Optical micro-manipulation has seen a resurgence of interest in recent years which has been due in part to new application areas and the use of tailored forms of light beams particularly using holographic optical tweezers technology. Particle dynamics in 2D and 3D light patterns and the transport properties therein are of interest. We show the ability to sort and separate biological matter in a three dimensional optical lattice.

We demonstrate how photoinduced electron transfer (PET) reactions can be successfully applied to monitor conformational dynamics in individual biopolymers. Single-pair fluorescence resonance energy transfer (FRET) experiments are ideally suited to study conformational dynamics occurring on the nanometer scale, e.g. during protein folding or unfolding. In contrast, conformational dynamics with functional significance, for example occurring in enzymes at work, often appear on much smaller spatial scales of up to several Angströms. Our results demonstrate that selective PET-reactions between fluorophores and amino acids or DNA nucleotides represent a versatile tool to measure small-scale conformational dynamics in biopolymers on a wide range of time scales, extending from nanoseconds to seconds, at the single-molecule level under equilibrium conditions. That is, the monitoring of conformational dynamics of biopolymers with temporal resolutions comparable to those within reach using new techniques of molecular dynamic simulations. We present data about structural changes of single biomolecules like DNA hairpins and peptides by using quenching electron transfer reactions between guanosine or tryptophan residues in close proximity to fluorescent dyes. Furthermore, we demonstrate that the strong distance dependence of charge separation reactions on the sub-nanometer scale can be used to develop conformationally flexible PET-biosensors. These sensors enable the detection of specific target molecules in the sub-picomolar range and allow one to follow their molecular binding dynamics with temporal resolution.

Using single molecule fluorescence spectroscopy we have investigated fluorescence resonance energy transfer (FRET) occurring between two peryleneimide (PI) chromophores in a series of synthetic systems: PI end-capped fluorene trimers, hexamers and polymers for which the interchromophoric distance vary from 3.4 to 5.9 and 42 nm, respectively. By monitoring in parallel the fluorescence intensity and the number of independent emitting chromophores from each molecule, we could discriminate between competitive Foerster-type energy transfer processes such as energy hopping, singlet-singlet annihilation and singlet-triplet annihilation for the PI end-capped fluorine compounds. Due to different energy transfer efficiencies, variations in the interchromophoric distance enable switching between these processes. The single molecule fluorescence data reported here suggest that similar energy transfer pathways have to be considered in the analysis of single molecule trajectories of donor/acceptor pairs, as well as in the case of more complex systems like natural multichromophoric systems, such as light harvesting antennas or oligomeric fluorescent proteins.

Symbolic nucleotide sequences are converted into digital genomic signals by using a complex representation derived from a tetrahedral vector representation of nucleotides. The study of complex genomic signals using signal processing methods reveals large scale features of chromosomes that would be difficult to grasp by using the statistical and pattern matching methods for the analysis of symbolic genomic sequences. On the other hand, in the context of operating with a large volume of data at various resolutions and visualizing the results to make them available to humans, the problem of data representability becomes critical. A novel mathematical description of data representability, based on the data scattering ratio on a pixel is defined and is applied for several typical cases of standard signals and for genomic signals. It is shown that the variation of genomic data along nucleotide sequences, specifically the cumulated and unwrapped phase, can be visualized adequately as simple graphic lines for low and large scales, while for medium scales (thousands to tens of thousands of base pairs) the statistical descriptions have to be used.

Without exceptions, in all living cells NADH is a key metabolite linking a large number of metabolic pathways. Flux rates through such pathways are an essential component in the understanding of the functioning of living cells. Knowledge about the way these fluxes depend on the concentrations of the metabolites involved (including NADH/NAD⁺) allows calculation of these fluxes. Therefore, a method to determine the concentration of free NADH is necessary. A distinction between the free and protein-bound NADH can be made on the basis of fluorescence emission spectra and fluorescence lifetimes. A method for such measurements using a microscopic set-up for time-gated fluorescence spectroscopy has been introduced by Schneckenburger and co-workers (Paul RJ, Schneckenburger H. Naturwissenschaften 83, pp. 32-35, 1996). We further improve this method by first characterizing NADH binding to model proteins by isothermal titration calorimetry and fluorescence. This allows a precise calculation of bound and free NADH and their respective spectra. An analysis of experimental data is advanced by applying two-component deconvolution and subsequent fitting. Using this method we can detect a significant proportion of free NADH in isolated potato tuber mitochondria respiring malate. Taken together these improvements allow a more accurate characterization of the NADH turnover in biological systems.

Image cytometry has made possible the collection and analysis of multiparameter cellular information. The wider use of image cytometry in drug screening will depend on its throughput, efficiency, repeatability, and on the added benefits compared with less sophisticated but faster methods. Throughput (number of datapoints per unit of time) and efficiency (number of datapoints from the given amount of reagents or plate area) are addressed here by screening multiple cell lines simultaneously using encoded carriers (CellCards). CellCards are rectangular particles with an expandable color barcode and a transparent section for cellular readout. Before performing the assay, each cell line is grown on a different class of carriers. CellCards, with attached cells, are mixed and dispensed into a microtiter plate where the assay is performed. Next the plates are imaged, decoded and the cells associated with each CellCard class are analyzed. Using CellCards the efficiency is increased by the multiplexing factor (the number of cell lines analyzed in each well). We routinely run assays with a multiplex factor of ten. Throughput is additionally addressed by working at the lowest possible magnification for a given assay. Decoding of CellCards requires one image per well in 96-well microtiter plate format. The system provides the added benefit of internal consistency since the data can be normalized to controls within each well.

We present a procedure for a precise power spectral analysis of optical tweezers data. This procedure uses the entire frequency range of the experimental power spectrum. We apply this procedure to experimental data from a biological system, data for the motion of a microsphere attached with a biotin-streptavidin linker to a single protein, the λ-receptor of E. coli. As in [Oddershede et al, Biophys. J. 83, 3152 (2002)] and [Oddershede et al, J. Phys.: Condens. Mat. 15, S1737 (2003)], we find that the λ-receptor moves diffusively in the outer membrane, but is confined to a particular region by a harmonic potential. Here, we show that since the power spectrum is known with precision up to its Nyquist frequency of 8 kHz, one can resolve the relative motion of the λ-receptor and the microsphere attached to it. We find that the biotin-streptavidin link between them can be described as a Hookean spring. Since we can see only the microsphere, these results are based on an interpretation of the power spectrum of its motion. This interpretation is based on a model. We use the simplest model that is necessary and sufficient to interpret the power spectrum in its full range. This model fits the spectrum well and the fit yields the model's parameters with some precision. These results indicate that optical tweezers have the potential to become a tool of precision. We discuss improvements of experiments and analysis that are necessary in order to realize this potential.

The morphology of cytoskeletal microtubules has been analyzed by fractal, direct and spectral methods. Sets of images were obtained from the epifluorescence microscopy of primary cultures of rat hepatocytes treated with fungicide concentrations of 5O and 25 µg/ml for 2h. The morphological descriptors extracted by said methods included contour and mass fractal dimension, total variation, the L¹-norm of the Laplacian and properties of the "enhanced spectrum". The latter is obtained by suitably processing the logarithm of power spectral density with the aim of separating image structure (low spatial frequency) from texture (high spatial frequency). Descriptors were fused by principal components analysis. A classification algorithm was trained to tell undisturbed (control) cytoskeletal structures from those treated at the higher dose. The eigenvector matrix of the trained classifier was used to rank structures treated at the lower dose: from regression on the set centroid coordinates a tentative relation between the first principal component (the "response") and dose has been obtained. The same ranking procedure was applied to structures recovering from injury (24h after exposure to the higher dose) and the extent of recovery has been quantified. The paper includes a possible interpretation of some morphological descriptors and their role in automatic classification.

We demonstrate the use of independently controlled doughnut beams for trapping and manipulation of an array of low-index microstructures. These types of particles are generally difficult to handle using conventional optical traps due to the inverse effect of the gradient force, which repels the particles from the region of stronger light intensity. The configurable multiple doughnut beams are generated using a nearly loss-less phase-to-intensity conversion of a phase-encoded collimated coherent light source. The two-dimensional phase distribution corresponding to the optical trap pattern is encoded using a computer-programmable spatial light modulator enabling each trap to be moved arbitrarily along the transverse direction. Experiments show trapping and manipulation of hollow “air-filled” glass micro-spheres suspended in aqueous medium.

We report a new method for generating multiple optical tweezers that enables beam-steering functionality and polarization state control for each trapping beam. The method employs a spatial light modulator (SLM) that imparts a two-dimensional (2-D) phase distribution onto a linearly polarized input beam from a CW-laser (λ = 830nm). A microlens array is used to divide the beam resulting in a matrix of point sources that are subsequently imaged onto the sample plane of a microscope as trapping beams. For beam steering, the plane of polarization of the input field is aligned parallel to the slow axis of the SLM, on which an array of phase grating patterns is encoded. The blazed grating parameters dictate the positional deflections of the optical traps. When the input polarization plane makes a 45°-angle with the SLM slow axis, an elliptic polarization state is produced for each optical trap and the degree of ellipticity depends on the phase difference introduced between the extraordinary and ordinary output field components at each SLM sub-aperture. It is known that elliptically polarized light changes its angular momentum when focused to a birefringent particle thereby inducing an axial rotation of the trapped object. We experimentally demonstrate the two modes of operation: (1) controlled lateral deflection of simultaneous trapped polystyrene microspheres and (2) polarization-induced rotation of simultaneously trapped calcite crystals.

By employing the total internal reflection fluorescence (TIRF) microscope with an ultra high NA (1.65) objective lens, we demonstrated detailed dynamics of exocytosis in various types of secretory vesicles. However, the TIRF microscopy could be applied to observations only on the plasma membrane and its immediate vicinity. To observe the vesicles in the deeper region of cytoplasm, we modified the TIRF optics to project a slit beam thinner than 1 μm in width to the cell. The slit beam illumination spotted single secretory vesicles inside the cell better and their movement and exocytosis easier. By scanning the slit beam, a fluorescence microscopy was possible at a high signal-to-noise ratio useful for measurement and analysis of single exocytosis in neurons and endocrine cells.

Trapping and active movement of the sample in a microscope is gently performed by applying optical forces. Optical tweezers, including the recently developed dynamic multiple-beam optical tweezers, have been shown to be indispensable tools in several fields. We demonstrate an optical trap that is formed by four laser beams. In our Differential Active Optical Manipulator (DAOM) the beams are arranged along the axes of a tetrahedron and are approximately collimated within the vicinity of their point of intersection. The DAOM makes use of a distinctive advantage of collimated light: in contrast to optical tweezers, this instrument allows movement of the confined particle over long distances without mechanical scanning. The beams' scattering forces superimpose in their common volume, and the total force (magnitude and direction) on the particle can be set by adjusting the beams' intensities. This can be done quickly and independently for each beam, allowing us to move a particle along arbitrary paths within a volume of about 1nℓ. Active feedback ensures that the sample can be held in place or transported to a specified position as desired. This allows focusing and lateral translation of the microscopic sample, which are common tasks in microscopy. During manipulation the particle is simultaneously observed from four sides. This opens new perspectives for 3D microscopy combined with contactless manipulation of the sample under observation.

The immobilization and hybridization processes of DNA strands on poly-l-lysine (PL) covered surfaces have been studied using the atomic force microscopy (AFM) in a topographic mode. The statistical analysis of topographic surfaces showed an increase in the Z-threshold with additions of single strand DNA (ssDNA) and the complimentary DNA (ccDNA). Also no drastic change of statistical fractal dimension (slope of the log-log perimeter-area plot) is observed when comparing the PL-surfaces coated with ssDNA and ccDNA. These two results suggest that ssDNA strands are successfully immobilized and spatially hybridized with ccDNA on the PL surface and the growth of hybridized ccDNA occurs mainly in the vertical dimension. The methods described here are good candidates for the detection of DNA hybridization, especially in the context of DNA nanoarrays.

The identification, fabrication and evaluation of biomimetic templates hold great potential for a variety of applications. The generation of templates on these substrates is achieved using an appropriately modified atomic force microscope (AFM) tip. The fabrication process of tissue and cell based templates reported in this paper is accomplished using Dip-Pen Nanolithography. This report identifies critical parameters necessary for the patterning process: initial surface quality, temperature and humidity conditions. The results of this proof of concept experiment can be utilized in future in vitro and in vivo studies to test the templates bio-compatibility and specificity.

We previously demonstrated in vitro that the simultaneous application of cellular heating and a laser-induced stress wave (LISW) enhanced the uptake of porfimer sodium (Photofrin) by cells. In this study, we attempted to apply this technique to gene transfer to cultured cells. LISW and/or a transient mild (~43°C) heating been applied to deliver plasmid coding for green fluorescent protein (EGFP) to NIH-3T3 cells. It was found that simultaneous application of an LISW and the heating significantly increased the transfection efficiency by a factor of 2.5 when compared with that for the cells treated with an LISW alone.

In neurons of patients with Alzheimers's disease (AD) signs of cell cycle re-entry as well as polyploidy have been reported, indicating that the entire or a part of the genome of the neurons is duplicated before its death but mitosis is not initiated so that the cellular DNA content remains tetraploid. It was concluded, that this imbalance is the direct cause of the neuronal loss in AD³. Manual counting of polyploidal cells is possible but time consuming and possibly statistically insufficient. The aim of this study was to develop an automated method that detects the neuronal DNA content abnormalities with Laser Scanning Cytometry (LSC). Frozen sections of formalin-fixed brain tissue of AD patients and control subjects were labelled with anti-cyclin B and anti-NeuN antibodies. Immunolabelling was performed using Cy5- and Cy2-conjugated secondary antibodies and biotin streptavidin or tyramid signal amplification. In the end sections of 20µm thickness were incubated with propidium iodide (PI) (50μg/ml) and covered on slides. For analysis by the LSC PI was used as trigger. Cells identified as neurons by NeuN expression were analyzed for cyclin B expression. Per specimen data of at least 10,000 neurons were acquired. In the frozen brain sections an automated quantification of the amount of nuclear DNA is possible with LSC. The DNA ploidy as well as the cell cycle distribution can be analyzed. A high number of neurons can be scanned and the duration of measuring is shorter than a manual examination. The amount of DNA is sufficiently represented by the PI fluorescence to be able to distinguish between eu- and polyploid neurons.

For immunophenotypic analysis more measurable parameters for the discrimination of leukocyte subsets are necessary. With a single scan six fluorochromes can be distinguished with the Laser Scanning Cytometer (LSC). Due to the number of PMTs the amount of simultaneously measurable fluorescences per scan is limited. Nevertheless, the amount of measurable colors can be improved to eight by appropriate change of the filter settings and two scans per specimen. Aim of this study was to use the special features of Slide based Cytometry (SBC) beyond filter change, remeasurement and merging to distinguish fluorochromes with similar emission spectra. The photosensitivity of fluorochromes that are excited and emit in a similar wavelength range may be very different. The number of measurable parameters per PMT was increased using photosensitivity of different fluorochromes as additional criteria. Peripheral blood leukocytes were stained with antibodies conjugated to the fluorochromes APC, APC-Cy5.5 and Alexa-Fluor 633 and mounted on conventional uncoated glass slides with Fluorescence mounting medium. Specimens were excited in the LSC with the HeNe (633nm) Laser and measured at different filter settings (670/20nm-filter for APC/ALEXA 633 and 710/20nm-filter for APC-Cy5.5). At this point, APC-Cy5.5 and APC/ALEXA633 were already distinguishable. In order to differentiate between APC and ALEXA633 photobleaching was performed by repeated excitation with the laser at 633nm. Control measurements proved that APC is much more sensitive against laser excitation, i.e. looses much more fluorescence intensity than ALEXA633. The separate measurements (before/after filter change and before/after bleaching) were merged into one file. The photostability of Alexa-Fluor 633 (1.02% bleach per scan) and APC (5.74% bleach per scan) are substantially different. Therefore, after bleaching and merging both fluorochromes can be distinguished and are regarded by the software as separate parameters. The fluorochromes APC/ALEXA633 and APC-Cy5.5 can be discriminated by changing the emission filters before bleach. By sequential photobleaching, change of filters and subsequent merging of the data the number of simultaneously measurable “colors” is substantially increased.

Highly focused laser microbeams are being used with increasing regularity for targeted cell lysis, cellular microsurgery and molecular delivery via transient cell membrane permeabilization. To examine the mechanisms of laser induced cell lysis, we performed time-resolved imaging of confluent PtK2 cell cultures following the delivery of a single 6 ns, 532 nm Nd:YAG laser pulse. The laser pulse energies employed correspond to 1x and 3x threshold for plasma formation. The resulting plasma formation, pressure wave propagation and cavitation bubble dynamics were imaged over a temporal range spanning 5 orders of magnitude (0.5 ns - 50 µs). Time-resolved imaging enabled determination of process characteristics including pressure wave speed and amplitude and cavitation bubble energies. The time-resolved images also revealed the onset of cellular damage to occur on nano-second time scales and complete within 1 µs. Moreover, the size of the damage zone was larger than the plasma but smaller than the maximum cavitation bubble size. This indicated that mechanisms apart from plasma vaporization namely pressure wave propagation and cavitation bubble expansion are contributors to cellular damage. Dye exclusion assays showed that the majority of cells experiencing considerable deformation due to fluid flow generated by the cavitation bubble expansion remain viable over 24 hours.

Efficient computer-aided cervical cancer detection can improve both the accuracy and the productivity of cytotechnologists and pathologists. Nuclear segmentation is essential to automated screening, and is still a challenge. We propose and demonstrate a novel approach to improving segmentation performance by multispectral imaging followed by unsupervised nuclear segmentation relying on selecting a useful subset of spectral or derived image features. In the absence of prior knowledge, feature selection can be negatively affected by the bias, present in most unsupervised segmentation, to erroneously segment out small objects, yielding ill-balanced class samples. To address this issue, we first introduce a new measurement, Criterion Vector (CV), measuring the distances between the segmentation result and the original data. This efficiently reduces the bias generated by feature selection. Second, we apply a novel recursive feature selection scheme, to generate a new feature subset based on the corresponding CV, ensuring that the correct part of the initial segmentation results is used to obtain better feature subsets. We studied the speed and accuracy of our two-step algorithm in analyzing a number of multispectral Pap smear image sets. The results show high accuracy of segmentation, as well as great reduction of spectral redundancy. The nuclear segmentation accuracy can reach over 90%, by selecting as few as 4 distinct spectra out of 30.

Improvements in the lanthanide enhanced luminescence (LEL) protocol have facilitated the use of the recently synthesized Eu(III)-macrocycle-mono-isothiocyanate, Quantum Dye, as a label. It was discovered that a homogeneous solution in ethanol or other solvent could be used to produce the lanthanide enhanced luminescence (LEL) effect, provided that the solution was permitted to evaporate. This protocol has been applied to the direct staining of cells in S phase, and was optimized for solid phase assays with Quantum Dye labeled streptavidin. Preliminary studies indicate that cells stained with the europium Quantum Dye can be observed both by conventional UV laser excitation and by infrared two-photon confocal microscopy. An enhancer has been found that enables the observation of simultaneous emissions from both the europium and terbium Quantum Dyes.

Optical imaging in cartilage is challenging due to the high levels of intra- and inter-cellular autofluorescence. We report here on high-resolution confocal and two-photon imaging of endogenous fluorescence of cartilage and of exogenous fluorescence of filamin A and B protein markers. Confocal laser scanning microscopy offers the advantage of quasi-theoretical spatial resolution and minimizes the autofluorescence contribution by eliminating the out-of-focus light. In non-labeled cartilage, we observe mostly intracellular autofluorescence that, due to the uniform distribution within the cell, can be further effectively minimized by careful choice of experimental parameters. The fluorescence of the exogenous markers AlexaFluor 488 and AlexaFluor 568 labeling Filamins A and B, respectively, could also be detected and quantitated using this procedure, revealing topologically different expression levels of filamin A and B proteins in the cartilage growth plate. Two-photon excited fluorescence imaging yielded further resolution improvements and structural and functional information.

We developed a new endoscope that allows for non-contact, rapid (sub-second) acquisition of polarized spectral images of tissue in vivo. The intent was to enable exploration of a variety of optical contrast mechanisms (such as light absorption, reflectance, scattering, and fluorescence) in a search for new methods of early cancer detection in a clinical setting. Our first new implementation for cancer detection is based on a body of spectroscopic work that employs elastic scattering (Mie) theory to estimate the size of bulk scatterers in a given medium - in our case, the epithelial tissue of lungs. This paper describes the novel design of the Hyperspectral Imaging Endoscope, and our initial experiences with employing it for the early detection of dysplasia and cancer in lung epithelia.