This PDF file contains the front matter associated with SPIE Proceedings Volume 8587, including the Title Page, Copyright information, Table of Contents, and the Conference Committee listing.

Window chamber models have been utilized for many years to investigate cancer development and the tumor microenvironment. Orthotopic mammary window chamber model have been developed for detailed study of breast cancer. Orthotopic window chamber models, due to the native environment, support more realistic growth and tumor behavior than ectopic models. The work by other groups thus far utilizing mammary window chamber models has focused solely on optical imaging techniques, limited to probing the first millimeter or less of tissue. These techniques do not take full advantage of the unrestricted, three-dimensional tumor growth the model supports. We have developed a custom plastic structure compatible with multimodality imaging. We present in this work the implementation of our custom window chamber in a mouse model and the successful imaging of the window chamber cancer model with MRI, nuclear imaging, and optical techniques. MRI provides a full three-dimensional view of the tumor growth and allows for additional, potentially clinically translatable, approaches to be utilized in investigating the cancer microenvironment. Nuclear imaging is accomplished using the Beta Imager, which is a novel approach to nuclear imaging of window chambers. The Beta Imager detects photons after the interaction of a single positron with a scintillator, instead of the coincidence detection of annihilation gamma ray pairs. We utilized the radioisotope glucose analog, 2-deoxy-2- (¹⁸F)fluoro-D-glucose or FDG, with the Beta Imager to obtain information on the glycolytic metabolism of the tumor and surrounding region.

Despite being an important pigment in skin, hair, the eye and the brain, melanin remains one of the most enigmatic of pigments. Although the main constituents of melanin are known to be dihydroxyindoles, its photophysics is complex and its detailed structure remains unknown. In this work we have arrested prior to completion the usual synthesis of eumelanin formed via auto-oxidation of 3, 4-dihydroxy-L-phenylalanine (L-DOPA), by the addition of copper ions. Using fluorescence techniques we report how copper modifies the self assembly of eumelanin by reducing the time to the onset of aggregation at pH 10 and yet produces simplified photophysics in terms of a clearly-defined fluorescence spectrum and a fluorescence decay that is described well by a dominant single lifetime of ~ 6ns. This behavior is consistent with copper inducing an enhanced abundance of 5,5-dihydroxyindole-2-carboxylic acid (DHICA). Metal ion binding to melanin is of particular importance to neurology and has potential applications in optoelectronics.

To quantitatively obtain the phase map of Onion and human red blood cell (RBC) from white light interferogram we used Hilbert transform color fringe analysis technique. The three Red, Blue and Green color components are decomposed from single white light interferogram and Refractive index profile for Red, Blue and Green colour were computed in a completely non-invasive manner for Onion and human RBC. The present technique might be useful for non-invasive determination of the refractive index variation within cells and tissues and morphological features of sample with ease of operation and low cost.

We are developing adaptive optics systems for aberration corrections in retinal imaging based on digital holography. Compared to existing technologies of adaptive optics, our systems do not have hardware components such as lenslet arrays or deformable mirrors. Instead, wavefront sensing and correction are done by acquisition and numerical manipulation of optical phase by digital holography, thereby substantially reducing hardware complexity and introducing novel imaging capabilities. Experimental results are presented to demonstrate capabilities of this novel imaging system.

Owing to its simplicity, lensless imaging system is adept at continuous monitoring of adherent cells inside the incubator. The setup consists of a CMOS sensor with pixel pitch of 2.2 μm and field of view of 24 mm², LED with a dominating wavelength of 525 nm, along with a pinhole of 150 μm as the source of illumination. The in-line hologram obtained from cells depends on the degree of cell-substrate adhesion. Drastic difference is observed between the holographic patterns of floating and adherent cells. In addition, the well-established fact of reduction of cell-substrate contact during cell division is observed with our system based on corresponding spontaneous transition in the holographic pattern. Here, we demonstrate that by recognizing this specific holographic pattern, number of cells undergoing mitosis in a cell culture with a population of approximately 5000 cells, can be estimated in real-time. The method is assessed on comparison with Edu-based proliferation assay. The approach is straightforward and it eliminates the use of markers to estimate the proliferation rate of a given cell culture. Unlike most proliferation assays, the cells are not harvested enabling continuous monitoring of cell culture.

Autophagy is an intracellular recycling mechanism that helps cells to survive against environmental stress and nutritional starvation. We have recently shown that prostate cancers undergo metabolic stress and caspase-independent cell death following exposure to arginine deiminase (ADI, an enzyme that degrades arginine in tissue). The aims of our current investigation into the application of ADI as a novel cancer therapy are to identify the components mediating tumor cell death, and to determine the role of autophagy (stimulated by ADI and/or rapamycin) on cell death. Using advanced fluorescence microscopy techniques including 3D deconvolution and superresolution structured-illumination microscopy (SIM), we show that prostate tumor cells that are killed after exposure to ADI for extended periods, exhibit a morphology that is distinct from caspase-dependent apoptosis; and that autophagosomes forming as a result of ADI stimulation contain DAPI-stained nuclear material. Fluorescence imaging (as well as cryo-electron microscopy) show a breakdown of both the inner and outer nuclear membranes at the interface between the cell nucleus and aggregated autophagolysosomes. Finally, the addition of N-acetyl cysteine (or NAC, a scavenger for reactive oxygen species) effectively abolishes the appearance of autophagolysosomes containing nuclear material. We hope to continue this research to understand the processes that govern the survival or death of these tumor cells, in order to develop methods to improve the efficacy of cancer pharmacotherapy.

We report a microscopic approach for determining cell cycle stages by measuring the nuclear optical path length (OPL) with quantitative differential interference contrast (DIC) microscopy. The approach is validated by the excellent agreement between the proportion of proliferating-to-quiescent cancerous breast epithelial cells obtained from DIC microscopy, and that from a standard immunofluorescence assay.

Conformational changes of individual fluorescently labeled proteins can be followed in solution using a confocal microscope. Two fluorophores attached to selected domains of the protein report fluctuating conformations. Based on Förster resonance energy transfer (FRET) between these fluorophores on a single protein, sequential distance changes between the dyes provide the real time trajectories of protein conformations. However, observation times are limited for freely diffusing biomolecules by Brownian motion through the confocal detection volume. A. E. Cohen and W. E. Moerner have invented and built microfluidic devices with 4 electrodes for an Anti-Brownian Electrokinetic Trap (ABELtrap). Here we present an ABELtrap based on a laser focus pattern generated by a pair of acousto-optical beam deflectors and controlled by a programmable FPGA chip. Fluorescent 20-nm beads in solution were used to mimic freely diffusing large proteins like solubilized F_oF₁-ATP synthase. The ABELtrap could hold these nanobeads for about 10 seconds at the given position. Thereby, observation times of a single particle were increased by a factor of 1000.

Many diseases involve changes in cell signaling cascades, as seen commonly in drug resistant cancers. To better understand these intricate signaling events in diseased cells and tissues, experimental methods of probing cellular communication at a single to multi-cell level are required. We recently introduced a general platform for activation of selected signaling pathways by optically controlled delivery and release of water soluble factors using gold-coated liposomes. In the example presented here, we encapsulated inositol trisphosphate (IP3), a ubiquitous intracellular secondary messenger involved in GPCR and Akt signaling cascades, within 100 nm gold-coated liposomes. The high polarizability of the liposome’s unique gold pseudo-shell allows stable optical trapping for subcellular manipulation in the presence of cells. We take this optical manipulation further by optically injecting IP3-containing liposomes into the cytosol of a single cell to initiate localized cell signaling. Upon optical injection of liposomal IP3 into a single ovarian carcinoma cell, we observed localized activation as reported by changes in Indo-1 fluorescence intensity. With established gap junctions between the injected cell and neighboring cells, we monitored propagation of this signaling to and through nearby cells.

Fungi have been found to be an underlying cause of 70% of all plant and animal extinctions caused by infectious diseases. Fungal infections are a growing problem affecting global health, food production and ecosystems. Lipid metabolism is a promising target for antifungal drugs and since effective treatment of fungal infections requires a better understanding of the effects of antifungal agents at the cellular level, new techniques are needed to investigate this problem. Recent advances in nonlinear microscopy allow chemically-specific contrast to be obtained non-invasively from intrinsic chemical bonds within live samples using advanced spectroscopy techniques probing Raman-active resonances. We present preliminary data using Stimulated Raman Scattering (SRS) microscopy as a means to visualise lipid droplets within individual living fungi by probing Raman resonances of the CH stretching region between 2825cm^-1 and 3030cm^-1.

Ball-lens hollow fiber Raman Probe (BHRP) and FTIR spectroscopy were main tools in this study. Thus, both of equipments detected the alteration of antisymmetric and symmetric P=O stretching vibration within our mice colorectal tumor models. Some differences of spectra due to randomly the edge of each BHRP and FTIR attached the surface of tumor during measurements. Meanwhile, the application of FTIR potentially differentiates the grade levels of non-clinic samples colorectal tumor models at four different grades (normal, grade 1, grade 2 and grade 3). Detailed investigations were assignable to wave numbers that publicized to represent biochemical alteration. The whole of investigated spectra in the fingerprint region revealed some different peaks and shoulders, most of which were assignable to wave numbers that exposed to represent biochemical alteration within the tissue. Differences in peak heights and peak ratio indicated differences in biochemical composition of cancer from different grade level. However, all collected colorectal tumor model at different peak was distinguishable, where antisymmetric and symmetric P=O stretching vibration was imaged and mapped clearly by both equipments. Therefore, BHRP were comfortable for in vivo studies. Meanwhile FTIR spectral analysis in combination with calibration curve might be used to distinguish cancer grade within colorectal tumor model tissue for ex vivo study.

An angular domain spectroscopic imaging system was built and validated using fresh ex vivo breast tissue samples (~ 2 mm thick). The hyperspectral system consisted of a halogen lamp, collimation optics, scanning stage with controller, a silicon micro-machined micro-channel array, and a pushbroom spectral imager. As a proof of concept, spectral data cubes acquired from tissue samples were input into principal component analysis and Mahalanobis discriminant analysis to differentiate between spectral signatures of breast tumor and normal tissue. It is proposed that the results from training sets can be used to construct a set of classifiers to enable tumor detection in samples representative of the surgical margins.

We introduce a multimode dermoscope (SkinSpect^TM) we developed for early detection of melanoma by combining fluorescence, polarization and hyperspectral imaging. Acquired reflection image datacubes were input to a wavelength-dependent linear model to extract the relative contributions of skin chromophores at every pixel. The oxy-hemoglobin, deoxy hemoglobin, melanin concentrations, and hemoglobin oxygen saturation by the single step linear least square fitting and Kubelka-Munk tissue model using cross polarization data cubes were presented. The comprehensive data obtained by SkinSpect can be utilized to improve the accuracy of skin chromophore decomposition algorithm with less computation cost. As an example in this work, the deoxy-hemoglobin over-estimation error in highly pigmented lesion due to melanin and deoxy hemoglobin spectral cross talk were analyzed and corrected using two-step linear least square fitting procedure at different wavelength ranges. The proposed method also tested in skin with underlying vein area for validating the proof of concept.

To minimize the problem with scattering in deep tissues while increasing the penetration depth, we explored the feasibility of imaging in the previously unexplored extended NIR (exNIR) spectral region at 900 - 1400 nm with endogenous chromophores. This region, also known as second NIR window, is weakly dominated by absorption of water and lipids and free from other endogenous chromophores, with virtually no autofluorescence. To demonstrate the applicability of the exNIR in bioimaging, we analyzed optical properties of individual components and animal organs using InGaAs spectrophotometer and a multispectral InGaAs scanning imager featuring in transmission geometry.

We present a polarization-sensitive hyperspectral imaging system (SkinSpect) that employs a spectrally-programmable light source in the visible and NIR domains. Multiple tissue-mimicking phantoms were fabricated to mimic the optical properties of normal skin as well as pigmented light and dark moles. The phantoms consist of titanium dioxide and a mixture of coffee, red food dye, and naphthol green as the scattering and the three absorptive agents in a polydimethylsiloxane (PDMS) base. Phantoms were produced with both smooth and rough textured surfaces and tested using Spatial Frequency Domain Imaging (SFDI) and Spatially Modulated Quantitative Spectroscopy (SMoQS) for homogeneity as well as determining absorption and scattering variance, respectively. The reflectance spectral images were also recorded using the SkinSpect research prototype; the spectral signatures of the phantoms were calculated using a two-flux single-layer Kubelka-Munk model and non-negative least square fitting routine was applied to extract the relative concentrations of the individual phantom components.

We describe a real-time image processor that has enabled a new automated flow through microscope to screen cells in flow at 100,000 cells/s and a record false positive rate of one in a million. This unit is integrated with an ultrafast optical imaging modality known as serial time-encoded amplified microscopy (STEAM) for blur-free imaging of particles in high-speed flow. We show real-time image-based identification and screening of budding yeast cells and rare breast cancer cells in blood. The system generates E-slides (an electronic version of glass slides) on which particles of interest are digitally analyzed.

A new approach for generating high-speed multispectral images has been developed. The central concept is that spectra can be acquired for each pixel in a confocal spatial scan by using a fast spectrometer based on optical fiber delay lines. This concept merges fast spectroscopy with standard spatial scanning to create datacubes in real time. The spectrometer is based on a serial array of reflecting spectral elements, delay lines between these elements, and a single element detector. The spatial, spectral, and temporal resolution of the instrument is described, and illustrated by multispectral images of laser-induced autofluorescence in biological tissues.

An estimated 12,000 new cases of spinal cord injury (SCI) occur every year in the United States. A small oxidative molecule responsible for secondary injury, acrolein, is an important target in SCI. Acrolein attacks essential proteins and lipids, creating a feed-forward loop of oxidative stress in both the primary injury area and the surrounding areas. A small molecule used and FDA-approved for hypertension, hydralazine, has been found to "scavenge" acrolein after injury, but its delivery and short half-life, as well as its hypertension effects, hinder its application for SCI. Nanomedical systems broaden the range of therapeutic availability and efficacy over conventional medicine. They allow for targeted delivery of therapeutic molecules to tissues of interest, reducing side effects of untargeted therapies in unwanted areas. Nanoparticles made from silica form porous networks that can carry therapeutic molecules throughout the body. To attenuate the acrolein cascade and improve therapeutic availability, we have used a one-step, modified Stober method to synthesize two types of silica nanoparticles. Both particles are “stealth-coated” with poly(ethylene) glycol (PEG) (to minimize interactions with the immune system and to increase circulation time), which is also a therapeutic agent for SCI by facilitating membrane repair. One nanoparticle type contains an amine-terminal PEG (SiNP-mPEG-Am) and the other possesses a terminal hydrazide group (SiNP-mPEG-Hz). The former allows for exploration of hydralazine delivery, loading, and controlled release. The latter group has the ability to react with acrolein, allowing the nanoparticle to scavenge directly. The nanoparticles have been characterized and are being explored using neuronal PC-12 cells in vitro, demonstrating the potential of novel silica nanoparticles for use in attenuating secondary injury after SCI.

We describe a new technique based on the use of a high-resolution quadri-wave lateral shearing interferometry wave front sensor to perform quantitative linear birefringence measurements on biological samples. The system combines quantitative phase images with different excitation polarizations to create birefringence images. This technique is fast and compatible with living samples. It gives information about the local retardance and structure of their anisotropic components.

The combination of fluorescent contrast agents with microscopy is a powerful technique to obtain real time images of tissue histology without the need for fixing, sectioning, and staining. The potential of this technology lies in the identification of robust methods for image segmentation and quantitation, particularly in heterogeneous tissues. Our solution is to apply sparse decomposition (SD) to monochrome images of fluorescently-stained microanatomy to segment and quantify distinct tissue types. The clinical utility of our approach is demonstrated by imaging excised margins in a cohort of mice after surgical resection of a sarcoma. Representative images of excised margins were used to optimize the formulation of SD and tune parameters associated with the algorithm. Our results demonstrate that SD is a robust solution that can advance vital fluorescence microscopy as a clinically significant technology.

Fluorescence microscopy is characterized by low background noise, thus a fluorescent object appears as an area of high signal/noise. Thermal gradients may result in apparent motion of the object, leading to a blurred image. Here, we have developed an image processing methodology that may remove/reduce blur significantly for any type of microscopy. A total of ~100 images were acquired with a pixel size of 30 nm. The acquisition time for each image was approximately 1 second. We can quantity the drift in X and Y using the sub pixel accuracy computed centroid location of an image object in each frame. We can measure drifts down to approximately 10 nm in size and a drift-compensated image can therefore be reconstructed on a grid of the same size using the “Shift and Add” approach leading to an image of identical size as the individual image. We have also reconstructed the image using a 3 fold larger grid with a pixel size of 10 nm. The resulting images reveal details at the diffraction limit. In principle we can only compensate for inter-image drift – thus the drift that takes place during the acquisition time for the individual image is not corrected. We believe that our results are of general applicability in microscopy and other types of imaging. A prerequisite for our method is the presence of a trackable object in the image such as a cell nucleus.

High-throughput screening in histology and analysis need a necessary automatic cell or nucleus counting. Current methods and systems based on grayscale or color images give results with counting errors. We suggest to use multispectral imaging (with more than three bands) rather than color one for nucleus counting. A traditional acquisition chains uses a source of white light and a CCD camera in addition to the optical microscope. To pass to a multispectral acquisition, we use a Programmable Light Source (PLS) in the place of the white light source. This PLS is capable of generating different wavelengths in the visible spectrum. So, one color image and four multispectral images have been acquired from histological slices. The four multispectral images contain respectively 3 bands, 5 bands, 7 bands and 10 bands. To make a proper comparison of data, several considerations have been taken, like camera linearity, intensity difference between the wavebands from the PLS and non uniformity of the light intensity range in the images. So, a set of measures were done for calibrating the system. An automatic detection method based on segmentation by expectation-maximization and ellipse fitting is used. An extension of this method is proposed in order to be applied to multispectral images. The original and the extended method are then applied to the data previously acquired to have first results regarding the effect of using multispectral images rather than color ones.

Introduction: The development of cytometry standards is complicated by their being relevant to pathology and biological science, which already have standards. CytometryML, the cytometry markup language, is an XML standard for flow and image cytometry, which includes both objects and their relationships, and is based upon existing standards: the International Society for Advancement of Cytometry ( ISAC) FCS, Digital Imaging and Communication in Medicine ( DICOM), and International Digital Publishing Forum (EPUB). Methods: The CytometryML schemas are written in XML Schema Definition (XSD1.1). Object-oriented methodology was employed to create the CytometryML schemas, which were tested by translating specific XSD elements into XML and filling in the values. The attribute based syntax description of relationships in the Resource Description Framework (RDF) has been replaced by an XSD element based implementation. The ISAC Archival Cytometry Standard (ACS) concept of a zipped data container file was further refined to be a EPUB file. Since Table of Contents information is present in an EPUB container, it was minimized in the Relations schema, which replaced the ToC schema of the ACS and includes a modified and extended version of the ToC RDF capabilities. Results: An XML based system that includes the DICOM specified separation of series and instances and includes relationships has been created. Conclusions: CytometryML and EPUB could be used for the transmission of research and medical data and be extension some of the pathology part of DICOM. The CytometryML version of RDF in XSD could be extended to provide XSD with full RDF capabilities.

Confocal laser scanning microscopy (CLSM) has become one of the most important biomedical research tools today due to its noninvasive and 3-D abilities. It enables imaging in living tissue with better resolution and contrast, and plays a growing role among microscopic techniques utilized for investigating numerous biological problems. In some cases, the sample was phase-sensitive, thus we introduce a novel method named laser oblique scanning optical microscopy (LOSOM) which could obtain a relief image in transparent sample directly. Through the LOSOM system, mouse kidney and HeLa cells sample were imaged and 10x, 20x and 40x magnify objective imaging results were realized respectively. Also, we compared the variation of pinhole size versus imaging result. One major parameters of LOSOM is the distance between fluorescence medium and the sample. Previously, this distance was set to 1.2 mm, which is the thickness of the slide. The experiment result showed that decreasing d can increase the signal level for LOSOM phase-relief imaging. We have also demonstrated the application of LOSOM in absorption imaging modality, when the specimen is non-transparent.

We present the applications of parallel Raman/SERS microspectroscopy by active illumination for simultaneously obtaining the full Raman/SERS spectra from multiple points within a field of view ~100 x 100 μm2 and ~micron spatial resolution. Two schemes have been employed in these studies: line-scan and programmable active illumination. We demonstrate the rapid classification of microparticles with similar shape, size and refractive index based on their chemical composition. We also demonstrate the rapid microanalysis of self-assembled monolayers on various types of gold nanostructures via SERS.

We outline the main differences in the activity profile of bacterial cultures studied by dynamic laser speckle (or biospeckle) patterns. The activity is detected in two sorts of culture mediums. The optical setup and the experimental procedure are presented. The experimentally obtained images are processed by the temporal difference method and a qualitative assessment is made with the time history of speckle patterns of the sample. The main differences are studied after changing the culture medium composition. We conclude that the EC medium is suitable to detect the E. coli bacterial presence in early hours and that Mueller Hinton agar delays some additional hours to make possible the assessment of bacteria in time.

LED-induced chlorophyll fluorescence analysis is exploited to observe, and monitor the time evolution of silicon-induced alleviation of toxicity in maize (Zea mays L.) seedlings in cadmium contaminated soil. Red, and far-red emissions were examined as a function of cadmium-silicon concentrations, during the 20 days period of the seedlings growing process under stress. The chlorophyll fluorescence spectral analysis provided detection, and evaluation of the damage imposed by the metal stress in the early stages of the plant growing process. The technique also provided the time evolution evaluation of the silicon-induced tolerance enhancement of maize plants to cadmium, which is not viable using conventional in vitro spectral analysis techniques

It is commonly believed that intrinsic skin aging is associated with the change of the collagen structures. The influence of the diabetes on the skin collagen is also considered to be similar to aging. Moreover, second-harmonic-generation (SHG) in collagen fibers is known to reflect the detailed collagen structures. It is thus highly valuable to adopt the SHG intensity as a collagen structure indicator. With the help of SHG, recently one can achieve in vivo imaging which provides the information of what really happens beneath the human skin. However, when analyzing the images, the SHG brightness of each pixel highly depends on the illumination condition, the depth of the SHG source, and the voltage of PMT. Therefore, it is important to calibrate these factors before statistical analysis. In this paper, we present our recent development that calibrates the in vivo SHG images by using noises. We first determine the regions of signals and noises by setting a threshold relating to the standard deviation of the image. By using the assumption that the noise was amplified by PMT with an amplification ratio the same as the SHG signal, we can define the brightness of the noise region as a parameter representing the voltage of PMT, and use this parameter to calibrate all SHG images. After calibrating, we can then compare different images from volunteers and analyze the influence of aging and diabetes on the SHG intensity from collagen fibers, even if the voltage of PMT was not fixed.

Recently, great deal of interest is investigation the function of the stem cells (SC) in tumors. In this study, we studied «recipient–tumor– fluorescent stem cells » system using the methods of in vivo imaging and laser scanning microscopy (LSM). We used adipose-derived adult stem (ADAS) cells of human lentiviral transfected with the gene of fluorescent protein Turbo FP635. ADAS cells were administrated into nude mice with transplanted tumor HeLa Kyoto (human cervical carcinoma) at different stages of tumor growth (0-8 days) intravenously or into tumor. In vivo imaging was performed on the experimental setup for epi – luminescence bioimaging (IAP RAS, Nizhny Novgorod). The results of the imaging showed localization of fluorophore tagged stem cells in the spleen on day 5-9 after injection. The sensitivity of the technique may be improved by spectral separation autofluorescence and fluorescence of stem cells. We compared the results of in vivo imaging and confocal laser scanning microscopy (LSM 510 META, Carl Zeiss, Germany). Internal organs of the animals and tumor tissue were investigated. It was shown that with i.v. injection of ADAS, bright fluorescent structures with spectral characteristics corresponding to TurboFP635 protein are locally accumulated in the marrow, lungs and tumors of animals. These findings indicate that ADAS cells integrate in the animal body with transplanted tumor and can be identified by fluorescence bioimaging techniques in vivo and ex vivo.

Wavelength-dependent light penetration depth in the measurement of water distribution of the skin tissue is analyzed. Near-infrared (NIR) imaging enables 2-D water content map on the skin tissue because water absorbs light strongly in the NIR region particularly around the wavelengths of 1450 and 1920 nm. However, the depth of the light penetration depends largely on wavelength as the absorption coefficient of water changes considerably in the NIR range. We investigate the measurement depth of the water content mapping with a NIR camera and bandpass filters at the wavelengths of 1300, 1450 and 1920 nm. Analysis is performed with Monte Carlo light scattering simulation adopting the optical parameters which is derived from the depth profile of the water contents measured by the confocal Raman spectroscopy. It is found that the NIR image in 1920 nm gives the highest sensitivity to the water content in the surface layer of the skin tissue.

Optical tweezers is a technique that can trap and manipulate small objects using a highly focused laser beam. Because optical tweezers can also be used to measure small forces, it has been extensively used for the measurement of the mechanical forces of cells. Previous research works typically study particle manipulation and cell force measurement in the lateral direction, hence excluding valuable insights about the axial mechanical properties of cells. Other works that investigate axial cell force measurements utilize spatial light modulators and other devices that are expensive and complicate the setup. Thus, in our study, we designed a simple scheme that can axially manipulate particles by adjusting the position of one lens, called L1-lens, in our setup. Image processing techniques were utilized to determine the changes in the axial particle translation, providing nanometer sensitivity. We investigated the capability of our system using two different-sized particles and results show that for a given L1-lens default position and movement, a 2-micron particle and a 4.26-micron particle were moved axially for 7.68 µm and 4.83 µm, respectively. Axial trapping stiffness was also measured for the stated bead sizes in different magnification. Using the computed trapping sti_ness, we will investigate the axial reactive forces of cells.

Drug discovery begins by identifying protein-small molecule binding pairs. Afterwards, binding kinetics and biofunctional assays are performed, to reduce candidates for further development. High-throughput screening, typically employing fluorescence, is widely used to find protein ligands in small-molecule libraries, but is rarely used for binding kinetics measurement because: (1) attaching fluorophores to proteins can alter kinetics and (2) most label-free technologies for kinetics measurement are inherently low-throughput and consume expensive sensing surfaces. We addressed this need with polarization-modulated ellipsometric scanning microscopes, called oblique-incidence reflectivity difference (OI-RD). Label-free ligand screening and kinetics measurement are performed simultaneously on small-molecule microarrays printed on relatively inexpensive isocyanate-functionalized glass slides. As a microarray is reacted, an OI-RD microscope tracks the change in surface-bound macromolecule density in real-time at every spot. We report progress applying OI-RD to screen purified proteins and virus particles against a 51,200-compound library from the National Cancer Institute. Four microarrays, each containing 12,800 library compounds, are installed in four flow cells in an automated OI-RD microscope. The slides are reacted serially, each giving 12,800 binding curves with ~30 sec time resolution. The entire library is kinetically screened against a single probe in ~14 hours and multiple probes can be reacted sequentially under automation. Real-time binding detection identifies both high-affinity and low-affinity (transient binding) interactions; fluorescence endpoint images miss the latter. OI-RD and microarrays together is a powerful high-throughput tool for early stage drug discovery and development. The platform also has great potential for downstream steps such as in vitro inhibition assays.

Using hybrid TPEF-SHG imaging and immunocytological techniques, we studied dedifferentiation of adult cardiomyocytes. First, the myofibrils shrank to shorten the sarcomere length. At the cell ends, the striated pattern of myosin filaments began to dissociate; at the center of the cell, the striated pattern of alpha-actinin first faded away and reappeared near the cell membrane during dedifferentiation. The results suggest that when freshly isolated adult cardiomyocytes are used to model cardiac muscle, the end-to-end connection may be important to maintain their striated myofibrillar structure and rod-shape morphology.

Problem of viruses is very actual for nowadays. Some viruses, which are responsible for human of all tumors, are about 15 %. Main purposes this study, early detection virus in live cell without labeling and in the real time by Raman spectroscopy. Micro Raman spectroscopy (mRs) is a technique that uses a Raman spectrometer to measure the spectra of microscopic samples. According to the Raman spectroscopy, it becomes possible to study the metabolites of a live cultured cell without labeling. We used mRs to detect the virus via HEK 293 cell line-infected adenovirus. We obtained raman specters of lives cells with viruses in 24 hours and 7 days after the infection. As the result, there is some biochemical changing after the treatment of cell with virus. One of biochemical alteration is at 1081 cm^-1. For the clarification result, we use confocal fluorescent microscopy and transmission electron microscopy (TEM).

The clinical diagnosis of most cancers is based on evaluation of histology microscopic slide to view the size and shape of cellular nuclei, and morphological structure of tissue. To achieve this goal in vivo and in deep tissue, near infrared (NIR) dyes-bovine serum albumin (BSA) and immunoglobulin G (IgG) conjugates were synthesized. The spectral study show that the absorption and fluorescence of the dye-conjugates are in the “tissue optical window” between 650 nm and 1100 nm. The internalization and pinocytosis of the synthesized compound were investigated in cell level using fluorescence microscopy to obtain the optimal concentration and staining time scale.

Raman spectral imaging has become a more and more popular technique in biological studies because it can extract chemical information from living cells in a label-free manner. One of the most challenging issues in the label-free Raman imaging of biological samples is to increase the molecular specificity in the spectra for better chemical contrast. Usually, the Raman spectrum from a cell is dominated by a few strong Raman bands such as the amide I band around 1650 cm^-1, CH₂ bend around 1445 cm^-1 or the amide III band around 1300 cm^-1 and it is not easy to get chemical contrast from other Raman bands that overlap with them. In this study, we aim to manipulate the chemical contrast in a living cell by exploiting the polarisation effects in Raman spectroscopy. By simply putting an analyser before the spectrometer, we can take the Raman image at the parallel and perpendicular polarisation against the incident light at the sample. The Raman spectra at the two orthogonal polarisations represent the Raman signals with different molecular orientation and symmetry of vibrations. Our experimental results demonstrate that at certain Raman shifts the two orthogonal polarisations indeed present different chemical contrasts. This indicates that polarized Raman imaging can help us visualise the different chemical contrasts that overlap at the same Raman shift and therefore increase the amount of chemical information we can get from cells.

In recent years there has been increasing interest in the use of molecular motors and cytoskeletal filaments in nanotechnological applications, particularly in the production of biomedical microdevices. In order for this to be possible it is important to exert a high level of control over the movement of the filaments. Chemical patterning techniques are often used to achieve this but these methods are often complex and the surface chemistry can be unstable. We investigated whether microfabricated silicon oxide lines of different widths with z-nanoscale heights of 20, 40 and 80 nm coated with heavy meromyosin (HMM) molecular motors could be used to control the motility of actin filaments by topographical means. Results demonstrated that filaments were confined by structures exceeding 20 nm in height regardless of the width of the channel indicating that topographical confinement offers a simple and possibly more cost-effective alternative to chemical patterning.

The bioinspired approaches to tissue strengthening and preservation rely on non-toxic cross-linking agents one of which is glyceraldehyde. In this study we used multiphoton microscopy that employs second harmonic generation (SHG) contrast to evaluate collagen microstructures and two-photon fluorescence (TPF) contrast to monitor progression of cross-linking upon treatment of tissues with glyceraldehyde. We examined collagen hydrogels assembled at 37 °C and 27 °C, bovine scleral and corneal tissues, skin as well as rat tail tendons. The results show a different effect of glyceraldehyde on collagen microstructures within the above tissues. This effect depends on the original microstructural assembly of collagen within a specific tissue. Our data suggests that epidermis (in skin and cornea) will protect collagen from cross-linking with glyceraldehyde. The work highlights benefits of monitoring progression of collagen cross-linking and effects of cross-linking on fiber microstructures as imaged with SHG and TPF signals.