F_oF₁-ATP synthases couple proton translocation with the synthesis of ATP using two rotary motors within the enzyme. To monitor inter-subunit movements during catalysis, we selectively attached two fluorophores to the F₁ part, sulforhodamine B at one of three β-subunits and Cy5 at the γ-subunit. Reassembly with F_o parts embedded in liposomes yielded functional holoenzymes. Fluorescence resonance energy transfer (FRET) was investigated in photon bursts of freely diffusing liposomes with reconstituted ATP synthases using a confocal set-up for single-molecule detection. Incubation with AMPPNP resulted in stable intensity ratios within a burst and three different FRET efficiencies. Upon ATP addition, a repeating sequence of three distinct FRET efficiencies was observed, indicating the stepwise movement of the γ-subunit during ATP hydrolysis. With this single-molecule FRET approach we detected a stepwise rotation of the γ-subunit under conditions for ATP synthesis (i.e. energization of the proteoliposomes by an acid-base-transition). The direction of rotation is opposite to the direction observed during ATP hydrolysis.

We report a new approach to an unidirectional photonic wire based on fluorescent dyes as chromophores and DNA as a rigid scaffold. The physical functioning of the wire is realized by dipole-dipole intreraction, i.e. resonant energy transfer, between chromophores. The use of four dyes (Alexa 430, TAMRA, Cy3.5, and Cy5) with different excited state energies creates an energy cascade constituting the driving force of the energy current and providing the unidirectionality of the device. The unique molecular properties of DNA, its scaffold-like structure, combined with straightforward synthesis methods allowed the engineering of a 30 base pair double-stranded DNA with inter-dye distances of 10 base pairs (3.4 nm), respectively, a range where electronic interactions between the chromophores can be neglected but dipole-dipole induced fluorescence resonance energy transfer (FRET) is expected to be still highly efficient. Steady-state and time-resolved ensemble spectroscopic measurements show an overall energy transfer efficiency of approximately 0.60. That is, the unidirectional transport of photonic energy over a distance of approximately 10 nm and a spectral separation of approximately 250 nm. Furthermore, pulsed diode laser excitation at 440 nm in combination with spectrally resolved fluorescence lifetime imaging microscopy (SFLIM) was applied to characterize the effectiveness of individual photonic wires dispersed on glass coverslips.

We present a new technique for high-resolution colocalization of fluorescent dyes. The technique is based on polarization modulated excitation and spectrally-resolved fluorescence lifetime imaging microscopy (SFLIM) as well as on coincidence analysis of the detected photon counts following pulsed laser excitation. The method takes advantage of single fluorescent dyes that can be efficiently excited by a single pulsed diode laser emitting at 635 nm but differ in their emission maxima, and in their fluorescence lifetime. A combined analysis of the fractional intensities and fluorescence lifetimes recorded on two spectrally-separated detectors enables the classification of the portion of each dye per pixel in a point-spread-function (PSF) image with high accuracy, even though only a limited number (generally a few thousand) photons are detected per single dye. From these portions two separate PSF images are calculated and fitted to two-dimensional (2D) Gaussian functions to localize their centers with a precision of a few nanometers. To reveal the number of absorbing and emitting molecules polarization modulated excitation and coincidence analysis of the detected photon counts is used. We demonstrate that by the use of appropriately selected dyes, the presented technique permits (1) the counting of the number of molecules present in the observation volume, and (2) the determination of the distance between two single molecules down to approximately 30 nm with a precision of approximately 10 nm without any chromatic aberrations. The developed techniques are promising for applications in molecular biology, e.g. to determine the number of polymerase molecules active within a transcription factory and/or to measure their distances to nanscent transcripts.

New strategies for analyzing molecular signatures of disease states in real time using single pair fluorescence resonance energy transfer (spFRET) were developed to rapidly detect point mutations in unamplified genomic DNA (DNA diagnostics). The assay was carried out using allele-specific primers, which flanked the point mutation in the target gene fragment and were ligated using a thremostable ligase enzyme only when the genomic DNA carried this mutation (ligase detection reaction, LDR). We coupled LDR with spFRET to identify a single base mutation in codon 12 of a K-ras oncogene that has high diagnostic value for colorectal cancers. A simple diode laser-based fluorescence system capable of interrogating single fluorescent molecules undergoing FRET was used to detect photon bursts generated from the MB probes formed upon ligation. We demonstrated the ability to rapidly discriminate single base differences in heterogeneous populations having as little as 600 copies of human genomic DNA without PCR amplification. Single base difference in the K-ras gene was discriminated in less than 5 min at a frequency of 1 mutant DNA per 10 normals using only a single LDR thermal cycle of genomic DNA. Real time analyses of point mutations were also performed in PMMA microfluidic device.

Fluorescence correlation spectroscopy (FCS) has developed into a widely used and very successful spectroscopic technique for detecting and quantifying minute concentrations of fluorescing dyes in solution, as well as for obtaining information about molecular parameters such as diffusion coefficients, photophysical transition rates, or chemical reaction kinetics. The standard evaluation method of FCS data is based on the simplified assumption that the convolution of the excitation intensity distribution and the light collection efficiency function is a three-dimensional Gaussian distribution. Although that assumption leads to satisfactory fits of experimental data, the adjustable parameters of this standard model have a rather unphysical meaning. In the present paper, an ab initio approach to modeling FCS curves is developed that is based on exact wave-optical calculations of fluorescence excitation and detection. Comparison with the standard model is made, and the ab initio calculated model curves are used for fitting experimental data and deriving absolute values of diffusion coefficient and concentration.

Here, we report our results on excitation intensity and nanoscale Ag cluster dependent spectral fluctuation dynamics of surface enhanced Raman scattering. We have studied single-Ag-cluster surface enhanced Raman scattering (SERS) intensity fluctuations under low molecule surface coverage of rhodamine 6G (R6G) and cytochrome c. By applying both experimental and theoretical approaches, we observed that spectral fluctuation phenomena are associated with SERS not only from single-molecule loaded nanoclusters but also from submonolayer molecule loaded nanoclusters. The nanoscale confinement of the local electric field enhancement under the laser excitation defines the SERS fluctuation. A new AFM-coupled two-channel photon time-stamping system, enabling in situ correlation of the topographic and spectroscopic information for single nanoparticle clusters, was used to record Raman intensity fluctuation trajectories at sub-μs resolution. Experimentally, we found that SERS fluctuation dynamics are highly inhomogeneous amongst nanocluster interstitial sites. Although the fluctuation above ~50 W/cm² excitation is dominated by photoinduced processes, spontaneous fluctuation can be observed at lower excitation intensity. Although a single Raman-active molecule confined within the volume of an electric field excitation gives a significant Raman spectral fluctuation, observation of the fluctuation alone may not be sufficient in identifying a single-molecule origin of a Raman spectrum. The Raman signal comes predominately from the localized electric field enhancement at interstitial sites, occuring in a very small volume at nanoscale (capable of holding only one or a few molecules), as estimated from finite-element methods simulations of an electric field enhancement using a classical electrodynamics approach. Such a small number of molecules, which are presumably under discrete diffusion and exposed to interactions with a locally strong electric field, results in the observed Raman fluctuation. The fluctuation autocorrelation amplitude is proportional to the reverse number of molecules confined at the volume of the electric field enhancement.

We apply cavity-enhanced frequency modulation absorption spectroscopy (also known as NICE-OHMS) to perform highly sensitive high-resolution spectroscopy of the sixth overtone band of NO near 797 nm. Our spectrometer provides a sensitivity corresponding to a minimum detectable absorption coefficient of 2.5×10^-10 cm^-1Hz^-1/2, which is ~80 times worse than the ideal shot-noise limited sensitivity. Line intensities are measured for the R(7.5), R(9.5), and R(10.5) rovibrational lines in the ²Π _1/2- ²Π_1/2 sub-band. From these line intensities we calculate a total vibrational band intensity of 2.2×10^-6 cm^-2atm^-1 (at 296 K) and a vibrational transition dipole moment of 4.1 μDebye. The measured values are estimated to be accurate to within ±25%.

We propose a new method to detect ultrasmall nonreciprocal phase shifts in solids based on the Sagnac interferometer combined with internal optical modulation of the absorption. The Sagnac interferometer with heterodyne balanced detection is expected to enable shot-noise-limited detection of phase shift as a result of its insensitivity to frequency and amplitude fluctuations of the laser. The low-concentration molecular sample is internally modulated by optical saturation with a pulsed laser. This internal modulation makes the molecular absorption time-dependent, and also removes the possibility of amplitude modulation feedthrough. We describe the design of this experiment and present preliminary characterizations of the noise performance.

The FCS⁺plus family of evaluation tools for confocal fluorescence spectroscopy, which was developed during recent years, offers a comprehensive view to a series of fluorescence properties. Originating in fluorescence correlation spectroscopy (FCS) and using similar experimental equipment, a system of signal processing methods such as fluorescence intensity distribution analysis (FIDA) was created to analyze in detail the fluctuation behavior of fluorescent particles within a small area of detection. Giving simultaneous access to molecular parameters like concentration, translational and rotational diffusion, molecular brightness, and multicolor coincidence, this portfolio was enhanced by more traditional techniques of fluorescence lifetime as well as time-resolved anisotropy determination. The cornerstones of the FCS⁺plus methodology will be shortly described. The inhibition of a phosphatase enzyme activity gives a comprehensive industrial application that demonstrates FCS⁺plus' versatility and its potential for pharmaceutical drug discovery.

Extracellular polymeric substances (EPS) secreted by bacteria have a key role in adhesion and aggregation of bacterial cells on solid surfaces. In the present study, atomic force microscopy (AFM) has been used to study the adhesion propensity of bacterial strain St. guttiformis, and the ultrastructure and distribution of the EPS materials, on hydrophobic poly(tert-butylmethacrylate)(PtBMA) and hydrophilic polystyrene maleic acid (PSMA) surfaces. The results showed that bacterial attachment to the PSMA surface over incubation periods of 24-72 h was insignificant, whereas there was a strong propensity for the bacterial cells to attach to the PtBMA surface, forming multi-layered biofilms. For the PSMA surface, planktonic EPS adsorbed onto the polymeric surface and formed a continuous surface layer. For the PtBMA surface, non-contact mode imaging revealed that capsular EPS on the cell surface exhibited granular structures with the lateral dimensions of 30-50 nm and the vertical roughness of 7-10 nm. Lateral force imaging showed inter-connected elongated features which had lower frictional property compared to the surrounding EPS matrix, suggesting possible segregation of hydrophobic fractions of the EPS materials. The planktonic EPS adsorbed onto the PtBMA surface also showed similar nanometer-scale granular structures and could form stacks up to 150 nm in height. However, lateral force imaging did not show frictional differences, as in the case of capsular EPS. This is attributed to possible differences in the composition of the two EPS materials, and/or greater deformation of the planktonic EPS in the contact imaging mode which may obscure the fine surface features.

We are developing a multi-modal miniature microscope (4M device) for imaging morphology and cytochemistry in vivo and providing better delineation of tumors. The 4M device is designed to be a complete microscope on a chip, including optical, micro-mechanical, and electronic components. It has advantages such as compact size and capability for microscopic-scale imaging. This paper presents the recent imaging experiment of 4M device including trans-illumination imaging, TIR illumination imaging and fluorescent imsging. We built a multi-modal imaging test-bed to demonstrate multi-modality of 4M device. In this paper, we present imaging experiment results by implementing various imaging modality with cervical cancer cells. In order to enhance image contrast, some imaging modality uses cells attached with contrast agency such as silver nano-particles. Imaging results indicate that the 4M prototype can resolve cellular detail necessary for detection of precancer.

The lack of structure-specific contrast limits the usefulness of confocal reflectance microscopy to morphologic investigations at the cellular- and nuclear-level in human and animal skin in vivo. Morphologic and functional imaging at specific organelle- and ultrastructure-levels will require contrast agents that may be used and detected in vivo. High-resolution confocal reflectance imaging is based on the detection of singly back-scattered photons, where contrast is provided by variations in the refractive indices of microstructures. We carried out a quantitative Mie back-scatter analysis and imaging experiments to understand signal detectability of reflectance contrast agents for visualizing human skin and animal microcirculation. When imaging at video-rate with illumination of 10 milliwatts at 1064 nm, we detect 100-10⁴ photons/pixel from the epidermis to dermis, relative to a background of 100 photons; this provides a signal-to-noise ratio of 3-40 and signal-to-background of 1-100. Organelles of size (d) 0.1-1.0 μm with refractive indices (n) of 1.34-1.45 (relative to n=1.34 for epidermis, n=1.38 for dermis) back-scatter 10-10⁴ photons/pixel. Exogenous contrast agents such as liposomes (n=1.41, d=0.7 μm) and polystyrene microspheres (d=0.2-1.0 μm, n=1.57; 100-10⁵ photons/pixel) are detectable and they strongly enhance the contrast of microcirculation in the dermis of Sprague-Dawley rats. Topically applied 5% acetic acid causes the intra-nuclear 30-100 nm-thin chromatin filaments to condense into 1-5 μm-thick strands, increasing back-scattered signal from 100 to 10⁴ photons/pixels, making the nuclei appear bright and easily detectable in basal cell cancers. Such analyses provide a basis for optimizing confocal microscope design for detectability of contrast agents in vivo.

Glioblastoma Multiforme is the most common form of malignant brain tumors and accounts for approximately 25% of all primary brain tumors. Only 5% of these patients survive longer than 2 years. The standard form of treatment is radiation therapy and surgery if the site is accessible. Different forms of adjuvant chemotherapy have been largely proven unsuccessful. Another form of adjuvant therapy, Photodynamic Therapy (PDT), has undergone preliminary trials showing some promising results but at the cost of increased side effects like rise in intracranial blood pressure and neurological deficiency. Apoptotic cell kill used as a biological treatment endpoint can possibly ameliorate these side effects. This study evaluates the significance of apoptotic cell death in the 9L rat gliosarcoma using the aminolevulinic acid (ALA) induced endogenous photosensitizer Protophorphyrin IX (PpIX). A strong influence of drug incubation time with cell kill was observed. The percentage of apoptotic cell death was less than 10% for 2 and 4 hours incubation times and irradiation times ensuring up to 70 and 80% cell kill respectively. Accumulation of PpIX in the mitochondria and cytoplasm was quantified by confocal fluorescence microscopy showing a linear relationship of PpIX fluorescence with concentration. The possibility of an in vitro threshold in the PDT dose is discussed, above which cell repair mechanisms may become exhausted. In conclusion for the range of parameters investigated, apoptotic cell kill may be hard to exploit therapeutically in this tumor model.

The paper presents some new results in the analysis of genomic information at the scale of whole chromosomes or whole genomes based on the conversion into genomic signals. Mainly, the phase analysis -- phase, cumulated phase and unwrapped phase, and the sequence path analysis are presented. The unwrapped phase displays an almost linear variation along whole chromosomes. The property holds for all the investigated genomes, being shared by both prokaryotes and eukaryotes, while the magnitude and sign of the unwrapped phase slope is specific for each taxon and chromosome. Such a behavior proves a rule similar to Chargaff's rule, but reveals a statistical regularity of the succession of the nucleotides -- a second order statistics, not only of the distribution of nucleotides -- a first order statistics. The cumulated phase of the genomic signal of certain prokaryotes also shows interesting specific behavior. The comparison between the behavior of the cumulated phase and of the unwrapped phae across the putative origins and termini of the replichores suggests an interesting model for the structure of chromosomes.

Manipulation of micron-scale silicon particles has been investigated with optical tweezers implemented using a two-dimensional scanning trap driven with acousto-optic modulators. Spheres of latex, Poly(methyl methacrylate) (PMMA), silica, and silver-coated PMMA have been utilized to calibrate transverse trapping forces. The goal of this work is to non-invasively manipulate 10-20 μm silicon-based devices in and around cells.

Fluorescence spectra and lifetime images of cultivated CHO cells incubated with the membrane marker laurdan are reported. The plasma membrane and intracellular membranes were distinguished using illumination of either the cell surface (by an evanescent wave) or of the whole cell. Parameters of membrane dynamics, probed by the generalized prolarization and the effective fluorescence lifetime of laurdan, were correlated with temperature, age and growth phase of the cells.

Both hemostasis and thrombosis occur as a result of platelet adhesion to the subendothelial matrix, platelet activation, and platelet aggregation. The first stage in hemostasis and thrombosis is the binding of the platelet membrane receptor, glycoprotein (GP) Ib-IX complex, to its ligand, von Willebrand factor (VWF), in the subendothelium. In particular, the A1 domain of VWF is responsible for binding GP Ib-IX. After immobilizing A1 on a 2.0 μm diameter polystyrene bead, we optically trapped the bead using a titanium-sapphire laser tuned to 830 nm. The A1-coated bead was then moved towards a transfected Chinese hamster ovary cell which expressed the GP Ib-IX complex, and allowed to adhere to the cell. We subsequently detached the cell from the bead at different constant loading rates, ranging over three orders of magnitude, by using a piezoelectrically-driven translational stage. Displacement of the bead was simultaneously monitored from the trapping center using a quadrant photodetector to determine the force required to detach A1 from GP Ib-IX. These dynamic measurements of unbinding force emphasize the important role that shear rate plays in the initial stage of thrombus formation.

Bacterial adhesion to host tissue is an initial step in the infectious process. Staphylococcus aureus, a major human pathogen, has covalently anchored cell surface adhesins called microbial surface components recognizing adhesive matrix molecules (MSCRAMMs) which mediate specific adhesion to extracellular matrix (ECM) molecules. Understanding MSCRAMM binding is potentially useful in developing effective antibacterial drugs. In this study, optical tweezers were used in conjunction with a quadrant photodetector to measure adhesive forces between MSCRAMMs and surfaces coated with the ECM molecule fibronectin. Using a piezoelectrically driven stage, a fibronectin-coated microsphere adherent to a coverslip was brought into contact with a cell optically trapped at 830 nm. The microsphere was subsequently moved away from the cell, and a quadrant photodiode monitored the cell displacement from the trap center during the detachment process. The photodetector voltage signals were subsequently converted into the adhesive forces between MSCRAMMs and fibronectin based on a calibration using Stoke’s law for viscous drag. Optical detection of the trapped bead displacement allowed us to study both the dynamics of the detachment process and observe the effects of various loading rates. This technique can be extended to identify the contributions of various MSCRAMM domains to adhesion in order to develop new methods of treating infections.

In this work we analyzed the calibration of optical trapping forces. One calibration technique utilizes the controlled motion of a trapped object in a fluid with known viscosity where the trapping force is calculated from the Stokes’ Law based on inertia-free assumptions (i.e., neglecting velocity and acceleration of the trapped object). In our study, we calculated the displacement of the trapped object from the trapping center using Fourier analysis of the equation of motion. Waveforms of different frequencies were used both in theoretical modeling and experiments to control the motion of the trapped object in an aqueous solution. Calibration data obtained experimentally were compared to theoretical results. The dynamic analysis of the trapped object showed that trapping force can significantly differ from theoretically predicted values under inertia-free assumptions. Various factors including type of the waveform used to control the motion of a trapped object during calibration, its frequency, viscosity of the calibration fluid, mass and dimensions of the trapped object, stiffness of the optical trap and frequency response of the equipment used to control the motion of the trapped object contribute to the differences.

Three-dimensional confocal laser scanning microscopy (CLSM) and two-photon excitation microscopy (TPEM) were used to study the response of cellular systems to fuzzy organized nanostructured polyelectrolytes used both as microcontainers and microcarriers for drug delivery. These nanostructured systems are named Nanocapsules and represent a new class of controllable colloids. CLSM and TPEM uniquely allow to follow the fate of encapsulated living cells and to track the pathway of nanocapsules introduced into cellular systems. For the former situation, it will be shown how living cells can be encapsulated and demonstrated the preservation of the metabolic and duplicating activity. In this case the role of the Nanocapsule is as microcontainer endowed of functionalized surface and of protective ability. The latter situation, is related to feeding living cells with Nanocapsules. This experiment serves in elucidating the comprehension of the potential cytotoxicity and of the ability of Nanocapsules to reach specific targets where active compounds can be released. Cellular systems used within this research are Saccharomyces cerevisiae and Paramecium primaurelia living cells. In the case of encapsulation of Saccharomyces cerevisiae living cells, the most relevant result is that, after encapsulation, cells preserve their metabolic activities and they are still able to divide. At this stage is also relevant the utilization of spectroscopic methods like fluorescence lifetime and second harmonic imaging. These hybrid polyelectrolyte-cells can provide a cheap model system in a wide range of biophysical and biotechnological applications, thanks to the tunable properties of the polyelectrolyte shell.

The choice of detector is based on tradeoffs between a variety of requirements such as performance, size, stability, robustness and, of course, price. A large number of detectors are now available, giving the design engineer an ever-increasing number of choices. We will briefly discuss the key performance issues that arise in the selection of a biotechnology detector. This will be followed by a discussion of new high sensitivity photomultipliers, photomultiplier arrays, APDS, APD arrays and CCDs.

The cytoskeletal microtubules (MTs) of rat hepatocytes treated by Benomyl (a fungicide) were imaged by means of immunofluorescent staining and optical microscopy. Images of untreated, or control (C), and of treated (T) cells were processed both by fractal and Fourier analysis. The C-MTs had contour fractal dimensions higher (≥ 1.4) than those of T-MTs (≤1.3). Fourier analysis included computation of the anisotropy of power spectral density, angle averaging and "spectrum enhancement," which corresponds to the application of a (pseudo)differential operator to the image. Enhanced spectra were interpolated by a polynomial, q, of degree 39, from which morphological descriptors were extracted. Descriptors from Fourier analysis made image classification possible. Principal components analysis was applied to the descriptors. In the plane of the first two components, {z₁,z₂}, the minimum spanning tree was drawn. Images of T-MTs formed a single cluster, whereas images of C-MTs formed two clusters, C1 and C2. The component z₁ correlated positively with the local maxima and minima of q, which reflected differences between T and C in power spectral density in the 1 to 2000 cycles/mm spatial frequency band. The difference between C1 and C2 was ascribed to anisotropy of the MT bundles. The implemented image classifier is capable of telling differences in cytoskeletal organization. As a consequence the method can become a tool for testing cytotoxicity and for extracting quantitative information about intracellular alterations of various origin.

The main disadvantage in ordinary agglutination immunoassay is difficulties to distinguish between specific and non-specific particle aggregation. We proposed to use Scanning Flow Cytometry to the kinetic study of the initial stages of agglutination process. The main advantage of the Scanning Flow Cytometry is a possibility to measure angular dependency of the light scattered by a single particle, an indicatrix. The most promising field for application of the indicatrix technology is a characterization of non-spherical particles. Validity of proposed method was verified by simultaneous measurements of the light scattering and fluorescence signal. We used Wentzel-Kramers-Brillouin approximation to simulate light scattering from two glued spheres and to explain the results obtained from measured indicatrices. To show an applicability of the proposed technique, the kinetic experiments were performed on latex particle covered with BSA (diameter 1.8 μm). Kinetics of dimer fraction growth initiated by mixing BSA-covered latex particles with anti-BSA immuoglobulins IgG was studied. In order to evaluate kinetic rate constant simple kinetic model involved only dimer growth reaction was applied for data treatment. Two kinetic rate constant for dimer fraction growth k_B=2.88•10^-12 cm³s^-1 and k_A=0.85•10^-12 cm³s^-1 were evaluated for two samples with the same origin but with different prehistory.

The request for a more profound immunophenotyping and sometimes the lack of material demands more measurable fluorescence colors to increase the number of detectable antigens per specimen. Six different fluorescences are distinguishable in the Laser Scanning Cytometer (LSC). In the present study we wanted to increase this number to eight colors per measurement. Combined with an earlier study it is likely possible to measure n fluorescences i.e. n leukocyte subsets by a series of measurements followed by subsequent restraining steps. The new method is realized by s-ing the combination of filter change and a subsequent re-measurement for the distinction between the fluorescent dyes Cy5 and Cy5.5. The optical filters are replaced after the first measurement and the same specimen is remeasured without removing it from the microscope. For the second measurement a filter is inserted that detects Cy5.5 but not Cy5 (710/10nm). After the second measurement of the same specimen both data files are combined. With the aid of this feature it is possible to line out the differences between both measurements. If the data from the second measuring (Cy5.5 only) is subtracted from the first, Cy5 data is the result. After the first two measurements when eight different fluorescences (i.e. antigens or leukocyte subsets) were analyzed, the same cells are restained and a new measurement is performed. In theory, one can perform n re-measurements with eight fluorescences respectively. The information gained per specimen is only limited by the number of available antibodies and b sterical hindrance.

Immunophenotyping of peripheral blood leukocytes (PBL) is a very well documented application of Slide Based Cytometry (SBC). As for any other assay it is of highest importance to ensure that all cells which are relevant for an analysis are recognized. Unlike assays for cultured cells which have homogenous morphology immunophenotyping of PBLs is performed on cells with heterogeneous size and shape. Therefore, triggering on parameters related to cell morphology might lead to an incomplete analysis of just a subset of cells especially in pathological conditions. Several dyes stain DNA specifically in a wide variety of emission spectra. Many of them show some influence of the chromatin condensation and organization on the staining intensity. DNA dyes therefore can be used to differentiate between cell types having the same ploidy. This can be exploited for immunophenotyping since some dyes therefore can partially replace antibody staining. The concept of using DNA dyes in the setting of immunostaining has the following advantages: (1) nuclear staining provides a stable and easy triggering signal that guarantees both, that neither cells are excluded nor that debris or polluting particles are included into the analysis; (2) some DNA dyes differentiate between mononuclear and polymorphonuclear cells. A disadvantage of DNA dyes is that mostly cells have to be permeabilized. Because of this only one set of immunophenotypic markers can be stained, cells are fixed and permeabilized, and then nuclei are stained with the appropriate DNA dye. In the study we demonstrate the use of the most commonly available DNA dyes (7-AAD, To-Pro, To-To, PI etc.) in their applicability in immunophenotyping. An overview of spectral properties, fluorescence spill-over and optimal combinations with surface antigen staining will be shown. As in general for SBC only very small sample volumes are needed. This allows to serially analyze PBL in clinical settings that up to now could not be studied in detail such as in the critical ill patient, during major surgery, and in new-borns and infants.

Increasing evidence suggests, that endothelial progenitor cells (EPC) play an important role in postnatal neovascularization. The formation of new blood vessels is important in many procedures, e.g. embryogenesis, wound healing, tumor growth and neovascularization of ischemic tissue. Aim of this study was to evaluate an assay which is able to detect EPCs qualitatively as well as quantitatively. This was done by Flo cytometry (FCM) and Laser Scanning Cytometry (LSC). Peripheral blood was drawn out of healthy control persons. In Flow Cytometry mononuclear cells of the peripheral blood, KDR and CD34 double positive cells were defined as precursors of EPCs. Cells from the same specimen were cultured and measured by LSC. While measuring with the LSC it was possible to exclude artifacts o debris by controlling the triggering. The specimen measured with FCM and LSC were examined serologically too, regarding cytokines which usually appear with EPCs (e.g. vascular endothelial growth factor [VEGF]). However, measured results had a good correlation, i.e. a higher amount of precursor cells were accompanied with higher EPC counts.

Laser emission analysis is an efficient method used for determination of quantitative and qualitative composition of chemical elements in materials. For the analyzer designs a proper choice of a laser type, stabilization of its pulse parameters and performance of a high-resolution spectrometer of sufficient responsivity are crucial problems. The correlation-based method of laser induced breakdown spectrometry for soil contamination was presented. A system of laser induced breakdown spectroanalyzer and the investigation of a portable laser emission spectro-analyzer for analysis of chemical composition of materials are described. The results and examples of its applications and optimizations are given.

Fluorescence lifetime imaging microscopy (FLIM) using near-UV excitation is being developed to probe endogenous fluorophores in cells and tissues. Here, we describe such a UV-FLIM system and present a useful method for measuring the fluorescence lifetime discrimination of the system using the viscosity dependent lifetime of 1,4-Bis(5-phenyloxazol-2-yl)benzene (POPOP). The time-domain UV-FLIM system employed a nitrogen laser (337.1 nm) fiber-optic coupled to an inverted microscope. Wide-field fluorescence images were obtained at controlled time delays with a 200 ps gated, intensified-CCD camera. Lifetimes were calculated from the intensity decay on a pixel-by-pixel basis. The system was capable of imaging endogenous fluorescence in living cells using UV excitation. POPOP is a nanosecond lifetime standard suitable for UV excitation (325-375 nm). Its single-exponential lifetime (1.4 ns in ethanol) is comparable to endogenous lifetime values measured in living cells. Increasing solvent viscosity via the incremental addition of glycerol produced a series of POPOP lifetime standards having a range of 1 ns. The FLIM system’s ability to discriminate lifetime differences of 50 ps was demonstrated using the POPOP series. Thus, POPOP’s viscosity dependent lifetime represents a useful and convenient resolution standard for UV-FLIM calibration.

The overall object of proteomics is to characterize all of the proteins expressed in a given cell type. With the rapid development of random gene tagging technology and high resolution fluorescence microscopy, it has become possible to generate libraries of digital images depicting the location patterns of most proteins in any given cell type. While the subcellular location of a protein is important to its function, no established methods exist for the systematic description, comparison or organization of protein location patterns. We have previously described classification methods that accurately recognize all major subcellular location patterns in both 2D and 3D images, as well as methods for rigorous statistical comparison of such patterns. We describe here the application of the numerical features from the previous work to images obtained by random tagging of proteins. Spinning disk confocal microscopy was used to collect images depicting the location patterns of 46 NIH 3T3 cell clones expressing proteins randomly tagged with a fluorescent protein. A set of 42 numerical features describing both image texture and object morphology were calculated and used to build subcellular location trees that group the tagged proteins by similarity of location pattern.

The central goal of proteomics is to clarify the mechanism by which each protein in a given cell type carries out its function. Automated protein subcellular location determination by fluorescence microscopy can play an important role in fulfilling this goal. The subcellular location of a protein is critical to understanding its function because each subcellular compartment has a unique biochemical environment. We have previously shown that neural network classifiers using sets of numerical features computed from fluorescence microscope images were able to recognize all major subcellular location patterns with reasonable accuracy. Current classifiers are limited by under-determined classification boundaries due to the limited number of available images compared to the number of features. In this paper, we compare various feature reduction methods that can address this problem. Specifically, principal component analysis, kernel principal component analysis, nonlinear principal component analysis, independent component analysis, classification trees, fractal dimensionality reduction, stepwise discriminant analysis, and genetic algorithms are used to select feature subsets that are evaluated using support vector machine classifiers. The best results were obtained using stepwise discriminant analysis and we found that as few as eight features can provide good classification accuracy for all major subcellular patterns in HeLa cells.

We have developed a photothermal method for far-field optical detection of nanometer-sized metal particles, combining high-frequency modulation and polarization interference contrast. We can image gold colloids down to 5 nm in diameter, with a signal-to-noise ratio higher than 10. This is a considerable improvement over commonly used optical methods based on resonance plasmon scattering which, for background reasons, are limited to particles of more than about 40 nm in diameter. We also show that in addition to its intrinsic sensitivity, our photothermal method is totally insensitive to non-absorbing scatterers as 10 nm nanoparticles can be imaged in cells.

Total internal reflection fluorescence (TIRF) microscopy is a powerful technique to investigate surface bound emitters exclusively, i.e. with low interference of the fluorescing bulk in the adjacent aqueous solution. Confocal microscopy is a powerful technique to detect low emission intensities, like the emission of a single molecule, with high temporal resolution and high signal-to-noise ratio. We present a confocal total internal fluorescence microscope, which combines the virtues of diffraction limited confocal imaging and TIRF. Annular supercritical focusing and fluorescence collection through standard glass cover slips is accomplished by a parabolic mirror lens. Tight focusing and supercritical excitation reduce the detection volume for fluorescent analyte molecules well below attoliters. Beyond, the large aperture of the element leads to a high collection efficiency of surface bound emitters (~50%). The system is characterized in detail by calculations of the electric fields in the focus region and simulated confocal imaging. Single molecule experiments demonstrate the performance of the microscope in practice.

A setup consisting on a laser scanning microscope equipped with appropriate detection units was developed for time-resolved intracellular fluorescence spectroscopy and fluorescence lifetime imaging (FLIM) for on-line detection of structural changes of various biomolecules. Short-pulsed excitation was performed with a diode laser which emits pulses at 398 nm with 70 ps duration. The laser was coupled to the laser scanning microscope. For time resolved spectroscopy a setup consisting of an Czerny Turner spectrometer and a MCP-gated and -intensified CCD camera was used. Time-gated spectra within the cells were acquired by placing the laser beam in "spot scan" mode. In addition, a time-correlated single photon counting module was used to determine the fluorescence lifetime from single spots and to record lifetime images (τ-mapping). The time-resolved fluorescence characteristics of 5-ALA (5-aminolevulinic-acid), as well as 5-ALAhe (5-aminolevulinic-acid-hexylester)- induced protoporphyrine IX (PPIX) were investigated before and during PDT with subcellular resolution. For cells which were incubated with 5-ALA, a component with a fluorescence lifetime of about 7 ns was correlated with a structured fluorescence, which probably coincides with mitochondria, whereas a shorter lifetime was found in the cytoplasm. In the case of 5-ALAhe the lifetime of PPIX was longer, which could be due to different localization. During PDT the component with the longer lifetime completely vanished, whereas the shorter liftime was retained. It seems that FLIM is a valuable method to selectively identify and localize the photodynamically active photosensitizer.

Background/problem. Monitoring of tumor response to cancer chemotherapy and dose optimization for specific patients are the key factors for successful application of anti-tumor drugs. Using patient's tumor cells for preliminary in vitro drug screening may allow optimal selection of drug type and dose. Method. Single cell state was studied with photothermal microscope. Carcinoma cells were irradiated at 427 nm with 8 ns laser pulse with energy 30 - 40 μJ. Cell photothermal (PT) response amplitude and shape from each cell were analyzed and amount of cells that produced specific PT response was used as PT parameter. Parallel experiment included cell viability control. Results were obtained for two cytotoxic chemotherapy agents -- Platinol-aq and Adrucil. Incubation of cell suspensions for 90 min at 20 and 37°C caused changes in cell PT parameters. Reaction of carcinoma cells to the drug was very similar to reaction of hepatocytes to respiratory chain inhibition and reaction of RBC to osmotic pressure decrease. PT effect was found to be dose-dependent. PT method allows detecting drug-induced changes before cell death or morphological changes and therefore can be fast and sensitive modality for control of chemotherapy.

Cytometry Markup Language, CytometryML, is a proposed new analytical cytology data standard. CytometryML is a set of XML schemas for encoding both flow cytometry and digital microscopy text based data types. CytometryML schemas reference both DICOM (Digital Imaging and Communications in Medicine) codes and FCS keywords. These schemas provide representations for the keywords in FCS 3.0 and will soon include DICOM microscopic image data. Flow Cytometry Standard (FCS) list-mode has been mapped to the DICOM Waveform Information Object. A preliminary version of a list mode binary data type, which does not presently exist in DICOM, has been designed. This binary type is required to enhance the storage and transmission of flow cytometry and digital microscopy data. Index files based on Waveform indices will be used to rapidly locate the cells present in individual subsets. DICOM has the advantage of employing standard file types, TIF and JPEG, for Digital Microscopy. Using an XML schema based representation means that standard commercial software packages such as Excel and MathCad can be used to analyze, display, and store analytical cytometry data. Furthermore, by providing one standard for both DICOM data and analytical cytology data, it eliminates the need to create and maintain special purpose interfaces for analytical cytology data thereby integrating the data into the larger DICOM and other clinical communities. A draft version of CytometryML is available at www.newportinstruments.com.

A Eu(III)-macrocycle-mono-isothiocyanate, Quantum Dye, has been synthesized that has minimal contamination with the Eu(III)-macrocycle-di-isothiocyanate, which cross-links proteins. The mono-isothiocyanate has been conjugated to streptavidin (EuMac-Strept). An indirect assay with EuMac-Strept and biotinylated anti5BrdU has been used to observe apoptotic cells. This system and cells directly labeled with the Eu(III)-macrocycle-di-isothiocyanate have been employed in fading studies and reagent stability tests. The fading of cells mounted in a plastic medium was much slower than that observed when the cells were in the aqueous, micellar Lanthanide Enhanced Luminescence (LEL) solution. The fading was not the result of the photo-destruction of the Eu(III)-macrocycle, since the luminescence returned after a second addition of the LEL solution. A time-gated, peltier cooled, monochrome CCD camera has been combined with a flashlamp to eliminate imaging of the emission of fluorescein while maintaining the images of EuMAc staining. This was demonstrated with both separate preparations of fluorescein and EuMac stained cells and mixtures thereof. Time-gating was employed to produce an EuMac image of cells that were stained with both the EuMac and DAPI.

Experiments using single-molecules of TOTO-1 intercalated into dsDNA were performed to investigate the DNA sequence dependence on the fluorescence detectable with single-molecule fluorescence spectroscopy. Previous work has shown that there is a difference in the fluorescence lifetime when TOTO-1 is intercalated in poly-AT DNA or in poly-GC DNA. The fluorescence detected from single-molecules in this work for poly-GC and poly-AT DNA showed fluorescence lifetimes of 2.1 and 1.8 nsec, respectively. Analysis of the fluorescence intensity detected from single-molecules of TOTO-1 was performed by fluorescence cross-correlation spectroscopy. TOTO-1 is shown to spend large amounts of time in dark states. These dark states reduce the detectable fluorescence intensity to approximately 10 photons per millisecond on average.

Multichromophoric systems play a key role in biological systems (light harvesting antenna complexes, fluorescent proteins...) and are equally important in material science applications (e.g. light emitting devices (LED) based on conjugated polymers). Our approach to get insight in the excited state processes of such systems is to make use of dendrimers labeled with photostable perylene dyes. Dendrimers synthesis indeed allows changing the number, relative position and orientation of attached chromophores in a controlled way. In the present contribution, excited state processes such as energy hopping, singlet-singlet annihilation, singlet-triplet annihilation are identified in individual tetrachromophoric dendrimers immobilized in a polymer matrix. Similar processes are then demonstrated to occur as well in immobilized tetramers of a red fluorescent protein from a coral of the Discosoma genus (DsRed).

Single-molecule fluorescence techniques are used to detect a specific nucleic acid sequence in a mixture of unrelated sequences. Co-hybridization of a pair oligonucleotide hybridization probes, each labeled with a spectrally-distinct fluorophore and complementary to a specific sub-sequence of the target nucleic acid, forms a fluorescent adduct containing both fluorophores. The presence of the specific sequence is signaled by the simultaneous detection of both fluorophore labels on a single target fragment. We demonstrate quantitative detection of target nucleic acid sequences at fragment concentrations as low as 100 fM with a simple instrument that uses low-power, continuous-wave laser excitation. Furthermore, we show that a cross-correlation analysis of the arrival times of individual single-molecule fluorescence photon bursts detected in spectrally separate channels permits quantitative detection of the dual-color labeled species at concentrations approximately 1000x lower than can be quantitatively detected using the photon cross-correlation between the two detection channels. We also demonstrate that a pair of quencher-labeled oligonucleotides each complementary to the fluorescent hybridization probes can be used to reduce unbound probe fluorescence, substantially improving the sensitivity of the assay. We use this approach to detect β-actin messenger RNA (mRNA) transcripts.

It has recently been shown that the combination of Raman spectroscopy and optical tweezers constitute a powerful tool for biological studies. Raman spectra of single cells immobilized in a sterile surrounding can then be recorded without the risk of surface-induced morphological cell changes. Further, the complete cellular environment can be changed while measuring dynamics in real time. We here introduce a novel Raman tweezers set-up ideal for resonance Raman studies of single cells. The system differs from earlier set-ups in that two separate laser beams, used for trapping and Raman excitation, are combined in a double-microscope configuration. This has the advantage that the wavelength and power of the trapping and probe beam can be adjusted individually, for example in order to optimize the functionality of the set-up or to record resonance Raman profiles from the same trapped cell. Further, the tweezers can be removed from the system without affecting the spectrometer configuration. Trapping is achieved by tightly focusing IR diode laser radiation (830 nm) through an inverted oil immersion objective with high numerical aperture (NA = 1.25), while Raman scattering is excited by the lines of an ArKr ion-laser. The backscattered Raman signal is collected by a single-grating spectrometer equipped with a microscope and a 60x water-immersion objective (NA = 0.9). The functionality of the system is demonstrated by measurements of trapped single functional erythrocytes using differen excitation lines (488, 514.5 568.2 nm) in resonance with the heme moiety and by studying the spectral evolution during illumination.

The back-illuminated Electron Multiplying Charge Coupled Device (EMCCD) camera stands to be one of the most revolutionary contributions ever to the burgeoning fields of low-light dynamic cellular microscopy and single molecule detection, combining extremely high photon conversion efficiency with the ability to eliminate the readout noise detection limit. Here, we present some preliminary measurements recorded by a vary rapid frame rate version of this camera technology, incorporated into a spinning disk confocal microscopy set-up that is used for fast intracellular calcium flux measurements. The results presented demonstrate the united effects of (1) EMCCD technology in amplifying the very weak signal from these fluorescently labelled cells above the readout noise detection limit, that they would otherwise be completely lost in; (2) back-thinned CCD technology in maximizing the singal/shot noise ratio from such weak photon fluxes. It has also been shown how this innovative development can offer significant signal improvements over that afforded by ICCD technology. Practially, this marked advancement in detector sensitivity affords benefits such as shorter exposure times (therefore faster frame rates), lower dye concentrations and reduced excitation powers and will remove some of the barriers that have been restricting the development of new innovative low-light microscopy techniques.

Optical tweezers together with a position sensitive detection system allows measurements of forces in the pN range between micro-sized biological objects. A prototype force measurement system has been constructed around in inverted microscope with an argon-ion pumped Ti:sapphire laser as light source for optical trapping. A trapped particle in the focus of the high numerical aperture microscope-objective behaves like an omni-directional mechanical spring if an external force displaces it. The displacement from the equilibrium position is a measure of the exerted force. For position detection of the trapped particle (polystyrene beads), a He-Ne laser beam is focused a small distance below the trapping focus. An image of the bead appears as a distinct spot in the far field, monitored by a photosensitive detector. The position data is converted to a force measurement by a calibration procedure. The system has been used for measuring the binding forces between E-coli bacterial adhesin and their receptor sugars.