All of the different modes of microscopy deliver light in a controlled fashion to the object to be examined and collect as much of the light containing the desired information about the object as possible. The system being presented replaces the simple circular or annular diaphragms of a conventional microscope with digital micromirror devices to enable digital light microscopy. The DMDs are placed in the optical path at positions corresponding to the field and aperture diaphragms of a conventional microscope. This allows for more precise and flexible control over the spatial location, amount, and angles of the illumination light, and the light to be collected. Digital light microscopy enables the improvement of existing modes of microscopy, specifically for quantitative microscopy applications. Confocal microscopy has been performed, realizing improvements in resolution, flexibility, and cost. Three different combinations of image acquisition and post- processing algorithms have ben sued to generate confocal images, as well as a tomographic reconstruction image.

Confocal microscopy is used widely for 3D biological imaging, but can be too slow for many applications. The limitations arise from scanning a single spot across the specimen at high speeds. Singe-spot confocal imaging usually works at a 1-2 Hz frame rate and faster systems tend to be signal/noise limited. Many live cellular events require both high speed and high SNR. Some parallel confocal systems have been developed to collect light from many points simultaneously to obtain high SNR and/or speed. Nipkow disks are compared of many pinholes, but have a fixed pattern and low light efficiency. Slit scanning systems collect an entire video line at a time, but compromise resolution. The TI Digital Micromirror Device (offers an alternative via large arrays of rapidly re-configurable micromirrors that can form arrays of reflection 'pinholes'. The prototype presented here exhibited 0.4 X 0.4 X 0.8 micrometers ³ resolution with a 100X 0.90 NA objective. An array of 10,000 or more neighborhoods, each compromising a single ON mirror in a group of OFF mirrors, creates the confocal parallelism. Alternating the ON mirror in each neighborhood until the image is completely formed on the CCD sensor enables transverse scanning. With 10,000-fold parallelism, for example, light collection efficiency and frame rate can both be 100X higher than in typical spot scanning. The high sensitivity allows high-speed confocal imaging at intensities below the cellular fluorotoxicity threshold. This was demonstrated in a hamster window preparation scanned daily in one-week longitudinal studies. Vessel geometry and localized blood flow were reconstructed to measure perfusion. High frame rate and sensitivity allowed real-time visualization of DiI stained intravascular red blood cells with no apparent tissue damage, supporting the tremendous potential advantages over current confocal technologies.

MLSC is an alternative to flow cytometry that has many advantages in clinical environments such as minimal sample preparation, low sample volume, and direct measurement of absolute cell counts. However, MLSC requires an added image- processing step to produce the industry-standard FCS output format. The image processing program needs to handle multiple binary images, representing different detection channels; it needs to determine the background fluorescence level in each channel; the overall noise in each channel such that it can enumerate cell from noise; it needs to ignore extraneous signal such as bubbles, dust particles and other artifacts; and it needs to characterize each recognized cell to report parameters such as weighted flux, size, ellipticity, and ratios and correlations between the signal in other channels at the same location. We have developed an image processing solution, SurroImage that meets the above criteria and performs well in a clinical research setting.

Simultaneous multi planar microscope imaging enables parallel computation of autofocus for high-sped image cytometry. Although image cytometry exhibits many potential advantages over flow cytometry, substantially slower speed has limited use to fewer applications. In commercial image cytometry instruments, long scanning times have typically been circumvented by identification of small areas of interest during high speed, low resolution scans for subsequent analysis at high resolution. This two-pass strategy of analyzing only a few cells at high resolution is a disadvantage and often cannot be used at all where dim fluorescence demands higher numerical aperture (NA) objectives. Continuous stage motion synchronized with line array or time-delay-and-integrate (TDI) CCD image acquisition is capable of increasing scan speed by an order of magnitude or more, but until recently lacked the autofocus required for higher resolution objectives where depth of field is about the thickness of a cell monolayer. Here we describe an improved design for simultaneous multi planar acquisition and on-the-fly autofocus. This new system replaces more complicated and less light efficient fiberoptic imaging bundles with beamsplitters and mirrors. This new image-splitting design also enables addition of magnification correction optics not easily added to the earlier fiberoptic version. The result is a simplified, high sensitivity, magnification-matched, parallel multi planar acquisition module containing an array of CCD sensors for high-speed focus tracking and 3D imaging.

This paper introduces a new object-oriented software framework suitable for scanning image cytometry application development. The goal of scanning image cytometry is the development of an instrument that locates and measures every cell on a microscope slide at high speed without requiring operator interaction. This capability extends traditional quantitative techniques, such as flow cytometry, through the availability of image data that can provide similar intensity measurement as well as detailed image morphometry and population geometry. Domain concepts are identified, abstracted and categorized into three thematic families: (1) physical cytometer hardware components, (2) image and image- derived measurement data, and (3) runtime dynamic and extendible image processing components. Additional abstractions are provided for the image table, an embedded database for disk archival and general access to metaimage areas-of-interest, and for the cytometer, which manages physical hardware abstractions and organizes their collaboration into high-level scanning operations. Detailed examples of framework mechanisms are given to illustrate use.

In clinical cytology, nuclear features play an important role in cell and tissue classification. To increase efficiency and decrease subjectivity of cytological results, automation of the analytic process has been proposed and discussed by many authors. This automation can be achieved by estimating the probability of occurrence of a certain class given particular features of a microscope specimen. In this paper, feature sets that might be used as inputs for mathematical cytological classification algorithms are reviewer. The primary goal was to determine the important properties of these features sets, i.e., are there mathematically efficient features that provide a more or less compete description of the cell. Under what conditions will these feature then result in optimal classification of the cells using quantitative fluorescence staining. And how would these mathematical features relate to conventional features that a human observer understands. Example human observer features are size, shape, and chromaticity o the cell nucleus while example mathematical features are image moments. If the cell image can be completely reconstructed from the feature set, then it should be possible to derive the conventional features used by human observers from the mathematical feature set for presentation to clinicians. Finally, the suitability of different mathematical decision making algorithms like probabilistic reasoning, clustering or neural networks are also briefly evaluated in the context of a mathematically complete feature set.

Advancements in image analysis shave recently made it possible to segment the cells and nuclei, of a wide variety of tissues, from 3D images collected using fluorescence confocal microscopy. This has made it possible to analyze the spatial organization of individual cells and nuclei within the natural tissue context. We present here a spatial statistical method which examines an arbitrary 3D distribution of cells of two different types and determines the probability that the cells are randomly mixed, cells of one type are clustered, or cells of different types are preferentially associated. Beginning with a segmented 3D image of cells, the Voronoi diagram is calculated to indicate the nearest neighbor relationships of the cells. Then, in a test image of the same topology, cells are randomly assigned a type in the same proportions as in the actual specimen and the ratio of cells with nearest neighbors of the same type versus the other types is calculated. Repetition of this random assignment is used to generate a distribution function which is specific for the tissue image. Comparison of the ratios for the actual sample to this distribution assigns probabilities for the conditions defined above. The technique is being used to analyze the organization of genetically normal versus abnormal cells in cancer tissue.

Most cytometry data analysis routines focus on individual data sets and require human decision-making in determining what is important to compare between two or more dat sets. As cytometry data becomes more complex, the experimenter could benefit from data mining techniques which can help guide the experimenter to the major similarities and differences between data sets. These similarities and differences are not always obvious, particularly in complex, multi parameter data and those involving rare-events. 'Subtractive clustering', a novel form of exploratory dat analysis/data mining we have recently developed compares the similarities and differences between two ro more Listmode data files. It represents a powerful new tool for cytometry. The program uses variable sized multidimensional data bins from test and control files while making no assumptions about the data. This presentation deals with subtractive clustering data mining of flow cytometry data, but the technique is entirely general and could be easily adapted to image cytometry and to any other situation involving comparison of large multidimensional data sets. This software allows for 'subtractive clustering' of data between files of varying sizes and permits the analysis of very large files. Various controllable parameters in the program allow for definition of 'similarity' of data clusters in multidimensional space. Subtraction is not a simple subtraction of multidimensional data points, but rather a subtraction of 'similar' dat objects which may or may not have the same data values. In addition, since the multidimensional coordinates of each cell are not stored in a multidimensional array, the program is not limited by data dimensionality or resolution. Data are processed and binned form Lismore data into sub-lists that permit rapid comparison between test and control files. By utilizing a Windows interface, the input of the experienced researcher is taken into account.

HT-PS is a fluidics-based pharmacology platform that uses viable cells and test compounds to rapidly identify active compounds and immediately determine their potency and specificity. Axiom employs this proprietary flow-through fluidics system coupled to a flow cytometer (FCM) as a detection system. Integration of FCM was enabled through a Plug-Flow Coupler (PFC) device that allows mixtures of cells and test compounds to be delivered to the FCM as discrete plugs of samples under positive air pressure. An FCM detector provides the advantages of multi parametric measurements and multiplexed, single cell analyses. Assays that combine two or more compatible, fluorescent bioresponse indicators simultaneously, such as measurements of intracellular pH and Ca²⁺, are possible. Alternatively, measurements of one or more bioresponses can be performed on several distinct cell populations individually stained with uniquely addressable fluorescent chromophores. These formats enable multiple experiments on a single sample and provide high content information thereby greatly increasing decision-making power regarding the activity, potency and selectivity of a test compound. Development of significant data with several hundred cells enables reduction in all requisite sample volumes. The PFC enables FCM sample analysis rates of at least 10 samples/minute. The data will illustrate HT-PS/PFC/FCM utility in the drug discovery arena.

Molecular rotors are fluorescent probes that change quantum yield with the viscosity of their environment. When integrated into the cell membrane, they can be used to probe viscosity changes of the membrane. Fluid shear stress is hypothesized to increase membrane fluidity in the membrane of endothelial cells, a change that leads to the activation of heterotrimetric G proteins, thus activating a signal transduction cascade. This hypothesis was examined using a molecular rotor, 9-dicyanovinyl-julolidine (DCVJ) as membrane probe. The principal response, a decease of fluorescence intensity caused by increased membrane fluidity, was obtained by adding a fluidity-increasing agent to the cells. In a parallel-plate flow chamber, a confluent layer of DCVJ-labeled human umbilical cord venous endothelial cells were exposed to different levels of fluid shear stress. With increased shear, a reduced fluorescence intensity was observed, indicating an increase of membrane fluidity. Step changes of fluid shear stress caused an approximately linear drop of fluorescence within 5 seconds, showing fast and almost full recovery after shear stopped. A linear relationship between shear stress and membrane fluidity changes was also observed. This study not only shows the suitability of the molecular rotor DCVJ as a membrane fluidity probe, but also provides evidence for the direct link between fluid shear stress and membrane fluidity, and suggests that the membrane is the primary flow mechanosensor of the cell.

We introduce a compact and portable photometric system for measurements of the calcium dynamics in cells. The photometer is designed for applications in centrifuges or in zero-gravity environment and thus extremely compact and reliable. It operates with the calcium-sensitive dye Indo-1. The excitation wavelength of 345nm is generated by frequency doubling of a laser diode. Two compact photomultiplier tubes detect the fluorescent emission. The electronics provides the sensitivity of photon counting combined with simultaneous measurement of the temperature, of air pressure, and of gravitational force. Internal data storage during the experiment is possible. A newly developed cell chamber stabilizes the cell temperature to 37.0 percent C +/- 0.1 degree C and includes a perfusion system to supply the cells with medium. The system has a modular set-up providing the possibility to change light source and detectors for investigation of other ions than calcium. Quantitative measurements of the intracellular calcium concentration are based on a comprehensive calibration of our system. First experiments show that the calcium dynamics of osteosarcoma cells stimulated by parathyroid hormone is observable.

Simultaneous detection of both a Eu(III) and a Sm(III) Quantum Dye is now possible because the enhanced luminescence of the Eu(III) and Sm(III) macrocycles occurs in the same solution and with excitation at the same wavelengths between 350 to 370 nm. Since DAPI is also excited between 350 to 370 nm, it is possible to use common excitation optics and a single dichroic mirror for measuring two molecular species and DNA. The narrow emissions of these macrocycles can be detected with negligible overlap between themselves or with DAPI-stained DNA. This will permit precise pixel by pixel ratio measurements of the Eu(III) macrocycle to Sm(III) macrocycle, and of each macrocycle to DNA> This technology should be applicable to antibodies, FISH, comparative genomic hybridization, and chromosome painting. Cofluorescence of the Tb(III)-macrocycle has also been obtained under different conditions. The luminescence of these lanthanide macrocycles can be observed with conventional fluorescence instrumentation previously unattainable low levels. Thus, it will be possible to employ narrow bandwidth lanthanide luminescent tags to identify three molecular species with a conventional microscope.

The wavelet tomography is used for statistical reconstruction of a lymphocyte nucleus for various diseases. The restored parameter is a probability density function (PDF) of radial distribution of condensed chromatin. In this content, PDF describes the cross-section of the nuclear structure of chromatin. Initial dat for reconstruction is the histogram of the chromatin granules, i.e. the number of granules versus the distance for the center of projection. This work is concerned with lymphocyte nuclei of peripheral blood for four groups of patients: control group; those who live in the area affected by the Chernobyl accident; mieloid group; leucose group. In contrast to the previous work, the new criteria of regularization is developed and used.

The problem of bacterial enumeration in different samples is of great importance in many fields of research. Construction of recombinant fluorescent and luminescent bacteria that can be easily detected by nondestructive instrumental methods proves us with an opportunity to monitor bacteria in a wide variety of clinical, environmental and food samples in real time. Three different labels were employed: Green Fluorescent Protein (GFP), Bacterial luciferase (BL) and Firefly Luciferase (FFL). Both plasmid and chromosomal transformants of different strains of E. coli, P. putida and S. enteritidis were used. For the detection of the in vivo GFP the Shimadzu RF 540 spectrofluorimeter, Labsystems FL- 500 plate fluorimeter and Night Owl LB 98 CCD-camera from EG and G Berthold supplied with excitation light source and proper spectral filters both in macroscopic and microscopic mode were used. For the detection of in vivo luminescence of BL and FFL, tube luminometer BG-P from GEM Biomedical Inc., luminometric plate reader from BioOrbit, BIQ Bioview CCD camera from Cambridge Imaging Ltd and Night Owl LB 98 CCD camera both in macroscopic and microscopic mode were used. The expression levels of the labels, their stability, stability of the signal and detection limits of tagged bacteria were investigated. The detection limits for GFP tagged bacteria were 5 X 10⁴ - 6 X 10⁶, for BL tagged bacteria 5 X 10² - 2 X 10⁵, and for FFL tagged bacteria - 4 X 10³ - 10⁶ CFU/ml, depending on the instrument used. Single bacteria could be detected with the help of the Night Owl in the microscopic mode.

We performed a multi parameter image analysis assessment of breast cancer cell population nuclear grade (NG), which is regarded as one of the main prognostic factors for treatment efficacy and survival of the patients and compared it with light microscopic estimation of NG. Cytological imprint slides from 20 ductal carcinomas were stained according to Leischmann-AzureII-eosine method, and NG was estimated by light microscopic observation according to Black in Fisher's modification. Simultaneously, using specially elaborated software, in each patient 100 cancer cells were analyzed for nuclear perimeter, diameter, area, nucleolar area, and average intensity of staining. The chromatin structure was assessed using mean diameter of chromatin grains and relatively chromatic are within the nucleus. Light microscopic estimation revealed 4/15 grade 2 and 7/15 grade 3 tumors out of 15 filtrating ductal carcinomas, with 4/15 classified as intermediate between grade 2-3. Multifactoral linear correlation coefficient r equals 0.39, p < 0.001 for ductal cancer, higher NG comes with increasing nucleolar area, nuclear roundness factor, nuclear are, and chromatin area within the cell nucleus. Image analysis may yield precise information on NG as a prognostic factor in breast cancer patients.

This study is focused on the modifications in erythrocytes of Chernobyl nuclear power plant accident clean-up workers as a late health effect of short-term impact of high level radioactive contamination. As a result, a new method based on erythrocyte refractive index properties at different pH has been elaborated.

We present the design of a magnetic tweezers microscope for cellular manipulation. Our design allows versatile and significant 3D stress application over a large sample region. For linear force application, forces up to 250 pN per 4.5 micrometers magnetic bead can be applied. Finite element analysis shows that variance in force level is around 10 percent within an area of 300 X 300 micrometers ². Our eight-pole design potentially allows 3D liner force application and exertion of torsional stress. Furthermore, our design allows high resolution imaging using high numerical aperture objective. Both finite element analysis of magnetic field distribution and force calibration of our design are presented. As a feasibility study, we incubated fibronectin coated 4.5 micrometers polystyrene beads with Swiss 3T3 mouse fibroblast cells. Under application around 250 pN of force per magnetic particle, we observed relative movement between attached magnetic and polystyrene beads to be on the order of 1 micrometers . Elastic, viscoelastic, and creeping responses of cell surfaces were observed. Our results are consistent with previous observations using similar magnetic techniques.

Recently, JPL has invented and developed a miniature optical microscope, microscope-on-chip using micro-channel and solid state image sensors. It is lightweight, low-power, fast sped instrument, it has no image lens, does not need focus adjustment, and the total mass is less than 50g. A prototype has been built and demonstrated at JPL.

Using an in vitro wound repair model of the respiratory epithelium, we have previously shown that cell migration is perfectly unidirectional during wound repair. Among the mechanisms potentially involved in the regulation of cell migration, we were particularly interested in the involvement of endogenous electric fields in the repair of wounded respiratory epithelium. Compared to non-injured confluent cultures of airway epithelial cell sin which a constant electric field was observed, we have measured very large variations of the electric field in wounded cultures during the repair process. Immediately after injury, the electric field was zero and progressively increased during wound closure. By using a potentiometric fluorescent probe, we also demonstrated an hyperpolarization of the membrane of migratory cells, as compared with stationary cells located far from wound. In addition, we observed that isolated respiratory epithelial cells, placed in a 10V/cm electric field, acquired a uniform and constant direction of migration in contrast to the random migration of cells not subjected to an electric field. These results suggest that an endogenous electric field could be one of the mechanisms triggering cell migration, thereby leading to the reepithelialization of the respiratory epithelium after injury.

The different binding behavior of some fluorescence dyes to DNA has been primarily investigated by absorption, fluorescence and atomic force microscopy (AFM). The results indicate that the AFM study of the complexes of DNA in their native conformation can be explored to reveal the special nanostructural information of the complexation of the biopolymer supramolecular system, which may directly provide some fresh structural evidence in relation to the binding modes of the ligand-DNA complex.

In cell biology, one of the great mysteries, which has bene only superficially 8investigate,d is the integration of cytoplasmic and nuclear organelles as part of the intracellular regulatory mechanism. The methodology used for the exploration of such intracellular processes is the pixel-by-pixel scanning by means of fluorescence spectral imaging and excitation emission fluorescence spectroscopy. While several of the steps required by this method are still in the process of implementation, the Michelson interferometer, the Sagnac interferometer and the related 'pentaferometer' are possible components of the instrumental design. One of the illustrative experimental models to begin the study of intracellular integrative processes is based on the hypothesis of a 'nuclear pump' in conjunction with cell treatment by chemotherapeutic agents such as adriamycin. Preliminary observations initiated in cultured fibroblasts, and to be pursued in Cloudman's melanoma cells, suggest that this cytotoxic agent first moves into the nucleus, form which it is subsequently ejected to be incorporated into the lysosomes and Golgi apparatus, possibly prior to exclusion via the multiple drug resistance pathway. The timetable of such a process is under investigation. This subject has obvious implications for diagnostic, prognostic and therapeutic studies of organelles integration.

Membrane traffic between the endoplasmic reticulum (ER) and the Golgi complex is regulated by two vesicular coat complexes, COPII and COPI. COPII has been implicated in selective packaging of anterograde cargo into coated transport vesicles budding from the ER. COPI-coated vesicles are proposed to mediate recycling of proteins from the Golgi complex to the ER. We have used multi spectral 3D imaging to visualize COPI and COPII behavior simultaneously with various GFP-tagged secretory markers in living cells. This shows that COPII and COPI act sequentially whereby COPI association with anterograde transport complexes is involved in microtubule-based transport and the en route segregation of ER recycling molecules from secretory cargo within TCS in transit to the Golgi complex. We have also investigated the possibility to discriminate spectrally GFP fusion proteins by fluorescence lifetime imaging. This shows that at least two, and possibly up to three GFP fusion proteins can be discriminated and localized in living cells using a single excitation wavelength and a single broad band emission filter.

Intracellular ion concentrations can be measured using ratiometric fluorophores. These fluorophores often show, in addition to the change in fluorescence spectrum, a change in fluorescence lifetime for different ion strengths. Accordingly, it has been suggested that using the fluorescence lifetime would make it possible to replace the intracellular calibration with a simplified extracellular calibration step. However, there are contradictory results on whether probe binding to membranes and macromolecules has influence on lifetime measurement. We have addressed this problem by systematically investigating three different probes used for intracellular pH determination. All probes were studied in vivo and in vitro. Influence of different factors such as protein and lipid concentration on the lifetime of the probes was measured. Our results give a possible explanation as to why earlier investigators have recorded constant pH throughout the whole cell.

Two-photon fluorescence spectroscopy has been performed on rat skeletal muscles to investigate the effect of fixation processes on the micro-environments of the endogenous fluorophores in rat skeletal muscles. The two-photon fluorescence spectra measured for different fixation periods show a differential among those samples that were fixed in water, formalin and methanol, respectively. The result imply that two-photon fluorescence spectroscopy can be a potential technique for identification of healthy and malignant biological tissues.

This study aimed to observe liposome uptake by leukocytes in vivo. The study was performed on skin by using a dorsal skin-fold chamber implanted in golden hamsters using intravital microscopy. 5,6-CF-encapsulated PEGylated liposomes were injected intravenously. The skin microcirculation was observed with an intravital Eclipse E800 Nikon microscope fitted with a Xenon light source and an epi-fluorescence assembly. An ultra-high sensitivity video-camera mounted on the microscope projected the image onto a monitor, and the images were recorded for playback analysis with a digital video cassette recorder. An acute inflammatory response was obtained by removing one complete layer of skin and the underlying fascia and avascular tissue on the opposing side of the flap corresponding to an area equivalent to the window aperture. Using these model and set-up, leukocyte rolling and adhesion were easily observed and the entry of PEGylated liposomes into hamster blood leukocytes was studied for a period of 6 hours. PEGylated liposomes were clearly identified alone inside the blood flow and inside the leukocytes as soon as the inflammatory reaction appeared. This study shows for the first time that blood leukocytes in their natural milieu of whole blood are capable of interacting with, and taking up liposomes. This observation is in accordance with previous in vitro studies.

We report here a new approach to genetically engineering tumors to become fluorescence such that they can be imaged externally in freely-moving animals. We describe here external high-resolution real-time fluorescent optical imaging of metastatic tumors in live mice. Stable high-level green flourescent protein (GFP)-expressing human and rodent cell lines enable tumors and metastasis is formed from them to be externally imaged from freely-moving mice. Real-time tumor and metastatic growth were quantitated from whole-body real-time imaging in GFP-expressing melanoma and colon carcinoma models. This GFP optical imaging system is highly appropriate for high throughput in vivo drug screening.

A dedicated small animal x-ray computed tomography system has been developed to screen mutagenized mice for anatomical phenotypes. The key components of the data acquisition instrumentation are described along with the system performance parameters. Image reconstruction, visualization and segmentation software algorithms are described. Two contrast media regimens are described and representative studies of mice with adipose, soft and skeletal tissue abnormalities are presented.

Of increasing interest is miniature and portable instrumentation for non- or minimally-invasive functional microscopy. Two-photon fluorescence confocal microscopy is one optical image modality not yet exploited in this context primarily because of the large physical size of the laser light sources needed to stimulate the two-photon absorption process. As a possible alternative, we report on the successful application of light at 1064 nm from a passively, Q-switched Nd:YAG microchip laser for two-photon fluorescence microscopy of biological samples. The high peak power in a small optomechanical package makes the microchip laser an attractive starting point for development of portable two-photon fluorescence imaging system for medical endoscopy and other remote sensing applications.

Fluorescence resonance energy transfer (FRET) imaging is a unique tool used to visualize the spaciotemporal dynamics of protein-protein interactions in living cells. We used FRET to study the dimerization of the pituitary-specific transcription factor of Pit-1 fused with blue flourescent protein and green fluorescent protein. Transcriptional activity of the GFP- and BFP-Pit-1 fusion proteins was demonstrated by their ability to activate the prolactin gene promoter. The energy transfer in the conventional fluorescence microscopy was less efficient due to photobleaching of the BFP-Pit-1 donor molecules. In our studies we developed two-photon excitation energy transfer microscopy, where the photobleaching of blue flourescent protein was considerably reduced. This 2p-FRET imaging system was used to acquire the donor and acceptor images for a living HeLa cell nucleus. We selected 732 nm from the tunable Verdi pumped ti:sapphire laser, in a way that only excites the BFP-Pit-1 and not the GFP-Pit-1 proteins. The efficiency of the 2p-FRET signal increased to 30 percent compared to the conventional FRET imaging, which clearly demonstrates that there is considerable reduction in photobleaching of donor molecules in the 2p-FRET microscopy.

A real-time confocal multiphoton fluorescence microscope was developed to observe Ca²⁺ dynamics in living rat- cardiac muscle cells. The real-time imaging was achieved by multifocus excitation of a specimen with a rotating microlens-array disk. A pinhole-array disk for confocal detection was introduced in the microscope to improve the spatial resolution and the contrast of fluorescence images. Ca²⁺ wave and Ca²⁺ transient in cultured rat- cardiac cells were successfully observed with the developed microscope.

In recent years, confocal Laser-Scanning microscopy became the most sophisticated microscopic technique for 3D-imaging in biomedical microstructure research. Experimental evidence, however, showed that under optical conditions relevant e.g. for nuclear genome analysis, the resolution of such an instrument as given by the Full Width at Half Maximum of the confocal point-spread function (PSF) is limited to about >= 0.3 micrometers laterally and to about >= 0.7 micrometers axially. A recently introduced light- microscopic approach, termed Spectral Precision Distance Microscopy (SPDM), allows the precise measurement of distances and angles between specifically labeled target sites far below the above mentioned resolution limit. SPDM is based on the use of 'point-like' objects labeled with different spectral signatures. Since in most cases, spectral signature differences have been realized by variation of excitation/fluorescence emission spectra, the calibration of chromatic aberrations is of utmost importance. Here, an improved procedure for the correction of chromatic shifts is presented. Statistical errors introduced by the localization accuracy can be minimized by the multiple measurement of the 3D-distances between the same specifically labeled target sites in a number of cases and subsequent averaging. However, the thus obtained mean distance, the estimate for the 'true' distance may be biased and therefore limited by the localization accuracy. Virtual microscopy simulations of test objects using an experimentally obtained PSF, showed that a few thousand detected fluorescence photons are sufficient to measure reliably distances down to about 20 nm, if other sources of error apart from voxelization, digitization and photon noise are negligible.

The determination of the 3D nanostructure of specific chromatic regions is highly relevant for an improved understanding of the functional topology of the genome. The use of different spectral signatures for the labeling and high accuracy nanodistance measurements in the spectral precision distance microscopy mode allows the investigation of the topology of such targets in 3D conserved nuclei. To obtain the required high-accuracy nanolocalization of small targets, interferometric illumination is a well established and reliable tool. New approaches use spatially modulated illumination (SMI) in various ways. In our laboratory a stage controlled optical sectioning through the object was applied. In this case the SMI point spread function is the product of the axial illumination modulation and of the conventional PSF of the microscope objective. Using this approach and an appropriate analysis algorithm, the position of 'point-like' fluorescent objects was determined with an axial localization precision in the range of 2nm. To provide a reliable high precision localization performance also for long time measurements, thermally invariant mounting devices have been developed for the SMI-system. Using this improved system, it was possible to measure the thermal shift induced by the microscope objective itself.

Laser irradiation of hyaline cartilage result in stable shape changes due to temperature dependent stress relaxation. In this study, we determined the structural changes in chondrocytes within rabbit nasal septal cartilage tissue over a 12-day period using a two-photon laser scanning microscope (TPM) following Nd:YAG laser irradiation. During laser irradiation surface temperature, stress relaxation, and diffuse reflectance, were measured dynamically. Each specimen received one or two sequential laser exposures. The cartilage reached a peak surface temperature of about 61 degrees C during irradiation. Cartilage denatured in 50 percent EtOH was used as a positive control. TPM was performed to detect the fluorescence emission from the chondrocytes. Images of chondrocytes were obtained at depths up to 150 microns, immediately following laser exposure, and also following 12 days in culture. Few differences in the pattern or intensity of fluorescence was observed between controls and irradiated specimens imaged immediately following exposure, regardless of the number of laser pulses. However, following twelve days in tissue culture, the irradiated specimens increase, whereas the native tissue diminishes, in intensity and distribution of fluorescence in the cytoplasm. In contrast, the positive control shows only extracellular matrices and empty lacuna, feature consistent with cell membrane lysis.

A focused laser beam can be used to trap and manipulate a particle in liquid, which has found applications in studying living cells. The characteristics of the interaction between the particle and the laser beam can give information of the particle. We put forward a method for determination the spring constant of an optical trap through external excitation and lock-in detection. The external sinusoidal excitations of the trapped particle can be introduced by electrostatic force through applying a sinusoidal voltage upon the transparent conductive glass substrates composing the sample cell. A sensor using the feedback effect of a laser diode with two external cavities is also proposed for monitoring the particle oscillation. This self-mixing interferometry has the capability of absolute measurement of the particle displacement, which is important to determine the interaction strength of a laser-particle trapping system.

Real-time monitoring of reorganization and molecular interactions of live cellular components on a precise spatial and temporal scale, requires a high-speed and high- sensitivity imaging system. Theoretically, excited state fluorescence lifetime imaging techniques provide high temporal resolution for dynamics studies of biological samples. In order to provide a comprehensive foundation for the development of the technique, we simulated images using the decay and normal equations for a single component with different windowing schemes. In this model we varied several parameters involved in the simulation to produce images under different theoretical conditions. To obtain a realistic result, we considered equations for noise due to readout, dark current, photon shot noises, and other factors. In addition, the simulant was validated using sample decay images with known lifetimes, which were obtained with current fluorescent decay systems. The software developed through this research is intended to create a compete tutorial describing the theory and procedures of lifetime image acquisition. This theoretical simulation is verified with experiments using known fluorophore lifetimes.

Considerable variation exists among pathologist in the interpretation of intraepithelial neoplasia making it difficult to determine the natural history of these lesion and to establish management guidelines for chemoprevention. The aim of the study is to evaluate architectural features of pre-neoplastic progression in lung cancer, and to search for a correlation between architectural index and conventional pathology. Quantitative architectural analysis was performed on a series of normal lung biopsies and Carcinoma In Situ (CIS). Centers of gravity of the nuclei within a pre-defined region of interest were used as seeds to generate a Voronoi Diagram. About 30 features derived from the Voronoi diagram, its dual the Delaunay tessellation, and the Minimum Spanning Tree were extracted. A discriminant analysis was performed to separate between the two groups. The architectural Index was calculated for each of the bronchial biopsies that were interpreted as hyperplasia, metaplasia, mild, moderate or severe dysplasia by conventional histopathology criteria. As a group, lesions classified as CIS by conventional histopathology criteria could be distinguished from dysplasia using the architectural Index. Metaplasia was distinct from hyperplasia and hyperplasia from normal. There was overlap between severe and moderate dysplasia but mild dysplasia could be distinguished form moderate dysplasia. Bronchial intraepithelial neoplastic lesions can be degraded objectively by architectural features. Combination of architectural features and nuclear morphometric features may improve the quantitation of the changes occurring during the intra-epithelial neoplastic process.

Cellular and non-cellular constituents of skin contain fundamental morphometric features and structural patterns that correlate with tissue function. High resolution digital image acquisitions performed using an automated system and proprietary software to assemble adjacent images and create a contiguous, lossless, digital representation of individual microscope slide specimens. Serial extraction, evaluation and statistical analysis of cutaneous feature is performed utilizing an automated analysis system, to derive normal cutaneous parameters comprising essential structural skin components. Automated digital cutaneous analysis allows for fast extraction of microanatomic dat with accuracy approximating manual measurement. The process provides rapid assessment of feature both within individual specimens and across sample populations. The images, component data, and statistical analysis comprise a bioinformatics database to serve as an architectural blueprint for skin tissue engineering and as a diagnostic standard of comparison for pathologic specimens.