UV lasers for flow cytometric analysis: HeCd versus argon laser excitation.

Applying flow cytometric single cell analysis, we compared the performance of UV excitation from argon ion and HeCd lasers using various UV-excitable fluorochromes of cell kinetic and cell physiological relevance. The AT-specific DNA fluorochromes DAPI, Hoechst 33258, and Hoechst 33342 showed no significant differences of G1-phase resolution and cell cycle distribution. With the HeCd laser, high-resolution cell kinetic analysis applying the novel BrdU/Hoechst-PI quenching technique showed superior resolution and an almost normalized G2M/G1 channel ratio of the first cell cycle. Indo-1 analysis for detection of intracellular free calcium gave similar results for both excitation sources, although the indo-1 ratio of activated cells was lower for HeCd excitation. Monochlorobimane as an indicator fluorochrome of glutathione content could not be excited sufficiently with the 325-nm line of the HeCd laser and exhibited poor resolution between positive and negative cells. However, the second glutathione-specific fluorochrome o-phtalaldehyde gave even better results with the HeCd laser. Our data indicate that air-cooled HeCd lasers are cheap and reliable UV-excitation sources for most UV-excitable fluorochromes, and might be an alternative to the expensive water-cooled argon and krypton laser.     

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.     

Photoconversion of DAPI and Hoechst dyes to green and red-emitting forms after exposure to UV excitation

The fluorescent dye 4′-6-Diamidino-2-phenylindole (DAPI) is frequently used in fluorescence microscopy as a chromosome and nuclear stain because of its high specificity for DNA. Normally, DAPI bound to DNA is maximally excited by ultraviolet (UV) light at 358 nm, and emits maximally in the blue range, at 461 nm. Hoechst dyes 33258 and 33342 have similar excitation and emission spectra and are also used to stain nuclei and chromosomes. It has been reported that exposure to UV can convert DAPI and Hoechst dyes to forms that are excited by blue light and emit green fluorescence, potentially confusing the interpretation of experiments that use more than one fluorochrome. The work reported here shows that these dyes can also be converted to forms that are excited by green light and emit red fluorescence. This was observed both in whole tissues and in mitotic chromosome spreads, and could be seen with less than 10-s exposure to UV. In most cases, the red form of fluorescence was more intense than the green form. Therefore, appropriate care should be exercised when examining tissues, capturing images, or interpreting images in experiments that use these dyes in combination with other fluorochromes.     

Assessment of fluorochromes for two-photon laser scanning microscopy of biofilms

A major limitation for the use of two-proton laser scanning microscopy (2P-LSM) in biofilm and other studies is the lack of a thorough understanding of the excitation-emission responses of potential fluorochromes. In order to use 2P-LSM, the utility of various fluorochromes and probes specific for a range of biofilm constituents must be evaluated. The fluorochromes tested in this study included classical nucleic acid-specific stains, such as acridine orange (AO) and 4",6"-diamidino-2-phenylindole (DAPI), as well as recently developed stains. In addition, stains specific for biofilm extracellular polymeric substances (EPS matrix components) were tested. Two-photon excitation with a Ti/Sapphire laser was carried out at wavelengths from 760 to 900 nm in 10-nm steps. It was found that autofluorescence of phototrophic organisms (cyanobacteria and green algae) resulted in strong signals for the entire excitation range. In addition, the coenzyme F(420)-related autofluorescence of methanogenic bacteria could be used to obtain images of dense aggregates (excitation wavelength, 780 nm). The intensities of the emission signals for the nucleic acid-specific fluorochromes varied. For example, the intensities were similar for excitation wavelengths ranging from 780 to 900 nm for AO but were higher for a narrower range, 780 to 810 nm, for DAPI. In selective excitation, fading, multiple staining, and combined single-photon-two-photon studies, the recently developed nucleic acid-specific fluorochromes proved to be more suitable regardless of whether they are intended for living or fixed samples. Probes specific for proteins and glycoconjugates allowed two-photon imaging of polymeric biofilm constituents. Selective excitation-emission was observed for Calcofluor White M2R (780 to 800 nm) and SyproOrange (880 to 900 nm). In addition, fluor-conjugated concanavalin A lectins were examined and provided acceptable two-photon emission signals at wavelengths ranging from 780 to 800 nm. Finally, CellTracker, a fluorochrome suitable for long-term labeling of microbial eucaryote cells, was found to give strong emission at wavelengths ranging from 770 to 810 nm. If fluorochromes have the same two-photon excitation cross section, they are suitable for multiple staining and multichannel recording. Generally, if an appropriate excitation wavelength and fluorochrome were used, it was possible to obtain more highly resolved images for thick biofilm samples with two-photon laser microscopy than with conventional single-photon laser microscopy. Due to its potential for higher resolution in light-scattering tissue-like material, such as biofilms, and extremely localized excitation, 2P-LSM is a valuable addition to conventional confocal laser scanning microscopy with single-photon excitation. However, further development of the method and basic research are necessary to take full advantage of nonlinear excitation in studies of interfacial microbial ecology.     

Analysis of UV-excited fluorochromes by flow cytometry using near-ultraviolet laser diodes.

INTRODUCTION: Violet laser diodes have become common and reliable laser sources for benchtop flow cytometers. While these lasers are very useful for a variety of violet and some ultraviolet-excited fluorochromes (e.g., DAPI), they do not efficiently excite most UV-stimulated probes. In this study, the next generation of InGaN near-UV laser diodes (NUVLDs) emitting in the 370-375-nm range have been evaluated as laser sources for cuvette-based flow cytometers. METHODS: Several NUVLDs, ranging in wavelength from 370 to 374 nm and in power level from 1.5 to 10 mW, were mounted on a BD Biosciences LSR II and evaluated for their ability to excite cells labeled with the UV DNA binding dye DAPI, several UV phenotyping fluorochromes (including Alexa Fluor 350, Marina Blue, and quantum dots), and the fluorescent calcium chelator indo-1. RESULTS: NUVLDs at the 8-10-mW power range gave detection sensitivity levels comparable to more powerful solid-state and ion laser sources, using low-fluorescence microsphere beads as measurement standards. NUVLDs at all tested power levels allowed extremely high-resolution DAPI cell cycle analysis, and sources in the 8-10-mW power range excited Alexa Fluor 350, Marina Blue, and a variety of quantum dots at virtually the same signal-to-noise ratios as more powerful UV sources. CONCLUSIONS: These evaluations indicate that near-UV laser diodes installed on a cuvette-based flow cytometer performed nearly as well as more powerful solid-state UV lasers on the same instrumentation, and comparably to more powerful ion lasers on a jet-in-air system, and. Despite their limited power, integration of these small and inexpensive lasers into benchtop flow cytometers should allow the use of flow cytometric applications requiring UV excitation on a wide variety of instruments.     

Effect of fluorochromes on bacterial surface properties and interaction with granular media.

Simple, efficient, and safe tagging methods are desired in short-term microbial transport studies such as in the study of filtration systems for water and wastewater treatment. Suitability of selected fluorochromes as bacterial tagging agents in transport studies was evaluated on the basis of stability of stained cells and the effect of staining on bacterial surface characteristics and interaction with granular media. Surface properties were characterized by zeta potential and microbial adhesion to hydrocarbons. The effect of staining on interactions between bacteria and porous media was evaluated in terms of removal of bacteria in batch adsorption tests using sand coated with aluminum hydroxide to enhance adsorption. The DNA-specific fluorochrome 4',6-diamidino-2-phenylindole (DAPI) had generally negligible effects on bacterial surface properties and interaction with sand, as indicated in batch adsorption tests using pure cultures (Escherichia coli or Acinetobacter sp.) and wastewater bacteria. Cells stained with DAPI were stable for 48 h at 4 or 20 degrees C. Other nucleic acid fluorochromes tested had different but significant effects on bacterial cells and produced less stable fluorescence. Since transport through porous media is modulated by surface properties, it may be concluded based on these results that the choice of fluorochromes is critical in microbial transport studies. DAPI appeared to be a promising tagging agent. Time dependence of fluorescence of stained cells may limit the use of fluorochrome-tagged cells in long-term transport studies.     

Cryptomonad algal phycobiliproteins as fluorochromes for extracellular and intracellular antigen detection by flow cytometry

BACKGROUND: Phycobiliproteins play an important role in fluorescent labeling, particularly for flow cytometry. The spectral properties of R-phycoerythrin (R-PE) and allophycocyanin (APC) have made them the dominant reagents in this class of fluorochromes. In this study, we evaluate a lesser-known but potentially important series of low-molecular weight cryptomonad-derived phycobiliproteins (commercially termed the CryptoFluortrade mark dyes) for their applicability to flow cytometry, both in extracellular and intracellular labeling applications. METHODS: Several cell lines were labeled with biotin-conjugated antibodies against expressed extracellular surface proteins, followed by streptavidin conjugates of three cryptomonad phycobiliproteins (CryptoFluor-2, CryptoFluor-4, and CryptoFluor-5). Cells were then analyzed by flow cytometry using a variety of laser lines and emission filters to establish the optimal excitation/emission characteristics for each fluorochrome. Some cells were permeabilized and labeled for intracellular antigens, also using the cryptomonad fluorochromes. Where appropriate, parallel samples were labeled with other fluorochromes (including R-PE, APC, the cyanin dyes Cy3 and Cy5, and others) to gauge the performance of the cryptomonad fluorochromes against fluorescent labels previously evaluated for flow cytometry. RESULTS: CryptoFluor-2 possessed excitation/emission maxima similar to those of APC and Cy5, with good excitation in the red (HeNe laser 632 nm) and strong emission in the far red (660 nm). CryptoFluor-4 possessed excitation/emission maxima similar to those of Cy3, with optimal excitation in the green (Kr 530 nm) and strong emission in the yellow/orange (585 nm). CryptoFluor-5 possessed excitation/emission maxima similar to those of lissamine rhodamine, with optimal excitation in the yellow (Kr 568 nm) and emission in the orange (610 nm). All cryptomonad fluorochromes gave satisfactory results for both intracellular and extracellular labeling, with detection sensitivities that were comparable or better than traditional phycobiliproteins and low- molecular weight synthetic fluorochromes such as the cyanin dyes. CONCLUSIONS: The CryptoFluor fluorochromes were applicable to flow cytometric immunodetection, with excitation and emission conditions commonly found on multilaser instruments. Performance of several of these dyes was at least comparable to existing fluorescent labels. The low molecular weights (30-60 kd) of phycobiliproteins may make them particularly useful in intracellular antigen detection. Cytometry 44:16-23, 2001. Published 2001 Wiley-Liss, Inc.     

Improved double immunofluorescence for confocal laser scanning microscopy.

Reliable double immunofluorescence labeling for confocal laser scanning microscopy requires good separation of the signals generated by the fluorochromes. We have successfully overcome the limitation of a single argon ion laser in achieving effective excitation of dyes with well-separated emission spectra by employing the novel sulfonated rhodamine fluorochromes designated Alexa 488 and Alexa 568. The more abundant antigen was visualized using the red-emitting Alexa 568, with amplification of the signal by a biotinylated bridging antibody and labeled streptavidin. This was combined with the green-emitting Alexa 488, which yielded brighter images than fluorescein but exhibited comparable photodegradation. With appropriate controls to ensure the absence of crosstalk between fluorescence channels, these dyes permitted unequivocal demonstration of co-localization. This combination of fluorochromes may also offer advantages for users of instruments equipped with alternative laser systems.     

Receptor-mediated calcium signal playing a nuclear third messenger in the activation of antigen-specific B cells.

We have studied receptor-mediated calcium signals in antigen-specific B cells (trinitrophenol-specific B cell clone, TP67.21) using a confocal fluorescence microscope with an argon ion laser (488 nm) and a He-Cd laser (325 nm). Confocal fluorescence images of fluo-3 loaded B cells, excited by an argon ion laser, became much brighter and more nonhomogeneous than those before antigen stimulation. Time-dependent fluorescence changes in intensities were abrupt and quite similar to the patterns of the intracellular calcium ion concentration [Ca2+]i observed by a conventional fluorescence microscope using fura-2. From the morphological patterns of the calcium images, the parts of the bright fluorescence seemed to belong to the nucleus in B cells. To confirm the above events we measured the confocal fluorescence images of the nucleus. From the fluorescence images of co-loaded Hoechst 33342 (a DNA-specific fluorescent probe), which excited by a He-Cd laser, the brighter parts of the fluo-3 fluorescence intensities were identified to the nucleus in B cells. This suggested the possibility that the increased intranuclear calcium ions may play a nuclear third messenger in B cells.     

Near-infrared dyes for six-color immunophenotyping by laser scanning cytometry

BACKGROUND: To adequately analyze the complexity of the immune system and reduce the required sample volume for immunophenotyping in general, more measurable colors for the discrimination of leukocyte subsets are necessary. Immunophenotyping by the laser scanning cytometer (LSC), a slide-based cytometric technology, combines cell detection based on multiple colors with their subsequent visualization without the need for physical cell sorting. In the present study, the filter setting of the LSC was adapted for the measurement of the far-red emitting dye cyanine 7 (Cy7), thereby increasing the number of measurable commercially available fluorochromes. METHODS: The optical filters of the LSC were replaced-photomultiplier (PMT) 3/allophycocyanin (APC): 740-nm dichroic long pass, and 670-/55-nm bandpass; PMT 4/Cy7: 810-/90-nm bandpass. Peripheral blood leukocytes were stained directly by fluorochrome-labeled antibodies or by indirect staining. The tandem dyes of Cy7 (phycoerythrin [PE]-Cy7, APC-Cy7) and the fluorochromes fluorescein isothiocyanate (FITC), PE, PE-Cy5, and APC were tested alone and in different combinations. RESULTS: With the new filter combination and tandem fluorochromes, Cy7 was measurable at 488-nm (argon laser) or 633-nm (helium-neon laser) excitation. Resolution was in the range of FITC for PE-Cy7 but approximately 30% lower for APC-Cy7; spillover into the respective donor fluorochrome channel for both tandem dyes was prominent. A six-color panel for leukocyte subtyping was designed. CONCLUSIONS: With this adaptation, it is possible to measure the tandem conjugates PE-Cy7 and APC-Cy7. This new setup opens the way for six-color immunophenotyping by LSC.     

Detection of endogenous and antibody-conjugated alkaline phosphatase with ELF-97 phosphate in multicolor flow cytometry applications

BACKGROUND: The fluorogenic alkaline phosphatase (AP) substrate 2-(5'-chloro-2'-phosphoryloxyphenyl)-6-chloro-4-(3H)-quinazolinone (ELF(R)-97 phosphate, for Enzyme-Labeled Fluorescence) has been used primarily in microscope-based imaging applications to detect endogenous AP activity, antigens and various ligands in cells and tissues, and nucleic acid hybridization. In a previous study, we demonstrated the applicability of ELF-97 phosphate for detecting endogenous AP activity by flow cytometry. In this study, we show that the spectral characteristics and high signal-to-noise ratio provided by the ELF-97 phosphate make it a useful label for immunodetection via flow cytometry. It can be combined with a variety of other fluorochromes for multiparametric flow cytometry analysis of both endogenous AP activity and intracellular and extracellular immunolabeling with AP-conjugated antibodies. METHODS: ELF-97 phosphate detection of endogenous AP activity in UMR-106 rat osteosarcoma cells was combined with intracellular antigen detection using Oregon Green 488 dye-conjugated secondary antibodies and DNA content analysis using propidium iodide (PI) or 7-aminoactinomycin D (7-AAD). ELF-97 phosphate detection of endogenous AP was also tested for spectral compatibility with a variety of other commonly used fluorochromes. ELF-97 phosphate was then used to directly label intracellular antigens via AP-conjugated antibodies, again combined with the analysis of DNA content using PI and 7-AAD. ELF-97 phosphate was also used to directly detect extracellular antigens. It was combined with Oregon Green 488 dye, phycoerythrin (PE), and PE-Cy5 dye-labeled antibodies for simultaneous four-color analysis. All samples were analyzed on a dual-beam flow cytometer, with UV excitation of the ELF-97 alcohol reaction product. RESULTS: Application of the ELF-97 phosphate to detect AP was found to be compatible with immunodetection and DNA staining techniques. It was also spectrally compatible with a variety of other fluorochromes. Endogenous AP activity could be detected simultaneously with both intracellular antigens labeled using Oregon Green 488 dye, PE, Cy5 dye and Alexa Fluor 568 dye-conjugated antibodies, and DNA content analysis with PI or 7-AAD. This multiparametric assay accurately delineated the distribution of AP in cycling cells and was able to identify cell subsets with varying endogenous AP levels. The ELF-97 alcohol reaction product was found to be an effective label for intracellular antigen immunolabeling with AP-conjugated reagents, and could also be combined with PI and 7-AAD. ELF-97 phosphate was also found to be a useful label for extracellular antigen immunolabeling with AP conjugates, and was compatible with Oregon Green 488 dye, PE, and PE-Cy5 dye-labeled antibodies for four-color surface labeling with minimal spectral overlap and color compensation. CONCLUSIONS: ELF-97 phosphate was shown to be a useful label for both endogenous and antibody-conjugated AP activity as detected by flow cytometry. Its spectral characteristics allow it to be combined with a variety of fluorochromes for multiparametric analysis. Cytometry 43:117-125, 2001. Published 2001 Wiley-Liss, Inc.     

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.     

Detection of endogenous alkaline phosphatase activity in intact cells by flow cytometry using the fluorogenic ELF-97 phosphatase substrate.

BACKGROUND: The alkaline phosphatase (AP) substrate 2-(5'-chloro-2'-phosphoryloxyphenyl)-6-chloro-4-(3H)-quinazolinone (ELF((R))-97 for enzyme-labeled fluorescence) has been found useful for the histochemical detection of endogenous AP activity and AP-tagged proteins and oligonucleotide probes. In this study, we evaluated its effectiveness at detecting endogenous AP activity by flow cytometry. METHODS: The ELF-97 phosphatase substrate was used to detect endogenous AP activity in UMR-106 rat osteosarcoma cells and primary cultures of chick chondrocytes. Cells were labeled with the ELF-97 reagent and analyzed by flow cytometry using an argon ultraviolet (UV) laser. For comparison purposes, cells were also assayed for AP using a Fast Red Violet LB azo dye assay previously described for use in detecting AP activity by flow cytometry. RESULTS: The ELF-97 phosphatase substrate effectively detected endogenous AP activity in UMR-106 cells, with over 95% of the resulting fluorescent signal resulting from AP-specific activity (as determined by levamisole inhibition of AP activity). In contrast, less than 70% of the fluorescent signal from the Fast Red Violet LB (FRV) assay was AP-dependent, reflecting the high intrinsic fluorescence of the unreacted components. The ELF-97 phosphatase assay was also able to detect very low AP activity in chick chondrocytes that was undetectable by the azo dye method. CONCLUSIONS: The ELF-97 phosphatase assay was able to detect endogenous AP activity in fixed mammalian and avian cells by flow cytometry with superior sensitivity to previously described assays. This work also shows the applicability of ELF-97 to flow cytometry, supplementing its previously demonstrated histochemical applications.     

Schistosoma mansoni: a diamidinophenylindole probe for in vitro death of schistosomula

The following fluorochromes were studied as probes for discrimination between living and dead Schistosoma mansoni schistosomula: ethidium bromide (EB), propidium iodide (PI), diamidinophenylindole (DAPI), and carboxyfluoresceine diacetate (C-FDA). While schistosomula stained with EB, PI, or C-FDA showed leakage of fluorochrome into the medium, this was not the case with DAPI. Dead schistosomula, which were stained with DAPI, showed an intense blue fluorescence, while living schistosomula were not stained even after prolonged incubation. In addition, the low DAPI concentration (1 microgram/ml) in the medium proved not to be toxic to the schistosomula, nor did it cause any background fluorescence. These properties make DAPI an ideal probe: the viability of S. mansoni schistosomula in cytotoxicity tests can be continuously monitored in tissue culture trays, using an inverted microscope with simultaneous transmitted light and incident fluorescent light illumination.     

A comparison of low-power helium-cadmium and argon ultraviolet lasers in commercial flow cytometers

The feasibility of installing a low power ultraviolet (UV) laser in a commercial flow cytometer was evaluated by testing an Ortho Cytofluorograf 50HH and a Coulter Epics V. Both instruments were equipped with two argon ion lasers, one emitting at 488 nm and the other in the UV region and were tested by measuring the DNA content of cells stained with Hoechst 33342 or DAPI. The coefficient of variation (CV) of the G0/G1 peak of the DNA histograms produced by each instrument did not deteriorate markedly when results obtained at 100-125 mW were compared to those obtained at 10 mW. These tests indicated that a helium-cadmium laser (He-Cd) which can produce 10 mW at 325 nm should work well as a UV laser in these instruments. An Ortho Cytofluorograf IIs was purchased with a 10 mW He-Cd laser installed in the forward position. Studies of DNA content have confirmed that this low power UV laser can produce CVs of 2.2% with DAPI stained fixed cells and 3.6% with Hoechst 33342 stained viable lymphocytes. Thus, the He-Cd laser should provide a reasonable alternative as a UV source for flow cytometers.     

Extended application of flow microfluorometry by means of dual laser excitation

Summary A dual laser beam excitation device for flow analysis of biological particles has been developed. The aid of this arrangement is to increase the range of fluorescent agents employed so far in quantitative and qualitative cytochemistry. Combining an argon ion and a helium-cadmium laser two color fluorescence measurements were performed employing propidium iodide as a DNA stain and fluorescamine which stains total protein in fixed cells. Energy transfer processes between the antibiotic and DNA specific dye mithramycin and propidium iodide both being bound to nuclear chromatin were analyzed. Utilization of energy transfer processes is generally discussed as a mean to extract information about the structure and conformation of nuclear chromatin in situ. The application of a crypton ion laser with three lines near 400 nm and a single line at 350 nm having a light output in each range of nearly one Watt gives the opportunity of utilizing DNA fluorochromes which have an excitation maximum in the deep blue region. DNA spectra are shown employing mithramycin, the benzimidazol derivative 33258 (Hoechst) and the indol compound DAPI which has a high DNA specifity combined with a great stability under UV illumination. By separating two focussed laser beams at their intereecting points with the liquid sample stream the trajectory of each flowing cell crosses the beams sequentially, which causes a solitary dual excitation of each cell. The advantages of a solitary excitation device compared with a simultaneous one is discussed.This work has been supported by the ministry of research and technology (FRG), contract No. 01VH015-B13MT 225a     

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.     

Relationship between DAPI-fluorescence fading and nuclear DNA content: An alternative method to DNA quantification?

In observations by confocal or conventional fluorescence microscopy, important factors should be considered in order to obtain accurate images. One of them, such as the fluorescence bleaching from highest intensity to lowest signal of fluorescence is a common problem with several DNA fluorochromes and especially for DAPI stain. The fluorescence of DAPI fades rapidly when it is exposed to UV light, under optimal conditions of observation. Although the fading process can be retarded using a mounting medium with antifading reagents, the photochemical process underlying the fluorescence decay has not yet been fully explained. In addition, no relationship between fluorescence fading and nuclear DNA content has been tested. In order to test this relationship, we measured by means of image analysis the DAPI-fluorescence intensity in several cellular types (spermatozoa, erythrocytes and haemocytes) during their fluorescence bleaching. An algorithm specifically built in MATLAB software was used for this approach. The correlation coefficient between nuclear DNA content and DAPI-fluorescence fading was found equal to 99%. This study demonstrates the feasibility to measure nuclear DNA content by fluorescence fading quantification, as an alternative method concurrently with image analysis procedures.     

Estimating percentage constitutive heterochromatin by flow cytometry.

Flow cytometry is a powerful method for the assessment of both plant and animal genomes. One of the most interesting aspects is the analysis of chromatin structure. By using intercalating and base pair-specific fluorochromes, the chromatin structure in various cell cultures and microorganisms has been determined. In this study, several maize lines of known heterochromatic composition were analyzed. The nuclei of each line were isolated and stained with DAPI (base pair specific) and PI (intercalator) separately. For each maize line, the PI/DAPI ratio was determined. A significant negative correlation was observed between C-band number and PI/DAPI ratio (r = 0.920) and between percentage heterochromatin and PI/DAPI ratio (r = 0.997). Flow cytometry with use of the fluorochromes DAPI and PI was found to be a rapid and efficient method of determining heterochromatin amount in maize.     

Dynamic imaging of the metaphase II spindle and maternal chromosomesin bovine oocytes: implications for enucleation efficiency verification, avoidanceof parthenogenesis, and successful embryogenesis

Manipulations of DNA and cellular structures are essential for the propagation of genetically identical animals by nuclear transfer. However, none of the steps have been optimized yet. This study reports a protocol that improves live dynamic imaging of the unfertilized bovine oocyte's meiotic spindle microtubules with microinjected polymerization-competent X-rhodamine-tubulin and/or with vital long-wavelength excited DNA fluorochrome Sybr14 so that the maternal chromosomes can be verifiably removed to make enucleated eggs the starting point for cloning. Suitability of the new fluorochromes was compared to the conventional UV excitable Hoechst 33342 fluorochrome. Enucleation removed the smallest amount of cytoplasm (4-7%) and was 100% efficient only when performed under continuous fluorescence, i.e., longer fluorescence exposure. This was in part due to the finding that the second metaphase spindle is frequently displaced (60.7 +/- 10%) from its previously assumed location subjacent to the first polar body. Removal of as much as 24 +/- 3% of the oocyte cytoplasm underneath the polar body, in the absence of fluorochromes, often resulted in enucleation failure (36 +/- 6%). When labeled oocytes were exposed to fluorescence and later activated, development to the blastocyst stage was lowest in the group labeled with Hoechst 33342 (3%), when compared to Sybr14 (19%), rhodamine-tubulin (23%), or unlabeled oocytes (37%). This suggests that longer wavelength fluorochromes can be employed for live visualization of metaphase spindle components, verification of their complete removal during enucleation, and avoidance of the confusion between artifactual parthenogenesis versus "cloning" success, without compromising the oocyte's developmental potential after activation.