic information for tumor differentiation and monitoring. Among the methods of achieving pharmacokinetic parameters, adaptive extended Kalman filtering (AEKF) as a nonlinear filter method demonstrates the merits of quantitativeness, noise-robustness, and initialization independence. In this paper, indirect and direct AEKF schemes based on a commonly used two-compartment model were studied to extract pharmacokinetic parameters from simulation data. To assess the effect of metabolic rate on the reconstruction results, a series of numerical simulation experiments with the metabolic time range from 4.16 min to 38 min were carried out and the results obtained by the two schemes were compared. The results demonstrate that when the metabolic time is longer than 18 min, the pharmacokinetic-rate estimates of two schemes are similar; however, when the metabolic time is shorter than 5 min, the pharmacokinetic parameters obtained by the indirect scheme are far from the true value and even unavailable.

In recent years, MRI using a rare gas has attracted attention. Rare gas MRI can perform imaging of lungs with few protons and can also evaluate gas dynamics and hemodynamics. Attempts have been made to enhance the signal intensity by hyperpolarizing nuclear spins to obtain NMR signals from the gas. This hyperpolarization technology is also expected to be effective when combined with low-field MRI including permanent magnet type. In this study, a xenon (¹²⁹Xe) hyperpolarization system using a 795nm laser was constructed and evaluated using a 0.3T NMR system. As a result, it was shown that the NMR signal intensity from ¹²⁹Xe can be enhanced 1.5 times with a polarization of 30 minutes.

Patients with B-ALL (Ph +ve) acute lymphoblastic leukemia are at high risk of relapse and mortality. We seek to establish a mechano-biological testing technique to assess biophysical properties of cell motility, potentiating the ability to distinguish between high and low risk leukemia populations and behaviors. Two experiments were performed to test the mechano-biological behavior of B-ALL using the ThorLabs modular optical tweezers and a microfluid chemotaxis chamber. The first experiment measured the relative mechanical energy carried by a cell in the optical trap, which was performed on B-ALL in control conditions and under SDF1 chemotaxis. The relative mechanical energy was found through an extension of the back-focal-plane calibration method for optical tweezers, and assumed that increased cellular activity manifests as random movements. There was no discernable difference in the relative mechanical energies between the control B-ALL, B-ALL under SDF1 chemotaxis, and B-ALL in the presence of mesenchymal stem cells. The second experiment quantified the real-time migrational force of B-ALL under SDF1 chemotaxis. This was found through prior calibration of the optical tweezers through determination of the terms κ and β. Of the viable measurements, 3 of 8 cells exhibited a significant force towards the SDF1 gradient. Further experimentation is necessary to normalize the experimental set-up conditions, and increase the number of viable measurements during cell migration.