This PDF file contains the front matter associated with SPIE Proceedings Volume 6441, including the Title Page, Copyright information, Table of Contents, and the Conference Committee listing.

We demonstrated the direct and noninvasive imaging of functional neurons,¹ as well as auricular heart muscle electrical activity² by Ionic Contrast Terahertz (ICT) near-field microscopy. This technique provides quantitative measurements of ionic concentrations in both the intracellular and extracellular compartments and opens the way to direct noninvasive imaging of neurons during electrical, toxin, or thermal stresses. Furthermore, neuronal activity results from both a precise control of transient variations in ionic conductances and a much less studied water exchange between the extracellular matrix and the intraaxonal compartment. The developed ICT technique associated with a full three-dimensional simulation of the axon-aperture near-field system allows a precise measurement of the axon geometry and therefore the direct visualization of neuron swelling induced by temperature change or neurotoxin poisoning. This technique should then provide grounds for the development of advanced functional neuroimaging methods based on diffusion anisotropy of water molecules.

In vivo diagnostics of skin diseases as well as understanding of the skin biology constitute a field demanding characterization of physiological and anatomical parameters. Biomedical optics has been successfully used, to qualitatively and quantitatively estimate the microcirculatory conditions of superficial skin. Capillaroscopy, laser Doppler techniques and spectroscopy, all elucidate different aspects of microcirculation, e.g. capillary anatomy and distribution, tissue perfusion and hemoglobin oxygenation. We demonstrate the use of a diffuse reflectance hyperspectral imaging system for spatial and temporal characterization of tissue oxygenation, important to skin viability. The system comprises: light source, liquid crystal tunable filter, camera objective, CCD camera, and the decomposition of the spectral signature into relative amounts of oxy- and deoxygenized hemoglobin as well as melanin in every pixel resulting in tissue chromophore images. To validate the system, we used a phototesting model, creating a graded inflammatory response of a known geometry, in order to evaluate the ability to register spatially resolved reflectance spectra. The obtained results demonstrate the possibility to describe the UV inflammatory response by calculating the change in tissue oxygen level, intimately connected to a tissue's metabolism. Preliminary results on the estimation of melanin content are also presented.

We present an optical system for small animal imaging that can combine various in vivo imaging modalities, including fluorescence (intensity and lifetime), spectral, and trans-illumination imaging. This system consists of light-tight box with ultrafast pulsed or cw laser light excitation, motorized translational and rotational stages, a telecentric lens for detection, and a cooled CCD camera that can be coupled to an ultrafast time-gated intensifier. All components are modular, making possible laser excitation at various wavelengths and pulse lengths, and signal detection in a variety of ways (multimode). Results of drug nanoconjugate carrier delivery studies in mice are presented. Conventional and spectrally-resolved fluorescence images reveal details of in vivo drug nanoconjugate carrier accumulation within the tumor region and several organs in real time. By multi-spectral image analysis of ex vivo specimens from the same mice, we were able to evaluate the extent and topology of drug nanoconjugate carrier distribution into specific organs and the tumor itself.

Electron Paramagnetic Resonance is an emerging technique finding applications in functional physiological imaging. Traditionally EPR imaging developed as a CW (continuous wave) technique involving the measurement of free radical distribution in vivo using constant frequency and field-sweep modality almost identical to the early developments of MRI. As in CT and PET this involved the generation of projections in presence of gradients and the reconstruction of images via filtered back-projection. The large line-width and the concomitant short relaxation times posed a serious challenge for the development of time-domain methods akin to modern pulsed NMR & MRI. With the recent availability of narrow line stable non-toxic radicals based on triarylmethyl (TAM), ultra fast data acquisition systems (signal digitizer and summer), very fast electronic switches and low-noise amplifiers, we have developed time-domain imaging schemes in EPR operating in the radiofrequency region Using a novel pure-phase encoding scheme, we are able to generate 2 and 3 dimensional spatial images and spectral-spatial images that adds an additional functional dimension to these images. The special space-encoding scheme with fast gradient ramping allow rapid in vivo imaging of small animals with superior spatial and functional information with good temporal resolution that can provide valuable physiological and pharmacokinetic insight. Our main thrust has been in the investigation of tumor hypoxia and tumor reoxygenation for the purpose of minimizing the radiation dose for maximum tumor cell killing. These and some of the allied imaging methods, and results from tumor investigation will be presented.

Apoptosis, also known as programmed cell death, is a process in which cells initiate a series of events to trigger their own demise. Normal cells use this mechanism in the regulation of their life cycle. On the contrary, abnormal or cancer cells have lost the ability to regulate themselves by this process. Because of this, there is much interest in the study of the apoptotic process. Currently, there are many commercial assays available to detect apoptosis in cells, most of which are fluorescence based. Limitations of such fluorescent assays lead to arbitrary or inclusive results. Raman spectroscopy is a powerful technique that yields specific molecular information on samples under study. The Raman spectra obtained from cell samples are very complex, yet the differences in the complex Raman spectra analyzed using chemometric techniques can identify chemical and physiological information about cells. Furthermore, Raman spectroscopy is a sensitive, rapid, reagentless, low-cost technique, making it a superior alternative to traditional fluorescence based apoptosis assays. In this study, we have employed Raman spectroscopy and Raman chemical imaging, along with chemometric techniques, to distinguish apoptotic cells from non-apoptotic cells in two prostate cancer cell lines, PC3 and LnCAP. Initial results indicate that Raman spectra of apoptotic and non-apoptotic cells are different in both cell lines. Furthermore, chemometric analysis of the data shows that the spectra separate into two distinct populations, apoptotic and non-apoptotic. Traditional fluorescence based apoptotic assays confirm the results. This work provides ample evidence that Raman spectroscopy is a valuable tool in biomedical imaging.

Phase-sensitive detection has long been recognized as a mechanism for increasing imaging contrast. The proliferation of quantitative phase contrast techniques and the breadth of emerging applications reflects the potential for achieving subdiffraction- limited resolution of cellular structure and dynamic phenomena with phase. Our laboratory developed spectral domain phase microscopy (SDPM) as a simple, phase-stable tool for studying cell dynamics and structure. As a functional extension of optical coherence tomography (OCT), SDPM inherited the high-resolution depth-sectioning capabilities for which OCT is well known, but adds to this an ability to discriminate sub-coherence length changes in optical pathlength within target samples at discrete axial positions. Early demonstrations of SDPM showed it to be extremely sensitive to thickness changes in biological and non-biological samples; the results of our previous studies investigating cell surface motion in cardiomyocyte contractility, cytoplasmic streaming rates in single-celled organisms, and rheological properties of the cytoskeleton suggest that SDPM can contribute insights of biological relevance. The principal aim of this work is to refine SDPM to enable imaging, interrogation, and quantification of parameters of interest in developing cardiomyocytes. In this manuscript, we report on the technology advances that enable multidimensional SDPM, and the results of new inotropic imaging studies of chick embryo cardiomyocytes.

A series of hyperspectral transmission images of hematoxylin and eosin stained tissue sections from cervical biopsies were acquired at 10 nm intervals and assembled into a hyperspectral image cube. Custom software providing extraction of spectra at each pixel allows selection of images with maximum contrast for determination of selected features and differentiation of tissue features. Illumination profiles were created using a spectrally and temporally programmable light engine based on a spatial light modulator that can dynamically create any narrow or broadband spectral profile was used to select illumination wavelengths. Images were acquired with a monochrome CCD camera. Several methods of combining images from individual or composite spectral bands to recreate color images for pathologist review are shown. Unlike current "mechanical" illumination systems employing optical filters, filter wheels, motors, shutters and multiple control interfaces, the light engine integrates the lamp, wavelength control, intensity control and exposure control in a simple MEMS based system, where the only moving part is the lamp cooling fan. Illumination can now be programmed dynamically with digital control of all illumination parameters allowing wavelengths and intensities to be changed much faster than with filter wheels, and providing exposure control orders of magnitude more precise than mechanical shutters. This system can be integrated with digital imaging systems. Digitally controlled illumination is bit additive with image data providing high dynamic range imaging with monochrome or with color imaging devices. Performance of image analysis software for nuclear morphometric and tissue architecture analysis are compared for different wavelength regions.

HPLC combined with laser induced fluorescence provides a very sensitive method for the separation and identification of the many proteins present in clinical samples. Protein profiles of clinical samples like Pap smear and tissue samples, from subjects with cervical cancer and normal volunteers, were recorded using HPLC-LIF. The protein profiles were analyzed by Principal Component Analysis (PCA). The profiles were characterized by parameters like scores of the factors, sum of squared residuals, and Mahalanobis Distance, derived from PCA. Parameters of each sample were compared with those of a standard set and Match/ No Match results were generated. Good discrimination between normal and malignant samples was achieved with high sensitivity and specificity.

A confocal time-resolved fluorescence anisotropy imaging set-up is presented. It combines a confocal laser scanning microscope equipped with a pulsed laser and two time gated detection systems with 4 gates each (LiMo, originally developed for FLIM). The anisotropy decays obtained with the time gating system yield results that compare well with the high time-resolution (non-imaging) decays recorded using Time Correlated Single Photon Counting. Time resolved anisotropy imaging experiments on cells expressing GPI-GFP were carried out. Clear distinction could be made between the anisotropy in the plasma membrane and in the interior of the cell.

Total internal reflection fluorescence microscopy (TIRFM) and non-radiative energy transfer (FRET) measurements have been combined in order to examine co-localization of the amyloid precursor protein (APP) and the ?-site APPcleaving enzyme (BACE) in human glioblastoma cells. So far, these proteins have been co-localized within whole cells (depending on the intracellular amount of cholesterol) and in some cases also within their plasma membranes. This supports the present hypothesis of localization within lipid domains on the cell surface and co-internalization via endocytosis.

Quantification of the oligomerisation state and conformational changes of the epidermal growth factor receptor in its constitutive and active states are critical to fully understand their signal transduction pathway. We show the ability of single-molecule fluorescence microscopy to determine this information and its implementation by combining multidimensional emission optics with total internal reflection fluorescence microscopy. Importantly, the signalling process is analysed in live cells under physiological conditions.

Measurable change in the sensory motor machinery of growth cones are induced by non contact femtosecond laser. The focused laser beam with an average power of 3 mW was positioned at some distance away from the closest fillopodia of cortical neurons from primary cell cultures (mice E15). By identifying a set of preliminary parameters we were able to statistically analyze the phenomenological behavior of the fillopodia and classify the effects different conditions of laser light has on the growth cone. Results show that fillopodia become significantly biased towards the focused femtosecond laser light. The same experiment performed with continuous wave (CW) produced results which were indistinguishable from the case where there is no laser light present (placebo condition) indicating no clear effects of the CW laser light on the fillopodia at a distance. These findings show the potential for ultrashort pulsed light to become a new type of pathfinding cue for neuronal growth cones.

Multiplexed high-content cytometric analysis of cells is a prerequisite for Cytomics and Systems Biology. Slide Based Cytometry (SBC) analysis yields quantitative cell related data on various cell constituents. It allows to measure and identify in high-throughput hundred-thousands of objects and obtain cytometric data on light absorption, scatter and fluorescence signals. Selected cells of interest can be rescanned and morphologically evaluated. To be cytometric SBC measurement needs high focal depth in order to acquire the fluorescence of the whole cell. For tissue analysis section thickness of >30μm is needed to reduce cell sectioning leading in multiple labelled specimens to an overestimation of multiple stained cells due to stereology, mimicking co-expression or elevated expression that is in fact due to coincidences in the z-axis direction. By confocal sectioning and 3D-reconstruction these overlays could be eliminated but confocal 3D imaging is slow and the resulting data are not cytometric. To overcome this obstacle, we combined SBC analysis with confocal imaging using a Laser Scanning Cytometer (iCys, Compucyte Corp., MA). Single to triple labelled 30-120μm thick human brain sections were scanned cytometrically (up to three laser 405nm, 488nm, 633nm) and double and triple labeled cells were identified. In the second step these objects were relocated, scanned confocally and 3D-reconstructed (Mathematica®, MathGL3d). This combination of high-throughput SBC and high-resolution confocal imaging enables for unequivocal identification of multiple labelled objects and is a prerequisite for Cytomic tissue analysis, Tissomics. (Support: HBFG 036/379-1)

Purpose: An in vivo flow cytometer was developed recently, providing quantification of fluorescently labeled cells in live animals without extracting blood samples. This non-invasive procedure allows continuously tracking a cell population of interest over long periods of time to examine its dynamic changes in the circulation. However, it has not been shown that counting signals arise from individual cells. Furthermore, cell morphology and cell-cell interaction in the blood stream (e.g. aggregation) are not visualized. Here we describe an imaging in vivo flow cytometer. Material and Methods: Fluorescence images are obtained simultaneously with quantitative information on a DiD-labeled cell population. As fluorescent cells pass through the slit of light focused across a blood vessel, the excited fluorescence is detected confocally. This cell counting signal triggers a strobe beam and an intensified CCD camera to capture a snapshot image of the cell as it moves down-stream from the slit. Results: Nearly all peaks counted as circulating T-cells originate from individual cells, while cell aggregates were rarely observed (<2%). Counting signal amplitude variation is attributed to uneven dye-loading among cells. We identify non-T-cells by their abnormal shape and size. Cell velocity was measured by determining the traveled distance from the slit within the delay of the strobe pulse or by applying multiple strobe pulses during the integration time of the CCD camera. Conclusions: An improved in vivo imaging flow cytometer can be a useful tool for studying cell populations in circulation.

We explore an optical spectroscopy approach to monitor the progression of ischemia and reperfusion in situ using a rat model. The system utilizes the sensitivity of NADH emission to changes in cell metabolism during ischemia and reperfusion. In addition, the emission from tryptophan is employed as a normalization against changes in other optical properties of the tissue. Ischemia was induced in one kidney followed by at least 60 minutes of reperfusion. During both phases, autofluorescence images of the exposed surfaces of both the ischemic kidney and the normal (control) kidney were acquired and the respective average emission intensities were determined. Preliminary results indicate that the kinetics of the ratio of the emissions under these two excitations is related to the injury time.

Digital holographic microscopy (DHM) is an interferometric technique, providing quantitative mapping of the phase shift induced by semi-transparent microscopic specimens, such as cells, with sub-wavelength resolution along the optical axis. Thanks to actual PCs and CCDs, DHM provides nowadays cost-effective instruments for real-time measurements at very high acquisition rates, with sub-micron transverse resolution. However, DHM phase images do not reveal the threedimensional (3D) internal distribution of refractive index, but a phase shift resulting from a mean refractive index (RI) integrated over the cellular thickness. Standard optical diffraction tomography (ODT) techniques can be efficiently applied to reveal internal structures and to measure 3D RI spatial distributions, by recording 2D DHM phase data for different sample orientations or illumination beam direction, in order to fill up entirely the Ewald sphere in the Fourier space. The 3D refractive index can then be reconstructed, even in the direct space with backpropagation algorithms or from the Fourier space with inverse Fourier transform. The presented technique opens wide perspectives in 3D cell imaging: the DHM-based micro-tomography furnishes invaluable data on the cell components optical properties, potentially leading to information about organelles intracellular distribution. Results obtained on biological specimens will be presented. Morphometric measurements can be extracted from the tomographic data, by detection based on the refractive index contrast within the 3D reconstructions. Results and perspectives about sub-cellular organelles identification inside the cell will also be exposed.

Shear stress is known to have a significant effect on the state of cellular differentiation. It also induces morphologic responses including changes to cytoskeletal organization subsequently leading to changes in cell shape. In fact, fluid shear stress caused by blood flow is a major determinant of vascular remodeling and can lead to development of atherosclerosis. The morphological changes are usually evaluated using boundary-based shape descriptors or binary geometrical moments on manually segmented cells. Although any one of the many automated segmentation methods could be employed, these techniques are known to be complex and time consuming, and often require user input to operate properly, which is especially problematic for HCS systems. Therefore, development of robust, quantitative morphological measurements that are not dependent on precision and reproducibility of segmentation is extremely important for a substantial improvement of shear-stress analysis. The goals of this study were to find simple morphological descriptors that could be applied to cells isolated by tessellation in order to enable a high-throughput screening of morphological shear-stress response, and to determine the amount of fluid shear stress to which endothelial cells were exposed on the basis of changes in their morphology. The proposed technique is based on the monitoring of changes in cytoskeleton organization using texture descriptors, rather than on quantifying cell-boundary modifications. We showed that objects identified by Voronoi tessellation carried enough information about cytoskeleton texture of individual cells to create a robust classifier. Our approach provided higher discriminant and predictive powers, and better classification capability, than traditional boundary-based methods. The robustness of classification in the presence of segmentation difficulties makes the proposed approach particularly suitable for automated HCS systems.

Tissue-specific biomarkers have been studied to identify micrometastases in bone marrow and/or peripheral blood. Many studies, however, have shown conflicting results for sensitivity and specificity of detection, forestalling translation of these findings into routine clinical use for prognosis or diagnosis. Genetic instability and heterogeneity of cancers may make using an absolute set of differential expression markers difficult, if not impossible, for accurate detection of rare cancer cells via a simple blood test. The literature is rich with examples of pathologists using morphology to identify cancer in tissue sections. We hypothesize that morphological features based on fluorescent staining of common subcellular compartments, in particular, the nucleus, may be useful for detection and classification. High-throughput/ high-content image cytometry and computer-automated classification can aid pathologists to find suspicious cells, independent of biomarkers. Feature data are collected from an in vitro spiked model of breast cancer in the circulation; prestaining with CellTracker Orange creates a gold standard for assessing cancer origin. A neural network classifier is designed using seven nuclear morphology features thought a priori to be important for classification. With adequate training data, sensitive and specific detection may be achieved. Neural networks may be robustly trained to assist pathologists in detecting a wide variety of cancers.

Most flow/image cytometric data analysis methods look for clusters in the data corresponding to specific cell subpopulations. Comparisons between different cytometry datafiles often use human pattern recognition visualization of all the different combinations of variables ("parameters") two at a time in so-called bivariate scattergrams. Not only is this tedious, but it can miss potential clusters due to projection of higher dimensional dataspaces down onto two dimensional planes making them indiscernible as separate clusters. Novel data mining algorithms, implemented in software allow for the comparison of two or more higher dimensional datafiles without the requirement for reduction of dimensionality for human visualization. Of equal importance is the comparison of higher dimensional clusters which may move around slightly in space, yet still be "similar" according to algorithms which provide measures of similarity. This software, written in C/C++ and currently implemented in software with a Windows graphical user interface, allows for direct reading of FCS2.0 format flow cytometry datafiles of any number of parameters. In a few minutes or less, complex multiparameter data of two or more files can be compared on a personal computer or workstation. The software operates in either supervised or unsupervised mode, depending on whether the user wishes to include prior user knowledge or in a data mining discovery mode. Differences between these files can be exported as sub-datafiles which can be further analyzed using any other software that can read FCS2.0 data format.

Biological microparticles scatter light in all directions when illuminated. The complex scatter pattern is dependent on particle size, shape, refraction index, density, and morphology. Commercial flow cytometers allow measurement at two nominal angles (2°⩽θ₁⩽20° and 70°⩽θ₂⩽110°) of scattered light intensity from individual microparticles with a speed varying from 10 to 10000 particles per second. The choice of angle is dictated by the fact that scattered light in the small-angle region is primarily influenced by cell size and refractive index, whereas side scatter intensity is related to the granularity of cellular structures. These rudimentary measurements cannot be used to separate populations of cells of similar shape, size, or structure. Hence, there have been several attempts in cytometry to measure the entire scatter patterns. However, the published concepts required use of unique custom-built cytometers and could not be applied to existing instruments. The presented work demonstrates application of pattern-recognition techniques to classify particles on the basis of their discrete scatter patterns collected at just five different angles, and accompanied by the measurement of axial light loss. Our approach can be used with existing instruments and requires only the addition of a custom-built scatter-detector. Our analytical model of scatter of laser beams by individual bacterial cells suspended in a fluid was used to determine the location for scatter sensors. Experimental results were used to train the pattern recognition system. It has been shown that information provided just by six scatter-related parameters was sufficient to recognize various bacteria with 90-99% success rate.

Because of the differences in the requirements, needs, and past histories including existing standards of the creating organizations, a single encompassing cytology-pathology standard will not, in the near future, replace the multiple existing or under development standards. Except for DICOM and FCS, these standardization efforts are all based on XML. CytometryML is a collection of XML schemas, which are based on the Digital Imaging and Communications in Medicine (DICOM) and Flow Cytometry Standard (FCS) datatypes. The CytometryML schemas contain attributes that link them to the DICOM standard and FCS. Interoperability with DICOM has been facilitated by, wherever reasonable, limiting the difference between CytometryML and the previous standards to syntax. In order to permit the Resource Description Framework, RDF, to reference the CytometryML datatypes, id attributes have been added to many CytometryML elements. The Laboratory Digital Imaging Project (LDIP) Data Exchange Specification and the Flowcyt standards development effort employ RDF syntax. Documentation from DICOM has been reused in CytometryML. The unity of analytical cytology was demonstrated by deriving a microscope type and a flow cytometer type from a generic cytometry instrument type. The feasibility of incorporating the Flowcyt gating schemas into CytometryML has been demonstrated. CytometryML is being extended to include many of the new DICOM Working Group 26 datatypes, which describe patients, specimens, and analytes. In situations where multiple standards are being created, interoperability can be facilitated by employing datatypes based on a common set of semantics and building in links to standards that employ different syntax.

Three dimensional imaging provides high-content information from living intact biology, and can serve as a visual screening cue. In the case of single cell imaging the current state of the art uses so-called "axial through-stacking". However, three-dimensional axial through-stacking requires that the object (i.e. a living cell) be adherently stabilized on an optically transparent surface, usually glass; evidently precluding use of cells in suspension. Aiming to overcome this limitation we present here the utility of dielectric field trapping of single cells in three-dimensional electrode cages. Our approach allows gentle and precise spatial orientation and vectored rotation of living, non-adherent cells in fluid suspension. Using various modes of widefield, and confocal microscope imaging we show how so-called "microrotation" can provide a unique and powerful method for multiple point-of-view (three-dimensional) interrogation of intact living biological micro-objects (e.g. single-cells, cell aggregates, and embryos). Further, we show how visual screening by micro-rotation imaging can be combined with micro-fluidic sorting, allowing selection of rare phenotype targets from small populations of cells in suspension, and subsequent one-step single cell cloning (with high-viability). Our methodology combining high-content 3D visual screening with one-step single cell cloning, will impact diverse paradigms, for example cytological and cytogenetic analysis on haematopoietic stem cells, blood cells including lymphocytes, and cancer cells.

A microscale cell culture analog (μCCA) is a cell-based lab-on-a-chip assay that, as an animal surrogate, is applied to pharmacological studies for toxicology tests. A μCCA typically comprises multiple chambers and microfluidics that connect the chambers, which represent animal organs and blood flow to mimic animal metabolism more realistically. A μCCA is expected to provide a tool for high-throughput drug discovery. Previously, a portable fluorescence detection system was investigated for a single μCCA device in real-time. In this study, we present a fluorescence-based imaging system that provides quantitative real-time data of the metabolic interactions in μCCAs with an emphasis on measuring multiple μCCA samples simultaneously for high-throughput screening. The detection system is based on discrete optics components, with a high-power LED and a charge-coupled device (CCD) camera as a light source and a detector, for monitoring cellular status on the chambers of each μCCA sample. Multiple samples are characterized mechanically on a motorized linear stage, which is fully-automated. Each μCCA sample has four chambers, where cell lines MES-SA/DX- 5, and MES-SA (tumor cells of human uterus) have been cultured. All cell-lines have been transfected to express the fusion protein H2B-GFP, which is a human histone protein fused at the amino terminus to EGFP. As a model cytotoxic drug, 10 μM doxorubicin (DOX) was used. Real-time quantitative data of the intensity loss of enhanced green fluorescent protein (EGFP) during cell death of target cells have been collected over several minutes to 40 hours. Design issues and improvements are also discussed.

We report on a new generation, commercial prototype of a programmable array optical sectioning fluorescence microscope (PAM) for rapid, light efficient 3D imaging of living specimens. The stand-alone module, including light source(s) and detector(s), features an innovative optical design and a ferroelectric liquid-crystal-on-silicon (LCoS) spatial light modulator (SLM) instead of the DMD used in the original PAM design. The LCoS PAM (developed in collaboration with Cairn Research, Ltd.) can be attached to a port of a(ny) unmodified fluorescence microscope. The prototype system currently operated at the Max Planck Institute incorporates a 6-position high-intensity LED illuminator, modulated laser and lamp light sources, and an Andor iXon emCCD camera. The module is mounted on an Olympus IX71 inverted microscope with 60-150X objectives with a Prior Scientific x,y, and z high resolution scanning stages. Further enhancements recently include: (i) point- and line-wise spectral resolution and (ii) lifetime imaging (FLIM) in the frequency domain. Multiphoton operation and other nonlinear techniques should be feasible. The capabilities of the PAM are illustrated by several examples demonstrating single molecule as well as lifetime imaging in live cells, and the unique capability to perform photoconversion with arbitrary patterns and high spatial resolution. Using quantum dot coupled ligands we show real-time binding and subsequent trafficking of individual ligand-growth factor receptor complexes on and in live cells with a temporal resolution and sensitivity exceeding those of conventional CLSM systems. The combined use of a blue laser and parallel LED or visible laser sources permits photoactivation and rapid kinetic analysis of cellular processes probed by photoswitchable visible fluorescent proteins such as DRONPA.

An optical biochip is being developed for monitoring the sensitivity of biological cells to a range of environmental changes. Such changes may include external factors such as temperature but can include changes within the suspending media of the cell. The ability to measure such sensitivity has a broad application base including environmental monitoring, toxicity evaluation and drug discovery. The device under development, capable of operating with both suspension and adherent cell populations, employs electrokinetic processes to monitor subtle changes in the physicochemical properties of cells as environmental parameters are varied. As such, the device is required to maintain cells in a viable condition for extended periods of time. The final device will employ integrated optical illumination of cells using red emitting LED or laser devices with light delivery to measurement regions achieved using integrated micro-optical components. Measurements of electrokinetic phenomena such as dielectrophoresis and electrorotation will be achieved through integrated optical detectors. Environmental parameters can be varied while cells are actively retained within a measurement structure. This enables the properties and sensitivity of a cell population to be temporally tracked. The optical biochip described here uses a combination of microfabrication techniques including photolithographic and laser micromachining processes. Here we describe the design and manufacturing processes to create the components of the environmental monitoring strutures of the optical biochip.

We have developed a range of optical biochip devices for conducting live and fixed cell-based assays. The devices encompass the ability to process an entire assay including fluorescently labelling cells, a microfluidic system to transport and maintain cells to deliver them to an optical area of the device for measurement, with the possibility of a incorporating a sorting step in between. On-chip excitation provided by red emitting LED and lasers define the excitation wavelength of the fluorophore to be incorporated into the assay readout. The challenge for such an integrated microfluidic optical biochip has been to identify and characterise a longterm fluorescent label suitable for tracking cell proliferation status in living cells. Traditional organic fluorophores have inherent disadvantages when considering their use for an on-chip device requiring longterm cellular tracking. This has led us to utilise inorganic quantum dots (QDots) as fluorophores for on- chip assays. QDs have unique properties such as photostability, broad absorption and narrow emission spectra and are available in a range of emission wavelengths including far red. They also have much higher quantum efficiencies than traditional organic fluorophores thus increasing the possible dynamic range for on-chip detection. Some of the QDots used have the added advantage of labelling intact cells and being retained and distributed among daughter cells at division, allowing their detection for up to 6 generations. The use of these QDs off-chip has suggested that they are ideal for live cell, nonperturbing labelling of division events, whereby over time the QD signal becomes diluted with each generation. Here we describe the use of quantum dots as live cell tracers for proliferating populations and the potential applications in drug screening and optical biochip environments.

We demonstrate complete integration of a fluorescence-based assay in that the analyte well is also an optical emitter. Laser machining is used to create 'active micro-wells' within semiconductor light emitting diode and laser structures. These are then used to optically excite fluorescently-labelled beads in solution within the well. The results show efficient illumination on a par with traditional lamp-based excitation. This technology therefore provides active microwell plates with completely localized excitation, confined to the analysis well, that can be engineered via the micro-well geometry. The micro-wells have also been machined within the cavity of lasing semiconductor structures and coherent emission maintained. Thus lasing multi-well plates are also realizable.

It is difficult to introduce a specific amount of a substance into cells by existing injection methods because there is no appropriate method of directly measuring the quantity of the injected substance. Although radioisotopes can be used, there is currently no apparatus that can practically handle such radioisotopes. The measurement of the diameter of a liquid droplet in air or oil is affected by surface tension if the liquid droplet is very small; but this issue does not occur with microinjection, in which a water solution is discharged under pressure through a capillary and into a cell. It is also difficult to measure the density or mass of the injected substance because of the low discharge rate, unlike the case of inkjet printers. To solve these problems, we propose a method of precise microinjection by summation of fluorescence intensity. In addition, we developed a new pressure pulse injection device that generates pressure with a rectangular waveform and a precise amplitude and pulse width to improve controllability of the discharge amount. Lastly, when the above device and method are combined, the coefficient of correlation between the specified number of pressure pulses per unit of time and the actual discharge amount exceeded 0.999. This research paper describes in detail the measurement system, standalone performance, and quantities of substances introduced into living cells.

Using a single phase-only spatial light modulator (SLM), we present a compact GPC-based optical trapping system for interactively manipulating microscopic particles in three dimensions (3D) and in real-time. We employ only one GPC 4f setup, which transforms 2D phase into intensity patterns, and utilize the SLM to form two phase-encoding regions defined by two equally sized apertures - one centered at x = x₀ and the other at x = -x₀ (with the optical axis centered at x = 0). Reconfigurable intensity patterns associated with the two independently addressable SLM-apertures are relayed to the sample volume to form a dynamic array of counterpropagating-beam traps. We discuss the experimental demonstrations showing 3D trapping of microparticles using the presented optical setup.

There has been considerable current interest in rotational behavior of red blood cells (RBC) in optical tweezers. However, the mechanism of rotation in polarized tweezers is still not well understood and there exists conflicts in the understanding of this phenomenon. Therefore, we re-examined the underlying phenomenon by use of confocal fluorescence microscopy. Under different osmolarities of the buffer, the three dimensionally reconstructed images showed that the trapped RBC maintains its discotic shape and is oriented in vertical direction. Using dual optical tweezers, the RBC could also be oriented three-dimensionally in a controlled manner. Since, no folding of the RBC was observed under optical trapping beam, the rotational mechanism based on optical birefringence caused by folding of RBC can be ruled out. The alignment of RBC with polarization of the tweezers beam can be attributed to its formbirefringence. We also present the mechanism for possible rotational behavior of RBC in circularly polarized beam.

The investigation of mechanical properties of living biological cells and biomaterials is challenging because they are inhomogeneous and anisotropic at microscopic scales, and often time-dependent over a broad time scale. Through three case studies of biomaterials and living cells, we demonstrate that a novel, oscillating optical tweezer-based imaging microrheometer developed recently in our laboratory has overcome many technical barriers posed by the complexity of biological systems. In this paper, we present the working principle, system setup and calibration of the imaging microrheometer, and report the groundbreaking results of the three applications: gelation dynamics of cross-linkable hyaluronan acid (HA) hydrogels; Mechanical in-homogeneity and anisotropy in purified microtubule networks; and effects of drug treatment and temperature variation on the mechanical properties of in vitro human alveolar epithelial cells. In each case, micro beads inserted in the materials, or attached to the cell membrane were used as probes for optical trapping. The probe particle was set into a forced harmonic oscillation by oscillating optical tweezers. Position sensing optics and phase lock-in signal processing allow the determination of the amplitude and phase shift of the particle motion at high sensitivity. The complex mechanical modulus G^* is then calculated from the amplitude and the phase shift. The rheometer system is capable of measuring dynamic local mechanical moduli in the broad frequency range of 1.3-1000 Hz at a sampling rate of 2 data point per second across a wide dynamic range (1~20,000 dyne/cm²). Integration of the rheometer system with spinning disk confocal microscopy enables the study of micromechanical properties and the microstructure of the sample simultaneously. Combination of dual-axis, piezo-electric activated mirror and 2-D position sensing detector gives the rheometer system the capability of investigating mechanical anisotropy in highly structured biological samples.

In this paper, we will report our recent development of a new type of OptoFluidic Microscope (OFM) that is capable of delivering resolution beyond the diffraction limit of light. Accurate control of the sample translation is accomplished by adopting an optical tweezer scanner into the system. During the image acquisition, a two-dimensional nanoaperture array defined on a thin aluminum film acts as an array of ultra-fine illumination sources. The imaging system is tested and demonstrated by using polystyrene beads and green algae (Chlamydomonas). Properties of the system are reported and discussed.

We report using a fast-shuttering CCD camera to determine the transverse stiffness of an optical tweezers trap. By utilizing the relation between the particle position variance and the trap stiffness we are able to circumvent the sampling requirements of the position power spectrum approach. We find that for trap stiffness of order 10^-5 N/m exposures shorter than 0.001 sec seem to be required in order to give valid measurements. For longer exposures (~0.01 sec) the trap stiffness can be overestimated by a factor of three or more.

We present a cost-effective and power efficient approach for on-chip large-scale trapping and sorting of particles in microchamber. Based on the Talbot self-imaging effect in Fresnel region, we make use of a 2D chessboard structure to create a 3D interconnected optical lattice near the emergent surface of the element without adopting an external optical projection configuration. The chessboard structure is designed to be a binary phase grating and fabricated with electron-beam lithography. As no focusing lens projections system is employed, the presented system enables a larger working area without sacrificing the advantage of high resolution. Theoretically the created optical lattice allows exponential size selectivity for particles sorting. We have experimentally demonstrated simultaneous trapping of hundreds of microparticles in a large regular array. Furthermore, in microfluidic chamber we proved the all-optical continuous separation of microparticles with different sizes.

Trapping of microscopic objects using fiber optical traps is gaining considerable interest since it has the potential to manipulate objects inside turbid medium such as tissue, thus removing the limitation of short working distance of the conventional optical tweezers based on high numerical aperture microscope objective. Here, we show that scattering force of an output beam from a single fiber can be reduced as compared to the axial gradient force when an axicon is built on the tip of the fiber, thus enabling single beam fiber-optic tweezers. Trapping of wide range of objects in size range of few hundreds of nanometers to tens of micrometers could thus be achieved. This fiber optic tweezers could be easily maneuvered in all three directions by moving the mechanical manipulator holding the axicon tip fiber. Further, chain of upto 40 particles could be trapped along the axial direction, which can be attributed as longitudinal optical binding where each trapped object acts as lens to trap subsequent object near its focal point. Apart from miniaturization capability, axicon tipped optical fiber can be used in multi-functional mode for cellular manipulation, as well as for two-photon fluorescence excitation for biomedical diagnosis.

Numerous investigations in the last years focused on chromosome and gene arrangements through the application of statistical methods that analyze the non randomness of spatial distributions of fluorescence in situ hybridization (FISH) labeled nucleic acid sequences in terms of their distance to the nuclear centers and their proximity to each other. However, existing imaging processing methods are rather limited in extracting sufficient number of nuclei with FISH label sequences, and manual analysis is unreasonably time-consuming and subjective. This paper presents an automated system that integrates a series of advanced image processing methods to over come this rate-limiting step. Evaluation results show that the proposed method is efficient, robust, and effective in extracting individual nuclei with FISH labels.

We describe a simple fluorescence microscope based on wide-field two-photon excitation. While still taking advantage of some inherent properties of non-linear (two-photon) microscopy, such as increased penetration depth through tissue and reduced phototoxicity, this approach provides video frame rate imaging, can be easily coupled to fluorescence spectral and lifetime detection modules, and makes efficient use of the high average power currently available from ultrashort pulsed lasers. For a standard histopathology specimen, we were able to identify different structures based on spectral and fluorescence lifetime detection and analysis. We examined the use of 200fs and 2ps pulses from Spectra Physics MaiTai and Tsunami lasers, respectively, with average power ranging from 50mW to 500mW.

One the major difficulties of microarray technology relate to the processing of large and - importantly - error-loaded images of the dots on the chip surface. Whatever the source of these errors, those obtained in the first stage of data acquisition - segmentation - are passed down to the subsequent processes, with deleterious results. As it has been demonstrated recently that biological systems have evolved algorithms that are mathematically efficient, this contribution attempts to test an algorithm that mimics a bacterial-"patented" algorithm for the search of available space and nutrients to find, "zero-in" and eventually delimitate the features existent on the microarray surface.

Imaging of small biological specimens and microorganisms that are living and moving is often hampered by a traditional microscope's small field of view at high resolution. This paper discusses a new optical microscope design, called the Adaptive Scanning Optical Microscope (ASOM), which uses a deformable mirror combined with a custom scanner lens to effectively enlarge the field of view. Using a high speed scanning mirror in a post-objective configuration, the ASOM captures a complete image (not a single point) at each scan position and assembles image mosaics on the fly. Consequently, this microscope offers advantages when compared to moving stage based approaches or confocal microscopes. Whereas previous work on imaging motile organisms has primarily focused on tracking only one temporally challenging specimen at a time within a single field of view, this microscope is well suited for tracking multiple moving organisms or monitoring larger organisms at both the full animal and single cell levels simultaneously. In studies requiring manipulation, probing, or sensing, the ability of the microscope to automatically monitor several regions of the specimen without agitating the workspace is particularly advantageous. Using a low cost prototype of the ASOM, we illustrate the basic capabilities of the instrument by imaging multiple living and freely moving Caenorhabditis elegans nematode worms. In addition to transmitted, reflected, and epifluorescent illumination modes, we have also integrated an LED light source that can be rapidly turned on and off in synchronization with the scanning to minimize unnecessary light exposure to the specimens.

Intensity modulation was introduced to total internal reflection fluorescence microscopy (TIRFM). This permits to obtain information about the axial distribution of fluorescent dyes in close vicinity to the surface of adherent cells and allows the application of (commercial) objective based TIRFM systems at a fixed angle of illumination. However, this new method seems to be less suitable for quantitative measurements of cell-substrate topology as compared to variableangle prism based TIRFM.

In this article, we will present a novel differential interference contrast (DIC) phase imaging device based on Young's interference. It is mainly based on either two or four nano apertures defined in an optically opaque aluminum film on a CMOS imaging sensor chip. It provides linear and disentangled differential phase and intensity images simultaneously. Furthermore, it's simple, free of bulky optical elements and compatible to the planar micro fabrication process. All of these features make it a promising device for the on-chip high resolution DIC phase imaging and beam profiling. The fabrication and operation of the device is explained in details. The performance is evaluated theoretically and is verified experimentally by examining the phase and intensity profile of a Gaussian beam and an optical vortex. The 2D quantitive differential phase distribution of an optical vortex has been recorded directly by our device with 1μm resolution.

The design of confocal Raman microscope, which achieves efficient temporal discrimination of Raman signal against fluorescence, is proposed and experimentally realized using TiO₂ as a material for Kerr-gating. The new instrument is tested for the variety of biological systems. The possible applications are outlined together with the routes for further improvement.

The recent emergence of bright, inexpensive colored LEDs offers several advantages over traditional light sources, including reduced size and increased portability, low power consumption and heat production, increased durability and longer life, and high temporal resolution. We assembled a modular array of different Phillips LUMILED LUXEON LEDs, white and seven colors with peak wavelengths between 450 and 640 nm and bandwidths of 20-30 nm. LED illumination was fiber-optically coupled to the transmitted light path of an inverted microscope, and digital images of sectioned human tissue stained with absorbing dyes were acquired using combinations of the white and color LEDs. The LED array was also coupled to an endoscope and used to image human and mouse tissue in situ. Image contrast was assessed (1) qualitatively by looking down the microscope and by viewing the digital images, and (2) quantitatively by using entropy analysis in the real and frequency domains to assess the dependence of contrast enhancement on spatial frequency. Contrast in image features of a given color range was enhanced by LEDs conjugate to that color, whereas LED colors spanning a wider range enhanced contrast in the entire image, with white LEDs often maximizing contrast of tissue. This analysis demonstrates the utility of LED illumination in modulating contrast in light microscopy and endoscopy, which may facilitate spectral segmentation and classification of image features.

Microscopy of fluorescently labeled proteins has become a standard technique for live cell imaging. However, it is still a challenge to systematically extract quantitative data from large sets of images in an unbiased fashion, which is particularly important in high-throughput or time-lapse studies. Here we describe the development of a software package aimed at automatic quantification of abundance and spatio-temporal dynamics of fluorescently tagged proteins in vivo in the budding yeast Saccharomyces cerevisiae, one of the most important model organisms in proteomics. The image analysis methodology is based on first identifying cell contours from bright field images, and then use this information to measure and statistically analyse protein abundance in specific cellular domains from the corresponding fluorescence images. The applicability of the procedure is exemplified for two nuclear localized GFP-tagged proteins, Mcm4p and Nrm1p.

We present the design, characterization and application of a novel, rapid, optically sectioned hyperspectral fluorescence lifetime imaging (FLIM) microscope. The system is based on a line scanning confocal configuration and uses a highspeed time-gated detector to extract lifetime information from many pixels in parallel. This allows the full spectraltemporal profiles of a fluorescence decay to be obtained from every pixel in an image. Line illumination and slit detection also gives the microscope a confocal optical sectioning ability. The system is applied to test samples and unstained biological tissue. In future, this microscope will be combined with recently-developed continuously electronically tunable, pulsed light sources based on tapered, micro-structured optical fibers. This will allow hyperspectral FLIM to be combined with the advantages of excitation spectroscopy to gain further insight into complex biological specimens including tissue and live cell imaging.

We describe a new DIC technique, which records phase gradients within microscopic specimens independently of their orientation. The proposed system allows the generation of images representing the distribution of dry mass (optical path difference) in the specimen. Unlike in other forms of interference microscopes, this approach does not require a narrow illuminating cone. The orientation-independent differential interference contrast (OI-DIC) system can also be combined with orientation-independent polarization (OI-Pol) measurements to yield two complementary images: one showing dry mass distribution (which is proportional to refractive index) and the other showing distribution of birefringence (due to structural or internal anisotropy). With a model specimen used for this work -- living spermatocytes from the crane fly, Nephrotoma suturalis --- the OI-DIC image clearly reveals the detailed shape of the chromosomes while the polarization image quantitatively depicts the distribution of the birefringent microtubules in the spindle, both without any need for staining or other modifications of the cell. We present examples of a pseudo-color combined image incorporating both orientation-independent DIC and polarization images of a spermatocyte at diakinesis and metaphase of meiosis I. Those images provide clear evidence that the proposed technique can reveal fine architecture and molecular organization in live cells without perturbation associated with staining or fluorescent labeling. The phase image was obtained using optics having a numerical aperture 1.4, thus achieving a level of resolution never before achieved with any interference microscope.

Phosphorylation of the chromatin protein H2AX (forming γH2AX) is implicated in the repair of DNA double strand breaks (DSB's); a large number of H2AX molecules become phosphorylated at the sites of DSB's. Fluorescent staining of the cell nuclei for γH2AX, via an antibody, visualises the formation of these foci, allowing the quantification of DNA DSB's and forming the basis for a sensitive biological dosimeter of ionising radiation. We describe an automated fluorescence microscopy system, including automated image processing, to count γH2AX foci. The image processing is performed by a Hough transform based algorithm, CHARM, which has wide applicability for the detection and analysis of cells and cell colonies. This algorithm and its applications for cell nucleus and foci detection will be described. The system also relies heavily on robust control software, written using multi-threaded cbased modules in LabWindows/CVI that adapt to the timing requirements of a particular experiment for optimised slide/plate scanning and mosaicing, making use of modern multi-core processors. The system forms the basis of a general purpose high-content screening platform with wide ranging applications in live and fixed cell imaging and tissue micro arrays, that in future, can incorporate spectrally and time-resolved information.

Multi-spectral imaging provides digital images of a scene or object at a large, usually sequential number of wavelengths, generating precise optical spectra at every pixel. We use the term "spectral signature" for a quantitative plot of optical property variations as a function of wavelengths. We present here intelligent spectral signature bio-imaging methods we developed, including automatic signature selection based on machine learning algorithms and database search-based automatic color allocations, and selected visualization schemes matching these approaches. Using this intelligent spectral signature bio-imaging method, we could discriminate normal and aganglionic colon tissue of the Hirschsprung's disease mouse model with over 95% sensitivity and specificity in various similarity measure methods and various anatomic organs such as parathyroid gland, thyroid gland and pre-tracheal fat in dissected neck of the rat in vivo.

Microscopic objects can be manipulated in a more complex and effective way by use of static aberrated tweezers. We theoretically studied dynamics of interaction of Rayleigh particles with such asymmetric tweezers. Microscopic objects are pulled at the high intensity gradient end of the asymmetric tweezers, get accelerated and are ejected from the other end. Thus objects from two locations could be transported by two asymmetric line tweezers and mixed at another place where the low intensity gradient regions of the two beams meet. It is pertinent to note here that the speed of transport is determined by the laser beam power and the degree of asymmetry in the intensity profile. And since for a fixed asymmetry and laser power, the speed is dependent on the refractive index and size of the objects, sorting of these objects could be made possible. Sorting could be achieved either by scanning the asymmetric line tweezers across the sample or without scanning the stage by use of two asymmetric line tweezers. In the all-optical approach (use of two asymmetric line tweezers), we exploited the fact that when the speed of objects transported by one tweezers crossed a threshold value, these did not interact with the second tweezers and therefore moved undeviated; whereas slower moving objects were collected by the second tweezers and transported to a well-separated location.

In fluorescence imaging studies of biological mechanisms, cyanine dyes have been employed as fluorescent labels. In particular, tricarbocyanines have the advantage that light at their emission and absorption maxima in the near-infrared (NIR) region around 650-900 nm can penetrate deeply into tissues. We successfully developed two types of cyanine dyes whose fluorescence properties change upon specific reaction with nitric oxide (NO) or zinc ion. The mechanism of fluorescence modulation of the NO probes involves photoinduced electron transfer, and the fluorescent intensity can change at the same wavelengths. We synthesized a series of amine-substituted tricarbocyanines in order to examine the correlation between the electron-donating ability of the amine and the fluorescence peak wavelength. We found that changing the electron-donating ability of the amine substituent altered the absorption and emission wavelengths. Then, we synthesized dipicolylcyanine (DIPCY), consisting of tricarbocyanine as a fluorophore and dipicolylethylenediamine as a heavy metal chelator, and investigated its response to various heavy metal ions. DIPCY can work as a ratiometric fluorescent sensor for zinc ion. This fluorescence modulation of amine-substituted tricarbocyanines should be applicable to dual-wavelength measurement of various biomolecules or enzyme activities. Thus, we have established two mechanisms for modulating the fluorescence properties of cyanines.

Lucilia cuprina pericardial cells are primarily involved in the filtration of hemolymph. Ratio images using fluorescent pH indicator, DM-Nerf, were collected using a confocal microscope. The results support suggestions that there is zonation of cellular activity that reflect organelle distribution. Statistical analysis of the excitation ratios indicate significant spatial differences in pH of the three major zones- cortex, vacuole zone and endoplasm in pericardial cells. The outer cortex was estimated to have a pH between 5.5 and 6.8, the vacuole zone between 4.5 and 5.5 and the endoplasm between 4.0 and 5.0.

Chemotaxis is the mechanism microorganisms use to sense the environment surrounding them and to direct their movement towards attractive, or away from the repellent, chemicals. The biochemical sensing is almost the only way for communication between unicellular organisms. Prokaryote and Eukaryote chemotaxis has been mechanically studied mainly by observing the directionality and timing of the microorganisms movements subjected to a chemical gradient, but not through the directionality and strength of the forces it generates. To observe the vector force of microorganisms under a chemical gradient we developed a system composed of two large chambers connected by a tiny duct capable to keep the chemical gradient constant for more than ten hours. We also used the displacements of a microsphere trapped in an Optical Tweezers as the force transducer to measure the direction and the strength of the propulsion forces of flagellum of the microorganism under several gradient conditions. A 9μm diameter microsphere particle was trapped with a Nd:YAG laser and its movement was measured through the light scattered focused on a quadrant detector. We observed the behavior of the protozoa Leishmania amazonensis (eukaryote) under several glucose gradients. This protozoa senses the gradient around it by swimming in circles for three to five times following by tumbling, and not by the typical straight swimming/tumbling of bacteria. Our results also suggest that force direction and strength are also used to control its movement, not only the timing of swimming/tumbling, because we observed a higher force strength clearly directed towards the glucose gradient.

Oleic acid (OA)-Pluronic-coated iron oxide nanoparticles were synthesized and characterized by Fourier Transform Infrared Spectroscopy (FT-IR) and Atomic Force Microscopy (AFM). FT-IR confirmed the bonding of oleic acid and Pluronic (surfactant) to the nanoparticles. AFM measurements on these nanoparticles indicated a root mean square (RMS) roughness, a measure of nanoparticle size of (50 ± 20) nm. The efficiency of these functionalized nanoparticles was investigated by loading with 5-Fluorouracil (5-FU) in aqueous solution. AFM measurements were used to characterize modified iron oxide nanoparticles and pancreatic MIA PaCa-2 cells, including size distribution, stability and cellular uptake. Nanoparticles were added to MIA PaCa-2 cells and assayed for their cytotoxic effects after 24 and 48 hours. The outcome of this study demonstrated the effectiveness of oleic acid (OA)-Pluronic-coated iron oxide nanoparticles as a non-toxic drug delivery agent for pancreatic cancer.

Second harmonic generation (SHG) is a well-known physical phenomenon of important in nonlinear optics, which has been widely used in tissue morphology and pathology. In this thesis, the influences on backward SHG intensity were studied. A two-photon laser scanning confocal microscope coupled with a mode-locked femtosecond Ti: sapphire laser was used to carry out the experiments. Rat-tails were used as experimental objects, there were 40 slices including transverse and longitudinal. All slices were un-dyed. The changes of backward SHG intensities of rat-tails versus the excited wavelengths, excited powers, exploring-depths and so on were obtained. It was clear that different irradiation conditions had evident influences on backward SHG intensity. Subsequently, the backward SHG imaging and two-photon excited fluorescence (TPEF) imaging through two independent channels were also compared preliminarily. This study demonstrates that SHG is a promising tool for more accurate information in biomedicine fields.

Gerbera hybrida (Shenzhen No.5) seedlings' inflorescence development was divided into six stages (P1-P6). With these six stages petal, delayed luminescence (DL) were observed during petal development using lab-made detector system, fluorescence spectrum and confocal imaging were also observed. The results showed that the intensity of DL were increased during P1-P4 and decreased in P5 and P6; with the excitation wavelength of 488 nm, fluorescence spectra were obviously different during P1-P6 stages; imaging of chlorophyll autofluorescence by confocal laser scanning microscopy (CLSM) showed that the intensity were stronger in P3 than in P1, while P6 stage autofluorescence only displayed in guard cell of epidermis. Our results suggested that the DL technique, combining with fluorescence spectra and CLSM imaging, might be useful for the rapid and noninvasive evaluation of chlorophyll content and degradation in petal development in Gerbera hybrida.

A liquid crystal based biosensor for the detection and diagnosis of sepsis is currently in development. Sepsis, a major clinical syndrome with a significant public health burden in the US due to a large elderly population, is the systemic response of the body to a localized infection and is defined as the combination of pathologic infection and physiological changes. Bacterial infections are responsible for 90% of cases of sepsis in the US. Currently there is no bedside diagnostic available to positively identify sepsis. The basic detection scheme employed in a liquid crystal biosensor contains attributes that would find value in a clinical setting, especially for the early detection of sepsis. Utilizing the unique properties of liquid crystals, such as birefringence, a bedside diagnostic is in development which will optically report the presence of biomolecules. In a septic patient, an endotoxin known as lipopolysaccharide (LPS) is released from the outer membrane of Gram-negative bacteria and can be found in the blood stream. It is hypothesized that this long chained molecule will cause local disruptions to the open surface of a sensor containing aligned liquid crystal. The bulk liquid crystal ampli.es these local changes at the surface due to the presence of the sepsis marker, providing an optical readout through polarizing microscopy images. Liquid crystal sensors consisting of both square and circular grids, 100-200 μm in size, have been fabricated and filled with a common liquid crystal material, 5CB. Homeotropic alignment was confirmed using polarizing microscopy. The grids were then contacted with either saline only (control), or saline with varying concentrations of LPS. Changes in the con.guration of the nematic director of the liquid crystal were observed through the range of concentrations tested (5mg/mL - 1pg/mL) which have been confirmed by a consulting physician as clinically relevant levels.