Improved confocal characterization of targets in nuclei of cytogenetic preparations

OBJECTIVE: To show that cellular preparations requiring depth analysis of different domains stained by molecular cytogenetic methods (fluorescence in situ hybridization and primed in situ) can be improved by regularized factor analysis of medical image sequences (FAMIS) to isolate fluorescent probes by means of intensity depth profiles of fluorochromes, to track relevant DNA sequences (cosmids and centromeres) in cell nuclei during interphase and to improve the use of cytogenetic techniques resulting in flat preparations of whole cells that are assumed to preserve probe access to their targets. STUDY DESIGN: 3D sequences of images obtained by depth displacement in a confocal microscope were first analyzed by the FAMIS algorithm, which provides factor curves. Factor images then resulted from regularization methods that improve signal/noise ratio while preserving target contours. RESULTS: Factor curves and regularized factor images helped analyze targets inside nuclei. CONCLUSION: It is possible to process preparations containing numerous spots (even when they are on different planes) to differentiate stained targets, to investigate depth differences and to improve visualization and detection.     

Factor analysis of confocal image sequences of human papillomavirus DNA revealed with fast red in cervical tissue sections stained with TOTO-iodide

OBJECTIVE: To visualize and localize specific DNA sequences by fluorescence in situ hybridization, confocal laser scanning microscopy (CLSM) and factor analysis of biomedical image sequences (FAMIS). STUDY DESIGN: Human papillomavirus (HPV) DNA was identified in cervical tissue sections with biotinylated DNA probes recognizing the whole genome of HPV DNA types 18 and 16, and DNA-DNA hybrids were revealed by streptavidin-alkaline phosphatase and Fast Red (FR). Cell nuclei were counterstained with TOTO-iodide. Image sequences were obtained using successive dynamic or spectral sequences of images on different optical slices from CLSM. The location of fluorescent signals inside tissue preparations was determined by FAMIS and/or selection of filters at emission. Image sequences were summarized into a reduced number of images, called "factor images," and curves, called "factors." Factors estimate spectral patterns and depth emission profiles. Factor images correspond to spatial distributions of the different factors. RESULTS: We distinguished between FR and nucleus staining in HPV DNA hybridization signals by taking into account differences in their spectral patterns and improved visualization by taking into account differences in their focus (depth emission profiles). CONCLUSION: FAMIS, together with CLSM, made possible the detection and characterization of HPV DNA sequences in cells of cervical tissue sections.     

Confocal image characterization of human papillomavirus DNA sequences revealed with Eu in HeLa cell nuclei stained with Hoechst 33342

OBJECTIVE: To visualize and localize specific viral DNA sequences revealed with Eu by fluorescence in situ hybridization, confocal laser scanning microscopy (CLSM) and factor analysis of biomedical image sequences (FAMIS). STUDY DESIGN: Human papillomavirus DNA (HPV-DNA) was identified in HeLa cells with biotinylated DNA probes recognizing HPV-DNA types 16/18. DNA-DNA hybrids were revealed by a three-step immunohistochemical amplification procedure involving an antibiotin mouse monoclonal antibody, a biotinylated goat antimouse polyclonal antibody and streptavidin-Eu. Cell nuclei were counterstained with Hoechst 33342. Image sequences were obtained using a CLSM that made possible ultraviolet excitation. The location of fluorescent signals inside cellular preparations was determined by FAMIS and selection of filters at emission. Image sequences were summarized into a reduced number of images, or factor images, and curves, or factors. Factors estimate spectral or temporal patterns and depth emission profiles. Factor images correspond to spatial distributions of the different factors. RESULTS: We distinguished between Eu corresponding to HPV-DNA hybridization signals and nuclear staining by taking into account differences in their spectral and temporal patterns and (using their decay rates). CONCLUSION: FAMIS, together with CLSM and Eu, made possible the detection and characterization of viral papillomavirus DNA sequences in HeLa cells.     

Confocal-multilabeling, ultrasensitive TUNEL analysis of DNA breaks in individual cells

OBJECTIVE: To visualize and localize fragmented DNA strands within apoptotic cells by means of fluorescence using TdT-mediated dUTP-biotin nick end labeling (TUNEL) techniques, laser scanning confocal microscopy (CLSM) and factor analysis of biomedical image sequences (FAMIS). STUDY DESIGN: For this experiment, lymphoid reverted cells were used as a model. Characteristic DNA breaks inside apoptotic cells were detected using TUNEL techniques by a reaction involving tetramethyl rhodamin isothyocyanate (TRITC). The DNA from cell nuclei was counterstained using chromomycin A3 (CA3). The tandem TRITC-CA3 in CLSM was applied to investigate the ability to detect DNA breaks in individual cells using TUNEL techniques and its amplified variants (TUNEL-CARD). FAMIS was applied on dynamic sequences of images of TUNEL preparations and on four-dimensional (4-D) sequences of images of TUNEL-CARD preparations. RESULTS: Distribution and amplitude of fluorescent structures were characterized on dynamic sequences of images. Characterization was improved when FAMIS was applied on 4-D sequences of images, taking into account differences in photobleaching and/or spectrum of TRITC and CA3. CONCLUSION: It is possible to discriminate targets from CA3. FAMIS and TUNEL methods can be used to visualize and localize multiple DNA breaks in lymphoid reverted cells in improved methods of experimentation.     

FRET multiphoton spectral imaging microscopy of 7-ketocholesterol and Nile Red in U937 monocytic cells loaded with 7-ketocholesterol

OBJECTIVE: To show the effect of 7-ketocholesterol (7KC) on cellular lipid content by means of flow cytometry and the interaction of 7KC with Nile Red (NR) via ultraviolet fluorescence resonance energy transfer (FRET) excitation of NR on U937 monocytic cells by means of 2-photon excitation confocal laser scanning microscopy (CLSM). STUDY DESIGN: Untreated and 7KC-treated U937 cells were stained with NR and analyzed by flow cytometry and CLSM. 3D sequences of images were obtained by spectral analysis in a 2-photon excitation CLSM and analyzed by the factor analysis of medical image sequences (FAMIS) algorithm, which provides factor curves and images. Factor images are the result of the FAMIS image processing method, which handles emission spectra. In FRET analysis, preparations are screened at selected UV wavelengths to avoid emission of NR in the absence of 7KC. RESULTS: During 7KC-induced cell death,flow cytometry and CLSM revealed a modification of the cellular lipid content. Factor images show FRET occurrence and subsequent colocalization of 7KC and NR. CONCLUSION: This investigation established the utility of 2-photon excitation CLSM to assess colocalization of 7KC with NR by FRET and to identify and distinguish polar and neutral lipids stained by NR that accumulate from the effect of 7KC.     

Analysis of CD36 expression on human monocytic cells and atherosclerotic tissue sections with quantum dots: investigation by flow cytometry and spectral imaging microscopy

OBJECTIVE: To demonstrate CD36 expression with quantum dots (QDs) 525 and/or 605 on human monocytic U937 cells and atherosclerotic tissue sections by means of flow cytometry (FCM) and/or confocal laser scanning microscopy (CLSM). STUDY DESIGN: U937 cells and tissue sections were analyzed by means of FCM and/or CLSM. FCM was performed, using different ultraviolet (UV) and visible (488/532 nm) excitation modes. In the visible mode, fluorescence intensities of QDs, phycoerythrin (PE) and fluorescein isothiocyanate (FITC) were compared. Three-dimensional (3-D) sequences of images were obtained by spectral analysis in a CLSM and analyzed by the factor analysis of medical image sequences (FAMIS) algorithm, providing factor curves and images. Factor images are the result of the FAMIS image processing method, which differentiates emission spectra from 3D sequences of images. In CLSM analysis, preparations are screened in a UV excitation mode to optimize the possibilities of QDs and have the benefit of 4',6-diamino-2-phenylindole or Hoechst 33342 counterstaining of nuclei. RESULTS: FCM and CLSM revealed CD36 expression by means of QDs 525 and/or 605. Fluorescence intensity of PE and of FITC was higher than that of QDs 525 and of 605. As factor curves and images show the red emission of QDs 605 only, subsequent reliable identification and localization of CD36 was obtained. CONCLUSION: QDs 525 and 605 are useful to analyze antigenic expression. Following FCM, which is well adapted to detect fluorescence emission of QDs in the UV or visible excitation mode, CLSM and subsequent spectral analysis assess more specific characterization of QD fluorescent emissions.     

Analysis of the fluorescence of monodansylcadaverine-positive cytoplasmic structures during 7-ketocholesterol-induced cell death

OBJECTIVE: To analyze multilamellar cytoplasmic structures by confocal laser scanning microscopy (CLSM) combined with factor analysis of biomedical image sequences (FAMIS). STUDY DESIGN: After treatment of U937 cells with 7-ketocholesterol (7-keto), cytoplasmic alterations were assessed with monodansylcadaverine (MDC). By ultraviolet excitation of a confocal laser scanning microscope (UV-CLSM), spectral sequences were performed to characterize 7-keto and MDC distribution inside cells. FAMIS was used to transform the image sequences in factor curves and images. RESULTS: By UV-CLSM, 7-keto fluorescence was detected together with MDC, which revealed morphologic cytoplasmic changes in cells. The factor images obtained from confocal image sequences emphasized the view of these results. These data are in agreement with biochemical characterizations of MDC-positive structures. CONCLUSION: The combined use of confocal microscopy and FAMIS allowed us to detect MDC-positive cytoplasmic structures in 7-keto-treated cells and to colocalize MDC and 7-keto distribution. This new method confirms the usefulness of MDC as a marker of oxysterol-induced cell death.     

Analysis of fluorescent MRI contrast agent behavior in the liver and thoracic aorta of mice

OBJECTIVE: To characterize the behavior of magnetofluorescent products injected in mice intravenously. STUDY DESIGN: The magnetic resonance imaging (MRI) products were labelled with fluorescent molecules to examine the biodistribution process in vivo and observe them at the cellular level by means of confocal microscopy. Three-dimensional (3D) sequences of images were obtained by spectral analysis of sample preparations in a multiphoton confocal microscope and analyzed by the factor analysis of medical image sequence algorithm, which provides factor curves. Factor images are the result of image-processing methods that utilize information from emission spectra. Preparations are also screened in the counting mode to provide fluorescent lifetime imaging microscopy (FLIM) characterizations. RESULTS: Factor images and FLIM images can help to analyze MRI targeting inside the liver and thoracic aorta of mice. They show positive detection of Fe-Texas red and BOPTA-Eu in the liver and positive detection of Fe-Texas red and negative detection of BOPTA-Eu inside the thoracic aorta. CONCLUSION: This investigation established the utility of fluorescent MRI contrast agents as in vivo staining tools for cellular sites.     

Flow cytometry and factor analysis evaluation of confocal image sequences of morphologic and functional changes occurring at the mitochondrial level during 7-ketocholesterol-induced cell death

OBJECTIVE: To analyze functional and morphologic alterations that occur at the mitochondrial level by flow cytometry and laser scanning confocal microscopy (CLSM) combined with factor analysis of biomedical image sequences (FAMIS). STUDY DESIGN: Under treatment of U937 cells with 7-ketocholesterol, functional alterations that occur at the mitochondrial level (especially loss of transmembrane mitochondrial potential [delta psi m]) were assessed with 3,3'-dihexyloxacarbocyanine iodide (DiOC6(3)) and mitotracker red (CMXRos), whereas morphologic changes were analyzed with nonyl acridine orange (NAO). By flow cytometry, these different dyes were excited at 488 nm, whereas on CLSM, excitation of NAO and CMXRos was performed by lines of an argon laser. By CLSM, spectral sequences were performed to characterize NAO and CMXRos. FAMIS was used to transform the image sequences in factor images. RESULTS: By flow cytometry, rapid loss of delta psi m induced by 7-ketocholesterol was detected with both DiOC6(3) and CMXRos, which gave similar results. Morphologic alterations of mitochondria were revealed with NAO. The factor images obtained from confocal image sequences confirmed these results. CONCLUSION: The simultaneous use of NAO, CMXRos and FAMIS constitutes a new method to detect morphologic and functional alterations occurring at the mitochondrial level during cell death.     

Confocal analysis of phosphatidylserine externalization with the use of biotinylated annexin V revealed with streptavidin-FITC, -europium, -phycoerythrin or -Texas Red in oxysterol-treated apoptotic cells

OBJECTIVE: To analyze externalization of phosphatidylserine via annexin V on apoptotic cells by laser scanning confocal microscopy and factor analysis of biomedical image sequences (FAMIS). STUDY DESIGN: Streptavidin-fluorescein isothiocyanate (FITC), -europium (Eu), -phycoerythrin (PE) and -Texas Red (TR) were chosen to reveal the binding of biotinylated annexin V on apoptotic U937 human leukemic cells and ECV-304 human endothelial cells induced under treatment with 7-ketocholesterol or 7 beta-hydroxycholesterol. Excitation of each fluorochrome was obtained by selection of specific lines (351 + 364 nm, 488 nm) of the argon laser of a confocal microscope. Temporal and spectral series were performed to characterize each fluorochrome. FAMIS was applied to these series to estimate images corresponding to stains. RESULTS: Each fluorochrome was clearly distinguished, and images showed localization of phosphatidylserine, which was improved by image analysis. CONCLUSION: On apoptotic cells it is possible to analyze differences in the improved visualization of phosphatidylserine in series processed by FAMIS with the use of biotinylated annexin V revealed with streptavidin-FITC, -Eu, -PE or -TR.     

Confocal multilaser focusing and single-laser characterization of ultraviolet excitable stains of cellular preparations

BACKGROUND: The aims of this study were (1) to realign cellular preparations when spots and structures are excited by different lasers of a confocal laser scanning microscope (multilaser studies); (2) to avoid the use of realigment methods by selecting fluorochromes that can be excited by only one laser (single-laser experiments). METHODS: In multilaser studies, we used propidium iodide fluorescent beads, as well as tetramethyl rhodamine isothiocyanate (TRITC), fluorescein isothiocyanate (FITC), and 4'-6 diamidino-2-phenylindole (DAPI)-stained human cancer lines. They were excited using HeNe, argon, and ultraviolet (UV) argon laser lines of a confocal laser scanning microscope. Single-laser experiments using UV excitation only were performed using europium as a model for magnetic resonance paramagnetic contrast agents. Nuclei of human cancer lines and tissue were counterstained by DAPI and cytoplasms were labeled with ELF-97 substrates. Factor analysis of medical images (FAMIS) and correlation methods were used to realign shifted images, focus images, and characterize each fluorochrome when necessary. RESULTS: In multilaser studies, superimposition of factor images corrected Z shifts and correlation methods provided X, Y correction values. In single-laser experiments, each fluorochrome was clearly distinguished in the group of fluorochromes. Estimated images in both studies showed colocalizations of structures. CONCLUSIONS: It is possible to characterize differences in the focus and alignment of fluorescent probes and to correct them. It is also possible to study colocalization of UV excitable fluorochromes (DAPI, ELF-97, europium) in cellular and tissular preparations via multilaser or single-laser experiments.     

Flow cytometry and spectral imaging multiphoton microscopy analysis of CD36 expression with quantum dots 605 of untreated and 7-ketocholesterol-treated human monocytic cells

OBJECTIVE: To evaluate CD36 expression with quantum dots 605 (QDs 605) on untreated and 7-ketocholesterol (7KC)-treated monocytic U937 cells by flow cytometry (FCM) and confocal and multiphoton laser scanning microscopy (CLSM). STUDY DESIGN: Cells were analyzed by CLSM, following flow cytometric quantification of CD36 expression and 7KC uptake. Image sequences were obtained by spectral analysis in monophoton and multiphoton CLSM and analyzed by the factor analysis of medical image sequences (FAMIS) algorithm to differentiate emission spectra. In CLSM analysis, cell deposits were screened in ultraviolet excitation modes to optimize the possibilities of QDs 605 and have the benefit of nuclei counterstaining by DAPI. RESULTS: FCM and CLSM reveal the expression of CD36 by means of QDs 605. FCM provides information on 7KC uptake. CLSM provides the localization of 7KC vs. DAPI. As factor curves and images show the red, narrow emission of QDs 605 vs. violet and blue emissions of 7KC and DAPI, respectively, a reliable identification of CD36 is obtained. CONCLUSION: QDs 605 are useful tools to perform antigenic expression in FCM and CLSM. Moreover, CLSM and subsequent spectral analysis provide a more specific characterization of QDs 605 fluorescent emission in the UV excitation mode and a simultaneous identification of 7KC.     

Effects of caspase inhibitors (z-VAD-fmk, z-VDVAD-fmk) on Nile Red fluorescence pattern in 7-ketocholesterol-treated cells: investigation by flow cytometry and spectral imaging microscopy.

BACKGROUND: The 7-ketocholesterol (7KC)-induced cell death has some characteristics of apoptosis and is associated with polar lipid accumulation. So, we investigated the effects of the broad-spectrum caspase inhibitor z-VAD-fmk and of the caspase-2 inhibitor z-VDVAD-fmk on lipid profile evaluated by staining with Nile Red (NR). METHODS: The 7KC-treated human monocytic U937 cells were cultured in the absence or in the presence of the caspase inhibitors z-VAD-fmk or z-VDVAD-fmk. When staining with NR is performed, neutral and polar lipids have yellow and orange/red emission, respectively, and fluorescence was then analyzed by flow cytometry (FCM) and by confocal laser scanning microscopy (CLSM) combined with subsequent image processing. The 3D-image sequences were obtained by means of CLSM using spectral analysis, and were analyzed by the factor analysis of medical image sequences algorithm to differentiate spectra inside mixed fluorescence emission and get corresponding specific images. RESULTS: By FCM, comparatively to untreated cells, higher percentages of red fluorescent cells were identified in 7KC-treated cells. Factor curves and images reveal orange and red fluorescence emissions in 7KC-treated cells and show yellow, orange, and red fluorescence emissions in 7KC-treated cells cultured in the presence of z-VAD-fmk or z-VDVAD-fmk. CONCLUSIONS: Our data support that investigation by FCM and by spectral analysis in CLSM associated with subsequent image processing provides useful tools to determine the effect of caspase inhibitors on lipid content evaluated with NR. They also favor the hypothesis of relationships between caspase activity and polar lipid accumulation.     

Confocal laser scanning microscopy of whole mouse ovaries: excellent morphology, apoptosis detection, and spectroscopy.

BACKGROUND: Ovaries consist of numerous follicles, oocytes, and granulosa cells in different stages of development. Many of these follicles will undergo an apoptotic process during the lifetime of the animal. By using proper tissue preparation methods, the events within the whole ovary can be observed by using 3D confocal microscopy. METHODS: Whole ovaries were stained with LysoTracker Red (LT), fixed with 4% paraformaldehyde (PF) and 1% glutaraldehyde (Glut), dehydrated with methanol (MEOH), and cleared with benzyl alcohol and benzyl benzoate (BABB). Using this tissue preparation technique, the ovary becomes relatively transparent, allowing its morphology to be observed with confocal microscopes. A spectral imaging system (PARISS) located on a conventional microscope was used to interpret the LT dye spectra and fixation products in the tissues with different excitation wavelengths. RESULTS: Apoptosis in the follicle was detected as clusters of intensely stained granulosa cells located in close proximity to the oocytes. The fixation with Glut and PF preserved morphological details, increased tissue fluorescence, thus increased the signal to noise of the background image. CONCLUSIONS: Thick tissues can be imaged after they are properly stained, aldehyde fixed, and BABB cleared. LT intensely stained single cells or clusters of apoptotic cells in the follicles and the nucleolus. Spectral differences between LT as an indicator of apoptosis and Glut-PF fixation was used to visualize ovarian morphology and apoptosis. The PARISS spectrophotometer revealed spectral peaks for LT at 609.6 nm and for Glut-PF at 471.3 nm. The proper use of the spectra from these fluorescence molecules is the foundation for high quality morphological images of apoptosis. By sequentially imaging the two probes with a 488 nm laser and a 543/568 nm laser, there was a reduction in fluorescent cross talk and an increase in image quality.     

Combined confocal and wide-field high-resolution cytometry of fluorescent in situ hybridization-stained cells

BACKGROUND: The recently developed technique of high-resolution cytometry (HRCM) enables automated acquisition and analysis of fluorescent in situ hybridization (FISH)-stained cell nuclei using conventional wide-field fluorescence microscopy. The method has now been extended to confocal imaging and offers the opportunity to combine the advantages of confocal and wide-field modes. METHODS: We have automated image acquisition and analysis from a standard inverted fluorescence microscope equipped with a confocal module with Nipkow disk and a cooled digital CCD camera. The system is fully controlled by a high-performance computer that performs both acquisition and related on-line image analysis. The system can be used either for an automatic two (2D) and three-dimensional (3D) analysis of FISH- stained interphase nuclei or for a semiautomatic 3D analysis of FISH-stained cells in tissues. The user can select which fluorochromes are acquired using wide-field mode and which using confocal mode. The wide-field and confocal images are overlaid automatically in computer memory. The developed software compensates automatically for both chromatic color shifts and spatial shifts caused by switching to a different imaging mode. RESULTS: Using the combined confocal and wide-field HRCM technique, it is possible to take advantage of both imaging modes. Images of some dyes (such as small hybridization dots or counterstain images of individual interphase nuclei) do not require confocal quality and can be acquired quickly in wide-field mode. On the contrary, images of other dyes (such as chromosome territories or counterstain images of cells in tissues) do require improved quality and are acquired in confocal mode. The dual-mode approach is two to three times faster compared with the single-mode confocal approach and the spectrum of its applications is much broader compared with both single-mode confocal and single-mode wide-field systems. CONCLUSIONS: The combination of high speed specific to the wide-field mode and high quality specific to the confocal mode gives optimal system performance.     

Two-dimensional fluorescence intensity distribution analysis: theory and applications

A method of sample analysis is presented which is based on fitting a joint distribution of photon count numbers. In experiments, fluorescence from a microscopic volume containing a fluctuating number of molecules is monitored by two detectors, using a confocal microscope. The two detectors may have different polarizational or spectral responses. Concentrations of fluorescent species together with two specific brightness values per species are determined. The two-dimensional fluorescence intensity distribution analysis (2D-FIDA), if used with a polarization cube, is a tool that is able to distinguish fluorescent species with different specific polarization ratios. As an example of polarization studies by 2D-FIDA, binding of 5'-(6-carboxytetramethylrhodamine) (TAMRA)-labeled theophylline to an anti-theophylline antibody has been studied. Alternatively, if two-color equipment is used, 2D-FIDA can determine concentrations and specific brightness values of fluorescent species corresponding to individual labels alone and their complex. As an example of two-color 2D-FIDA, binding of TAMRA-labeled somatostatin-14 to the human type-2 high-affinity somatostatin receptors present in stained vesicles has been studied. The presented method is unusually accurate among fluorescence fluctuation methods. It is well suited for monitoring a variety of molecular interactions, including receptors and ligands or antibodies and antigens.     

Comparison of the binding of anti-tubulin antibody and the fluorescent taxol derivative Flutax-1 to the microtubular system of Tetrahymena

Using confocal microscopic analysis, FITC-labelled anti-alpha-tubulin antibody and the fluorescent taxol derivative Flutax-1 in fixed and living Tetrahymena pyriformis GL, longitudinal microtubules, oral and somatic cilia, deep fibers, and contractile vacuole pores were equally labeled. While the antibody stained transversal microtubules, these were not labeled by Flutax-1. At the same time, oral cilia were more intensely stained by Flutax-1, than by the antibody. There were no differences in the staining of fixed preparations and living cells. The observations suggest (i) the difference between the MAPs of longitudinal and transversal microtubules which allow or inhibit the binding of the indicator molecules, and (ii) the different functions of these two types of microtubules.     

High-resolution three-dimensional images from confocal scanning laser microscopy. Quantitative study and mathematical correction of the effects from bleaching and fluorescence attenuation in depth

Three-dimensional images can be assembled by piling up consecutive confocal fluorescent images obtained by confocal scanning laser microscopy. The present work was based on three-dimensional (50-microns-deep) images at high (x, y) resolution obtained with an MRC-500 after en bloc staining of thick slices of rat liver by chromomycin A3 for nuclear DNA. The results of studies on bleaching, fluorescence excitation and emission intensities at various depths of histologic preparations are described. These effects could be evaluated separately by acquiring piled-up ("brick-stepping") and non-piled-up ("side-stepping") (x, y) images at consecutive depths and also (x, z) images. Empirical equations allowed the fitting of experimental plots of bleaching versus time, at different laser intensities and at different depths, and of fluorescence emission intensity versus depth. The main conclusions were that under our experimental conditions: (1) there was no attenuation by depth of the fluorochrome penetration, (2) there was no attenuation of the exciting beam intensity up to at least 50 microns deep, (3) there was an attenuation of the fluorescence emission intensity by depth, (4) bleaching happened equally on all planes above and below any confocal plane being studied, and (5) the fluorescence bleaching half-life was independent of depth. A mathematical correction scheme designed to compensate for bleaching and for attenuation of fluorescence emission in depth is presented. This correction is required for obtaining three-dimensional images of better quality, for optimal three-dimensional image segmentation and for any quantitative analysis based upon voxel-discretized emission intensities (gray levels)--e.g., estimating, by confocal image cytometry, textural chromatin parameters and nuclear DNA amounts.     

Automated classification and quantification of F-actin-containing ruffles in confocal micrographs

Membrane ruffles are actin-rich protrusions of the plasma membrane that can be observed on the surface of many cell types. Phase contrast and fluorescent microscopy are widely used in the analysis of ruffles, which are commonly identified in cells stained with fluorescently labeled phalloidin. Currently, comparison of cellular ruffle formation under different experimental conditions is generally qualitative or semiquantitative. Ruffle structures are often defined using manual tracing and thresholding methods. Here, we report the rapid and accurate segmentation of ruffles from two-dimensional confocal projections of cells using automated method based on well-established image processing and analysis methods. Line-shaped ruffles were detected using line detectors and were then separated from the filtered images. Automated categorizing of the segmented line structures enabled accurate quantification of the ruffles. This automated approach is efficient and reliable and hence can serve as a powerful tool in studies of the mechanism of ruffle formation.