We describe a technique based on the use of a high-resolution quadri-wave lateral shearing interferometer to perform quantitative linear birefringence measurements on biological samples [1] such as living cells and tissues. The system combines QPI with different excitation polarizations to create retardance images. This creates a new kind of image contrast based on the local retardance, reveals the structure of sample anisotropic components and adds specificity to label-free phase images. We implemented this technique allowing us to take retardance images in less than 1 second which allows us to make high speed acquisitions to reconstruct tissues virtual slides with different modalities (i.e intensity, phase and retardance). Comparisons between healthy and tumoral 10 µm thick skin tissues and collagen orientation studies in the latter will be presented. [1] S. Aknoun, P. Bon, J. Savatier, B. Wattellier, and S. Monneret, "Quantitative retardance imaging of biological samples using quadriwave lateral shearing interferometry," Opt. Express 23, 16383-16406 (2015).

Granulocytes are the major players in innate immunity and are prognostic markers in diseases. An in-depth phenotypic characterization of granulocyte subtypes and correlation with biometry or lifestyle is so far lacking. The reason is, that either preparation of mononuclear cells was analyzed or that cells in the neutrophil window were neglected in the analysis. Here we show for the first time lymphocyte- (LL) and monocyte-like (ML) cells within the granulocyte scatter gate as new, previously unknown cell subpopulation. Immunophenotyping of 905 healthy German adults from the LIFE study [1] was performed by 10-color flow cytometry [2]. Age of men (n=420): 56.5±14.0 years, women (n=485): 56.7±13.6 y (range of 18-81 y). Data analyzed by FlowJo v10.0.6. Values compared by Mann-Whitney-U test: men vs women, young (18-49 y) vs. elderly (50-81 y.) men, and young (19-49 y.) vs. elderly (50-81 y.) women; significance: p<0.05. Within the granulocyte gate four phenotypically distinct cell types were detected (all CD45+, SSCmid-high): LL1 CD3+,CD4+,CD8++,CD16/56+,CD38+,HLA-DR+ LL2 CD3+,CD4low,CD8+,CD38low LL3 CD3+,CD4+,CD8- ML1 CD3-,CD4low,CD14+,CD38+ LL2 counts were increased in men (p=0.042), as well as ML1 counts (p <0.001). Most of the cell counts were not dependent on age, except LL2 in women. In conclusion, new lymphocyte like cell types with the neutrophil scatter characteristics are reported. Counts correlate with age and gender. We plan to sort these new subtypes for further functional characterization and aim to establish them as cellular biomarkers for the early detection of various diseases. [1] BMC Public Health. 2015;15:691; [2] Cytometry A. 2014;85(9):781

Chemotherapy used for cancer treatment, due to the lack of specificity of drugs, is associated to various damaging side effects that have severe impact on patients’ quality of life. Over the past 30 years, increasing efforts have been placed on optimizing chemotherapy dosing with the main goal of increasing antitumor efficacy while reducing drug-associated toxicity. A novel research shows that stem cells may act as a reservoir for the anticancer agent, which will subsequently release some of the drug’s metabolites, or even the drug in its original form, in vicinity of the cancer cells. These cells may play a dual role in controlling drug toxicity depending on their capacity to uptake and release the chemotherapeutic drug. In our study, we show that Dental Pulp Stem Cells DPSCs are able to rapidly uptake Paclitaxel PTX, and to release it in the culture medium in a time-dependent manner. This resulting conditioned culture medium is to be transferred to breast cancer cells, the MCF-7. By applying Confocal Raman Microscopy, the anticancer drug uptake by the MCF-7 was measured. Surprisingly, the cancer cells -without any direct contact with PTX- showed a drug uptake. This proves that the stem cells carried and delivered the anticancer drug without its modification. It could be a revolution in chemotherapy to avoid the drug’s side effects and increase its efficacy.

There is a critical need to predict effective treatments for individual cancer patients. The goal of this work is to validate Optical Metabolic Imaging (OMI) of tumor-derived organoids as a predictive drug screening platform in breast and pancreatic cancer, by relating multiphoton fluorescence lifetime imaging (FLIM) data from these organoids to clinical patient outcomes. Three-dimensional organoids were generated from core needle biopsies of breast tumors and surgically resected pancreatic ductal adenocarcinomas (PDAC). These organoids were treated with the patient’s prescribed therapy, and early metabolic changes were measured using multiphoton FLIM of the metabolic co-enzymes NAD(P)H and FAD at the single-cell level. Changes were quantified using the OMI Index, a linear combination of the optical redox ratio (ratio of the fluorescence intensities of NAD(P)H to FAD), and the mean NAD(P)H and FAD fluorescence lifetimes. Organoids grew from a variety of untreated breast tumor subtypes including triple negative, HER2+, and ER+/PR+/HER2-, and early metabolic changes could be resolved at the single-cell level after only 24 hours of treatment in vitro. Surgical pathology 2-7 months after completing neoadjuvant treatment served as gold standard validation of breast cancer patient drug response. Organoids were also successfully grown from surgically resected PDAC samples, and included two subtypes of epithelial cells as well as stromal fibroblasts. Patient follow-up data after surgery and subsequent treatment was used as gold standard validation of PDAC patient drug response. This platform shows promise for predicting long-term response to therapy in breast and pancreatic cancer patients.

Current microscopy techniques are not optimal to image fluorescence in whole live animals. We present fluorescence lifetime optical projection tomography (FLIM OPT) applied to imaging enzyme activity in live transgenic zebrafish expressing Förster Resonance Energy Transfer (FRET) biosensors. OPT can be considered the optical equivalent to x-ray CT. Samples are rotated through 360 with images acquired at set intervals, and a back projection technique is applied to reconstruct the 3D image. It can be performed in transmission or fluorescence modes, allowing a wide range of visualisation techniques, including FLIM. Combination of OPT with FRET FLIM can therefore provide functional information in 3D. The optimal size range for OPT is mm-cm, which fills the size gap between confocal and MRI and is also the size range for zebrafish, making them an ideal model for imaging. Transgenic zebrafish expressing a Caspase 3 FRET biosensor were generated on the TraNac background (a transparent mutant) to provide live readouts of apoptosis. We have shown that using FLIM OPT we can detect changes in Caspase 3 activity in both embryo and adult Tg(Ubi:Caspase3biosensor) zebrafish. Apoptosis was induced using 25 Gy from a 137Cs source and post irradiation an increase in fluorescence lifetime was quantified in the head region indicative of biosensor cleavage and Caspase 3 activity. Though development of compressive sensing and multiplexed imaging with two imaging arms we have applied OPT and FLIM OPT to adult zebrafish, enabling us to quickly acquire datasets so the fish can be recovered and imaged longitudinally.

The intestinal epithelial barrier provides protection from external threats that enter the digestive system and persist beyond passage through the stomach. The effects of toxic agents on the intestinal epithelial cell monolayer have not been fully characterized at a cellular level as live imaging of this dynamic interplay at sufficient resolution to interpret cellular responses presents technological challenges. Using a high-resolution native contrast modality called Micro-Optical Coherence Tomography (μOCT), we generated real-time 3D images depicting the impact of the chemical agent EDTA on polarized intestinal epithelial monolayers. Within minutes following application of EDTA, we observed a change in the uniformity of epithelial surface thickness and loss of the edge brightness associated with the apical surface. These observations were measured by generating computer algorithms which quantify imaged-based events changing over time, thus providing parallel graphed data to pair with video. The imaging platform was designed to monitor epithelial monolayers prior to and following application of chemical agents in order to provide a comprehensive account of monolayer behavior at baseline conditions and immediately following exposure. Furthermore, the platform was designed to simultaneously measure continuous trans-epithelial electric resistance (TEER) in order to define the progressive loss of barrier integrity of the cell monolayer following exposure to toxic agents and correlate these findings to image-based metrics. This technological image-based experimental platform provides a novel means to characterize mechanisms that impact the intestinal barrier and, in future efforts, can be applied to study the impact of disease relevant agents such as enteric pathogens and enterotoxins.

Septic shock induces organ dysfunction by microcirculatory disturbance. Observation and quantification of microcirculation are expected to be effective for the diagnosis of septic shock. Sidestream dark-filed (SDF) imaging is a suitable technique for observation of microcirculation. It can noninvasively visualize red blood cells (RBCs) of microcirculation. We are developing early diagnostic criteria for septic shock from microcirculation SDF images. As an initial study, we use the blood flow velocity estimated from the images as a diagnostic criteria. However, low contrast quality and subject’s movement disturb the blood flow velocity estimation. In this paper, we present a procedure of image processing for a stable estimation of the blood flow velocity. In the procedure, we first perform a robust principal component analysis (RPCA) as a preprocessing. RPCA decomposes a motion picture into a low-rank (L) component and a sparse (S) component. The S component images clearly expresses RBCs flow and is used for the velocity estimation. The temporal change of the intensity profile along the vessel was analyzed by Hough transform to estimate the blood flow velocity is. The proposed procedure was examined with dorsal microcirculation of septic model rats and a sham rat. As a result, the decrease in blood flow velocity of the septic rats after 17 hours was greater than that of the sham. It was also suggested that blood flow velocity might be faster index of septic shock reaction earlier than lactic acid value. These results suggest that the velocity estimation is reasonable for diagnosis of septic shock.

Treatment options for head and neck cancer are limited, and can cause an impaired ability to eat, talk, and breathe. Therefore, optimized and personalized therapies could reduce unnecessary toxicities from ineffective treatments. Organoids are generated from primary tumor tissue and provide a physiologically-relevant in vitro model to measure drug response. Additionally, multiphoton fluorescence lifetime imaging (FLIM) of the metabolic cofactors NAD(P)H and FAD can resolve dynamic cellular response to anti-cancer treatment. This study applies FLIM of NAD(P)H and FAD to head and neck cancer organoids. Head and neck cancer tissue was digested and grown in culture as three-dimensional organoids. Gold standard measures of therapeutic response in vivo indicate stable disease after treatment with cetuximab (antibody therapy) or cisplatin (chemotherapy), and treatment response after combination treatment. In parallel, organoids were treated with cetuximab, cisplatin, or combination therapy for 24 hours. Treated organoids exhibit decreased NAD(P)H lifetime (p<0.05) and increased FAD lifetime (p<0.05) compared with control organoids. Additionally, analysis of cellular heterogeneity identifies distinct subpopulations of cells in response to treatment. A quantitative heterogeneity index predicts in vivo treatment response and demonstrates increased cellular heterogeneity in organoids treated with cetuximab or cisplatin compared with combination treatment. Mapping of cell subpopulations enables characterization of spatial relationships between cell subpopulations. Ultimately, an organoid model combined with metabolic fluorescence imaging could provide a high-throughput platform for drug discovery. Organoids grown from patient tissue could enable individualized treatment planning. These achievements could optimize quality of life and treatment outcomes for head and neck cancer patients.

Transient retinal phototropism (TRP) has been observed in rod photoreceptors activated by oblique visible light flashes. Time-lapse confocal microscopy and optical coherence tomography (OCT) revealed rod outer segment (ROS) movements as the physical source of TRP. However, the physiological source of TRP is still not well understood. In this study, concurrent TRP and electroretinogram (ERG) measurements disclosed a remarkably earlier onset time of the ROS movements (≤10 ms) than that (~38 ms) of the ERG a-wave. Furthermore, low sodium treatment reversibly blocked the photoreceptor ERG a-wave, which is known to reflect hyperpolarization of retinal photoreceptors, but preserved the TRP associated rod OS movements well. Our experimental results and theoretical analysis suggested that the physiological source of TRP might be attributed to early stages of phototransduction, before the hyperpolarization of retinal photoreceptors.

Distinguishing between intact cells, dead but still whole cells, and cell debris is an important but difficult task in life sciences. The most common way to identify dead cells is using a cell-impermeant DNA binding dye, such as propidium iodide. A healthy living cell has an intact cell membrane and will act as a barrier to the dye so that it cannot enter the cell. A dead cell has a compromised cell membrane, and it will allow the dye into the cell to bind to the DNA and become fluorescent. The dead cells therefore will be positive and the live cells will be negative. The dead cells later deteriorate quickly into debris. Different pieces of debris from a single cell can be incorrectly identified as separate dead cells. Although a flow cytometer can quickly perform numerous quantitative, sensitive measurements on each individual cell to determine the viability of cells within a large, heterogeneous population, it is bulky, expensive, and only large hospitals and laboratories can afford them. In this work, we show that the distance-dependent coupling of fluorophore light to surface plasmon coupled emission (SPCE) from fluorescently-labeled cells can be used to distinguish whole cells from cell debris. Once the fluorescent labels are excited by a laser, the fluorescently-labeled whole cells create two distinct intensity rings in the far-field, in contrast to fluorescently-labeled cell debris, which only creates one ring. The distinct far-field patterns can be captured by camera and used to distinguish between whole cells and cell debris.

Motivation and background: Melanoma, the fastest growing cancer worldwide, kills more than one person every hour in the United States. Determining the depth and distribution of dermal melanin and hemoglobin adds physio-morphologic information to the current diagnostic standard, cellular morphology, to further develop noninvasive methods to discriminate between melanoma and benign skin conditions. Purpose: To compare the performance of a multimode dermoscopy system (SkinSpect), which is designed to quantify and map in three dimensions, in vivo melanin and hemoglobin in skin, and to validate this with histopathology and three dimensional reflectance confocal microscopy (RCM) imaging. Methods: Sequentially capture SkinSpect and RCM images of suspect lesions and nearby normal skin and compare this with histopathology reports, RCM imaging allows noninvasive observation of nuclear, cellular and structural detail in 1-5 m-thin optical sections in skin, and detection of pigmented skin lesions with sensitivity of ~ 90-95% and specificity of ~ 70-80%. The multimode imaging dermoscope combines polarization (cross and parallel), autofluorescence and hyperspectral imaging to noninvasively map the distribution of melanin, collagen and hemoglobin oxygenation in pigmented skin lesions. Results: We compared in vivo features of ten melanocytic lesions extracted by SkinSpect and RCM imaging, and correlated them to histopathologic results. We present results of two melanoma cases (in situ and invasive), and compare with in vivo features from eight benign lesions. Melanin distribution at different depths and hemodynamics, including abnormal vascularity, detected by both SkinSpect and RCM will be discussed. Conclusion: Diagnostic features such as dermal melanin and hemoglobin concentration provided in SkinSpect skin analysis for melanoma and normal pigmented lesions can be compared and validated using results from RCM and histopathology.

Purpose: To determine the performance of a multimode dermoscopy system (SkinSpect) designed to quantify and 3-D map in vivo melanin and hemoglobin concentrations in skin and its melanoma scoring system, and compare the results accuracy with SIAscopy, and histopathology. Methods: A multimode imaging dermoscope is presented that combines polarization, fluorescence and hyperspectral imaging to accurately map the distribution of skin melanin, collagen and hemoglobin in pigmented lesions. We combine two depth-sensitive techniques: polarization, and hyperspectral imaging, to determine the spatial distribution of melanin and hemoglobin oxygenation in a skin lesion. By quantifying melanin absorption in pigmented areas, we can also more accurately estimate fluorescence emission distribution mainly from skin collagen. Results and discussion: We compared in vivo features of melanocytic lesions (N = 10) extracted by non-invasive SkinSpect and SIMSYS-MoleMate SIAscope, and correlate them to pathology report. Melanin distribution at different depths as well as hemodynamics including abnormal vascularity we detected will be discussed. We will adapt SkinSpect scoring with ABCDE (asymmetry , border, color, diameter, evolution) and seven point dermatologic checklist including: (1) atypical pigment network, (2) blue-whitish veil, (3) atypical vascular pattern, (4) irregular streaks, (5) irregular pigmentation, (6) irregular dots and globules, (7) regression structures estimated by dermatologist. Conclusion: Distinctive, diagnostic features seen by SkinSpect in melanoma vs. normal pigmented lesions will be compared by SIAscopy and results from histopathology.

Selective plane illumination microscopy (SPIM) is an optical sectioning technique that allows imaging of biological samples at high spatio-temporal resolution. Standard SPIM devices require dedicated set-ups, complex sample preparation and accurate system alignment, thus limiting the automation of the technique, its accessibility and throughput. We present a millimeter-scaled optofluidic device that incorporates selective plane illumination and fully automatic sample delivery and scanning. To this end an integrated cylindrical lens and a three-dimensional fluidic network were fabricated by femtosecond laser micromachining into a single glass chip. This device can upgrade any standard fluorescence microscope to a SPIM system. We used SPIM on a CHIP to automatically scan biological samples under a conventional microscope, without the need of any motorized stage: tissue spheroids expressing fluorescent proteins were flowed in the microchannel at constant speed and their sections were acquired while passing through the light sheet. We demonstrate high-throughput imaging of the entire sample volume (with a rate of 30 samples/min), segmentation and quantification in thick (100-300 μm diameter) cellular spheroids. This optofluidic device gives access to SPIM analyses to non-expert end-users, opening the way to automatic and fast screening of a high number of samples at subcellular resolution.

Locally advanced adenocarcinomas located in the distal rectum are commonly treated via 5-fluorouracil (5-FU)-based neoadjuvant chemoradiation therapy (CRT). The occurrence of pre-operative pathological complete response, or the absence of any histological evidence of residual cancer, is seen in 15-27% of rectal cancer cases. Response to chemotherapeutic agents varies between patients, introducing the need for a system to predict optimal drug combinations. We propose a method of utilizing optical metabolic imaging of in vitro, primary tumor-derived, three-dimensional organoid culture to create specific drug sensitivity profiles, and to rapidly assess a patient’s potential response to drugs. Murine xenografts were developed in Swiss athymic nude mice, using human colorectal adenocarcinoma cell lines, implanted in the flank (RKO, ATCC). Tumors were excised upon reaching a volume of 500mm3 and processed for organoid culture. Organoids were subjected to longitudinal metabolic imaging of metabolic cofactors FAD and NADH for seven days. The resulting images were used to yield an optical redox value on a cell-by-cell basis, determined by the fluorescence intensity ratio of FAD/(FAD+NADH). This data infers proliferative index of the organoids. Beginning on day three, a control vehicle dimethyl sulfoxide, or the cytotoxic agent 5-FU, was added to the organoid growth media in wells, with metabolic imaging performed the same as previously stated. The optical redox values decreased due to the addition of 5-FU, which targets rapidly dividing cells and induces apoptosis. The changes in the optical redox histograms were correlated to markers of cell proliferation (Ki-67) and apoptosis (cleaved caspase-3).

Spectral imaging is an important method that is used for a whole spectrum of applications, but measuring very large spectral images is a challenge that so far was not achieved. We present a novel system for scanning very large spectral images of microscopy samples in a rather short time. The system captures the information while the sample is continuously being scanned on the fly. It therefore breaks the size and speed limits that resulted from existing spectral imaging methods. The spectral separation is achieved through Fourier spectroscopy by using an interferometer mounted along the optical axis (no moving parts). We describe the system and its use for pathological samples.

Optical Coherence Tomography (OCT) makes viable acquisition of 3D fingerprints from both dermis and epidermis skin layers and their interfaces, exposing features that can be explored to improve biometric identification such as the curvatures and distinctive 3D regions. Scanned images from eleven volunteers allowed the construction of the first OCT 3D fingerprint database, to our knowledge, containing epidermal and dermal fingerprints. 3D dermal fingerprints can be used to overcome cases of Failure to Enroll (FTE) due to poor ridge image quality and skin alterations, cases that affect 2D matching performance. We evaluate three matching techniques, including the well-established Iterative Closest Points algorithm (ICP), Surface Interpenetration Measure (SIM) and the well-known KH Curvature Maps, all assessed using a 3D OCT fingerprint database, the first one for this purpose. Two of these techniques are based on registration techniques and one on curvatures. These were evaluated, compared and the fusion of matching scores assessed. We applied a sequence of steps to extract regions of interest named (ROI) minutiae clouds, representing small regions around distinctive minutia, usually located at ridges/valleys endings or bifurcations. The obtained ROI is acquired from the epidermis and dermis-epidermis interface by OCT imaging. A comparative analysis of identification accuracy was explored using different scenarios and the obtained results shows improvements for biometric identification. A comparison against 2D fingerprint matching algorithms is also presented to assess the improvements.

Polarized light is commonly used to detect optical anisotropies, such as birefringence, in tissues. This optical anisotropy is often attributed to underlying structural anisotropy in tissue, which may originate from regularly aligned collagen fibers. In these cases, the optical anisotropy, such as birefringence, is interpreted as a relative measure of the structural anisotropy of the collagen fibers. However, the relative amplitude of optical anisotropy depends on factors other than fiber orientation, and few models allow quantitative interpretation of absolute measures of true fiber orientation distribution from the optical signal. Our model uses the Mie solution to scattering of linearly polarized light from infinite cylindrical scatterers. The model is expanded to include populations of scatterers with physiologically relevant size and orientation distributions. We investigated the influences of fiber diameter, orientation distribution, and wavelength on the back-scattering signal with our computational model, and used these results to extract structural information from experimental fiber phantoms and bovine tendon. Our results demonstrated that by fitting our model to the experimental data using limited assumptions, we could extract fiber orientation distributions and diameters that were comparable to those found in scanning electron microscope images of the same fiber sample. We found a higher alignment of fibers in the bovine tendon sample, and the extracted fiber diameter was within the expected physiological range. Our model showed that the amplitude of optical anisotropy can vary widely due to factors other than the orientation distribution of fiber structures, including index of refraction, and therefore should not be taken as a sole indicator of structural anisotropy. This work highlights that the accuracy of model assumptions plays a crucial role in extracting quantitative structural information from optical anisotropy.

Light sheet fluorescence microscopy (LSFM) is a powerful optical resolution fluorescence microscopy technique which enables to observe the mouse brain vascular network in cellular resolution. However, micro-vessel structures are intensity inhomogeneity in LSFM images, which make an inconvenience for extracting line structures. In this work, we developed a vascular image segmentation method by enhancing vessel details which should be useful for estimating statistics like micro-vessel density. Since the eigenvalues of hessian matrix and its sign describes different geometric structure in images, which enable to construct vascular similarity function and enhance line signals, the main idea of our method is to cluster the pixel values of the enhanced image. Our method contained three steps: 1) calculate the multiscale gradients and the differences between eigenvalues of Hessian matrix. 2) In order to generate the enhanced microvessels structures, a feed forward neural network was trained by 2.26 million pixels for dealing with the correlations between multi-scale gradients and the differences between eigenvalues. 3) The fuzzy local information c-means clustering (FLICM) was used to cluster the pixel values in enhance line signals. To verify the feasibility and effectiveness of this method, mouse brain vascular images have been acquired by a commercial light-sheet microscope in our lab. The experiment of the segmentation method showed that dice similarity coefficient can reach up to 85%. The results illustrated that our approach extracting line structures of blood vessels dramatically improves the vascular image and enable to accurately extract blood vessels in LSFM images.

The segmentation of retinal morphology has numerous applications in assessing ophthalmologic and cardiovascular disease pathologies. The early detection of many such conditions is often the most effective method for reducing patient risk. Computer aided segmentation of the vasculature has proven to be a challenge, mainly due to inconsistencies such as noise, variations in hue and brightness that can greatly reduce the quality of fundus images. Accurate fundus and/or retinal vessel maps give rise to longitudinal studies able to utilize multimodal image registration and disease/condition status measurements, as well as applications in surgery preparation and biometrics. This paper further investigates the use of a Convolutional Neural Network as a multi-channel classifier of retinal vessels using the Digital Retinal Images for Vessel Extraction database, a standardized set of fundus images used to gauge the effectiveness of classification algorithms. The CNN has a feed-forward architecture and varies from other published architectures in its combination of: max-pooling, zero-padding, ReLU layers, batch normalization, two dense layers and finally a Softmax activation function. Notably, the use of Adam to optimize training the CNN on retinal fundus images has not been found in prior review. This work builds on prior work of the authors, exploring the use of Gabor filters to boost the accuracy of the system to 0.9478 during post processing. The mean of a series of Gabor filters with varying frequencies and sigma values are applied to the output of the network and used to determine whether a pixel represents a vessel or non-vessel.

Over the past 2 decades, hyperspectral imaging technologies have been adapted to address the need for molecule-specific identification in the biomedical imaging field. Applications have ranged from single-cell microscopy to whole-animal in vivo imaging and from basic research to clinical systems. Enabling this growth has been the availability of faster, more effective hyperspectral filtering technologies and more sensitive detectors. Hence, the potential for growth of biomedical hyperspectral imaging is high, and many hyperspectral imaging options are already commercially available. However, despite the growth in hyperspectral technologies for biomedical imaging, little work has been done to aid users of hyperspectral imaging instruments in selecting appropriate analysis algorithms. Here, we present an approach for comparing the effectiveness of spectral analysis algorithms by combining experimental image data with a theoretical “what if” scenario. This approach allows us to quantify several key outcomes that characterize a hyperspectral imaging study: linearity of sensitivity, positive detection cut-off slope, dynamic range, and false positive events. We present results of using this approach for comparing the effectiveness of several common spectral analysis algorithms for detecting weak fluorescent protein emission in the midst of strong tissue autofluorescence. Results indicate that this approach should be applicable to a very wide range of applications, allowing a quantitative assessment of the effectiveness of the combined biology, hardware, and computational analysis for detecting a specific molecular signature.

Single photon detectors allows us work with the weakest fluorescence signals. Single photon arrays, combined with ps-controlled gating allow us to create image maps of fluorescence lifetimes, which can be used for in-vivo discrimination of tissue activity. Here we present fluorescence lifetime imaging using the ‘SwissSPAD’ sensor, a 512-by-128-pixel array of gated single photon detectors, fabricated in a standard high-voltage 0.35 μm CMOS process. We present a protocol for spatially resolved lifetime measurements where the lifetime can be retrieved for each pixel. We demonstrate the system by imaging patterns of Fluorescein and Rhodamine B on test slides, as well as measuring mixed samples to retrieve both components of the decay lifetime. The single photon sensitivity of the sensor creates a valuable instrument to perform live cell or live animal (in vivo) measurements of the weak autofluorescent signals, for example distinguishing unlabelled free and bound NADH. Our ultimate goal is to create a real time fluorescence lifetime imaging system, possibly integrated into augmented reality goggles, which could allow immediate discrimination of in vivo tissues.

Multi-day tracking of cells in culture systems can provide valuable information in bioscience experiments. We report the development of a cell culture imaging system, named EmSight, which incorporates multiple compact Fourier ptychographic microscopes with a standard multiwell imaging plate. The system is housed in an incubator and presently incorporates six microscopes, imaging an ANSI standard 6-well plate at the same time. By using the same low magnification objective lenses (NA of 0.1) as the objective and the tube lens, the EmSight is configured as a 1:1 imaging system that, providing large field-of-view (FOV) imaging (5.7 mm × 4.3 mm) onto a low-cost CMOS imaging sensor. The EmSight improves the image resolution by capturing a series of images of the sample at varying illumination angles; the instrument reconstructs a higher-resolution image by using the iterative Fourier ptychographic algorithm. In addition to providing high-resolution brightfield and phase imaging, the EmSight is also capable of fluorescence imaging at the native resolution of the objectives. We characterized the system using a phase Siemens star target, and show four-fold improved coherent resolution (synthetic NA of 0.42) and a depth of field of 0.2 mm. To conduct live, long-term dopaminergic neuron imaging, we cultured ventral midbrain from mice driving eGFP from the tyrosine hydroxylase promoter. The EmSight system tracks movements of dopaminergic neurons over a 21 day period.

Radionuclides are used for sensitive and specific detection of small molecules in vivo and in vitro. Recently, radioluminescence microscopy extended their use to single-cell studies. Here we propose a new single-cell radioisotopic assay that improves throughput while adding sorting capabilities. The new method uses fluorescence-based sensor for revealing single-cell interactions with radioactive molecular markers. This study focuses on comparing two different experimental approaches. Several probes were tested and Dihydrorhodamine 123 was selected as the best compromise between sensitivity, brightness and stability. The sensor was incorporated either directly within the cell cytoplasm (direct approach), or it was coencapsulated with radiolabeled single-cells in oil-dispersed water droplets (droplet approach). Both approaches successfully activated the fluorescence signal following cellular uptake of 18F-fluorodeoxyglucose (FDG) and external Xrays exposure. The direct approach offered single-cell resolution and longtime stability ( > 20 hours), moreover it could discriminate FDG uptake at labelling concentration as low as 300 μCi/ml. In cells incubated with Dihydrorhodamine 123 after exposure to high radiation doses (8-16 Gy), the fluorescence signal was found to increase with the depletion of ROS quenchers. On the other side, the droplet approach required higher labelling concentrations (1.00 mCi/ml), and, at the current state of art, three cells per droplet are necessary to produce a fluorescent signal. This approach, however, is independent on cellular oxidative stress and, with further improvements, will be more suitable for studying heterogeneous populations. We anticipate this technology to pave the way for the analysis of single-cell interactions with radiomarkers by radiofluorogenic-activated single-cell sorting.

Dielectrophoresis (DEP) is a commonly used technique in biomedical engineering to manipulate biomolecules. DEP is defined as the force acting on dielectric particles when they are exposed to non-uniform electric fields. DEP effect can be divided in three categories: positive (dielectric particles are attracted to the electrodes), negative, and zero force DEP. The cross-over frequency is the frequency in which the DEP force is equal to zero. The cross-over frequency depends on the conductivity and the permittivity of the particles and of the suspended medium. The DEP cross-over frequency has been utilized in detecting/quantifying biomolecules. A manual procedure is commonly used to estimate the cross-over frequency of biomolecules. Therefore, the accuracy of this detection method is significantly limited. To address this issue, we designed and tested an automated procedure to carry out DEP spectroscopy in dielectric particles dissolved in a biological buffer solution. Our method efficiently measures the effect of the DEP force through a live video feed from the microscope camera and performs real-time image processing. It records the change in the fluorescence emission as the system automatically scans the electric frequency of the function generator over a specified time interval. We demonstrated the effectiveness of the method by extracting the crossover frequencies and the DEP spectrum of polystyrene beads with blue color dye (1000 nm diameter) and green fluorescent polystyrene beads with 500 nm diameter using this procedure. This approach can lead to the development of a biosensor with significantly higher sensitivity than existing detection methods.

Femtosecond-laser pulses can assist to transfect cells by creating transient holes in the cell membrane, thus making them temporarily permeable for extraneous genetic material. This procedure offers the advantage of being completely “virus free” since no viruses are used for the delivery and integration of gene factors into the host genome and, thereby, avoiding serious side effects which so far prevent clinical application. Unfortunately, focusing of the laser radiation onto individual cell membranes is quite elaborate and time consuming. Regarding these obstacles, we briefly review two optical setups for fast, efficient and high throughput laser-assisted cell transfection based on femtosecond laser pulse excitation. The first setup aims at assisting the transfection of adherent cells. It comprises of a modified laser-scanning microscope with beamshaping optics as well as home-made software to automate the detection, targeting and laser-irradiation process. The second setup aims at laser-assisted transfection of non-adherent cells in suspension which move in a continuous flow through the laser focus region. The setup allows to address a large number of cells, however, with much lower transfection efficiency than the individual-cell targeting approach.

The sidestream dark-field (SDF) imaging allows direct visualization of red blood cells in microvessels near tissue surfaces. We have developed an image-based oximetry method using two-band images obtained by SDF imaging (SDF oximetry) and a trial SDF device with light-emitting diodes to obtain band images. In this study, we propose a technique of producing oxygen saturation (SO₂) maps from SDF images and perform animal experiments in vivo. To produce SO₂ maps, we use spectral analysis using two band images obtained with our SDF device. As an image processing, the combination of both the Hessian-based and pixel value-based techniques as blood vessel extraction from an SDF image is used. From the experiment with the surface of rat small intestines, we can produce SO₂ maps and find that the map represents arterioles and venules those were determined based on the blood ow from SDF images. Moreover, we find the variation of SO₂ along a blood vessel running direction.

Hyperspectral imaging (HSI) can estimate the spatial distribution of skin blood oxygenation, using visible to near-infrared light. HSI oximeters often use a liquid-crystal tunable filter, an acousto-optic tunable filter or mechanically adjustable filter wheels, which has too long response/switching times to monitor tissue hemodynamics. This work aims to evaluate a multispectral snapshot imaging system to estimate skin blood volume and oxygen saturation with high temporal and spatial resolution. We use a snapshot imager, the xiSpec camera (MQ022HG-IM-SM4X4-VIS, XIMEA), having 16 wavelength-specific Fabry–Perot filters overlaid on the custom CMOS-chip. The spectral distribution of the bands is however substantially overlapping, which needs to be taken into account for an accurate analysis. An inverse Monte Carlo analysis is performed using a two-layered skin tissue model, defined by epidermal thickness, haemoglobin concentration and oxygen saturation, melanin concentration and spectrally dependent reduced-scattering coefficient, all parameters relevant for human skin. The analysis takes into account the spectral detector response of the xiSpec camera. At each spatial location in the field-of-view, we compare the simulated output to the detected diffusively backscattered spectra to find the best fit. The imager is evaluated for spatial and temporal variations during arterial and venous occlusion protocols applied to the forearm. Estimated blood volume changes and oxygenation maps at 512x272 pixels show values that are comparable to reference measurements performed in contact with the skin tissue. We conclude that the snapshot xiSpec camera, paired with an inverse Monte Carlo algorithm, permits us to use this sensor for spatial and temporal measurement of varying physiological parameters, such as skin tissue blood volume and oxygenation.

This paper proposes to apply nonlinear estimation of chromophore concentrations: melanin, oxy-hemoglobin, deoxyhemoglobin and shading to the real hyperspectral image of skin. Skin reflectance is captured in the wavelengths between 400nm and 700nm by hyperspectral scanner. Five-band wavelengths data are selected from skin reflectance. By using the cubic function which obtained by Monte Carlo simulation of light transport in multi-layered tissue, chromophore concentrations and shading are determined by minimize residual sum of squares of reflectance. When dark circles are appeared under the eyes, the subject looks tired and older. Therefore, woman apply cosmetic cares to remove dark circles. It is not clear about the relationship between color and chromophores distribution in the dark circles. Here, we applied the separation method of the skin four components to hyperspectral image of dark circle, and the separated components are modulated and synthesized. The synthesized images are evaluated to know which components are contributed into the appearance of dark circles. Result of the evaluation shows that the cause of dark circles for the one subject was mainly melanin pigmentation.

Little is currently known about the fluorescence excitation spectra of disparate tissues and how these spectra change with pathological state. Current imaging diagnostic techniques have limited capacity to investigate fluorescence excitation spectral characteristics. This study utilized excitation-scanning hyperspectral imaging to perform a comprehensive assessment of fluorescence spectral signatures of various tissues. Immediately following tissue harvest, a custom inverted microscope (TE-2000, Nikon Instruments) with Xe arc lamp and thin film tunable filter array (VersaChrome, Semrock, Inc.) were used to acquire hyperspectral image data from each sample. Scans utilized excitation wavelengths from 340 nm to 550 nm in 5 nm increments. Hyperspectral images were analyzed with custom Matlab scripts including linear spectral unmixing (LSU), principal component analysis (PCA), and Gaussian mixture modeling (GMM). Spectra were examined for potential characteristic features such as consistent intensity peaks at specific wavelengths or intensity ratios among significant wavelengths. The resultant spectral features were conserved among tissues of similar molecular composition. Additionally, excitation spectra appear to be a mixture of pure endmembers with commonalities across tissues of varied molecular composition, potentially identifiable through GMM. These results suggest the presence of common autofluorescent molecules in most tissues and that excitationscanning hyperspectral imaging may serve as an approach for characterizing tissue composition as well as pathologic state. Future work will test the feasibility of excitation-scanning hyperspectral imaging as a contrast mode for discriminating normal and pathological tissues.

One of the fundamental analytical measurements performed in microbiology is monitoring and characterizing cell concentration in culture media. Measurement error will give rise to reproducibility problems in a wide range of applications, from biomanufacturing to basic research. Therefore, it is critical that the generated results are consistent. Single wavelength optical density (OD) measurements have become the preferred approach. Here, we compare the conventional OD600 technique with a multi-wavelength normalized scattering optical spectroscopy method to measure the growth rates of Pseudomonas aeruginosa and Staphylococcus aureus, two of the leading nosocomial pathogens with proven abilities to develop resistance. The multi-wavelength normalization process minimizes the impact of bacteria byproducts and environmental noise on the signal, thereby accurately quantifying growth rates with high fidelity at low concentrations. In contrast, due to poor absorbance and scattering at 600 nm, the classic OD600 measurement method is able to detect bacteria but cannot quantify the growth rate reliably. Our wavelength-normalization protocol to detect bacteria growth rates can be readily and easily adopted by research labs, given that it only requires the use of a standard spectrophotometer and implementation of straightforward data analysis. Measuring and monitoring bacteria growth rates play a critical role in a wide range of settings, spanning from therapeutic design and development to diagnostics and disease prevention. Having a full understanding of the growth cycles of bacteria known to cause severe infections and diseases will lead to a better understanding of the pathogenesis of these illnesses, leading to better treatment and, ultimately, the development of a cure.

Hyperspectral imaging (HSI) systems collect both morphological and chemical characteristics from a sample by simultaneously acquiring spatial and spectral information. HSI has potential to advance cancer diagnostics by characterizing reflectance and fluorescence properties of a tissue, as well as extracting microstructural in- formation, all of which are altered through the development of a tumor. Illumination uniformity is a critical pre-condition for extracting quantitative data from an HSI system. Spatial, angular, or spectral non-uniformity can cause glare, specular reflection and unwanted shading, which negatively impact statistical analysis techniques used to extract abundance of different chemical species. This is further exacerbated when imaging three-dimensional structures, such as tumors, whose appearance can cast shadows and form other occlusions. Furthermore, as HSI can be used simultaneously for white light and fluorescence imaging, a flexible system, which multiplexes narrowband and broadband illumination is necessary to fully utilize the capabilities of a biomedical HSI system. To address these challenges, we modeled illumination systems frequently used in wide-field biological imaging with the software LightTools and FRED. Each system is characterized for spectral, spatial, and angular uniformity, as well as total efficiency. While all three systems provide high spatial and spectral uniformity, the highest angular uniformity is achieved using a diffuse scattering dome, yielding a contrast of 0.503 and average deviation of 0.303 with a 3.91% model error. Nonetheless, results suggest that conventional systems may not be suitable for low-light-level applications, where tailoring illumination to match spatial and spectral requirements may be the best approach to maximize the performance.

Psoriasis is a chronic skin disease affecting approximately 125 million people worldwide. Currently, dermatologists monitor changes of psoriasis by clinical evaluation or by measuring psoriasis severity scores over time which lead to Subjective management of this condition. The goal of this paper is to develop a reliable assessment system to quantitatively assess the changes of erythema and intensity of scaling of psoriatic lesions. A smartphone deployable mobile application is presented that uses the smartphone camera and cloud-based image processing to analyze physiological characteristics of psoriasis lesions, identify the type and stage of the scaling and erythema. The application targets to automatically evaluate Psoriasis Area Severity Index (PASI) by measuring the severity and extent of psoriasis. The mobile application performs the following core functions: 1) it captures text information from user input to create a profile in a HIPAA compliant database. 2) It captures an image of the skin with psoriasis as well as image-related information entered by the user. 3) The application color correct the image based on environmental lighting condition using calibration process including calibration procedure by capturing Macbeth ColorChecker image. 4) The color-corrected image will be transmitted to a cloud-based engine for image processing. In cloud, first, the algorithm removes the non-skin background to ensure the psoriasis segmentation is only applied to the skin regions. Then, the psoriasis segmentation algorithm estimates the erythema and scaling boundary regions of lesion. We analyzed 10 images of psoriasis images captured by cellphone, determined PASI score for each subject during our pilot study, and correlated it with changes in severity scores given by dermatologists. The success of this work allows smartphone application for psoriasis severity assessment in a long-term treatment.

Recently lower limit of detection (LOD) is necessary for rapid bacteria detection and analysis applications in clinical practices and daily life. A critical pre-conditioning step for these applications is bacterial concentration, especially for low level of pathogens. Sample volume can be largely reduced with an efficient pre-concentration process. Some approaches such as hollow-fiber ultra-filtration and electrokinetic technique have been applied to bacterial concentration. Since none of these methods can provide a concentrating method with a stable recovery efficiency, bacterial concentration still remains challenging Ceramic micro- filter can be used to concentrate the bacteria but the cross flow system keeps the bacteria in suspension. Similar harvesting bacteria using ultra-filtration showed an average recovery efficiency of 43% [1] and other studies achieved recovery rates greater than 50% [2]. In this study, an enhanced ceramic micro-filter with 0.14 μm pore size was proposed and demonstrated to optimize the concentration of E.coli. A high recovery rate (mean value >90%) and a high volumetric concentration ratio (>100) were achieved. Known quantities (10⁴ to 10⁶ CFU/ml) of E.coli cells were spiked to different amounts of phosphate buffered saline (0.1 to 1 L), and then concentrated to a final retentate of 5 ml to 10 ml. An average recovery efficiency of 95.3% with a standard deviation of 5.6% was achieved when the volumetric con- centration ratio was 10. No significant recovery rate loss was indicated when the volumetric concentration ratio reached up to 100. The effects of multiple parameters on E.coli recovery rate were also studied. The obtained results indicated that the optimized ceramic micro- filtration system can successfully concentrate E.coli cells in water with an average recovery rate of 90.8%.

We demonstrate ultrafast time-stretch microscopy in, to the best of our knowledge, the shortest wavelength regimes, i.e. 532 nm. This is enabled by a new all-optical ultrahigh-speed laser-scanning technique called free-space angular-chirpenhanced delay (FACED) that achieves a line-scan rate as high as 20 MHz. In contrast to the predominant fiber-based implementation, time-stretch imaging based on FACED allows wavelength-independent and low-loss operations, and more intriguingly reconfigurable all-optical laser-scanning rate. Using this technique, we present high-resolution single-cell images captured in an ultrafast microfluidic flow (1.5m/s). This could unleash numerous cell and tissue imaging applications, e.g. high-throughput image flow cytometry and whole-slide imaging.

It has been shown that Raman spectroscopy provides superb ability to differentiate individual cell types, and can also be used to detect circulating tumor cells (CTCs).1 CTCs have been recently identified as a main culprit for the development of cancer metastasis in cancer patients.2 It is also well known that the presence of CTCs is negatively associated with the development of metastasis and the progression of cancer. Hence, a reliable method for CTC identification will have a major impact on cancer diagnostic, monitoring of cancer progression, and cancer therapy. There are, however, two general problems of using Raman spectroscopy for the identification of cells. On the one hand, it is not clear from which cellular location a Raman spectrum that reliably represents the given cell should be acquired. On the other hand, the Raman signal intensity is weak, so that acquisition times of several seconds are required, prohibiting a high-throughput cell sampling. In this work we firstly show that by rapidly scanning a diffraction-limited spot over the cell and continuously acquiring a Raman spectrum it is possible to overcome the intracellular heterogeneity of a cell. And the resulting chemometric models provide a better and more robust cell classification. Secondly, we can show that the spectral resolution of a Raman spectrum is not as crucial to distinguish between different cell types. By reducing the spectral resolution 6-fold, we can achieve a signal gain 5-fold and still reliably identify single cells.

We present a cell image quantification method for image-based drug response prediction from patient-derived glioblastoma cells. Drug response of each person differs at the cellular level. Therefore, quantification of a patient-derived cell phenotype is important in drug response prediction. We performed fluorescence microscopy to understand the features of patient-derived 3D cancer spheroids. A 3D cell culture simulates the in-vivo environment more closely than 2D adherence culture, and thus, allows more accurate cell analysis. Furthermore, it allows assessment of cellular aggregates. Cohesion is an important feature of cancer cells. In this paper, we demonstrate image-based quantification of cellular area, fluorescence intensity, and cohesion. To this end, we first performed image stitching to create an image of each well of the plate with the same environment. This image shows colonies of various sizes and shapes. To automatically detect the colonies, we used an intensity based classification algorithm. The morphological features of each cancer cell colony were measured. Next, we calculated the location correlation of each colony that is appeal of the cell density in the same well environment. Finally, we compared the features for drug-treated and untreated cells. This technique could potentially be applied for drug screening and quantification of the effects of the drugs.

Biomarker screening for prostate-specific antigen (PSA) is the current clinical standard for detection of prostate cancer. However this method has shown many limitations, mainly in its specificity, which can lead to a high false positive rate. Thus, there is a growing need in developing a more specific detection system for prostate cancer. Using a Photonic- Crystal-based biosensor in a Total-Internal-Reflection (PC-TIR) configuration, we demonstrate the use of refractive index (RI) to accomplish label-free detection of prostate cancer cells against non-cancerous prostate epithelial cells. The PC-TIR biosensor possesses an open microcavity, which in contrast to traditional closed microcavities, allows for easier access of analyte molecules or cells to interact with its sensing surface. In this study, an imaging system was designed using the PC-TIR biosensor to quantify cell RI as the contrast parameter for prostate cancer detection. Non-cancerous BPH-1 prostate epithelial cells and prostate cancer PC-3 cells were placed on a single biosensor and measured concurrently. Recorded image data was then analyzed through a home-built MatLab program. Results demonstrate that RI is a suitable variable for differentiation between prostate cancer cells and non-cancerous prostate epithelial cells. Our study shows clinical potential in utilizing RI test for the detection of prostate cancer.

Non-invasive 3D imaging technique is essential for regenerative tissues evaluation. Optical coherence tomography (OCT) is one of 3D imaging tools with no staining and is used extensively for fundus examination. We have developed Phase-Diversity Homodyne OCT which enables cell imaging because of high resolution, whereas conventional OCT was not used for cell imaging because of low resolution. We demonstrated non-invasive imaging inside living spheroids with Phase-Diversity Homodyne OCT. Spheroids are spheroidal cell aggregates and used as regenerative tissues. Cartilage cells were cultured in low-adhesion 96-well plates and spheroids were manufactured. Cell membrane and cytoplasm of spheroid were imaged with OCT.

Auto fluorescence distribution of coenzymes NADH and FAD is investigated for the unstained tumor detection using an [?] originally designed confocal spectroscope. The tumor region in digestive organ can be determined by evaluating the redox index which is defined as the raio of NADH and FAD concentration. However, the redox index is largely influenced by the presence of collagen in the submucosal layer because its auto fluorescence spectrum overlaps considerably with that of NADH. Therefore, it is necessary to know in advance the distribution of NADH, FAD, and collagen in the mucosal layer. The purpose of our study is to investigate the vertical distribution of the redox index in tissue using depth-sensitive auto fluorescence spectroscopy. The experimental procedure and the results are presented.

Nuclear magnetic resonance imaging (MRI) is widely used as tomographic image inspection in clinical. MRI has many advantages such as obtaining tomographic images by non-exposure. However, it requires a strong magnetic field (0.4 to 3 T in general), so it also has disadvantages such as large size and high cost. If we use extremely low magnetic field as low as the earth B-field for MRI may be useful. In this study, we demonstrated the increase of nuclear magnetic resonance signals by spin hyperpolarization by spin exchange optical pumping for xenon in an extremely low magnetic field.

Object of study: The investigation is focused on development of personalized medicine. The determination of mechanical properties of bone tissues based on in vivo data was considered. Methods: CT, MRI, natural experiments on versatile test machine Instron 5944, numerical experiments using Python programs. Results: The medical diagnostics methods, which allows determination of mechanical properties of bone tissues based on in vivo data. The series of experiments to define the values of mechanical parameters of bone tissues. For one and the same sample, computed tomography (CT), magnetic resonance imaging (MRI), ultrasonic investigations and mechanical experiments on single-column test machine Instron 5944 were carried out. The computer program for comparison of CT and MRI images was created. The grayscale values in the same points of the samples were determined on both CT and MRI images. The Haunsfield grayscale values were used to determine rigidity (Young module) and tensile strength of the samples. The obtained data was compared to natural experiments results for verification.

To date, the incident rates of various skin diseases have increased due to hereditary and environmental factors including stress, irregular diet, pollution, etc. Among these skin diseases, seborrheic dermatitis and psoriasis are a chronic/relapsing dermatitis involving infection and temporary alopecia. However, they typically exhibit similar symptoms, thus resulting in difficulty in discrimination between them. To prevent their associated complications and appropriate treatments for them, it is crucial to discriminate between seborrheic dermatitis and psoriasis with high specificity and sensitivity and further continuously/quantitatively to monitor the skin lesions during their treatment at other locations besides a hospital. Thus, we here demonstrate a mobile multispectral imaging system connected to a smartphone for selfdiagnosis of seborrheic dermatitis and further discrimination between seborrheic dermatitis and psoriasis on the scalp, which is the more challenging case. Using the system developed, multispectral imaging and analysis of seborrheic dermatitis and psoriasis on the scalp was carried out. It was here found that the spectral signatures of seborrheic dermatitis and psoriasis were discernable and thus seborrheic dermatitis on the scalp could be distinguished from psoriasis by using the system. In particular, the smartphone-based multispectral imaging and analysis moreover offered better discrimination between seborrheic dermatitis and psoriasis than the RGB imaging and analysis. These results suggested that the multispectral imaging system based on a smartphone has the potential for self-diagnosis of seborrheic dermatitis with high portability and specificity.

We present here the control and measurement of temperature rise using femtosecond optical tweezers at near infrared (NIR) region. Based on our theoretical development, we have designed our experimental techniques. The high temporal sensitivity of position autocorrelation and equipartition theorem is simultaneously applied to elucidate temperature control and high precision measurement around focal volume. Experimentally we have made the benign NIR wavelength to induce local heating by adding very low fluorescent dye molecule with low average power. Local temperature control in aqueous solution exciting within optically absorbing window of the low quantum yield molecules can be possible due to non-radiative relaxation via thermal emission. The stochastic nature of Brownian particle has enough information of its surroundings. We have mapped the nano-dimension beam waist environment by probing the fluctuation of trapped particle. We have observed up to 30K temperature rise from room temperature at sub micro molar concentration. The gradient of temperature is as sharp as the fluence of pulsed laser focused by high numerical aperture objective. Thus, pulsed laser radiation always allows finer surgical techniques involving minimal thermal injuries. Our new techniques with multiphoton absorbing non-fluorescent dye can further be used to selective phototherapeutic diagnosis of cancer cells due to peak power dependent nonlinear phenomenon (NLO).

Embryonic stem cells have great promise in regenerative medicine because of their ability to self-renew and differentiate into various cell types. Delivery of therapeutic genes into cells has already been achieved using of chemical agents and viral vectors with high transfection efficiencies. However, these methods have also been documented as toxic and in the latter case they can cause latent cell infections. In this study we use femtosecond laser pulses to optically deliver genetic material in mouse embryonic stem cells. Femtosecond laser pulses in contrast to the conventional approach, minimises the risk of unwanted side effects because photons are used to create transient pores on the membrane which allow free entry of molecules with no need for delivery agents. Using an Olympus microscope, fluorescence imaging of the samples post irradiation was performed and decreased expression of stage specific embryonic antigen one (SSEA-1) consistent with on-going cellular differentiation was observed. Our results also show that femtosecond laser pulses were effective in delivering SOX 17 plasmid DNA (pSOX17) which resulted in the differentiation of mouse embryonic stem cells into endoderm cells. We thus concluded that laser transfection of stem cells for the purpose of differentiation, holds potential for applications in tissue engineering as a method of generating new cell lines.

Our lab has worked to develop high-speed hyperspectral imaging systems that scan the fluorescence excitation spectrum for biomedical imaging applications. Hyperspectral imaging can be used in remote sensing, medical imaging, reaction analysis, and other applications. Here, we describe the development of a hyperspectral imaging system that comprised an inverted Nikon Eclipse microscope, sCMOS camera, and a custom light source that utilized a series of high-power LEDs. LED selection was performed to achieve wavelengths of 350-590 nm. To reduce scattering, LEDs with low viewing angles were selected. LEDs were surface-mount soldered and powered by an RCD. We utilized 3D printed mounting brackets to assemble all circuit components. Spectraradiometric calibration was performed using a spectrometer (QE65000, Ocean Optics) and integrating sphere (FOIS-1, Ocean Optics). Optical output and LED driving current were measured over a range of illumination intensities. A normalization algorithm was used to calibrate and optimize the intensity of the light source. The highest illumination power was at 375 nm (3300 mW/cm²), while the lowest illumination power was at 515, 525, and 590 nm (5200 mW/cm²). Comparing the intensities supplied by each LED to the intensities measured at the microscope stage, we found there was a great loss in power output. Future work will focus on using two of the same LEDs to double the power and finding more LED and/or laser diodes and chips around the range. This custom hyperspectral imaging system could be used for the detection of cancer and the identification of biomolecules.

Surface enhanced Raman scattering (SERS) spectroscopy is a useful method for detection of trace amounts of molecules. It has already been successfully implemented for detection of explosives, food additives, biomarkers in blood or urine, etc. In the last decade, SERS spectroscopy was introduced into the field of health sciences and has been especially focused on early disease detection. In the recent years, application of SERS spectroscopy for detection of various types of human cancerous tissues emerged. Furthermore, SERS spectroscopy of extracellular fluid shows great potential for the differentiation of normal and cancerous tissues; however, due to high variety of molecules present in such biological samples, the experimental spectrum is a combination of many different overlapping vibrational spectral bands. Thus, precise assignment of these bands to the corresponding molecular vibrations is a difficult task. In most cases, researchers try to avoid this task satisfying just with tentative assignment. In this study, low temperature SERS measurements of extracellular fluid of cancerous and healthy kidney tissue samples were carried out in order to get a deeper understanding of the nature of vibrational spectral bands present in the experimental spectrum. The SERS spectra were measured in temperature range from 300 K down to 100 K. SERS method was implemented using silver nanoparticle colloidal solution. The results of the low temperature SERS experiment were analysed and compared with the results of theoretical calculations. The analysis showed that the SERS spectrum of extracellular fluid of kidney tissue is highly influenced by the vibrational bands of adenine and Lcystine molecules.

It is well known that platelets play a central role in hemostasis and thrombosis, they also mediate tumor cell growth, dissemination and angiogenesis. The purpose of the present experiment was to evaluate living platelet size, function and morphology simultaneously in unactivated and activated states using Phase-Interference Microscope “Cytoscan” (Moscow, Russia). We enrolled 30 healthy volunteers, who had no past history of aeteriosclerosis-related disorders, such as coronary heart disease, cerebrovascular disease, hypertention, diabetes or hyperlipidemia and 30 patients with oropharynx cancer. We observed the optic-geometrical parameters of each isolated living cell and the distribution of platelets by sizes have been analysed to detect the dynamics of cell population heterogeneity. Simultaneously we identified 4 platelet forms that have different morphological features and different parameters of size distribution. We noticed that morphological platelet types correlate with morphometric platelet parameters. The data of polymorphisms of platelet reactivity in tumor progression can be used to improve patient outcomes in the cancer prevention and treatment. Moreover morphometric and functional platelet parameters can serve criteria of the efficiency of the radio- and chemotherapy carried out. In conclusion the computer phase-interference microscope provides rapid and effective analysis of living platelet morphology and function at the same time. The use of the computer phase-interference microscope could be an easy and fast method to check the state of platelets in patients with changed platelet activation and to follow a possible pharmacological therapy to reduce this phenomenon.

Analog mean-delay (AMD) method is a new powerful alternative method in determining the lifetime of a fluorescence molecule for high-speed confocal fluorescence lifetime imaging microscopy (FLIM). Even though the photon economy and the lifetime precision of the AMD method are proven to be as good as the state-of-the-art time-correlated single photon counting (TC-SPC) method, there have been some speculations and concerns about the accuracy of this method. In the AMD method, the temporal waveform of an emitted fluorescence signal is directly recorded with a slow digitizer whose bandwidth is much lower than the temporal resolution of lifetime to be measured. We found that the drifts and the fluctuations of the absolute zero position in a measured temporal waveform are the major problems in the AMD method. As a referencing technique, we already proposed dual-channel waveform measurement scheme that may suppress these errors. In this study, we have demonstrated real-time confocal AMD-FLIM system with dual-channel waveform measurement technique.

Food spoilage is mainly caused by microorganisms, such as bacteria. In this study, we measure the autofluorescence in meat samples longitudinally over a week in an attempt to develop a method to rapidly detect meat spoilage using fluorescence spectroscopy. Meat food is a biological tissue, which contains intrinsic fluorophores, such as tryptophan, collagen, nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FAD) etc. As meat spoils, it undergoes various morphological and chemical changes. The concentrations of the native fluorophores present in a sample may change. In particular, the changes in NADH and FAD are associated with microbial metabolism, which is the most important process of the bacteria in food spoilage. Such changes may be revealed by fluorescence spectroscopy and used to indicate the status of meat spoilage. Therefore, such native fluorophores may be unique, reliable and nonsubjective indicators for detection of spoiled meat. The results of the study show that the relative concentrations of all above fluorophores change as the meat samples kept in room temperature (~19° C) spoil. The changes become more rapidly after about two days. For the meat samples kept in a freezer (~-12° C), the changes are much less or even unnoticeable over a-week-long storage.

Objective: This study utilizes fluorescence microscopy to assess the effect of the oxygen tension on the production of reactive oxygen species (ROS) in mitochondria of fetal pulmonary artery endothelial cells (FPAECs). Introduction: Hypoxia is a severe oxygen stress, which mostly causes irreversible injury in lung cells. However, in some studies, it is reported that hypoxia decreases the severity of injuries. In this study, ROS production level was examined in hypoxic FPAECs treated with pentachlorophenol (PCP, uncoupler). This work was accomplished by monitoring and quantifying the changes in the level of the produced ROS in hypoxic cells before and after PCP treatment. Materials and methods: The dynamic of the mitochondrial ROS production in two groups of FPAECs was measured over time using time-lapse microscopy. For the first group, cells were incubated in 3% hypoxic condition for 2 hours and then continuously were exposed to hypoxic condition for imaging as well. For the second group, cells were incubated in normal oxygen condition. Time lapse images of the cells loaded with Mito-SOX (ROS indicator) were acquired, and the red fluorescence intensity profile of the cells was calculated. Changes in the level of the fluorescence intensity profile while they are treated with PCP indicates the dynamics of the ROS level. Results: The intensity profiles of the PCP-treated cells in the first group showed 47% lower ROS production rate than the PCP-treated cells in the second group. Conclusion: Time lapse microscopy revealed that hypoxic cells have lower ROS generation while treated with PCP. Therefore, this result suggests that hypoxia decreased electron transport chain activity in uncoupled chain.