We characterised populations of wild type baking and brewing yeast cells using intrinsic fluorescence and fluorescence lifetime microscopy, in order to obtain quantitative identifiers of different strains. The cell autofluorescence was excited at 405 nm and observed within 440-540 nm range where strong cell to cell variability was observed. The images were analyzed using customised public domain software, which provided information on cell size, intensity and texture-related features. In light of significant diversity of the data, statistical methods were utilized to assess the validity of the proposed quantitative identifiers for strain differentiation. The Kolmogorov-Smirnov test was applied to confirm that empirical distribution functions for size, intensity and entropy for different strains were statistically different. These characteristics were followed with culture age of 24, 48 and 72 h, (the latter corresponding to a stationary growth phase) and size, and to some extent entropy, were found to be independent of age. The fluorescence intensity presented a distinctive evolution with age, different for each of the examined strains. The lifetime analysis revealed a short decay time component of 1.4 ns and a second, longer one with the average value of 3.5 ns and a broad distribution. High variability of lifetime values within cells was observed however a lifetime texture feature in the studied strains was statistically different.

Total internal reflection fluorescence microscopy (TIRFM) induces the evanescent field from an incident light with an incident angle greater than the critical angle selectively to excite fluorescent molecules on or near a surface. The TIRFM not only provides enhanced understanding of cellular function but also improves signal-to-noise ratio of detecting signal in real time. However, fluorescent emission need to be increased when a dynamic biomolecular image is requested at the frame rate of greater than 100 frames/s. Therefore, the fluorescent signal is enhanced via surface plasmons to match the requirements of better efficiency and larger quantity. In this study, a plasmon-enhanced TIRFM whose operation is based on the electromagnetic field enhancement via surface and particle plasmon effects offered by a nano-scalar silver thin film and particles has been presented. The developed microscopy has been successfully used in the real-time observation of the enhanced fluorescence from the thrombomodulin protein of a living cell membrane. The simulated and experimental results demonstrate that the plasmon-enhanced TIRFM can provide brighter living cell images through surface plasmon enhanced fluorescence.

To study cellular morphology and functions in vivo in realtime, we developed a fiber-coupled confocal microscope (FCM), and observed fluorescently-labeled cells inside the body of anesthetized rat. We developed an imaging fiber bundle (IFB), which consisted of an objective lens and a multi-fiber assembly (unit fiber: NA > 0.4, 3 micron in diameter). By combining the IFB with a real-time confocal scanner, we detected intracellular signals of the molecular messenger, and the death signals in the form of fluorescence changes even from cells located deep (> 2 mm) inside the solid organs. The FCM we developed is very promising for detailed studies in both the cell-based researches and clinical researches.

Combining light scatting spectroscopy (LSS) and spectroscopic optical coherence tomography (LS-SOCT) can provide a wealth of information. A theoretical model for analyzing the scattering in SOCT is proposed based on plane wave decomposition. Based on the model, we discuss the possibility of matching the physical characteristics of scatterers with observed spectroscopic signals. Many complicating factors are considered including the effects of scatterer size, incident light polarization, interference between the fields scattered from closely adjacent scatterers, and the numerical aperture of the OCT system. We found the modulation of the spectrum of the incident light by scattering of a plane wave from a single sphere is a good indicator of particle size and composition. We demonstrate that measuring wavelength-dependent scattering in SOCT can be used for particle sizing and contrast enhancement by differentiating cells in 3D cell culture.

Hyperspectral imaging provides complex image data with spectral information from many fluorescent species contained within the sample such as the fluorescent labels and cellular or pigment autofluorescence. To maximize the utility of this spectral imaging technique it is necessary to couple hyperspectral imaging with sophisticated multivariate analysis methods to extract meaningful relationships from the overlapped spectra. Many commonly employed multivariate analysis techniques require the identity of the emission spectra of each component to be known or pure component pixels within the image, a condition rarely met in biological samples. Multivariate curve resolution (MCR) has proven extremely useful for analyzing hyperspectral and multispectral images of biological specimens because it can operate with little or no a priori information about the emitting species, making it appropriate for interrogating samples containing autofluorescence and unanticipated contaminating fluorescence. To demonstrate the unique ability of our hyperspectral imaging system coupled with MCR analysis techniques we will analyze hyperspectral images of four-color in-situ hybridized rat brain tissue containing 455 spectral pixels from 550 - 850 nm. Even though there were only four colors imparted onto the tissue in this case, analysis revealed seven fluorescent species, including contributions from cellular autofluorescence and the tissue mounting media. Spectral image analysis will be presented along with a detailed discussion of the origin of the fluorescence and specific illustrations of the adverse effects of ignoring these additional fluorescent species in a traditional microscopy experiment and a hyperspectral imaging system.

Spectral imaging has recently been introduced in the biomedical field as a noninvasive, quantitative means of studying biological tissues. Many of its potential applications have been demonstrated (in vitro and, to a lesser degree, in vivo) with the use of stains or dyes. Successful translation to the clinical environment has been largely lagging, due to safety considerations and regulatory limitations preventing use of contrast agents in humans. We report experiments showing the feasibility of high-resolution spectral imaging of breast cancer without the use of contrast agents, thus completing the continuum of translational research, to in vivo imaging that will be directly applicable in the clinical environment. Our initial work focused on image acquisition using Fourier transform microinterferometry and subsequent segmentation of both stained and unstained breast cancer slides-derived image sets. We then applied our techniques to imaging fresh unstained ex vivo specimens of rat breast cancer and sentinel lymph nodes. We also investigated multiple methods of classification to optimize our image analyses, and preliminary results for the best algorithm tested yielded an overall sensitivity of 96%, and a specificity of 92% for cancer detection. Using spectral imaging and classification techniques, we were able to demonstrate that reliable detection of breast cancer in fixed and fresh unstained specimens of breast tissue is possible.

It is well established that hypoxia can influence tumor biology and physiology, gene expression, metastatic potential, treatment efficacy, and patient survival. Most human solid tumors have been shown to have some hypoxic regions, thus there is a strong motivation to understand the various causes of hypoxia. One key to understanding tumor hypoxia involves the study of oxygen transport to tumors, and the connection between hypoxia, tumor microvasculature, and the tumor microenvironment. Recent research has suggested that the causes of tumor hypoxia are much more complex than indicated by the classical paradigms ("chronic" and "acute" hypoxia), and several potential factors have been identified. Two such factors are temporal fluctuations in tissue pO₂ and longitudinal gradients in oxygen transport. Research has shown the existence of low frequency (<2 cycles per minute) fluctuations in tumor pO₂ without cessation of blood flow that can lead to transient hypoxia. In addition, longitudinal gradients in tumor pO₂ along the arteriolar afferent direction have been documented in window chamber tumors. However, the causes of the pO₂ temporal fluctuations and longitudinal gradients are not exactly known, and the clinical significance of these observations is not well understood. In this preliminary study, we demonstrate the potential of optical imaging measurements of hemoglobin saturation to add new information in these areas. Slow temporal fluctuations of hemoglobin saturation (HbSat) and gradients in the average HbSat were observed in some 4T1 mouse mammary carcinoma microvessels. With additional research, the mechanisms behind these phenomena and insights into their clinical significance may be revealed.

The protein substructure of skeletal muscle fibers forms a diffraction grating with repeating units, termed 'sarcomeres'. A laser scanning system is described that maps the lengths of sarcomeres (SL) and the widths of the first-order diffraction lines (DLW) of permeabilized single fibers in real-time. The apparatus translates a laser beam (λ = 670 nm and w₀ = ~75 μm) along the length of a fiber segment through 20 contiguous regions per sweep at 500 sweeps/s. The fiber segments (~1 mm long) were obtained from vastus lateralis muscles of humans by needle biopsy. During both passive stretches and maximum fixed-end activations, the mappings of SL and DLW of the fibers were extracted from the diffraction spectra. Heterogeneity of SLs was evaluated by computing the standard deviation ( σ_SL) of the 20 SLs measured during a single sweep. Compared with the σ_SL before a passive stretch, the increase of 5±0.5% in σ_SL after the passive stretch, indicated differences in passive length-tension relationships along the fiber. In contrast, no change, ~0.5±0.1%, was observed in DLW. Within 10s after the fiber was returned to its initial length, the shape of the SL profile returned close to pre-stretch conditions ( σ_SL = 1± 0.2%). Following maximum Ca²⁺ - activation of the fiber, the heterogeneity of the steady state SLs increased greatly (DLW up by ~300% and σ_SL up by ~100%). The scanning system provided high resolution tracking of sarcomere behavior single muscle fibers. Potential applications are for studies of the mechanisms of muscle fiber injury and injury propagation.

Spectral changes of lung cancer serum in the process of tumor evolution was investigated in this study. We kept close watch on the tumor progression of a group of patients, and measured their serum spectra using 488.0nm and 514.5nm excitation of an Ar-ion laser once a week. There was no apparent change observed in fluorescence spectrum in different period. However, the relative intensity of three Raman peaks (mode A, B and C) decreased every week later. For quantitative analysis of such changes, a parameter Ir (relative intensity of C Raman peak) was introduced and Ir-value was calculated. Calculation showed that Ir-value was degressive with tumor evolution, but β (Ir5145 /Ir4880) varied irregularly. To the end, no Raman peak was observed. We assumed that three Raman peaks were derived from beta carotene. It indicated that the content of beta carotene decreased with the aggravation of lung cancer.

Fluorescence Resonance Energy Transfer (FRET) - a process of nonradiative energy transfer from an optically excited molecule (donor, D) to an unexcited nearby molecule (acceptor, A) - is a powerful tool in studies of protein-protein interactions in living cells. FRET can be quantified, e.g., by measuring an increase in the donor fluorescence after inactivating the acceptor through photobleaching. In spite of its sheer simplicity, this method also introduces donor bleaching, which often complicates the interpretation of data. Correction methods are complicated by the fact that D photobleaching depends on whether a D molecule is free or coupled to an A molecule. In this communication we show that, instead of being a nuisance, donor bleaching actually can be harnessed to provide invaluable information about a population of interacting proteins in vivo. We present data on proteins tagged with fluorescent molecules, which indicate that donor and acceptor bleaching kinetics reveal quantitative information on the stoichiometry of proteinprotein interaction. Under appropriately chosen conditions, we are able to model the bleaching kinetics of D and A (both interacting and noninteracting) and determine the stoichiometry of the protein interaction. This provides a method for imaging protein complexes in living cells, which opens a way for testing the law of mass action in vivo.

Atypical lesions of the breast have potential to turn malignant. The diagnosis of these lesions has increased considerably with screening mammography. A good understanding of their progression to invasive cancer is yet to be proved. Using Raman spectroscopy to study their chemical finger printing at different stages of proliferation a clear picture of whether a progression exists between lesions could be made. At present there is no clear recognition of the biochemical changes that distinguish between the different proliferative lesions of the breast. Our aim is to understand these changes through Raman mapping studies. Raman spectroscopy is a highly sensitive and specific technique for demonstration of biochemical changes in different atypical proliferative lesions of the breast. The technique could be used to classify the different grades and analyse progression of pathology in the proliferative lesions of the breast. Breast pathologists carefully marked 50 ducts and classified the different pathology on H and E sections from biopsy samples. Raman spectra were measured, using a Renishaw Raman Spectrometer, on a 20-micron thick consecutive frozen section. Principal component analysis was undertaken using Matlab. Pseudocolor maps of the principal components scores have been generated. The peaks of the corresponding loads were identified enabling visualisation of the biochemical changes associated with proliferative lesions. Proliferative lesions of the duct were grouped according to the existing standard pathological classification and formed four major groups-HUT, ADH, DCIS and IDC. Spectra of biochemical constituents were fitted to mean spectra from selected regions, taken from maps of each pathology, to identify the relative concentration of the constituents. The study gave an insight into chemical make up of the ducts in each pathology group and showed similar results to earlier studies in progression but no clear-cut demarcation or continuum of the proliferative disease.

Structural and compositional analysis of normal and pathological tissues by OCT often is performed ex vivo and subsequently compared to the histology. Many of the tissues of interest require immediate fixation to prevent degradation of the sample. Frequently, samples are obtained up to a week prior to procuring images by OCT. We investigated whether fixation affects OCT image analysis by acquiring images of freshly isolated bovine ligament samples and repeating OCT imaging of the same area after fixation at 24 hours and at one week. Samples were divided into two groups: group one was fixed in 10% neutral buffered formalin for 24 hours and placed in normal saline while group two remained in formalin for one week. Tissue samples were processed for paraffin embedment and stained with Masson's trichrome or with picrosirius red. The banding pattern contrast ratio of the OCT images before and after fixation for both groups was measured and compared for possible differences. Histology was evaluated for tissue integrity and compared to the OCT images. The mean contrast ratio at time 0 was 5.41 ± 1.1 and 5.31 ± 0.6 for groups 1 and 2, respectively. Results at 1 week were slightly lower with 5.11 ± 0.3 and 5.20 ± 0.7, respectively. Statistical analysis of the data by ANOVA showed no difference in the contrast ratios with time or with treatment. This data indicates that 24 hours in formalin is sufficient to fix these small ligament samples with little effect on imaging up to one week after fixation.

We present a phase-shifting holographic set-up for the microscopic imaging of adherent cells. The superposition of an object wave field and a reference wave is recorded on a digital sensor with three reference wave phases. The reference phases are then recovered by statistical analysis of the recorded intensities. Subsequently, the object wave phase is calculated by the generalized phase shifting algorithm. After phase unwrapping and background subtraction, the phase shift introduced by the adherent cell culture is reconstructed. As the interferograms are recorded in the image plane of the microsope objective, the full lateral resolution is achieved in contrast to off-axis holography where the reconstruction requires numerical propagation for the separation of 0^th and 1^st order. Our approach uses three arbitrary unknown reference phases and poses thus minimum requirements on the mechanical and thermal stability of the set-up. We give preliminary results of images from a Vero cell line and pollen grains.

Fluorescent semiconductor nanocrystals in quantum confinement regime (quantum dots) present several well known features which make them very useful tools for biological labeling purposes. Low photo-bleaching rates, high chemical stability, active surface allowing conjugation to living cells, explains the success of this labeling procedure over the commonly used fluorescent dyes. In this paper we report the results obtained with high fluorescent core-shell CdTe-CdS (diameter = 3-7 nm) colloidal nanocrystals synthesized in aqueous medium and conjugated to glucose molecules, incubated with living yeast cells, in order to investigate their glucose up-take activity.

In situ spectral analysis can be used to understand the targeting and interaction of agents in cellular compartments. A range of novel red excitable fluorescent probes, related to the anthraquinone family of anti-cancer agents, were designed for their DNA affinic properties and their ability to enter and penetrate living cells. We report on the spectral features of these probes, both in solution and bound within intact cells, to identify unique fluorescent signatures that exploit their use in bioassays on optical biochip devices. The probes demonstrated red shifted emission spectra and increased 2 photon lifetime, with minimal fluorescent enhancement, upon binding to DNA. Spectral confocal laser scanning microscopy revealed complex emission profiles representing the bound (nuclear) and unbound (cytoplasmic) fractions of the DNA probes within live interphase, mitotic and apoptotic cells. Analysis of the emission peaks encoded the spectra to provide cell compartment recognition and profiles for cells in different cell states. Sampling the entire emission spectra of these probes for cell locating, even in the presence of unbound molecules, provides good signal-to-noise in biochip devices. Furthermore, by sampling the fluorescence output at specific spectral windows we can obtain high spatial information without imaging. The technological challenge is to integrate these fluorophores and appropriate detection capacity onto an optical biochip platform with microfluidic systems for cell handling.

Proliferation/apoptosis balance is an important information in gastrointestinal ulcerative and malignant diseases. Immunohistochemical staining and visual counting is routine procedure. Recently we reported a new scanning fluorescence technique for automated motorized microscopes (SFM). Development of triple fluorescent labeling method for proliferating/apoptotic/resting cells and application of SFM for the automated analysis and counting on gastric biopsy specimen. Routine antral biopsy specimens by gastroscopy were fresh frozen and 5 micron sections were prepared. Proliferation was detected using a PCNA antibody, anti-mouse-biotin and streptavidin-Texas-Red labeling system. Apoptotic cells was labeled using the TUNEL reaction with FITC bound nucleotids. DAPI nuclear counter staining was applied. Labeled sections were scanned and digitized in the three fluorescent channels. SFM was modified to detect epithelial surface, glands in the biopsy specimen. Automated nuclei detection, PCNA and TUNEL detection was performed, ratio was calculated. In parallel standard biopsies were labeled with PCNA and AEC. TUNEL reaction was performed. Up to 1000 epithelial cells were manually counted. The mean PCNA labeling in healthy samples were 45,3±12,4%, that significantly increased to 56.4±8.7% in H. Pylori positive cases. Positive TUNEL reaction was found in 2,9±1,1% in H. pylori negative cases, while in the H. Pylori positive cases the apoptotic ratio was significantly increased (14.1±3.2%, p<0.05). Significant correlation in apoptosis/proliferation ratio between the SFM and routine methods could be observed (p<0,05). SFM procedure proved to be more time efficient both in labeling, both in detection procedures. Triple fluorescent labeling and automated fluorescence microscopy is an applicable tool for the proliferation, apoptosis determination in fresh frozen samples.

There is a constant need for clinical diagnostic systems that enable to predict disease course for preventative medicine. Apoptosis, programmed cell death, is the end point of the cell's response to different induction and leads to changes in the cell morphology that can be rapidly detected by optical systems. We tested whether apoptosis of T-cells in the peripheral blood is useful as predictor and compared different preparation and analytical techniques. Surgical trauma is associated with elevated apoptosis of circulating leukocytes. Increased apoptosis leads to partial removal of immune competent cells and could therefore in part be responsible for reduced immune defence. Cardiovascular surgery with but not without cardiopulmonary bypass (CPB) induces transient immunosuppression. Its effect on T-cell apoptosis has not been shown yet. Flow-cytometric data of blood samples from 107 children (age 3-16 yr.) who underwent cardiac surgery with (78) or without (29) CPB were analysed. Apoptotic T-lymphocytes were detected based on light scatter and surface antigen (CD45/CD3) expression (ClinExpImmunol2000;120:454). Results were compared to staining with CD3 antibodies alone and in the absence of antibodies. T-cell apoptosis rate was comparable when detected with CD45/CD3 or CD3 alone, however not in the absence of CD3. Patients with but not without CPB surgery had elevated lymphocyte apoptosis. T-cell apoptosis increased from 0.47% (baseline) to 0.97% (1 day postoperatively). In CPB patients with complication 1.10% significantly higher (ANOVA p=0.01) comparing to CPB patients without complications. Quantitation of circulating apoptotic cells based on light scatter seems an interesting new parameter for diagnosis. Increased apoptosis of circulating lymphocytes and neutrophils further contributes to the immune suppressive response to surgery with CPB. (Support: MP, Deutsche Herzstiftung, Frankfurt, Germany)

The goal of predictive medicine is the detection of changes in patient's state prior to the clinical manifestation of the deterioration of the patients current status. Therefore, both the diagnostic of diseases like cancer, coronary atherosclerosis or congenital heart failure and the prognosis of the effect specific therapeutics on patients outcome are the main fields of predictive medicine. Clinical Cytomcs is based on the analysis of specimens from the patient by Cytomic technologies that are mainly imaging based techniques and their combinations with other assays. Predictive medicine aims at the recognition of the "fate" of each individual patients in order to yield unequivocal indications for decision making (i.e. how does the patient respond to therapy, react to medication etc.). This individualized prediction is based on the Predictive Medicine by Clinical Cytomics concept. These considerations have recently stimulated the idea of the Human Cytome Project. A major focus of the Human Cytome Project is multiplexed cy-tomic analysis of individual cells of the patient, extraction of predictive information and individual prediction that merges into individualized therapy. Although still at the beginning, Clinical Cytomics is a promising new field that may change therapy in the near future for the benefit of the patients.

Cytology automation and research will be enhanced by the creation of a common data format. This data format would provide the pathology and research communities with a uniform way for annotating and exchanging images, flow cytometry, and associated data. This specification and/or standard will include descriptions of the acquisition device, staining, the binary representations of the image and list-mode data, the measurements derived from the image and/or the list-mode data, and descriptors for clinical/pathology and research. An international, vendor-supported, non-proprietary specification will allow pathologists, researchers, and companies to develop and use image capture/analysis software, as well as list-mode analysis software, without worrying about incompatibilities between proprietary vendor formats. Presently, efforts to create specifications and/or descriptions of these formats include the Laboratory Digital Imaging Project (LDIP) Data Exchange Specification; extensions to the Digital Imaging and Communications in Medicine (DICOM); Open Microscopy Environment (OME); Flowcyt, an extension to the present Flow Cytometry Standard (FCS); and CytometryML. The feasibility of creating a common data specification for digital microscopy and flow cytometry in a manner consistent with its use for medical devices and interoperability with both hospital information and picture archiving systems has been demonstrated by the creation of the CytometryML schemas. The feasibility of creating a software system for digital microscopy has been demonstrated by the OME. CytometryML consists of schemas that describe instruments and their measurements. These instruments include digital microscopes and flow cytometers. Optical components including the instruments' excitation and emission parts are described. The description of the measurements made by these instruments includes the tagged molecule, data acquisition subsystem, and the format of the list-mode and/or image data. Many of the CytometryML data-types are based on the Digital Imaging and Communications in Medicine (DICOM). Binary files for images and list-mode data have been created and read.

In this paper, a common pulse shaping system is used to compress a femtosecond optical pulse beyond the spectrum limit. An effective optimization method is proposed to design the spectrum filter. Numerical examples are presented to illustrate the validity of the proposed method.

The high incidence and mortality rates of prostate cancer have stimulated research for prevention, early diagnosis and appropriate treatment. DNA ploidy status of tumour cells is an important parameter with diagnostic and prognostic significance. In the current study, DNA ploidy analysis was performed using image cytometry technique and digital image processing and analysis. Tissue samples from prostate patients were stained using the Feulgen method. Images were acquired using a digital imaging microscopy system consisting of an Olympus BX-50 microscope equipped with a color CCD camera. Segmentation of such images is not a trivial problem because of the uneven background, intensity variations within the nuclei and cell clustering. In this study specific algorithms were developed in Matlab based on the most prominent image segmentation approaches that emanate from the field of Mathematical Morphology, focusing on region-based watershed segmentation. First biomedical images were simplified under non-linear filtering (alternate sequential filters, levelings), and next image features such as gradient information and markers were extracted so as to lead the segmentation process. The extracted markers are used as seeds; watershed transformation was performed to the gradient of the filtered image. Image flooding was performed isotropically from the markers using hierarchical queues based on Beucher and Meyer methodology. The developed algorithms have successfully segmented the cell from its background and from cells clusters as well. To characterize the nuclei, we attempt to derive a set of effective color features. By analyzing more than 50 color features, we have found that a set of color features, hue, saturation-weighted hue, I₁=(R+G+B)/3, I₂=(R-B),I₃=(2G-R-B)/2, Karhunen-Loeve transformation and energy operator, are effective.

The paper presents results in the study of pathogen variability by using genomic signals. The conversion of symbolic nucleotide sequences into digital signals offers the possibility to apply signal processing methods to the analysis of genomic data. The method is particularly well suited to characterize small size genomic sequences, such as those found in viruses and bacteria, being a promising tool in tracking the variability of pathogens, especially in the context of developing drug resistance. The paper is based on data downloaded from GenBank [32], and comprises results on the variability of the eight segments of the influenza type A, subtype H5N1, virus genome, and of the Hemagglutinin (HA) gene, for the H1, H2, H3, H4, H5 and H16 types. Data from human and avian virus isolates are used.

Modern microscopic techniques, like high-content, high-throughput screening (HCS), may involve collection of thousands of images per experiment. Efficient image-compression techniques are indispensable to manage these vast amounts of data. Such compression may be obtained with lossy compression algorithms such as JPEG and JPEG2000. However, these algorithms are optimized to preserve visual quality but not necessarily the integrity of the scientific data. Here, we describe an observer-independent compression algorithm designed to preserve information contained in microscope images. We construct a model of noise as a function of signal in our imaging system, using the imaged specimen as the standard. The noise and signal are then calculated in the wavelet domain for each pixel of a single image. The SNR (signal-to-noise ratio) is used as a quality measure to establish which image components may be discarded. The denoised images, coded using reversible JPEG2000, require less storage space than their non-denoised counterparts. We used model images and microscope test patterns (grating arrays) to demonstrate that the proposed denoising scheme does not alter the effective microscope modulation transfer function (MTF) when used in conjunction with lossless JPEG2000. Furthermore, we confirm these findings by estimating the alterations introduced by compression of images of cell nuclei using brightness histograms (earth's mover distance algorithm) and several texture parameters. We demonstrate that the proposed denoising procedure reduces artifacts when used as a preprocessing step for irreversible JPEG2000 in model as well as in real biological images.

Prostate cancer is diagnosed by histopathology interpretation of hematoxylin and eosin (H and E)-stained tissue sections. Gland and nuclei distributions vary with the disease grade. The morphological features vary with the advance of cancer where the epithelial regions grow into the stroma. An efficient pathology slide image analysis method involved using a tissue microarray with known disease stages. Digital 24-bit RGB images were acquired for each tissue element on the slide with both 10X and 40X objectives. Initial segmentation at low magnification was accomplished using prior spectral characteristics from a training tissue set composed of four tissue clusters; namely, glands, epithelia, stroma and nuclei. The segmentation method was automated by using the training RGB values as an initial guess and iterating the averaging process 10 times to find the four cluster centers. Labels were assigned to the nearest cluster center in red-blue spectral feature space. An automatic threshold algorithm separated the glands from the tissue. A visual pseudo color representation of 60 segmented tissue microarray image was generated where white, pink, red, blue colors represent glands, epithelia, stroma and nuclei, respectively. The higher magnification images provided refined nuclei morphology. The nuclei were detected with a RGB color space principle component analysis that resulted in a grey scale image. The shape metrics such as compactness, elongation, minimum and maximum diameters were calculated based on the eigenvalues of the best-fitting ellipses to the nuclei.

A digital light modulation microscope (DLMM) using a digital micro-mirror device (DMD, Texas Instruments) has been developed to enable detection of O₂ concentration in micro-bioreactors using O₂-quenching porphyrin phosphorescent dyes. The emission intensity and phosphorescence lifetime of such dyes are both a function of O₂ concentration. While emission intensity can vary in these dye systems as a function of concentration and illumination intensity, phosphorescence lifetime is primarily sensitive to only O₂ concentration. In contrast to conventional phosphorescence lifetime imaging, the DLMM eliminates the need for a pulsed light source, scanning mirrors, or a high-speed camera for time-gated imaging. This technique can selectively address structured light illumination to each sensor location, which is a beneficial feature for analysis of large micro-sensor arrays within lab-on-a-chip devices. The mirrors on the DMD perform as electronically addressable optical switches, each having a ~15 μs switching time, shorter than the phosphorescence lifetimes of potential O₂ sensing dyes (~25-100 μs). The structured light pattern of the DMD and the switching rate of the mirrors are controlled by a PC. An arc lamp illuminates the DMD uniformly and then projects to the specimen through a filter cube for the selected phosphorescent sensor compound. The emitted light returns to the filter cube and is detected by a photo multiplier tube (PMT). An oscilloscope is used to record the emission signal waveform from the PMT. To demonstrate O₂ sensing with lab-on-a-chip devices, an array of 150-μm-diameter micro-wells coated with phosphorescent porphyrin were observed using the DLMM. The goal of this platform is to measure the O₂ consumption of individual cells trapped in the microwells.

We report on the development and application of a flow cytometer using a 16-channel avalanche photodiode (APD) linear detector array. The array is configured with a dispersive grating to simultaneously record emission over a broad wavelength range using the 16 APD channels of the linear APD array. The APD detector elements have a peak quantum efficiency of 80% near 900 nm and have at least 40% quantum efficiency over the 400-nm to 1000-nm wavelength range. The extended red sensitivity of the detector array facilitates the use of lower energy excitation sources and near IR emitting dyes which reduces the impact of autofluorescence in signal starved measurements. The wide wavelength sensitivity of the APD array permits the use of multiple excitation sources and many different fluorescent labels to maximize the number of independent parameters in a given experiment. We show the sensitivity and linearity measurements for a single APD detector. Initial results for the flow cytometer with the 16-element APD array and the 16-channel readout ASIC (application specific integrated circuit) are presented.

While fluorescence microscope systems remains an essential tool in modern biology and medical work, no compact instrumentation has been developed for the rapid calibration of such systems. Almost invariably results are presented in terms of the [AU], "arbitrary units". To remedy this situation we have developed a small, portable instrument - the size of a microscope slide - that uses low-power LEDs at different wavelengths to produce calibrated amounts of light. A computer controls the instrument--through a USB connector--so that the current to the selected LED can be swept through an increasing range of values. The amount of light measured by the microscope's total imaging system (lenses, filters, EO sensor, and digitizer) is then recorded to provide a "current in, digital value out" calibration. Further, the current can be translated easily to optical power and thus photons per second at the chosen LED wavelength. We have built and programmed such a system, tested it for accuracy and precision, and used it to calibrate several microscopes and microscope/lens combinations. The results will be presented.

Collecting microlymphatics play a vital role in promoting lymph flow from the initial lymphatics in the interstitial spaces to the large transport lymph ducts. In most tissues, the primary mechanism for producing this flow is the spontaneous contractions of the lymphatic wall. Individual units, known as lymphangion, are separated by valves that help prevent backflow when the vessel contracts, thus promoting flow through the lymphatic network. Lymphatic contractile activity is inhibited by flow in isolated lymphatics, however there are virtually no in situ measurements of lymph flow in these vessels. One of the difficulties associated with obtaining such measurements is the time consuming methods of manual particle tracking used previously by our group. Using an in situ preparation with mesenteric microlymphatics (~ 100 μm in diameter) and a high speed imaging system (500 fps), we have developed an image correlation method to measure lymphatic flow with a standard error of prediction of 0.3 mm/sec when compared with manual particle tracking.

A portable fluorescence cytometric system that provides a modular platform for quantitative real-time image measurements has been used to explore the applicability to investigating cellular events on multiple time scales. For a short time scale, we investigated the real-time dynamics of uptake of daunorubicin, a chemotherapeutic agent, in cultured mouse L-cells in a micro cell culture analog compartment using the fluorescent cytometric system. The green fluorescent protein (GFP) expression to monitor induction of pre-specified genes, which occurs on a much longer time scale, has also been measured. Here GFP fluorescence from a doxycycline inducible promoter in a mouse L-cell line was determined. Additionally, a system based on inexpensive LEDs showed performance comparable to a broadband light source based system and reduced photobleaching compared to microscopic examination.

The acousto-optic tunable filter (AOTF) is one example of a small number of commercially available optical filter technologies that lend themselves to imaging applications. In recent years the demand for high specification devices has increased significantly, and diffraction limited performance is being achieved.

Recent studies have shown that the cell is mechanically differentiated both spatially and temporally, leading to a regional approach in cell behaviour essays. Most experiments are based on spatially-controlled contacts between microbeads and cells. We here propose an apparatus based on holographic optical tweezers to put on a target cell a two- or three-dimensional custom-built pattern of beads, with respect to the target cell shape, with both temporal and spatial dynamic control of each contact. In order to avoid disturbance or contact from the excess beads with the target cell, we keep the beads under isolated condition, by placing them in a confinement chamber made by microstereolithography. Our system exploits a digital display to project binary images on a photocurable resin surface, and induce space-resolved photopolymerisation reactions, constructing three-dimensional micro structures with complex shapes, including reservoirs for the filling, outlets, and confinement chambers. Combination of microfluidics, holographic optical tweezers and one supplementary single manually steerable optical tweezers leads to several experimental procedures allowing the sequential or parallel deposition of beads onto a target, with both a spatial and temporal control.

A force-measuring optical tweezers instrumentation and long time measurements of the elongation and retraction of bacterial fimbriae from Uropathogenic E. coli (UPEC) under strain are presented. The instrumentation is presented in some detail. Special emphasis is given to measures taken to reduce the influence of noise and drifts in the system and from the surrounding, which makes long term force measurements possible. Individual P pili from UPEC bacteria were used as a biological model system for repetitive unfolding and refolding cycles of bacterial fimbriae under equilibrium conditions. P pili have evolved into a three-dimensional helix-like structure, the PapA rod, that can be successively and significantly elongated and/or unfolded when exposed to external forces. The instrumentation is used for characterization of the force-vs.-elongation response of the PapA rod of individual P pili, with emphasis on the long time stability of the forced unfolding and refolding of the helical structure of the PapA rod. The results show that the PapA rod is capable of withstanding extensive strain, leading to a complete unfolding of the helical structure, repetitive times during the life cycle of a bacterium without any noticeable alteration of the mechanical properties of the P pili. This function is believed to be importance for UPEC bacteria in vivo since it provides a close contact to a host cell (which is an initial step of invasion) despite urine cleaning attempts.

The red blood cell (RBC) viscoelastic membrane contains proteins and glycolproteins embedded in, or attached, to a fluid lipid bilayer and are negatively charged, which creates a repulsive electric (zeta) potential between the cells and prevents their aggregation in the blood stream. There are techniques, however, to decrease the zeta potential to allow cell agglutination which are the basis of most of the tests of antigen-antibody interactions in blood banks. This report shows the use of a double optical tweezers to measure RBC membrane viscosity, agglutination and zeta potential. In our technique one of the optical tweezers trap a silica bead that binds strongly to a RBC at the end of a RBCs rouleaux and, at the same time, acts as a pico-Newton force transducer, after calibration through its displacement from the equilibrium position. The other optical tweezers trap the RBC at the other end. To measure the membrane viscosity the optical force is measured as a function of the velocity between the RBCs. To measure the adhesion the tweezers are slowly displaced apart until the RBCs disagglutination happens. The RBC zeta potential is measured in two complimentary ways, by the force on the silica bead attached to a single RBC in response to an applied electric field, and the conventional way, by the measurement of terminal velocity of the RBC after released from the optical trap. These two measurements provide information about the RBC charges and, also, electrolytic solution properties. We believe this can improve the methods of diagnosis in blood banks.

Since the low index particles are repelled away from the highest intensity point, trapping them optically requires either a rotating Gaussian beam or optical vortex beams focused by a high numerical microscope objective. However, the short working distance of these microscope objectives puts a limit on the depth at which these particles can be manipulated. Here, we show that axicon like structure built on tip of a single mode optical fiber produces a focused beam that is able to trap low index particles. In fact, in addition to transverse trapping inside the dark conical region surrounded by high intensity ring, axial trapping is possible by the balance of scattering force against the buoyancy of the particles. The low-index particle system consisted of an emulsion of water droplets in acetophenone. When the fiber was kept horizontal, the low index spheres moved away along the beam and thus could be transported by influence of the scattering force. However in the vertical position (or at an angle) of the fiber, the particles could be trapped stably both in transverse and axial directions. Chain of such particles could also be trapped and transported together by translation of the fiber. Using escape force technique, transverse trapping force and thus efficiency for particle in Mie regime was measured. Details of these measurements and theory showed that trapping of Raleigh particle is possible with such axicon-tip fibers. This ability to manipulate low-index spheres inside complex condensed environments using such traps will throw new insights in the understanding of bubble-bubble and bubble-wall interactions, thus probing the physics behind sonoluminescence and exploring new applications in biology and medicine.

A combined microchannel-type erythrocyte deformability test with optical tweezers has been developed especially for more sensitive detection of cancerous diseases. To demonstrate the performance and sensitivity of the microchannel-type method, we measured the transit velocity of individual erythrocytes passing through a specific confinement region and calculated the modified elongation index defined by the ratio of the width of the microchannel to the elongated length of the squeezed erythrocytes. To know exactly the effect of optical tweezers on erythrocytes, we investigated several morphologies of optically deformed erythrocytes and measured the shape recovery time of erythrocytes in a static aqueous solution under various powers (~ 24 mW) of 1064-nm laser by a dual-trap optical tweezers. Finally we combined these two methods by considering the key parameters of erythrocyte deformability. The results show that the ambiguity of the overlapped experimental data from microchannel-type erythrocyte deformability test was conspicuously reduced, and that the subtle change (≈ 100-200 ms) in shape recovery time which is one of mechanical properties of erythrocyte membrane surface was remarkably amplified to readily discriminate the difference (≈ 2-3 s) between normal and cancerous blood. This suggests the combined method is more sensitive enough to pinpoint the minor quantitative differences between individual erythrocytes, especially in the field of cancer and cardiovascular diseases.

Optical tweezers have previously been used to characterize the force-vs.-elongation dependence of the PapA rod of uropathogenic E. coli P pili. It was found that the PapA rod elongates in several elongation regions. In the two first, the elongation originates from an elastic stretching and a sequential unfolding of the layer-to-layer bonds (and thereby of the helical structure). Region III is characterized by an elongation that originates from an elastic stretching and an opening of the head-to-tail bonds in the linearized PapA rod. The opening of these bonds takes place in a random order, wherefore the response in this region is affected by entropy. Since the entropic softening of a macromolecule depends on the number of units, the shape of this region can be used to assess the number of PapA units. We provide in this work a recipe for how this can be done solely from the form of region III. An advantage with this technique is that it does not require a continuous monitoring of the elongation of a single PapA rod from unstretched conditions, which often is difficult because of simultaneous multi-pili binding; it suffices to detect it in the third region at which binding often is mediated by only one pilus. Another advantage is that it does not require any prior knowledge about (or assessment of) any physical entity of the PapA rod; the number of PapA units can be assessed solely from the shape of the curve in the third elongation region.

We report an analysis of 2 yeast cell mutants using biocavity laser spectroscopy. The two yeast strains differed only by the presence or absence of mitochondrial DNA. Strain 104 is a wild-type (ρ⁺) strain of the baker's yeast, Saccharomyces cerevisiae. Strain 110 was derived from strain 104 by removal of its mitochondrial DNA (mtDNA). Removal of mtDNA causes strain 110 to grow as a "petite" (ρ^-), named because it forms small colonies (of fewer cells because it grows more slowly) on agar plates supplemented with a variety of different carbon sources. The absence of mitochondrial DNA results in the complete loss of all the mtDNA-encoded proteins and RNAs, and loss of the pigmented, heme-containing cytochromes a and b. These cells have mitochondria, but the mitochondria lack the normal respiratory chain complexes I, III, IV, and V. Complex II is preserved because its subunits are encoded by genes located in nuclear DNA. The frequency distributions of the peak shifts produced by wild-type and petite cells and mitochondria show striking differences in the symmetry and patterns of the distributions. Wild-type ρ⁺ cells (104) and mitochondria produced nearly symmetric, Gaussian distributions. The ρ^- cells (110) and mitochondria showed striking asymmetry and skew that appeared to follow a Poisson distribution.

In this article, we will demonstrate a novel optical imaging device that can be directly integrated into a microfluidic network, and therefore enables on-chip imaging in a microfluidic system. This micro imaging device, termed optofluidic microscope (OFM) is potentially free of bulk optics and is based on a nanohole array defined in a nontransmissive metallic layer that is patterned onto the floor of the microfluidic channel. The operation of the optofluidic microscope will be explained in details and its performance is examined by using a popular animal model, Caenorhabditis elegans (C. elegans). Images from a large population of nematode worms are efficiently acquired within a short time frame. The quality of the OFM images of C. elegans and the morphological characteristics revealed therein are evaluated. Two groups of early-stage C elegans larvae, wild-type and dpy-24 are successfully separated even though their morphological difference at the larval stage is subtle. The experimental results support our claim that the methodology described therein can be effectively used to develop a powerful tool for fulfilling high-resolution, highthroughput imaging task in microfluidics-based systems.

Laser-activated micro- and nano-bubbles (LAB) in cells may be used as universal and sensitive probes for measuring properties of individual cells. Such bubbles can be detected and imaged by using microscopy and flow cytometry. LABs in living blood and tumor cells were induced by pulsed (532 nm, 10 ns) laser radiation and were detected by a thermal lens optical method. LAB lifetime and maximal diameter varied, correspondingly, within the ranges 0.02-10 ms and 0.44-100 mm. LAB parameters - thresholds and probabilities - were found to depend upon the physiological state of cells. Specificity and sensitivity of LAB cytometry were increased by using light-absorbing nanoparticles conjugated to specific monoclonal antibodies.

The ability of cellular membranes to generate electrically-induced mechanical force (EMF) has been demonstrated in many cell types, including cochlear outer hair cells, axons, and some cultured mammalian cells. Models of membrane based EMF generation are based on an interaction between the transmembrane electric field and membrane surface charge. We use a technique that combines optical trapping with voltage clamping to investigate the effects of an electrically charged amphipathic agent on EMF by membrane tethers. Our preliminary results indicate that salicylate, a negatively charged amphipathic agent, which is also known to cause reversible hearing loss and reduce outer hair cell electromotility, reduces EMF in membrane tethers. These measurements provide a basis to better understand the role of membrane charge properties in EMF generation.

In eukaryotic cells, proper position of the mitotic spindle and the division plane is necessary for successful cell division and development. In this work the nature of forces governing the positioning and elongation of the mitotic spindle and the spatio-temporal regulation of the division plane positioning in fission yeast was studied. By using a mechanical perturbations induced by laser dissection of the spindle and astral microtubules, we found that astral microtubules push on the spindle poles. Further, laser dissection of the spindle midzone induced spindle collapse inward. This suggests that the spindle is driven by the sliding apart of antiparallel microtubules in the spindle midzone. Exploiting a combination of non-linear microscopy and optical trapping, we performed an optical manipulation procedure designed to displace the cell nucleus away from its normal position in the center of the cell. After the laser-induced displacement, the nucleus typically returned towards the cell center, in a manner correlated with the extension of a microtubule from the nucleus to the closer tip of the cell. This observation suggests that the centering of the nucleus is provided by microtubule pushing force. Moreover the cells in which the nucleus was displaced during interphase displayed asymmetric division, whereas when the nucleus was displaced during late prophase or metaphase, the division plane formed at the cell center as in non-manipulated cells. This result suggests that in fission yeast the division plane is selected before pro-metaphase and that the signal is not provided by the mitotic spindle.

The regulation of morphogenetic movements that shape an embryo during its development remains a challenging issue in developmental biology, and may in certain cases involve mechanical sensitivity. Addressing this issue requires novel experimental approaches. We show that the combination of femtosecond laser pulse-induced ablation and multiphoton microcopy can be used to modulate and quantify morphogenetic movements in Drosophila embryos. We characterized the effects of focused nanoJoule pulse trains in developing embryos. We used targeted ablations to locally modify the embryo structural integrity and modulate morphogenesis. Femtosecond-pulse induced ablation was combined with nonlinear microscopy based on two-photon-excited fluorescence (2PEF) and third-harmonic generation (THG).Correlation-based analysis of microscopy data allowed us to track the outcome of ablations and to analyze tissue deformations. These experiments provided insight into the interplay between gene expression and tissue deformations in developing embryos.

The rapid development of the optical tweezers technique has broadened the applicability of the technique from physics to biology. Although the construction of an optical tweezers system only requires basic skills in optics, the realization of an optical tweezers set up is not always as easy as it seems. A number of designs for moveable traps have been presented in the literature. It is not clear to the readers, however, how the movability of the trap affects its quality. We have therefore scrutinized and compared the most commonly used techniques for steering of an optical trap in terms of the aberrations they introduce. The study shows that a moveable trap based on the movement of a lens introduces significantly more aberration than the systems based on fiber optics or the tilting of a mirror.

The genus Saprolegnia in the phylum Oomycota have intracellular structures that are distinct from that of filamentous fungi. The vacuolar reticulum for example in Saprolegnia consists of fine static tubules that taper towards the apex of the hypha and are connected to a large vacuole in the basal region. This paper discusses the contribution of the different microscopic techniques in observing ultrastructural changes resulting from modulating GTP binding proteins associated with vesicle production and placement. TEM, DIC and fluorescent observations complemented each other and provided valuable detailed information as to changes in the vacuolar reticulum and the arrangement of organelles. The use of comparative imaging was essential for obtaining sufficient information to make an accurate assessment of changes resulting from perturbation. Without comparison of multiple imaging techniques the resulting conclusions would have been limited with the added potential of being inaccurate. Imaging properties such as cellular detail, overview and specificity from the various forms of microscopy confirmed and contributed information to the analysis. The argument of whether Saprolegnia use a tubular or a vesicular network system to transfer nascent membrane to the growing tip would have been difficult to determine using only one or two imaging techniques. Comparative analysis has indicated that the vacuolar reticulum, previously considered to be static, is a membrane reservoir that allows for membrane transfer to the apical and subapical regions.

Ultraweak bio-chemiluminescence (UBC) from germinating soybean (Glycine max L. Merr) cotyledon under mechanical wounding was observed by a high sensitivity imaging system based on an intensified charge couple device (ICCD) detector or a high sensitive single photon counter (SPC) device. The UBC imaging showed that the intensity of UBC at the injury location on a wounded green cotyledon was greater than on a wounded etiolated cotyledon. Based on results with a SPC UBC intensity of wounded green cotyledon was high at first and then gradually decreased. The emission spectrum of wounded green cotyledon had a greater proportion of red light. The increase in UBC of wounded etiolated soybean cotyledon was less than that of green ones. The emission spectrum of wounded etiolated cotyledon had a greater amount of orange light. The data suggest that most of the UBC in green cotyledon was due to damaged chloroplasts and mitochondria. Our data suggest that oxidation of damage's tissue lead to the production of ROS. Electronic excitation energy was transfered from the excited molecules by ROS to Chl-a in the thylakoid membranes, so the intensity of UBC in the wounded soybean green cotyledon was obviously higher than in the wounded etiolated soybean cotyledon.

We explore the Generalized Phase Contrast (GPC) approach for optical sorting in microfluidic systems. A microsystem is used in which two streams meet, interact and separate in an X-shaped channel. When the flow in the two arms of the X is balanced, the laminar flow that exists at very low Reynolds numbers ensures minimal stream blending and the fluid separates without mixing (i.e. diffusion is negligible). Optical forces due to an intensity pattern can be fashioned to induce a selective deflection of particles between the two streams. This method is known as optical fractionation (OF). In brief, OF uses the same mechanisms as optical tweezers to exert forces upon microscopic particles. OF has been shown to have an exponential size selectivity. This means that the interaction between the streams can be made to discriminate by particle size at a critical flow velocity. With correctly adjusted flow velocity, particles with a certain size will more often shift to the other stream than another particle size. One method for creating the light pattern is by interference of several beams that are variably attenuated using mechanical means. However, this approach offers low optical efficiency and is not easily reconfigured. The GPC method offers a solution that gives the possibility to instantaneously reconfigure the intensity pattern by a method that is inherently computer-controllable. This enables one to rapidly test various intensity patterns to optimize sorting of particles.

Changes in the intracellular Ca²⁺ concentration ([Ca²⁺]i) play a crucial role involved in the modulation of signal transduction, development, and plasticity in the CNS. Glial cells can respond to various stimuli with an increase in [Ca²⁺]i. In this paper, we used confocal microscopy to study calcium transient induced by glutamate in cultured astrocytes. Firstly, 100 μM glutamate induced long-time intracellular calcium oscillations in astrocytes and only a single spike under calcium-free solution. When the concentration of glutamate decreased to 1 μM, only a single spike could be induced. It shows that intracellular calcium oscillations depend on agonist concentration and extracellular Ca²⁺. Secondly, we investigated amplitude of responses under different stimulation. The amplitude of initial peak induced by 100 μM glutamate decreased in Ca²⁺-free condition, whereas the duration of kinetics was prolonged. But both the amplitude and area of a single spike induced by 1 μM Glu decreased in Ca²⁺-free condition. The results show that areaof peak is more accurate than amplitude to display transients of [Ca²⁺]i. All results above suggest that astrocytes are not passive, they display diverse temporal and spatial increases in [Ca²⁺]i in response to a variety of stimuli. These [Ca²⁺]i increases provide a possible means for information coding.

Intracellular calcium, as an important second messenger, plays a significant role in cell signaling transduction and metabolism. Glutamate can induce the intracellular calcium transient through triggering diverse signaling pathways. To test the effect of glutamate to neurons, we loaded Fluo-3/Am in cultured rat hippocampal neurons, and then acquired two-dimensional fluorescent image by confocal microscopy and the analyzed fluorescent intensity. In cultured neurons, we observed two types of neurons that have different morphology: bipolar-type and pyramidal-type. Inducing [Ca²⁺]_i transient by glutamate, we found the amplitude and time constant of the response curves of bipolar neurons are larger than those of pyramidal neurons. Further, we induced [Ca²⁺]_ii transient under different concentrations of glutamate. Two different types of kinetic of the [Ca²⁺]_i transient have been found, corresponded to the two kinds of neuron. The amplitude of [Ca²⁺]_i transient increased when applying higher concentration of glutamate in pyramidal neurons; while it decreased in bipolar ones. Responses of neurons bathing in calcium-free extracellular solution to glutamate were different from those bathing in normal solution. [Ca²⁺]_i transient of pyramidal neurons caused by any concentration were totally blocked; while [Ca²⁺]_i transient in bipolar neurons caused by high concentration of glutamate (500μM) were partly inhibited. All of the phenomena suggest that different types of cultured hippocampal neurons may have different mechanism of the response to glutamate.

Caspase-3 is a kind of cysteine proteases that plays an important role in cell apoptosis. We have constructed a FRET (fluorescence resonance energy transfer) probe fused with ECFP (enhanced cyan fluorescence protein) and DsRed (Discosoma red fluorescent protein) with a linker containing a caspase-3 cleavage sequence (CCS, DEVD).It could be observed much change in fluorescence emission ratio when the probe was cleaved by caspase-3. Therefore, application of this probe we can real-time detected the activation of caspase-3. It was already confirmed that caspase-3 was activated in HeLa cells treated by cisplatin or TRAIL (Tumor necrosis factor (TNF)-related apoptosis-inducing ligand). In the present study, we detected the activation of caspase-3 during cisplatin or TRAIL induced apoptosis in living HeLa cells, and also observed the activation of caspase-3 caused by both cisplatin and TRAIL combined treatment. Our results demonstrated a synergistic effect between cisplatin and TRAIL. Cisplatin is one of the most broadly used drugs in the Clinical applications of cancer chemotherapy, and TRAIL, which belongs to the TNF family proteins, can selectively induce apoptosis in many transformed cells but not in normal cells. Therefore, TRAIL is a very valuably prospective utility as its potential tumor-specific cancer therapeutic. Most of anticancer drugs can induce apoptosis which mediated by the activation of caspase pathway. We can select the best synergistic effect group by our FRET probe. This finding would be useful in the design of treatment modalities for patients.

In order to study the dynamic change in the cell, we modified the evanescence microscope with an ultra high NA objective lens so as to modulate the penetration depth of the evanescent wave. We employed a galvanomirror to aim and switch the laser beam rapidly at the back focal plane near the periphery of 1.45 or 1.65 NA objectives. Under this microscope equipped with a 1.45 NA objective, images of the fluorescent bead were clearly distinguishable by the modulation of the penetration depth of the evanescent wave. Thus, translocation dynamics of protein kinase Cα (PKCα) upon cell activation were compared every 0.5 s between two modes using HeLa cells expressing PKCα fused with the green fluorescent protein (GFP). Stimulation of the cell with phorbol ester induced a transient increase in GFP fluorescence images illuminated by the thin evanescent field, but not in the image illuminated by the thick evanescent field. Later, a persistent increase in fluorescence appeared at cell borders in the both images. Using a 1.65 NA objective, trafficking of secretory vesicles was studied in MIN6 cells expressing insulin-GFP. Occasionally, the change in fluorescence of a vesicle observed under one illumination mode appeared very different from the other, allowing unique assignments of the fluorescence change to a certain combination of vesicle movement and a chemical response of fluorescent molecules. The ultra high NA lens provides a large window for evanescent illumination with a wide range of penetration depth, thus is useful for analyzing 3D events in the cell.

In this work, we present spatially resolved pharmacokinetic rate images of indocyanine green (ICG) obtained from three breast cancer patients using near infrared imaging methods. We used a two-compartment model, namely, plasma and extracellular extravascular (EES), to model ICG kinetics around the tumor region. We introduced extended Kalman filtering (EKF) framework to estimate the ICG pharmacokinetic rate images. The EKF framework allows simultaneous estimation of pharmacokinetic rates and the ICG concentrations in each compartment. Based on the pharmacokinetic rate images, we observed that the rates from inside and outside the tumor region are statistically different with a p-value of 0.0001 for each patient. Additionally, we observed that the ICG concentrations in plasma and the EES compartments are higher around the tumors agreeing with the hypothesis that ICG may act as a diffusible extravascular flow in leaky capillary of cancer vessels. Our study shows that spatially resolved pharmacokinetic rate images can be potentially useful for breast cancer screening and diagnosis.