Human chromosome analysis is an essential task in cytogenetics, especially in prenatal screening, genetic syndrome diagnosis, cancer pathology research and mutagen dosimetry. Chromosomal analysis begins with the creation of a karyotype, which is a layout of chromosome images organized by decreasing size in pairs. Both manual and automatic classification of chromosomes are limited by the resolution of the microscope and imaging system used. One way to improve the results of classification and even detect subtleties now remaining undetected, is to enhance the resolution of the images. It is possible to achieve lateral resolution beyond the classical limit, by using spatially modulated illumination (SMI) in a wide-field, non-confocal microscope. In this case, the sample is illuminated with spatially modulated light, which makes normally inaccessible high-resolution information visible in the observed image by shifting higher frequencies within the OTF limits of the microscope. Although, SMI microscopes have been reported in the past, this manuscript reports the development of a transillumination microscope for opaque, non-fluorescent samples. The illumination path consisted of a light source illuminating a ruled grating which was subsequently imaged on the sample. The grating was mounted on a rotating and translating stage so that the magnification and rotation of the pattern could be adjusted. The imaging lens was a 1.25 NA oil immersion objective. Test samples showed resolution improvement, as judged from a comparison of the experimentally obtained FWHM. Further studies using smaller fringe distance or laser interference pattern illumination will be evaluated to further optimize the SMI results.

Microbial contamination has serious consequences for the industries that use fermentation processes. Common contaminants such as faster growing lactic acid bacteria or wild yeast can rapidly outnumber inoculated culture yeast and produce undesirable end products. Our study focuses on a rapid method of identification of such contaminants based on autofluorescence spectroscopy of bacterial and yeast species. Lactic acid bacteria (Lac-tobacillus casei), and yeast (Saccharomyces cerevisiae) were cultured under controlled conditions and studied for variations in their autofluorescence. We observed spectral differences in the spectral range representative of tryptophan residues of proteins, with excitation at 290 nm and emission scanned in the 300 nm - 440 nm range. Excitation scans between 240 nm and 310 nm were also performed for the emission at 340 nm. Moreover, we observed clearly pronounced differences in the excitation and emission in the visible range, with 410 nm excitation. These results demonstrate that bacterial and yeast species can be differentiated using their intrinsic fluorescence both in UV and in the visible region. The comparative spectroscopic study of selected strains of Saccharomyces yeast showed clear differences between strains. Spectrally-resolved laser scanning microscopy was carried out to link the results obtained using ensembles of cells with spectral properties of individual cells. Strongly fluorescent subpopulation were observed for all yeast strains with excitation at 405 nm. The fluorescence spectra showed variations correlated with cell brightness. The presented results demonstrate that using autofluorescence, it is possible to differentiate between yeast and lactic acid bacteria and between different yeast species.

We describe imaging the luminance of red fluorescent protein (DsRed₂)-expressing bacteria from outside intact infected animals. This simple, nonintrusive technique can show in great detail the temporal behavior of the infectious process. Fluorescence stereo microscope, laser and cooled CCD are expensive to many institutes, we set up an inexpensive compact whole-body fluorescent imaging tool, which consisted of a digital camera, fluorescence filters and a mercury 50-W lamp power supply as excitation light source. The bacteria, expressing the DsRed₂, are sufficiently bright as to be clearly visible from outside the infected animal and recorded with simple equipment. Introduced bacteria were observed in the abdomen. Instantaneous real-time images of the infectious process were acquired by using a digital camera by simply illuminating nude mice with mercury lamp. The development of infection over 48 hours and its regression after kanamycin treatment were visualized by whole-body imaging. The DsRed₂ was excited directly by mercury lamp with EF500/50 nm band-pass filter and fluorescence was recorded by digital camera with CB580 nm long-pass filter. By this easy operation tool, the authors imaged, in real time, fluorescent tumors growing in live mice. The imaging system is external and noninvasive. For one year our experiments suggested the imaging scheme was feasible, which affords a powerful approach to visualizing the infection process.

The understanding and control of cell growth in confined microenvironments has application to a variety of fields including cell biosensor development, medical device fabrication, and pathogen control. While the majority of work in these areas has focused on mammalian and bacterial cell growth, this study reports on the growth behavior of fungal cells in three-dimensionally confined PDMS microenvironments of a scale similar to that of individual hyphae. The general responses of hyphae to physical confinement included continued apical extension against barriers, resultant filament bending and increased rates of subapical branching with apparent directionality towards structure openings. Overall, these responses promoted continued extension of hyphae through the confined areas and away from the distal regions of the fungal colony. The induction of branching by apical obstruction provides a means of controlling the growth and branching of fungal hyphae through purposefully designed microstructures.

In this communication, we report the imaging of living glioma cells using fluorescence lifetime imaging (FLIM) technique. The growing interests in developing novel techniques for diagnosis and minimally invasive therapy of brain tumor have led to microscopic studies of subcellular structures and intracellular processes in glioma cells. Fluorescence microscopy has been used with a number of exogenous molecular probes specific for certain intracellular structures such as mitochondria, peripheral benzodiazepine receptor (PBR), and calcium concentration. When probes with overlapping emission spectra being used, separate samples are required to image each probe individually under conventional fluorescence microscopy. We have developed a wide-field FLIM microscope that uses fluorescence lifetime as an additional contrast for resolving multiple markers in the same essay. The FLIM microscope consists of a violet diode laser and a nitrogen-pumped dye laser to provide tunable sub-nanosecond excitation from UV to NIR. The detection system is based on a time-gated ICCD camera with minimum 80 ps gate width. The performance of the system was evaluated using fluorescence dyes with reported lifetime values. Living rat glioma C6 cells were stained with JC-1 and Rhodamine 123. FLIM images were acquired and their lifetimes in living cells were found in good agreements with values measured in solutions by a time-domain fluorescence spectrometer. These results indicate that imaging of glioma cells using FLIM can resolve multiple spectrally-overlapping probes and provide quantitative functional information about the intracellular environment.

A novel setup for fluorescence measurements of surfaces of biological samples, in particular cell membranes, is described. The method is based on multiple total internal reflection (TIR) of a laser beam on the surface of a multi-well plate, such that 96 individual samples are excited simultaneously. Main prerequisites are an appropriate thickness and high transmission of the glass bottom, a non-cytotoxic adhesive, and appropriate glass rods for TIR illumination. Fluorescence from cell surface is detected simultaneously using an integrating CCD camera and appropriate optical filters. For validation of the system, transfected cells expressing a fluorescent membrane protein are used. In addition, intracellular translocation of green fluorescent protein kinase c upon stimulation is examined. The method appears well suitable for high throughput screening (HTS), since neither washing of the samples nor any re-adjustment of the equipment after changing of individual plates are necessary.

Membranes of living cells are characterized by laser-assisted fluorescence microscopy, in particular a combination of microspectrofluorometry, total internal reflection fluorescence microscopy (TIRFM) and fluorescence decay kinetics. The generalized polarization (GP, characterizing a spectral shift which depends on the phase of membrane lipids), the time constant of fluorescence anisotropy (τ_r) as well as the fluorescence lifetime (τ) of the membrane marker laurdan revealed to be appropriate measures for membrane stiffness and fluidity. GP decreased with increasing temperature and was always higher for the plasma membrane than for intracellular membranes. The latter effect was correlated with the intracellular content of cholesterol, which could be modified using defined protocols of depletion or enrichment. Concomitant with generalized polarization the fluorescence lifetime τ increased with the content of cholesterol. Changes of cholesterol amounts in cell membranes have previously been related to specific diseases and may have some influence on the uptake of pharmaceutical agents.

Immunophenotyping of peripheral blood leukocytes (PBLs) is performed by flow cytometry (FCM) as the golden standard. Slide based cytometry systems for example laser scanning cytometer (LSC) can give additional information (repeated staining and scanning, morphology). In order to adequately judge on the clinical usefulness of immunophenotyping by LSC it is obligatory to compare it with the long established FCM assays. We performed this study to systematically compare the two methods, FCM and LSC for immunophenotyping and to test the correlation of the results. Leucocytes were stained with directly labeled monoclonal antibodies with whole blood staining method. Aliquots of the same paraformaldehyde fixed specimens were analyzed in a FACScan (BD-Biosciences) using standard protocols and parallel with LSC (CompuCyte) after placing to glass slide, drying and fixation by aceton and 7-AAD staining. Calculating the percentage distribution of PBLs obtained by LSC and by FCM shows very good correlation with regression coefficients close to 1.0 for the major populations (neutrophils, lymphocytes, and monocytes), as well as for the lymphocyte sub-populations (T-helper-, T-cytotoxic-, B-, NK-cells). LSC can be recommended for immunophenotyping of PBLs especially in cases where only very limited sample volumes are available or where additional analysis of the cells’ morphology is important. There are limitations in the detection of rare leucocytes or weak antigens where appropriate amplification steps for immunofluorescence should be engaged.

We have developed an automated microinjection system that captures many floating cells and controls capillary positions precisely. To capture many cells simultaneously, we constructed an array of holes on a 10 x 10 mm silicon-based substrate. The hole diameter is 3 μm because our target cells are 10 - 20 μm in diameter. A suction pump connected to the bottom of the multi-hole silicon chip draws the medium into the holes using a slight vacuum, so cells are caught there. Using an initial prototype chip having 121 holes, we captured over 90 cells in a single sweep. Automated microinjection requires precise control of the capillary positions, so images of the capillary and holes on the chip are observed using a microscope with a CCD camera located above the biological medium. The 3D positions of these elements are accurately measured by processing these images. The capillary and the chip are mounted on automatic stages individually controlled with this position data. Using these techniques, this system can microinject about one cell per second. Its success rate for microinjection is 61% for PC12 cells.

We report two novel applications of multiphoton microscopy for pharmacological studies of unstained cardiovascular tissue. First, we show that second harmonic generation (SHG) microscopy of unstained cardiac myocytes can be used to determine the sarcomere length with sub-resolution accuracy, owing to the remarkable contrast of the SHG signal originating from myosin filaments. A measurement precision of 20 nm is achieved, taking the sample variability into account. We used this technique to measure sarcomere contracture in the presence of saxitoxin, and results were in agreement with mechanical measurements of atrial tissue contracture. Second, we characterized multiphoton microscopy of intact unlabeled arteries. We performed simultaneous detection of two-photon-excited fluorescence (2PEF) from elastin laminae and SHG from collagen fibers upon 860 nm excitation. Combined 2PEF/SHG images provide a highly specific, micron scale description of the architecture of these two major components of the vessel wall. We used this methodology to study the effects of lindane (a pesticide) on the artery wall structure and evidenced structural alteration of the vessel morphology.

It has recently been shown that mutations in Filamin A and B genes produce a large spectrum of skeletal disorders in developing fetuses. However, high-resolution optical microscopy in cartilage growth plate using fluorescent antibody assays, which should elucidate molecular aspects of these disorders, is extremely difficult due to the high level of autofluoresce in this tissue. We apply multiphoton, confocal, lifetime and spectral microscopy to (i) image and characterize autofluorophores in chondrocytes and subtract their contributions to obtain a corrected antibody-marker fluorescence signal, and (ii) measure the interaction between Filamin A and B proteins by detecting the fluorescence resonance energy transfer (FRET) between markers of the two proteins. Taking advantage of the different fluorescence spectra of the endogenous and exogenous markers, we can significantly reduce the autofluorescence background. Preliminary results of the FRET experiments suggest no interaction between Filamin A and B proteins. However, developing of new antibodies targeting the carboxy-terminal immunoglobulin-like domain may be necessary to confirm this result.

We have developed a system for the direct spectroscopic identification of protein biomarkers in biological samples. By conjugating Raman dyes and biomolecular targeting agents to gold nanoparticles, we have produced highly selective optically encoded probes. The nanostructures are grown within a cellular sample to generate a surface that is highly active for surface enhanced Raman spectroscopy (SERS). Both in vitro characterizations of SERS detection as well as the sensitive and specific detection of cancer biomarkers in cultured cancer cells have been demonstrated.

Biological specimens are three-dimensional structures. However, when capturing their images through a microscope, there is only one plane in the field of view that is in focus, and out-of-focus portions of the specimen affect image quality in the in-focus plane. It is well-established that the microscope’s point spread function (PSF) can be used for blur quantitation, for the restoration of real images. However, this is an ill-posed problem, with no unique solution and with high computational complexity. In this work, instead of estimating and using the PSF, we studied focus quantitation in multi-spectral image sets. A gradient map we designed was used to evaluate the sharpness degree of each pixel, in order to identify blurred areas not to be considered. Experiments with realistic multi-spectral Pap smear images showed that measurement of their sharp gradients can provide depth information roughly comparable to human perception (through a microscope), while avoiding PSF estimation. Spectrum and morphometrics-based statistical analysis for abnormal cell detection can then be implemented in an image database where the axial structure has been refined.

Cell-based engineered tissue models have been increasingly useful in the field of tissue engineering, in in vitro drug screening systems, and in complex cell biology studies. While techniques for engineering tissue models have advanced, there have been few imaging technique capable of assessing the complex 3-D cell behaviors in real-time and at the depths that comprise thick tissues. Understanding cell behavior requires advanced imaging tools to progress from characterizing 2-D cell cultures to complex, highly-scattering, thick 3-D tissue constructs. Optical coherence tomography (OCT) is an emerging biomedical imaging technique that can perform cellular-resolution imaging in situ and in real-time. OCT, which uses near-infrared laser light, provides deep-tissue imaging up to several millimeters within highly-scattering tissue, thus permitting visualization of changes at depths previously unattainable. In this study, we demonstrate that it is possible to use OCT to evaluate dynamic cell behavior and function in a quantitative fashion in four dimensions (3-D space plus time). We investigated and characterized cell dynamics and processes in deep tissue models, such as cell de-adhesion, cell proliferation, cell chemotaxis migration, cell necrosis, and cell apoptosis. This optical imaging technique was developed and utilized in order to gain new insights into how chemical microenvironments influence cellular functions and dynamics in multi-dimensional models. In addition, by detecting the changes in cell dynamics, effective chemical concentration could be estimated. With high penetration depth and increased spatial and temporal resolution in 3-D space, OCT will be a useful tool for improving our understanding of cell dynamics in situ and in real-time, for elucidating the complex biological interactions, and for directing our designs toward functional and biomimetic engineered tissues.

Quantitative analysis in combination with fluorescence microscopy calls for innovative digital image measurement tools. We have developed a three-dimensional tool for segmenting and analyzing FISH stained telomeres in interphase nuclei. After deconvolution of the images, we segment the individual telomeres and measure a distribution parameter we call ρ_T. This parameter describes if the telomeres are distributed in a sphere-like volume (ρ_T ≈ 1) or in a disk-like volume (ρ_T >> 1). Because of the statistical nature of this parameter, we have to correct for the fact that we do not have an infinite number of telomeres to calculate this parameter. In this study we show a way to do this correction. After sorting mouse lymphocytes and calculating ρ_T and using the correction introduced in this paper we show a significant difference between nuclei in G2 and nuclei in either G0/G1 or S phase. The mean values of ρ_T for G0/G1, S and G2 are 1.03, 1.02 and 13 respectively.

Multispectral imaging has been in use for over half a century. Owing to advances in digital photographic technology, multispectral imaging is now used in settings ranging from clinical medicine to industrial quality control. Our efforts focus on the use of multispectral imaging coupled with spectral deconvolution for measurement of endogenous tissue fluorophores and for animal tissue analysis by multispectral fluorescence, absorbance, and reflectance data. Multispectral reflectance and fluorescence images may be useful in evaluation of pathology in histological samples. For example, current hematoxylin/eosin diagnosis limits spectral analysis to shades of red and blue/grey. It is possible to extract much more information using multispectral techniques. To collect this information, a series of filters or a device such as an acousto-optical tunable filter (AOTF) or liquid-crystal filter (LCF) can be used with a CCD camera, enabling collection of images at many more wavelengths than is possible with a simple filter wheel. In multispectral data processing the “unmixing” of reflectance or fluorescence data and analysis and the classification based upon these spectra is required for any classification. In addition to multispectral techniques, extraction of topological information may be possible by reflectance deconvolution or multiple-angle imaging, which could aid in accurate diagnosis of skin lesions or isolation of specific biological components in tissue. The goal of these studies is to develop spectral signatures that will provide us with specific and verifiable tissue structure/function information. In addition, relatively complex classification techniques must be developed so that the data are of use to the end user.

This paper describes a novel multispectral imaging microscope that can simultaneously record both spectral and spatial information of a sample, which can take advantage of spatial image processing and spectroscopic analysis techniques. A Liquid Crystal Tunable Filter device is used for fast wavelength selection and a cooled two-dimensional monochrome CCD for image detection. In order to acquire images that are not so dependent on imaging devices, a clever CCD exposure time control and a software based spectral and spatial calibration process is performed to diminish the influence of illumination, optic ununiformity, CCD’s spectral response curve and optic throughput property. A set of multispectral image processing and analysis software package is developed, which covers not only general image processing and analysis functions, and also provides powerful analysis tools for multispectral image data, including multispectral image acquisition, illumination and system response calibration, spectral analysis and etc. The combination of spatial and spectral analysis makes it an ideal tool for the applications to biomedicine. In this paper, two applications in biomedicine are also presented. One is medical image segmentation. Using multispectral imaging techniques, a mass of experiments on both marrow bone and cervical cell images showed that our segmentation results are highly satisfactory while with low computational cost. Another is biological imaging spectroscopic analysis in the study of pollen grains in rice. The results showed that the transmittance analysis of multispectral pollen images can accurately identify the pollen abortion stage of male-sterile rice, and can easily distinguish a variety of male sterile cytoplasm.

Recently, we have shown that single fluorescent dye molecules within the diffraction limited detection volume can be counted by coincidence analysis. In combination with spectrally resolved fluorescence lifetime imaging microscopy (SFLIM), polarization modulation and high-resolution colocalization we suggested to use these techniques for the structural and dynamic investigation of functional protein assemblies and molecular machines in cells. Here we present the application of these techniques within fixed and living cells since quantification and observation of protein assembly in-vivo is of great interest for biological research. We show that appropriately chosen dyes, e.g. ATTO 620, can be discriminated from autofluorescent background within the cells by determination of their spectral emission and their fluorescence lifetimes measured by time correlated single photon counting (TCSPC) under pulsed laser excitation on a confocal microscope. Whereas a lot of autofluorescent signal can be found in the cytoplasm especially in living cells, the nucleus contains almost no fluorescent signal. This offers the opportunity to apply the above methods to protein assemblies, e.g. transcription units, within the cell nucleus. By investigation of fluorescently labeled poly-T40-oligonucleotides hybridized to poly-A-termini of mRNA or tethered within the cell nucleus we demonstrate the feasibility of coincidence analysis for counting single fluorescent molecules within fixed and living cells as a fundamental step for structural investigation below the diffraction limit of optical resolution.

With the development of fluorescence correlation spectroscopy (FCS) in the 1990s, a fundamental milestone was set in the field of highly resolved and quantitative fluorescence detection. Moreover, the increasing knowledge about the meaning of confocal fluorescence detection and its experienced handling enabled unrivalled degrees of detection sensitivity. In the end of the decade, hence the possibility of detecting single fluorescent molecules initiated a productive scientific rush for a comprehensive exploitation of fluorescence properties on the single molecule level. Meanwhile, confocal fluorescence spectroscopy has overcome its predominantly scientific meaning in basic research, and rather found wide applications even in the life science industry. However, biological assay systems relevant for industrial dedication mainly require reagent concentrations above those of classical single molecule detection. They rather lie within the "molecular fluctuation range", which means that in the temporal average a plurality of fluorescent particles rather than only one is present within the confocal detection area at a time. Thus, although individual molecules may not longer be resolved, their diffusive fluctuations are furthermore visible and contain a valuable amount of information. On the other hand FCS, a typical fluctuation evaluation technique, is restricted to the quantification of translational molecular diffusion, which in a number of cases is not sufficient to characterize the biological system of interest. Hence, a series of additional complementary fluctuation and non-fluctuation based confocal spectroscopic techniques was developed during recent years and named FCS⁺plus. They provide simultaneous access to a multitude of molecular properties like concentration, translational and rotational diffusion, molecular brightness, coincidence and fluorescence lifetime and thus meet the needs of both scientific research and industrial application. In the following particular aspects of molecular polarization will be shortly described and illustrated by a comparison of stationary and time-resolved anisotropy. Another valuable subject in especially industrial application of fluorescence is that of artificially interfering effects. The most prominent of these disturbances is given with the potential "auto-fluorescence" of non-labeled biological molecules. In these - frequently appearing - cases the wanted signal of fluorescently labeled material will be superimposed by artifacts, making a proper data interpretation rather difficult. However, the FCS⁺plus' abilities of decomposing a fluorescence signal into the molecular species' fractional contributions enables a sophisticated consideration of unwanted interferences.

Fluorescence correlation spectroscopy (FCS) has evolved to a valuable tool for biomolecular analysis on the single molecule level. Measurements on a single molecule level can only be performed if the measurement volume is small enough to contain on average only very few molecules. Common FCS-systems are therefore based on a confocal geometry in which a laser spot is focused into a liquid sample. This illumination concept in combination with a pinhole in the detection path leads to an observation volume in the order of one femtoliter. On the other hand, many biomolecular interactions need to be measured on surfaces. To study such interactions or the fluctuating signal of surface bound molecules itself, as for instance during single molecule enzyme catalysis, evanescent field based excitation seems advantageous as compared to confocal FCS. We discuss different schemes for evanescent field FCS and present an efficient excitation-detection scheme in an objective-based TIR-FCS configuration.

Fluorescence correlation spectroscopy (FCS) is an important technique for studying analyte molecules on a single molecule level in solution. The core molecular characteristic that is addressed by FCS is the translational diffusion coefficient of the analyte molecules, which can be used for studying molecular binding interactions or conformational changes of macromolecules. We present a thorough theoretical analysis of the FCS technique, paying special attention to the various frequently occurring technical artifacts. Particularly, we consider the influence of refractive index mismatch, cover-slide thickness, fluorescence anisotropy, optical adjustment, and optical saturation on the measured autocorrelation curve (ACF). The impact of these factors on the apparently determined diffusion coefficient is quantitatively evaluated. Extensive experimental results are presented demonstrating the theoretically predicted effects and dependencies.

Synthesis of the cellular 'energy currency' ATP is catalyzed by membrane-bound F₀F₁-ATP synthases. The chemical reaction at three binding sites in the F₁ part is coupled to proton translocation through the membrane-integrated F₀ part by an internal rotation of subunits. We examined the rotary movements of the ε-subunit of the 'rotor' with respect to the b-subunits of the 'stator' by single-molecule fluorescence resonance energy transfer (FRET). Rotation of ε during ATP hydrolysis is divided into three major steps with constant FRET level corresponding to three binding sites. Different catalytic activities of the individual binding sites were observed depending on the relative orientation of the 'rotor'. Computer simulations of the FRET signals and non-equally distributed orientations of ε strongly corroborate asymmetry of catalysis in F₀F₁-ATP synthase.

Semiconductor quantum dots (QD) are nanometer size fluorophores with improved brightness, resistance against photobleaching and narrow emission bands. These properties make QDs ideal for ultrasensitive imaging of biomolecules in living cells, in multiplexed format. By conjugating QDs with a delivery agent such as TAT peptide and a target-recognition element such as an antibody, we have delivered and imaged target-specific fluorescent probes in living cells. In this work, we demonstrate staining of actin filaments in living Human Dermal Fibroblast (HDF) cells using QD probes functionalized with monoclonal actin antibody. Actin probes were developed by coupling streptavidin coated QDs (λ_em = 605 nm, QDC Corp.) to biotinylated monoclonal β-actin antibody. Antibody molecules on QDs were conjugated with the TAT peptide. Finally, HDF cells were incubated with the QD-actin antibody-TAT construct. As expected, the characteristic fine streaks of actin filaments were observed in the cells and on the periphery of the cells, similar to phalloidin staining of actin filaments in fixed cells. Using a similar approach, one may image cellular components, proteins or nucleic acids, in a living cell, in real time.

The effect of DC electric field strength on in vitro actomyosin motility was examined. Rabbit skeletal muscle heavy meromyosin (HMM) was adsorbed to nitrocellulose-coated glass, and the myosin driven movement of fluorescently labeled actin filaments was recorded in the presence of 0 to 9000 V m^-1 applied DC voltage. The applied electric field resulted in increased filament velocity and oriented actin movement, with leading heads of filaments directed towards the positive electrode. Velocity (v) was found to increase moderately with electric field strength at applied fields up to ~ 4500 V m^-1 (Δv/ΔE = 0.037 μm² V^-1sec^-1), and then increased at a more rapid rate (Δv/ΔE = 0.100 μm² V^-1sec^-1) at higher field strengths up to 9000 V m^-1. The electrophoretic effect caused up to 70% of actin motion to be oriented within 30 degrees of the positive electrode, with the largest effect observed using an applied field of 6000 V m^-1. Higher electric field strengths caused filament breakage.

We report on the application of a novel fluorescence-microscope based scanning device with single molecule sensitivity to microarray readout. The device is based on a CCD camera operated in time delay and integration (TDI) mode synchronized with the movement of a sample scanning stage, enabling continuous data acquisition. The implementation of a focus hold system keeps the sample in focus during scanning. Results from ultra-sensitive high-resolution microarray readout provide clear evidence for the superiority of the novel detection method over conventional microarray readout systems in particular regarding to sensitivity and dynamic range. Minute sample amount and lacking amplification methods will demand for this ultra-sensitive readout technology in future genomic and proteomic research.

Many biological processes are accompanied by changes in the orientation of membrane-embedded molecules. Fluorescence polarization microscopy (FPM) is an optical technique that can be used to determine how the transition dipole of a fluorophore is oriented with respect to the membrane and therefore may detect these changes. The FPM experimental setup is essentially an anisotropy measurement executed on a microscope. A linear polarizer is placed in both the excitation and emission light paths of an inverted microscope, and images are taken with the polarizers parallel or orthogonal. Intensity measurements are recorded at regions on the sample and compared with theoretical predictions to validate a model of dye orientation. We have applied FPM to determine the orientation of 1,2-Dioleoyl-sn-Glycero-3-Phosphoethanolamine-N-(Lissamine Rhodamine B Sulfonyl) (PE-rhodamine) in pure 1-Stearoyl-2-Oleoyl-sn-Glycero-3-Phosphocholine (SOPC) giant unilamellar vesicles (GUVs). Vesicles are widely used as model membranes, and GUVs are particularly useful as cellular models because of their large diameter size (~10-40 μm). A model for membrane dye orientation follows theory developed previously and indicates that PE-rhodamine molecules orient at ~31° with respect to the membrane. A computational routine was developed to minimize deviations between predicted and experimental results. We have also applied FPM to study the orientation of 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI) in GUVs. Preliminary results indicate that DiI is oriented at ~23°.

A microdroplet or a latex microsphere often acts as an optical cavity that supports Morphology Dependent Resonances (MDRs) at wavelengths where the droplet circumference is an integer multiple of the emission wavelength. Enhanced radiative energy transfer through these optical resonances can also be utilized as a transduction mechanism for chemical and biological sensing. Enhancement in radiative energy transfer is observed when a donor/acceptor pair is present in the resonant medium of a microcavity. Here, we demonstrate avidin-biotin binding and its detection through a FRET pair as a potential application for ultra-sensitive detection for fluoroimmunoassays. The binding interaction between the biotinylated fluorescent beads (donor) and streptavidin-Alexa Fluor 555 (acceptor) conjugate was used to observe the energy transfer between the dye pairs. Strong coupling of acceptor emission into optical resonances shows that the energy transfer is efficiently mediated through these resonances.

A capillary fluorometer was constructed using a 2 mW, 365 nm ultraviolet (UV) light emitting diode (LED) as the excitation source and a new-generation high-gain (3×10⁸) channel photomultiplier tube. The use of a LED permitted rapid pulsing of the excitation source so that the instrument could be employed for time-resolved fluorescence (TRF) applications. A detection limit of ~2×10⁸ molecules of BHHT (4,4’-bis (1",1",1",2",2",3",3"-heptafluoro-4",6"-hexanedion-6"yl)-o-tephenyl)-Eu (III) were resolved within a 1.25 nanoliter volume at a S/N ratio of 3:1. Ultimate sensitivity of the system was compromised due to visible luminescence emitted by the UV LED, centred around 550 nm extending to > 700 nm and 2nd-order exponentially decaying with lifetimes of 40 μs and 490 μs.

Hyper-Rayleigh scattering is used to measure the nonlinear optical properties of collagen type I in acetic acid solution. We find that the first hyperpolarizability unexpectedly drops with the increase of the concentration of collagen in solution. Circular dichroism absorption spectroscopy and UV absorption measurements indicate that the helical conformation remains stable, and the changes of nonlinear optical properties of collagen molecules are, most probably, due to the formation of some supramolelcular structures of collagen in solution.

Non-invasive monitoring of cellular metabolism offers promising insights into areas ranging from biomarkers for drug activity to cancer diagnosis. Fluorescence spectroscopy can be utilized in order to exploit endogenous fluorophores, typically metabolic co-factors nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FAD), and estimate the redox status of the sample. Fluorescence spectroscopy was applied to follow metabolic changes in epithelial ovarian cells as well as bladder epithelial cancer cells during treatment with a chemopreventive drug that initiates cellular quiescence. Fluorescence signals consistent with NADH, FAD, and tryptophan were measured to monitor cellular activity, redox status, and protein content. Cells were treated with varying concentrations of N-4-(hydroxyphenyl) retinamide (4-HPR) and measured in a stable environment with a sensitive fluorescence spectrometer. A subset of measurements was completed on a low concentration of cells to demonstrate feasibility for medical application such as in bladder or ovary washes. Results suggest that all of the cells responded with similar dose dependence but started at different estimated redox ratio baseline levels correlating with cell cycle, growth inhibition, and apoptosis assays. NADH and tryptophan related fluorescence changed significantly while FAD related fluorescence remained unaltered. Fluorescence data collected from approximately 1000 - 2000 cells, comparable to a bladder or ovary wash, was measurable and useful for future experiments. This study suggests that future intrinsic biomarker measurements may need to be most sensitive to changes in NADH and tryptophan related fluorescence while using FAD related fluorescence to help estimate the baseline redox ratio and predict response to chemopreventive agents.

Fluorescence lifetime imaging is independent of signal intensity and is thus efficient and robust. Additionally, lifetime can be used to differentiate fluorophores and sense fluorophore micro-environment change. A time-resolved optical system is usually used to measure fluorescent decay kinetics, and then one fits the decay to get lifetime. Since the system impulse response function (IRF) is finite, it impacts lifetime fitting. Deconvolution of the IRF can diminish its impact. In thick tissues, light diffusion due to scattering is also convolved with the fluorescence decay. One can recover the decay using an inversion algorithm. However, processing data in this way is computationally intensive and therefore not practical for real time imaging. We present here results of our studies on the IRF impact to fluorescence lifetime fitting in a turbid medium over a wide range of parameters, using a unique time-domain imaging system. Fluorophores were submerged inside a turbid medium that models tissue. Analytical analysis and computation show that when the lifetime is 1.5 times larger than the FWHM of system IRF, reasonable fluorescence lifetimes can be obtained by fitting the decay tail without taking into account IRF. For small source-fluorophore-detector separation, the effect of optical diffusion on the lifetime fitting is also negligible. This gives a guidance of system precision limit for fluorescence lifetime imaging by fast tail fitting. Experimental data using a fs laser with a streak camera and a pulsed diode laser with PMT-TCSPC for ICG, Cy5.5, and ATTO 680 support the theoretical results.

Microparticles including biological cells were trapped and manipulated using a continuous wave tweezer and a femtosecond laser tweezer. The difference of the optical trapping force between CW and femtosecond optical tweezers increased as the particle size decreased, possibly due to the self-focusing effect of the ultrashort pulses. Also, the white damage spots were generated near the focus during femtosecond optical trapping of biological cells even with extremely low average power. The instantaneous optical damage threshold was measured as a function of the trap depth as well. These results may help to optimize the optical trapping of biological cells using femtosecond lasers.

The aim of our work is to develop new optical tools to quantify parameters that may enter into models of cell motion in response to chemical gradients (chemotaxis). Dictyostelium discoidium is a well-known model organism for studying chemotaxis. We have developed a technique for manipulating Dictyostelium cells directly using a holographic laser tweezer array. Using this technique we have perturbed crawling Dictyostelium cells by changing their direction of motion. After tens of seconds, the cells generate protrusions perpendicular to the rotated polarization as they reorient in the direction of the local cAMP gradient. Here we describe how such micromanipulation may be used to test proposed biochemical pathways and their connection to mechanical deformations.

Optical tweezers (OT) rely on the radiation pressure to trap and manipulate microscopic particles and living microorganisms. Because the optical forces vary from hundreds of femto to tens of pico Newtons, OT can be used as an ultra sensitive force measurement tool to study interactions involving very small forces. We use a double tweezers to perform ultra sensitive measurement of the force due to the scattering of light as a function of its wavelength, in other words, to perform a Force Spectroscopy. Our results show not only the Mie resonances but also a selective coupling to either the TE, TM or both microsphere modes using the light polarization and the beam positioning. Mie resonances have usually been observed by scattering measurements. Very few reports of levitation experiments observed these resonances directly through the force. The double tweezers system has the advantage and flexibility of a stable restorative force measurement system. The experimental results show excellent agreement with Gaussian shaped beam partial wave decomposition theory. The understanding of the optical scattering forces in dielectric microspheres under different incident beam conditions is important as they have been used as the natural force transducer for mechanical measurements. Our results show how careful one has to be when using optical force models for this purpose. The Mie resonances can change the force values by 30-50%. Also the results clearly show how the usually assumed azimuthal symmetry in the horizontal plane no longer holds because the beam polarization breaks this symmetry.

The Anti-Brownian ELectrophoretic trap (ABEL trap) is a new device that allows a user to trap and manipulate fluorescent objects as small as 20 nm freely diffusing in solution. We describe in detail how to build an ABEL trap.

We use spatially sculptured light for user-interactive micromanipulation of mixed yeast cell populations to analyze growth behavioural patterns. There is negligible absorption in the near-infrared region of the light spectrum making it suitable for direct manipulation of individual cells in a growing population. Rather than using a single-beam optical trap, multiple cells are manipulated using a system based on the Generalized Phase Contrast (GPC) method, which allows arbitrary trapping configurations i.e. control over the number of traps, and the size/shape of each trap. This enables the cells to be selectively trapped in all three-dimensions (3D) and manipulated in real-time while under direct observation. Here, we impose controlled experiments using these multiple 3D optical traps to show the alteration of growth patterns in mixed cultures of Saccharomyces cerevisiae and Hanseniaspora uvarum experiencing spatially constrained conditions.

We present a combination of nonlinear microscopy, laser nanosurgery and optical trapping applied to the 3D imaging and manipulation of intracellular structures in live cells. We use Titanium-sapphire laser pulses for a combined nonlinear microscopy and nanosurgery on microtubules tagged with green fluorescent protein (GFP) in fission yeast. The same laser source is also used to trap small round lipid droplets naturally present in the cell. The trapped droplets are used as handles to exert a pushing force on the nucleus, allowing for a displacement of the nucleus away from its normal position in the center of the cell. We show that nonlinear nanosurgery and optical manipulation can be performed with sub-micrometer precision and without visible collateral damage to the cell. We present this combination as an important tool in cell biology for the manipulation of specific structures in alternative to genetic methods or chemical agents. This technique can be applied to several fundamental problems in cell biology, including the study of dynamics processes in cell division.

CytometryML is a proposed new Analytical Cytology (Cytomics) data standard, which is based on a common set of XML schemas for encoding flow cytometry and digital microscopy text based data types (metadata). CytometryML schemas reference both DICOM (Digital Imaging and Communications in Medicine) codes and FCS keywords. Flow Cytometry Standard (FCS) list-mode has been mapped to the DICOM Waveform Information Object. The separation of the large binary data objects (list mode and image data) from the XML description of the metadata permits the metadata to be directly displayed, analyzed, and reported with standard commercial software packages; the direct use of XML languages; and direct interfacing with clinical information systems. The separation of the binary data into its own files simplifies parsing because all extraneous header data has been eliminated. The storage of images as two-dimensional arrays without any extraneous data, such as in the Adobe Photoshop RAW format, facilitates the development by scientists of their own analysis and visualization software. Adobe Photoshop provided the display infrastructure and the translation facility to interconvert between the image data from commercial formats and RAW format. Similarly, the storage and parsing of list mode binary data type with a group of parameters that are specified at compilation time is straight forward. However when the user is permitted at run-time to select a subset of the parameters and/or specify results of mathematical manipulations, the development of special software was required. The use of CytometryML will permit investigators to be able to create their own interoperable data analysis software and to employ commercially available software to disseminate their data.

The Multifunctional Staring Mode Microscope was developed to permit three modes of imaging for cell counting in mouse embryos: Optical Quadrature, Differential Interference Contrast (DIC), and Fluorescence Imaging. The Optical Quadrature Microscope, consisting of a modified Mach-Zender Interferometer, uses a 632.8 nm laser to measure the amplitude and phase of the signal beam that travels through the embryo. Four cameras, preceded by multiple beamsplitters, are used to read the four interferograms, which are then combined to produce an image of the complex electric field amplitude. The phase of the complex amplitude is then unwrapped using a 2-D phase unwrap algorithm and images of optical path length are produced. To combine the additional modes of DIC and Fluorescence Imaging with the Optical Quadrature Microscope, a 632.8 nm narrow bandpass beamsplitter was placed at the output of the microscope. This allows the laser light to continue through the Mach-Zender while all other wavelengths are reflected at 90 degrees to another camera. This was effective in combining the three modes as the fluorescence wavelength for the Hoechst stain is well below the bandpass window of the beamsplitter. Both live and fixed samples have been successfully imaged in all three modes. Accuracy in cell counting was achieved by using the DIC image for detecting cell boundaries and the Optical Quadrature image for phase mapping to determine where cells overlap. The final results were verified by Hoechst fluorescence imaging to count the individual nuclei. Algorithms are currently being refined so larger cell counts can be done more efficiently.

Detailed information on cellular and sub-cellular interactions can be extracted from large-scale data sets through the application of image processing and analysis techniques from computer vision and pattern recognition. An automated, high-speed method for analysis of cellular systems in 2D includes boundary analysis of the cells and may be extended to texture (content) analysis or further. The overall goal of such analysis is to reach conclusions as to the physiological state and behavior of the cells. In this paper, we focus on shape analysis of cells, as shape is an effective factor for quantification of the many apparent physiological changes. We explore shape analysis techniques, including geometric (regular), Zernike, and Krawtchouk moment invariants. We also report on our investigation of the effects of resolution changes (in imaging systems) on the descriptors of cell shape in terms of stability and consistence of these moment invariants. Our results show that Krawtchouk moment invariants are better cell shape descriptors compared to geometric moment invariants in low resolution images.

A novel data mining program called “subtractive clustering” picks out the most important differences between two or more flow cytometry listmode data files. While making no assumptions about the data, the program uses a variable weight and skew metric in the determination of bin size allowing for subtractive clustering of data without the need for bit-reduction or projection. In contrast, other subtraction methods, such as channel-by-channel subtraction, are dependent upon dimensionality and resolution, which can lead to an overestimation of positive cells because they do not account for the overall distribution of the test and control data sets. By taking into account human visual inspection of the data it is possible for the experimenter to choose an optimal subtraction by choosing an appropriate weight and skew metric, but without allowing direct modification of the results. By maximizing a bin size which can still differentiate clusters, it is possible to minimize computation while still removing data. The choice of control weight allows for different levels of bin destruction during the subtraction stage, the smaller the number the more conservative the subtraction, the larger, the more liberal. Three data sets illustrate full dimensional subtraction, single step biological data and multi-stage subtraction to show definitive test results. Subtractive clustering was able to conservatively remove control information leaving populations of interest. Subtractive clustering provides a powerful comparison of clusters and is a first step for finding non-obvious (hidden) differences and minimizing human prejudice during the analysis.

The conversion of genomic sequences into digital genomic signals offers the possibility to use powerful signal processing methods for the analysis of genomic information. The study of genomic signals reveals local and global features of chromosomes that would be difficult to identify by using only the symbolic representation used in genomic data bases. The paper presents a study of HIV variability using standard 'wet' methods of nucleotide sequence analysis, corroborated with IT techniques based on the genomic signal approach. Specifically, Independent Component Analysis is used to characterize the variability defining the F subtype HIV strains isolated in Romania.

Recently, these authors developed a heterogeneous, one-level image classifier (C_H) based on morphological descriptors from direct domain analysis (spatial differentiation), fractal analysis and “spectrum enhancement” (a kind of non-linear filtering). Classifier C_H was applied to epi-fluorescence microscope images of cytoskeletal microtubules and was trained to recognize structural alterations of the cytoskeleton in various circumstances. The application dealt with images of rat hepatocytes (rh). The scope of this paper is twofold: a) to investigate different classifier architectures, which include the multiobjective optimization of some image analysis parameters by means of suitable algorithms; b) to apply said classifiers to new sets of images obtained from mouse fibroblasts (mf) and HepG2 (hg) cells. Image sets from control and treated cell cultures are analyzed. Classifier C_H is applied to mf microtubules. A new classifier entirely relying on “spectrum enhancement” (although on different descriptors) is developed and applied to rh and hg images. From the latter classifier, by bringing in descriptors from direct domain and fractal analysis, a hierarchical classifier is derived and applied to rh images. Results are compared. Classifier performance is expressed in terms of sensitivity, specificity and information contents of the first three principal components.

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 optimised to preserve visual quality but not necessarily the integrity of the scientific data. Here, we propose three observer-independent compression algorithms, designed to preserve information contained in the images. These algorithms were constructed using signal to noise ratio (SNR) computed from a single image as a quality measure to establish which image components may be discarded. Signal to noise ratio (SNR) was used in this study to construct three lossy compression techniques, which preserve information contained in the images. The compression efficiency was measured as a function of image brightness (and SNR). Furthermore, the alterations introduced by compression were estimated using brightness histograms (earth’s mover distance algorithm) and textures (Haralick parameters).

In previous publications we have shown that we can perform enzymatic reactions in nanoarrays by means of a microarray-reader based on a conventional microscope. In this publication we report on a modification of this system in order to monitor the aggregation kinetics of the natively unfolded protein α-synuclein. We describe the motivation for this development, the problems associated with the miniaturization of the aggregation assay, and the validation of our modifications.

Miniaturization and integration of biosensor platforms is appealing due to smaller reaction volumes, larger numbers of detection sites and integration of various functionalities. Proper design of integrated biosensors is crucial in such systems due to limitation in resources such as power, chip area and cost. The optimal design involves determining the required sensor metrics and achieving these metrics with minimum use of the available resources. The system-level requirements of various biosensor arrays are discussed in this paper. We will show here, that while in certain applications, the best sensor performance in terms of signal-to-noise ratio (SNR) or dynamic range (DR) is desirable, in others, these metrics can be traded off with power, area and ease of design and implementation. As a practical example, the design of a high DR sensor array for bioluminescence detection is considered. Various high DR schemes are qualitatively compared in order to determine the advantages and disadvantages of each scheme in terms of SNR and power consumption. Two schemes are shown to be most suitable for such applications: synchronous self-reset with residue readout and read-self reset. The SNR and suitable applications of these techniques are compared in greater detail through behavioral simulations.

Metal nanoparticles represent an interesting tool for bioanalytics. Due to their small size, attachment to biomolecules does not interfere significantly with specific molecular binding. Therefore particles can be applied as label in affinity assays (e.g., DNA hybridization), using setups with high parallelization. Beside this rather passive use of nanoparticles, these structures can also be utilized as 'nano antenna' for the conversion of laser light pulses into heat. Using DNA-modified particles sequence-specific bound to DNA, a novel restriction technique is in development that applies this conversion for local DNA destruction. Metal nanoparticles combine the ability for highly precise positioning (due to specific molecular binding) with the possibility of optical access in a bright-field mode. They exhibit an interesting potential for spanning the gap between the macroscopic technical environment and the molecular scale, thereby enabling a true integration of nanoscale constructs with today’s technology.

The objective of this research is to use the displacements of a polystyrene microsphere trapped by an optical tweezers (OT) as a force transducer in mechanical measurements in life sciences. To do this we compared the theoretical optical and hydrodynamic models with experimental data under a broad variation of parameters such as fluid viscosity, refractive index, drag velocity and wall proximities. The laser power was measured after the objective with an integration sphere because normal power meters do not provide an accurate measurement for beam with high numerical apertures. With this careful laser power determination the plot of the optical force (calculated by the particle displacement) versus hydrodynamic force (calculated by the drag velocity) under very different conditions shows an almost 45 degrees straight line. This means that hydrodynamic models can be used to calibrate optical forces and vice-versa. With this calibration we observed the forces of polystyrene bead attached to the protozoa Leishmania amazonensis, responsible for a serious tropical disease. The force range is from 200 femto Newtons to 4 pico Newtons and these experiments shows that OT can be used for infection mechanism and chemotaxis studies in parasites. The other application was to use the optical force to measure viscosities of few microliters sample. Our result shows 5% accuracy measurements.

Single molecule fluorescence microscopy is a relatively novel technique that is used, for example, to study the behavior of individual biomolecules in cells. Since a single molecule can move in all three dimensions in a cellular environment, the three dimensional tracking of single molecules can provide valuable insights into cellular processes. It is therefore of importance to know the accuracy with which the location of a single molecule can be determined with a fluorescence microscope. We study this performance limit of a fluorescence microscope from a statistical point of view by deriving the Fisher information matrix for the estimation problem of the location of the single molecule. In this way we obtain a lower bound on the standard deviation of any reasonable (unbiased) estimation method of the location parameters. This lower bound provides a fundamental limit on the accuracy with which a single molecule can be localized using a fluorescence microscope and is given in terms of such quantities as the photon detection rate of the single molecule, the acquisition time, the numerical aperture of the objective lens etc. We also present results that show how factors such as noise sources, detector size and pixelation deteriorate the fundamental limit of the localization accuracy. The present results can be used to evaluate and optimize experimental setups in order to carry out three dimensional single molecule tracking experiments and provide guidelines for experimental design.

DNA restriction is a basic method in today’s molecular biology. Besides application for DNA manipulation, this method is used in DNA analytics for 'restriction analysis'. Thereby DNA is digested by sequence specific restriction enzymes, and the length distribution of the resulting fragments is detected by gel electrophoresis. Differences in the sequence lead to different restriction patterns. A disadvantage of this standard method is the limitation to a small set of fixed sequences, so that the assay can not be adapted to any sequence of interest (e.g. SNP). We designed a scheme for DNA restriction in order to provide access to any desired sequence, based on laser light conversion on sequence-specific positioned metal nanoparticles. Especially gold nanoparticles are known for their interesting optical properties caused by plasmon resonance. The resulting absorption can be used to convert laser light pulses into heat, resulting in nanoparticle destruction. We work on the combination of this principle with DNA-modification of nanoparticles and the sequence-specific binding (hybridization) of these DNA-nanoparticle complexes along DNA molecules. Different mechanisms of light-conversion were studied, and the destructive effect of laser light on the nanoparticles and DNA is demonstrated.

Genomic research is nowadays based on high throughput analytical techniques. Microarray assays are commonly used to determine DNA content of heterogeneous mixtures up to full genome scale. For low amounts of sample material this method, however, requires time consuming and error prone PCR based amplification steps. Here, we present an assay with the ability to characterize the cDNA content of a low number of cells using ultra-sensitive fluorescence microscopy. For detection, a newly developed chip reader was used. The instrument is based on a modified fluorescence microscope with single dye sensitivity. The highly sensitive CCD detector is operated in TDI mode, which allows avoiding overhead times for sample positioning and signal integration. This enabled the scanning of areas of 1x0.2cm² within 50 seconds at a pixel size of 200nm. At this resolution, single dye molecules can be reliably detected with an average signal to background noise ratio of ~42. For DNA hybridization experiments, oligonucleotides were covalently linked to a newly developed aldehyde surface. Subsequently, fluorescence labeled complementary oligonucleotides were hybridized at various concentrations. Down to femto-molar oligonucleotide concentrations, specific signals were detected. At 10fM concentration signals of individual specifically hybridized oligonucleotide molecules were resolvable. This assay provides the conceptual basis for expression profiling of low amounts of sample material without signal amplification.

We have used single molecular-pair fluorescence resonance energy transfer (FRET) to probe the conformation of a RNA loop-loop “kissing complex” formed by two small RNA hairpins (R1inv and R2inv) derived from Escherichia coli (ColE1) plasmid-encoded transcripts, RNA I and RNA II. This RNA kissing complex is a critical intermediate in a multi-step hybridization pathway which controls plasmid replication. Biotinylated RNA molecules were labeled with donor and acceptor dyes on their 5' ends and immobilized on a biotinylated surface using streptavidin. Fluorescence from the donor and acceptor dyes was collected and measured by photon counting detectors in two spectrally separated channels in a customized confocal microscope. Quantitative measurement of intramolecular distances between 5' ends of the RNA was obtained using donor-only single molecule FRET. This donor-only single molecule FRET technique is described in detail and validated through determination of the distance between 5' ends of 8mer A-form RNA helices of known structure.

A new multi-focus multi-confocal set-up for performing fluorescence spectroscopy of single molecules in solution is presented. The ultimate goal of the set-up is to track individual molecules during diffusion in solution, when all standard methods of trapping such as optical tweezers or dielectrophoretic traps fail. We present here a detailed description of the experimental setup and show first experimental results.

Currently, pathologists rely on labor-intensive microscopic examination of tumor cells using century-old staining methods that can give false readings. Emerging BioMicroNanotechnologies have the potential to provide accurate, realtime, high throughput screening of tumor cells without invasive chemical reagents. These techniques are critical to advancing early detection, diagnosis, and treatment of disease. Using our award-winning Hyperspectral Inceptor to rapidly assess the properties of cells flown through a micro/nano semiconductor device, we discovered a method to rapidly assess the health of a single mammalian cell. The key discovery was the elucidation of biophotonic differences in normal and cancer cells by using intracellular mitochondria as biomarkers for disease. This technique holds promise for detecting cancer at a very early stage and could nearly eliminate delays in diagnosis and treatment.

A population of identical proteins has the same amino acid sequence, but there may be subtle differences in local folding that lead to variations in activity. Single molecule studies allow us to understand these subtle differences. Single molecule experiments are usually time consuming and difficult because only a few molecules are observed in one experiment. To address this problem, we have developed an assay where we can simultaneously measure the activity of multiple individual molecules of a protease, α-chymotrypsin. The assay utilizes a synthetic chymotrypsin substrate that is non-fluorescent before cleavage by chymotrypsin, but is intensely fluorescent after. To study the activity of individual enzymes, the enzyme and substrate are encapsulated in micron-sized droplets of water surrounded by silicone oil. On average, each micro-droplet contains less than one enzyme. The fluorescence of these droplets is recorded over time using a microscope and a CCD camera system. Software tracks individual droplets over time and records fluorescence. The kinetics of individual chymotrypsin molecules is calculated through the increase of fluorescence intensity of the same individual droplet over time. The activity profiles of the individual enzymes and the bulk sample of the enzyme are very similar. This validates the assay and demonstrates that the average of a few individual molecules can be representative of the behavior of the bulk population.

The completion of the human genome sequence enables the discovery of genes involved in common human disorders. The successful identification of these genes is dependent on the availability of informative sample sets, validated marker panels, a high-throughput scoring technology, and a strategy for combining these resources. We have developed a universal platform technology based on mass spectrometry (MassARRAY) for analyzing nucleic acids with high precision and accuracy. To fuel this technology, we generated more than 100,000 validated assays for single nucleotide polymorphisms (SNPs) covering virtually all known and predicted human genes. We also established a large DNA sample bank comprised of more than 50,000 consented healthy and diseased individuals. This combination of reagents and technology allows the execution of large-scale genome-wide association studies. Taking advantage of MassARRAY’s capability for quantitative analysis of nucleic acids, allele frequencies are estimated in sample pools containing large numbers of individual DNAs. To compare pools as a first-pass “filtering” step is a tremendous advantage in throughput and cost over individual genotyping. We employed this approach in numerous genome-wide, hypothesis-free searches to identify genes associated with common complex diseases, such as breast cancer, osteoporosis, and osteoarthritis, and genes involved in quantitative traits like high density lipoproteins cholesterol (HDL-c) levels and central fat. Access to additional well-characterized patient samples through collaborations allows us to conduct replication studies that validate true disease genes. These discoveries will expand our understanding of genetic disease predisposition, and our ability for early diagnosis and determination of specific disease subtype or progression stage.

We previously reported the application of spinning-disk interferometry, implemented in a compact optical sensor format called the BioCD, in the detection of antigen-antibody recognition. The BioCD consists of interferometers micro-fabricated on the surface of a 2” laser mirror disk, which can spin up to 6000 rpm resulting in high data acquisition rates. The interferometric elements are fabricated by evaporating gold ridges on the mirror substrate operating in the linear sensitivity regime of the interferometer defined as quadrature. Antibodies or proteins are immobilized on the gold interferometric structures through alkanethiols, and the target molecules are immobilized by application of reagents or samples to the disk while it is spinning. The centrifugal force distributes the sample over the sensor surface, causing a change in the optical phase of the interferometric elements, which is detected in real time using a lock-in amplifier with small detection bandwidth. We detected the binding of Mouse IgG by immobilized Anti-Mouse IgG using the BioCD with a detection limit of 1 ng/ml and low non-specific binding. Furthermore, the selectivity of specific binding was found to be greater than 1 in 10000, determined using the response curve of the BioCD to exposures of specific and non-specific analytes of varying concentrations. This opens up the possibility of simultaneous detection of several analytes with the same sensor while maintaining high selectivity. In this paper we demonstrate simultaneous detection of Rabbit and Mouse IgG on the same disk. The sensitivity limit for multi-analyte detection remains the same as that for a single analyte. In addition to the ability to do simultaneous detection, the current detection scheme presents a way to reference the results of one track with respect to others, thus increasing the reliability of the data. Used in conjunction with high-density protein patterning techniques, the BioCD has the potential to be a highly multiplexed label-free high-speed sensor.

Optical microspectrometers are potentially important components for improving the functionality of lab-on-a-chip systems used for detection and identification of cells and other biological material. For disposable and portable applications, monolithic integration of the microfluidic channels, optical waveguides and diffraction grating is desirable. This paper presents the design and simulation of a transmission grating that can be integrated with microfluidic channels for on-chip fluorescence detection. The grating design features two stigmatic points and large facet sizes that can be easily fabricated in low-cost polymers. Scalar simulations predict grating efficiencies greater than 74% for wavelengths from 500 nm to 700 nm in the -2 diffraction order.

Current proteomics experiments often rely upon printing techniques such as ink jet, pin, or quill arrayers that were developed for the creation of cDNA microarrays. These techniques often do not meet the spotting requirements needed for successful high throughput protein identification and profiling. The Naval Research Laboratory has developed an alternative to these commercially available arrayers that does not rely upon a solid pin or capillary-based fluidics. This presentation describes experiments demonstrating that biological laser printing, or BioLP, is capable of depositing microarrays of proteins rapidly and efficiently. This technique utilizes a focused laser pulse to obtain micron-scale resolution rather than a pin or orifice, thereby eliminating clogging and protein loss commonly encountered in commercially available printers. The speed and spot-to-spot reproducibility of the printer is comparable to other techniques, while the minimum spot diameter and volume per printed droplet is significantly less at 30 microns and ~500 fL, respectively. The transfer of fluid by BioLP occurs through a fluid jetting mechanism, as observed by high-speed images of the printing process. In addition, printed biotinylated bovine serum albumin is identified through immunoassay and observed by fluorescent detection. These results indicate that BioLP holds promise as a novel protein printer for use in a wide range of applications in the proteomics field.

An effective high throughput screening technique is described for the rapid analysis of solid-supported libraries to identify selective chelating agents for metals, focusing on those possibly present in radioactive dispersal devices. Micro X-ray Fluorescence (MXRF) is the method of choice. MXRF is a rapid and sensitive technique which allows the measurement of metal-chelator complexes without significant sample preparation. For this study we have employed commercial peptide libraries and have examined factors such as side-chain charge, oligopeptide length, hydrophobicity, and aromaticity in relation to metal binding. MXRF provides a rapid and quantitative means for screening chelator-ion binding. Initial experiments carried out successfully identified sequences that are selective for Co under certain binding conditions containing possible interferences (e.g. Ca, Fe, Al, Cs, Ir), which could be encountered in our application.

In this paper, we report the preliminary results from a microfabricated substrate system that is amenable to both electromagnetic field-enhanced spectroscopy such as surface enhanced Raman scattering (SERS) and analyte separation and detection. Substrates consisting of arrays of gold post-like and pit-like features of varying pitch on gold substrates were fabricated by electron beam lithography. These substrates were characterized and tested for reproducible SERS activity, as well as evaluated for incorporation into a microfluidic system for separation and identification of components of complex matrices. Identification of analytes relevant to biodetection and biological screening is reported.

Nanoparticles made of lanthanide oxides are promising fluorophores as a new class of tags in biochemistry because of their large Stokes shift, sharp emission spectra, long lifetime and lack of photobleaching. We demonstrate for first time the application of these nanoparticles to the visualization of protein micropatterns. Europium-doped gadolinium oxide (Eu:Gd₂O₃) nanoparticles were synthesized by spray pyrolysis and were characterized by means of laser-induced fluorescent spectroscopy and TEM. Their main emission peak is at 612 nm. And their size distribution is from 5 nm to 500 nm. The nanoparticles were coated with avidin through physical adsorption. Biotinylated Bovine Serum Albumin (BSA-b) was patterned on a silicon wafer using a micro-contact printing technique. The BSA-b - patterned wafer was incubated in a solution containing the avidin-coated nanoparticles. The specific interaction between biotin and avidin was studied by means of fluorescent microscopy and atomic-force microscopy (AFM). The fluorescent microscopic images revealed that the nanoparticles were organized into designated structures as defined by the microcontact printing process - non-specific binding of the avidin-coated nanoparticles to bare substrate was negligible. The fluorescent pattern did not suffer any photobleaching during the observation process which demonstrates the suitability of Eu:Gd₂O₃ nanoparticles as fluorescent labels with extended excitation periods - organic dyes, including chelates, suffer bleaching over the same period. More detailed studies were preformed using AFM at a single nanoparticle level. The specific and the non-specific binding densities of the particles were qualitatively evaluated.