Flow cytometry as an estimation tool for honey bee sperm viability

Flow cytometry is a method to conduct a multiparameter analysis of cells suspended in liquid and passing through a laser beam. Analyses of human and other mammal sperm using this method have already been performed but its application for insect semen is still the subject of investigation. Semen isolated from honey bee Apis mellifera seminal vesicles was dyed using SYBR-14 and propidium iodide (PI). The fluorescence of the SYBR-14 stained cells was analyzed in a green fluorescence channel (FL-1), while the PI fluorescence was analyzed in a red fluorescence channel (FL-3). Living and dead cell populations were separated using a density dot plot and the percentage of each in the sample was calculated. Flow cytometry seems to be an effective tool for assessing the viability of honey bee semen, solving the problems of distinguishing and counting the double-stained cells.     

Detection of human papillomavirus DNA in genital lesions by enzymatic in situ hybridization with Fast Red and laser scanning confocal microscopy

Human papillomavirus (HPV) infection with potentially oncogenic types 16 or 18 is common in genital lesions especially in uterine carcinomas. In such lesions, in situ hybridization with non-radioactive probes is a powerful tool for the histopathologist to detect and type HPV DNA either on cell deposits or on tissue sections. The use of an immunohistochemical method involving alkaline phosphatase and Fast Red TR salt/naphthol AS-MX phosphate is proposed for use with conventional bright-field or fluorescence microscopy as well as by laser scanning confocal microscopy. The alkaline phosphatase-Fast Red reaction has the advantage of producing a red precipitate that permits the detection of in situ hybridization signals by bright-field microscopy, and of obtaining a strong red fluorescence characterized by a lack of bleaching when excited by a green light. Therefore, the alkaline phosphatase-Fast Red reaction is well adapted for observations by fluorescence and confocal microscopy, the latter method allowing the detection, in tissue sections of cervical intraepithelial lesions, of small punctate and large diffuse hybridization signals, considered as integrated and episomal states of HPV DNA respectively. The combination of in situ hybridization with the alkaline phosphatase-Fast Red reaction and confocal microscopy is particularly convincing when hybridization signals are of small size and/or of low fluorescence intensity, especially if they are present in various focal planes; in such conditions, infected cells are easily detected by three-dimensional reconstruction. Therefore, this combination is a suitable method for identifying and characterizing HPV DNA in cells and tissue sections This revised version was published online in November 2006 with corrections to the Cover Date.     

Detection of Active Yeast Cells (Saccharomyces cerevisiae) in Frozen Dough Sections

A new method based on fluorescence microscopy was developed to detect active yeast cells in cryosections of wheat dough. The sections were stained with 4′,6-diamidino-2-phenylindole (DAPI) and counterstained with Evans blue. The active yeast cells in the sections appeared brilliant yellow and were readily distinguished from the red dough matrix. The dead cells allowed penetration of the Evans blue through the cell membrane, which interfered with the DAPI staining and caused the dead cells to blend into the red environment. The number of active yeast cells in fermenting dough sections containing different proportions of living and dead yeast cells correlated well with the gas-forming capability of the yeast in the dough but not with the results of the conventional plate count method. The new method allows the study of yeast activity not only during the different stages of frozen dough processing but also during the fermentation of doughs.     

Computer-Assisted Laser Scanning and Video Microscopy for Analysis of Cryptosporidium parvum Oocysts in Soil, Sediment, and Feces

A computer-assisted laser scanning microscope equipped for confocal laser scanning and color video microscopy was used to examine Cryptosporidium parvum oocysts in two agricultural soils, a barnyard sediment, and calf fecal samples. An agar smear technique was developed for enumerating oocysts in soil and barnyard sediment samples. Enhanced counting efficiency and sensitivity (detection limit, 5.2 x 10(sup2) oocysts(middot)g [dry weight](sup-1)) were achieved by using a semiautomatic counting procedure and confocal laser scanning microscopy to enumerate immunostained oocysts and fragments of oocysts in the barnyard sediment. An agarose-acridine orange mounting procedure was developed for high-resolution confocal optical sectioning of oocysts in soil. Stereo images of serial optical sections revealed the three-dimensional spatial relationships between immunostained oocysts and the acridine orange-stained soil matrix material. In these hydrated, pyrophosphate-dispersed soil preparations, oocysts were not found to be attached to soil particles. A fluorogenic dye permeability assay for oocyst viability (A. T. Campbell, L. J. Robertson, and H. V. Smith, Appl. Environ. Microbiol. 58:3488-3493, 1992) was modified by adding an immunostaining step after application of the fluorogenic dyes propidium iodide and 4(prm1),6-diamidino-2-phenylindole. Comparison of conventional color epifluorescence and differential interference contrast images on one video monitor with comparable black-and-white laser-scanned confocal images on a second monitor allowed for efficient location and interpretation of fluorescently stained oocysts in the soil matrix. This multi-imaging procedure facilitated the interpretation of the viability assay results by overcoming the uncertainties caused by matrix interference and background fluorescence.     

A method to measure the duration of DNA synthesis and the potential doubling time from a single sample

A method is described whereby the DNA synthesis time, Ts, can be calculated using data of a single sample of cells taken several hours after labelling with bromodeoxyuridine (BrdUrd). The method involves a simple calculation using flow cytometry data of BrdUrd incorporation (green fluorescence, FITC-labelled anti-BrdUrd-DNA antibody) and total DNA content (red fluorescence, propidium iodide). The movement of BrdUrd-labelled cells through the S phase can be quantified by measuring their mean red fluorescence relative to that of G1 and G2 cells. Assuming the movement of the labelled cells toward G2 is linear with time, Ts can be calculated by measuring their relative movement at any one time. The method was tested on cells in vitro and on bone marrow and tumor cells in vivo. Reasonable agreement was seen with published estimates of Ts for these tissues.     

Immunofluorescence technique for 100-nm-thick semithin sections of Epon-embedded tissues

An immunofluorescence staining method for Epon-embedded materials is described. Rat kidney and liver were fixed by perfusion with 1% glutaraldehyde for 10 min. Tissue slices were embedded in Epon. Semithin sections with thicknesses ranging from 1,000 to 100 nm were cut and mounted on clean glass slides. Epoxy resin was removed by treatment with 10% sodium ethoxide. Sections were digested with 0.05% trypsin and then treated with sodium borohydride. Sections were immunostained for leucine aminopeptidase (plasma membrane), catalase (peroxisomes), 3-ketoacyl-CoA thiolase (mitochondria), cathepsin D (lysosomes), and LGP107 (lysosomal membrane) using Cy(2)- or Alexa 546-labeled secondary antibodies. In 1,000-nm-thick sections, non-specific fluorescence remained and such fluorescence decreased as the sections became thinner. Clear specific fluorescence was obtained in the sections with thicknesses ranging from 250 to 100 nm with Alexa 546-labeled antibody (red fluorescence) but was not specific enough with Cy(2)- or Alexa 430-labeled antibody (green fluorescence). Sodium borohydride greatly abolished autofluorescence of glutaraldehyde. The present method made it possible to obtain signals in cross-sections of biological materials with a thickness of 250-100 nm, which are difficult to obtain in optical section using a confocal laser microscope.     

DNA labeling in living cells.

BACKGROUND: Live cell fluorescence microscopy experiments often require visualization of the nucleus and the chromatin to determine the nuclear morphology or the localization of nuclear compartments. METHODS: We compared five different DNA dyes, TOPRO-3, TOTO-3, propidium iodide, Hoechst 33258, and DRAQ5, to test their usefulness in live cell experiments with continuous imaging and photobleaching in widefield epifluorescence and confocal laser scanning microscopy. In addition, we compared the DNA stainings with fluorescent histones as an independent fluorescent label to mark chromatin. RESULTS: From the dyes tested, only Hoechst and DRAQ5 could be used to stain DNA in living cells. However, DRAQ5 had several advantages, namely low photobleaching, labeling of the chromatin compartments comparable to that of H2B-GFP fusion proteins, and deep red excitation/emission compatible with available genetically encoded fluorescent proteins such as C/G/YFP or mRFP. CONCLUSIONS: The DNA dye DRAQ5 is well suited for chromatin visualization in living cells and can easily be combined with other fluorophores with blue to orange emission.     

Three-Dimensional Labeling Program for Elucidation of the Geometric Properties of Biological Particles in Three-Dimensional Space

For all biological particles such as cells or cellular organelles, there are three-dimensional coordinates representing the centroid or center of gravity. These coordinates and other numerical parameters such as volume, fluorescence intensity, surface area, and shape are referred to in this paper as geometric properties, which may provide critical information for the clarification ofin situmechanisms of molecular and cellular functions in living organisms. We have established a method for the elucidation of these properties, designated the three-dimensional labeling program (3DLP). Algorithms of 3DLP are so simple that this method can be carried out through the use of software combinations in image analysis on a personal computer. To evaluate 3DLP, it was applied to a 32-cell-stage sea urchin embryo, double stained with FITC for cellular protein of blastomeres and propidium iodide for nuclear DNA. A stack of optical serial section images was obtained by confocal laser scanning microscopy. The method was found effective for determining geometric properties and should prove applicable to the study of many different kinds of biological particles in three-dimensional space.     

β-chemokine expression and distribution in paraffin-embedded transplant renal biopsy sections: analysis by scanning laser confocal microscopy

 Previous immunohistochemical and in situ hybridisation studies have shown that, in tubulitis associated with acute cellular rejection of human renal allografts, intratubular T cells proliferate and are fully activated in situ. In the immunohistochemical study reported here we have attempted to establish some understanding of the involvement of the β-chemokines RANTES, MCP-1, MIP-1α and MIP-1β in recruiting T cells to the intratubular site. Paraffin-embedded routine biopsy sections were treated for conventional indirect immunofluorescence to detect the selected chemokines. Scanning laser confocal microscopy was used to provide a measure of fluorescence intensity resulting from binding of FITC-labelled secondary antibody. Cells expressing chemokines could be identified and, within the limits of the staining method, it was possible to obtain a semi-quantitative assessment of individual chemokine activity at different points in biopsy sections by constructing a profile of fluorescence intensity. High concentrations of chemokines (especially RANTES, MIP-1β and/or MIP-1α) were localised to the basolateral surface of tubular epithelial cells (TEC). MCP-1 was also consistently present but at a lower level than RANTES except in one case identified as BANFF category 3. There was diffuse distribution of chemokines in the interstitial matrix and low intensity fluorescence outlined some endothelial cells of peritubular venules and interstitial fibroblast-like cells. Our results suggest a mechanism for specific chemotactic recruitment of inflammatory cells by TEC-produced chemokines. Accepted: 22 January 1998     

Fluorescent Method for Monitoring Cheese Starter Permeabilization and Lysis

A fluorescence method to monitor lysis of cheese starter bacteria using dual staining with the LIVE/DEAD BacLight bacterial viability kit is described. This kit combines membrane-permeant green fluorescent nucleic acid dye SYTO 9 and membrane-impermeant red fluorescent nucleic acid dye propidium iodide (PI), staining damaged membrane cells fluorescent red and intact cells fluorescent green. For evaluation of the fluorescence method, cells of Lactococcus lactis MG1363 were incubated under different conditions and subsequently labeled with SYTO 9 and PI and analyzed by flow cytometry and epifluorescence microscopy. Lysis was induced by treatment with cell wall-hydrolyzing enzyme mutanolysin. Cheese conditions were mimicked by incubating cells in a buffer with high protein, potassium, and magnesium, which stabilizes the cells. Under nonstabilizing conditions a high concentration of mutanolysin caused complete disruption of the cells. This resulted in a decrease in the total number of cells and release of cytoplasmic enzyme lactate dehydrogenase. In the stabilizing buffer, mutanolysin caused membrane damage as well but the cells disintegrated at a much lower rate. Stabilizing buffer supported permeabilized cells, as indicated by a high number of PI-labeled cells. In addition, permeable cells did not release intracellular aminopeptidase N, but increased enzyme activity was observed with the externally added and nonpermeable peptide substrate lysyl-p-nitroanilide. Finally, with these stains and confocal scanning laser microscopy the permeabilization of starter cells in cheese could be analyzed.     

Enhanced resolution of specific chromosome and nuclear regions by reflectance laser scanning confocal microscopy

 DNA sequences digested by HaeIII and reconstructed by in situ nick translation employing digoxigenin-labelled nucleotides are usually revealed either by horseradish peroxidase or FITC fluorescence. To obtain a significant improvement in terms of resolution, sensitivity and specificity, colloidal gold has been used instead of FITC (as the reporter molecule) to reveal the labelled DNA. Colloidal gold and propidium iodide were visualised by employing the reflectance mode and the 488-nm laser line of a confocal laser scanning microscope. In chromosomes, the fluorescent reaction pattern showed diffuse areas of labelling in which it was impossible to identify any specific kind of banding along the arms. In some chromosomes and, in particular, 1 and 9, a C-negative banding due to the negativity of the centromeric areas was seen. A more accurate localisation on chromosomes, including telomeric regions, often organised in spot pairs that resembled an R-like banding, was detected using 1-nm colloidal gold. A fine labelling was also demonstrated in nuclei, especially at their peripheral heterochromatin. The non-fading properties of colloidal gold combined with visualisation by reflectance confocal laser scanning microscopy demonstrated the possibility of obtaining a higher spatial resolution than when using conventional fluorophores or higher laser wavelength. This improved way to study the localization of HaeIII digestion sites in single chromosomes and in interphase nuclei made the reaction a valuable tool for the detection of antigens or of specific DNA sequences in biological preparations. Accepted: 5 September 1996     

Discrimination of depolarized from polarized mitochondria by confocal fluorescence resonance energy transfer

Mitochondrial depolarization promotes apoptotic and necrotic cell death and possibly other cellular events. Polarized mitochondria take up cationic tetramethylrhodamine methylester (TMRM), which is released after depolarization. Thus, TMRM does not label depolarized mitochondria. To identify both polarized and depolarized mitochondria in living cells, cultured rat hepatocytes, and sinusoidal endothelial cells were co-loaded with green-fluorescing MitoTracker Green FM (MTG) and red-fluorescing TMRM for imaging by laser scanning confocal microscopy. Like TMRM, MTG is a cationic fluorophore that accumulates electrophoretically into polarized mitochondria. Unlike TMRM, MTG binds covalently to intramitochondrial protein thiols and remains bound after depolarization. In cells labeled only with MTG, excitation with blue (488 nm) light yielded green but almost no red fluorescence. After subsequent loading with TMRM, green MTG fluorescence became quenched. Instead, blue excitation yielded red fluorescence. Mitochondrial de-energization restored green fluorescence and abolished red fluorescence. Conversely, when MTG was added to TMRM-labeled cells, red fluorescence excited by blue light was enhanced, an effect again reversed by de-energization. These observations of reversible quenching of donor fluorescence and augmentation of acceptor fluorescence signify fluorescence resonance energy transfer (FRET). In undisturbed hepatocytes, spontaneous depolarization of a subfraction of mitochondria was an ongoing phenomenon. In conclusion, confocal FRET discriminates individual depolarized mitochondria against a background of hundreds of polarized mitochondria.     

Distribution of actin-filament bundles in myoid cells,Sertoli cells,and tunica albuginea of rat and mouse testes

Summary Frozen sections of the rat and mouse testes were stained with either FITC-phalloidin or NBD-phallacidin and viewed with conventional fluorescence and confocal laser microscopes in order to demonstrate the arrangment of actin-filament bundles in myoid cells, Sertoli cells and tunica albuginea. Myoid cells are rich in actin-filament bundles crossing at right angles. These bundles running in different directions can also be visualized by means of electron microscopy. Nerve fibers occur in the vicinity of myoid cells, suggesting a neural control of the cell. At Sertoli cell junctions actin filaments occur at the circumference of the cell, where they show a honeycomb pattern. The ratio of the number of Sertoli cells per myoid cell can be calculated by means of confocal microscopy; this technique may provide a new parameter for determining spermatogenic activity. In the tunica albuginea of the juvenile mouse testis, actin filaments are arranged in an alternate fashion.     

Assessing photosystem I and II distribution in leaves from C4 plants using confocal laser scanning microscopy

Images of chlorophyll fluorescence emitted at wavelengths above and below 700 nm were recorded from leaf sections of C₄ species using confocal laser scanning microscopy (LSM). We investigated species exhibiting both NAD-malic enzyme (NAD-ME) C₄ photosynthesis and NADP-malic enzyme (NADP-ME) C₄ photosynthesis. Comparing LSM fluorescence of leaf sections with flow-cytometrically determined fluorescence from individual chloroplasts revealed that LSM fluorescence was distorted by the optical properties of leaf sections. Leaf section fluorescence, when corrected by transmission data derived from light transmission images, agreed with flow cytometry data. The corrected LSM fluorescence yielded information on the distribution of the individual photosystems in the C₄ leaf sections: PSII concentrations in bundle sheath cells were elevated in NAD-ME species but diminished in most of the NADP-ME species investigated. The NADP-ME species, Arundinella hirta, however, showed normal PSII and increased PSI concentration in bundle sheath chloroplasts. Finally, a gradient of PSI was observed within the bundle sheath cells from Euphorbia maculata.     

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.     

Alkaline phosphatase cytochemistry in confocal scanning light microscopy for imaging the bone marrow stroma

This paper reports on the use of alkaline phosphatase cytochemistry and combined conventional and confocal reflection and fluorescence scanning light microscopic modes in the study of human marrow stroma. It was found that the end product of the enzyme reaction using Napthol AS phosphate as substrate and Fast Blue BB as coupler reflected the 633 nm (red) light from a Helium-Neon laser. Serial optical sections suitable for 3-D reconstruction and selectively depicting the marrow reticulum cells could be obtained from thick glycol methacrylate sections reacted for Alkaline phosphatase. Furthermore, the yellow background of uncoupled diazonium salt over cytochemically unreactive structures in the same specimens and fields was used for imaging haemopoietic cell mass by operating the microscope at 488 nm (argon ion laser, blue-green). These methods may offer advantages in the investigation of the bone marrow stroma and its interplay with haemopoiesis and osteogenesis in normal and disease conditions.     

Application of the immunofluorescence technique and confocal laser scanning microscopy for studying the distribution of the luteinizing hormone/chorionic gonadotropin (LH/CG) receptor on rat luteal cells

We used confocal scanning microscopy to study the semi-quantitative distribution of luteinizing hormone/chorionic gonadotropin (LH/CG) receptors on rat luteal cells at both the two- and the three-dimensional level. The receptors were visualized in 6-microns sections of pseudopregnant rat ovaries using polyclonal rabbit antiserum to hCG-affinity-purified LH/CG receptor in conjunction with rhodamine-conjugated anti-rabbit immunoglobulins. Twenty to 30 optical sections were taken at different focal planes from representative luteal cells with a confocal laser scanning microscope and then processed digitally to two- and three-dimensional pseudocolored images. Distinct differences in fluorescence intensity could be demonstrated at both the two- and the three-dimensional level on the luteal cell surfaces, suggesting an uneven distribution of the LH/CG receptors on the cell membranes. This probably results in the compartmentalization and polarization of luteal cell function.     

Combining microtomy and confocal laser scanning microscopy for structural analyses of plant–fungus associations

The serious problem of extended tissue thickness in the analysis of plant–fungus associations was overcome using a new method that combines physical and optical sectioning of the resin-embedded sample by microtomy and confocal microscopy. Improved tissue infiltration of the fungal-specific, high molecular weight fluorescent probe wheat germ agglutinin conjugated to Alexa Fluor® 633 resulted in high fungus-specific fluorescence even in deeper tissue sections. If autofluorescence was insufficient, additional counterstaining with Calcofluor White M2R or propidium iodide was applied in order to visualise the host plant tissues. Alternatively, the non-specific fluorochrome acid fuchsine was used for rapid staining of both, the plant and the fungal cells. The intricate spatial arrangements of the plant and fungal cells were preserved by immobilization in the hydrophilic resin Unicryl?. Microtomy was used to section the resin-embedded roots or leaves until the desired plane was reached. The data sets generated by confocal laser scanning microscopy of the remaining resin stubs allowed the precise spatial reconstruction of complex structures in the plant–fungus associations of interest. This approach was successfully tested on tissues from ectomycorrhiza (Betula pendula), arbuscular mycorrhiza (Galium aparine; Polygala paniculata, Polygala rupestris), ericoid mycorrhiza (Calluna vulgaris), orchid mycorrhiza (Limodorum abortivum, Serapias parviflora) and on one leaf–fungus association (Zymoseptoria tritici on Triticum aestivum). The method provides an efficient visualisation protocol applicable with a wide range of plant–fungus symbioses.     

Targeted optical injection of gold nanoparticles into single mammalian cells

We present an all optical technique for the targeted delivery of single 100 nm diameter gold nanoparticles into a specified region of the interior of an individual mammalian cell through a combination of optical tweezing and optical injection. The internalisation of the nanoparticle is verified by confocal laser scanning microscopy and confocal laser scanning reflectance microscopy. This represents the first time that nano sized particles have been tweezed and optically injected into mammalian cells using only light, and provides a novel methodology for internalising nanosphere based biosensors within specific intracellular regions of a mammalian cell. (© 2009 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)