Quantitative fluorescence resonance energy transfer measurements using fluorescence microscopy.

Fluorescence resonance energy transfer (FRET) is a technique used for quantifying the distance between two molecules conjugated to different fluorophores. By combining optical microscopy with FRET it is possible to obtain quantitative temporal and spatial information about the binding and interaction of proteins, lipids, enzymes, DNA, and RNA in vivo. In conjunction with the recent development of a variety of mutant green fluorescent proteins (mtGFPs), FRET microscopy provides the potential to measure the interaction of intracellular molecular species in intact living cells where the donor and acceptor fluorophores are actually part of the molecules themselves. However, steady-state FRET microscopy measurements can suffer from several sources of distortion, which need to be corrected. These include direct excitation of the acceptor at the donor excitation wavelengths and the dependence of FRET on the concentration of acceptor. We present a simple method for the analysis of FRET data obtained with standard filter sets in a fluorescence microscope. This method is corrected for cross talk (any detection of donor fluorescence with the acceptor emission filter and any detection of acceptor fluorescence with the donor emission filter), and for the dependence of FRET on the concentrations of the donor and acceptor. Measurements of the interaction of the proteins Bcl-2 and Beclin (a recently identified Bcl-2 interacting protein located on chromosome 17q21), are shown to document the accuracy of this approach for correction of donor and acceptor concentrations, and cross talk between the different filter units.     

Telomere length measurements using digital fluorescence microscopy.

BACKGROUND: The ends of chromosomes (telomeres) are important to maintain chromosome stability, and the loss of telomere repeat sequences has been implicated in cellular senescence and genomic instability of cancer cells. The traditional method for measuring the length of telomeres (Southern analysis) requires a large number of cells (>10(5)) and does not provide information on the telomere length of individual chromosomes. Here, we describe a digital image microscopy system for measurements of the fluorescence intensity derived from telomere repeat sequences in metaphase cells following quantitative fluorescence in situ hybridization (Q-FISH). METHODS: Samples are prepared for microscopy using Q-FISH with Cy3 labeled peptide nucleic acid probes specific for (T(2)AG(3))(n) sequences and the DNA dye DAPI. Separate images of Cy3 and DAPI fluorescence are acquired and processed with a dedicated computer program (TFL-TELO). With the program, the integrated fluorescence intensity value for each telomere, which is proportional to the number of hybridized probes, is calculated and presented to the user. RESULTS: Indirect tests of our method were performed using simulated as well as defined tests objects. The precision and consistency of human telomere length measurements was then analyzed in a number of experiments. It was found that by averaging the results of less than 30 cells, a good indication of the telomere length (SD of 10-15%) can be obtained. CONCLUSIONS: We demonstrate that accurate and repeatable fluorescence intensity measurements can be made from Q-FISH images that provide information on the length of telomere repeats at individual chromosomes from limited number of cells.     

Anti-Candida albicans germ tube antibodies reduce in vitro growth and biofilm formation of C. albicans

Background

Invasive candidiasis by Candida albicans is associated with high morbidity and mortality, due in part to the late implementation of an appropriate antifungal therapy hindered by the lack of an early diagnosis.

FITC-Dextran tracers in microcirculatory and permeability studies using combined fluorescence stereo microscopy,fluorescence light microscopy and electron microscopy

Summary Coupling fluorescein-isothiocyanate to dextrans (FITC-D) extends the usefulness of the dextrans as electron microscopic tracer particles by permitting preceding fluorescence stereo microscopy and high-power light microscopy of the tissue specimens. The fate of the tracer may thus be studied in vivo during the experiment, during fixation, and during the succeeding tissue processing. A study of some simple physicochemical characteristics of the tracer, and the influence, if any, of the fixing agent are also made possible. FITC-D was found to be uncharged in the pH range from 6.5 to 8.5, more rapidly precipitated by acetone than by alcohol, and to react with glutaraldehyde and osmium tetroxide in an unknown way during tissue fixation. FITC-D with molecular weights 70,000 and 150,000 showed no signs of diffusion during tissue preparation with the methods reported in the paper, whereas FITC-D 40,000 did so to a slight degree, when the tissue was kept for several days in the fixative vehicle. Securing the preservation of the lower molecular weight FITC-Ds during tissue fixation and preparation is more difficult and the described methods are not adequate. Dextrans provoke an anaphylactic reaction in most rat strains, but are well tolerated by Wistar Furth rats. The introduction of FITC into the dextran molecule might alter the biological reactions, but was also well tolerated by Wistar Furth rats. Combined fluorescence stereo microscopy, fluorescence microscopy of sections, light microscopy of stained sections and electron microscopy made it possible to follow a particular microcirculatory area, selected in vivo, to the final study in the electron microscope.     

FITC-dextran tracers in microcirculatory and permeability studies using combined fluorescence stereo microscopy, fluorescence light microscopy and electron microscopy

Coupling fluorescein-isothiocyanate to dextrans (FITC-D) extends the usefulness of the dextrans as electron microscopic tracer particles by permitting preceding fluorescence stereo microscopy and high-power light microscopy of the tissue specimens. The fate of the tracer may thus be studied in vivo during the experiment, during fixation, and during the succeeding tissue processing. A study of some simple physicochemical characteristics of the tracer, and the influence, if any, of the fixing agent are also made possible. FITC-D was found to be uncharged in the pH range from 6.5 to 8.5, more rapidly precipitated by acetone than by alcohol, and to react with glutaraldehyde and osmium tetroxide in an unknown way during tissue fixation. FITC-D with molecular weights 70,000 and 150,000 showed no signs of diffusion during tissue preparation with the methods reported in the paper, whereas FITC-D 40,000 did so to a slight degree, when the tissue was kept for several days in the fixative vehicle. Securing the preservation of the lower molecular weight FITC-Ds during tissue fixation and preparation is more difficult and the described methods are not adequate. Dextrans provoke an anaphylactic reaction in most rat strains, but are well tolerated by Wistar Furth rats. The introduction of FITC into the dextran molecule might alter the biological reactions, but was also well tolerated by Wistar Furth rats. Combined fluorescence stereo microscopy, fluorescence microscopy of sections, light microscopy of strained sections and electron microscopy made it possible to follow a particular microcirculatory area, selected in vivo, to the final study in the electron microscope.     

Single large DNA molecule analysis using fluorescence microscopy.

A large DNA analysis method which enable to obtain spatial information of positions of specific sequences along DNA molecule has been developed. Making use of the phenomenon that large DNA molecule is elongated stably under alternative current field in a concentrated linear polymer solution, direct observation of elongated individual lambda DNA molecules with fluorescence probes was carried out using fluorescence microscopy. Then, the spatial positions of the fluorescence spot of the probe on the DNA molecule were determined by image analysis.     

Single long DNA molecule analysis using fluorescence microscopy.

Single long DNA molecule (T4 DNA) in agarose gel was visualized with a fluorescence microscope. We confirmed alternating current electric fields is effective for stretching of single DNA molecule in agarose gel. This stretching phenomenon was observed with wide range of agarose gel concentration from 0.5%(W/V) to 1.5%. From this observation, the presence of agarose gel fiber is essential for this stretching phenomenon. The stretching process of several DNA molecules in gel shows discontinuity, which is never observed in polymer systems. It would be based on topological restriction from gel fibers.     

Imaging proteins in vivo using fluorescence lifetime microscopy

Fluorescence lifetime imaging (FLIM) represents a key optical technique for imaging proteins and protein interaction in vivo. We review the principles and recent advances in the application of the technique, instrumentation and molecular probe development.     

Morphological and physiological variations between sectors isolated from giant colonies of Candida albicans and C. Stellatoidea

Summary Species of the genusCandida differ in their morphological and physiological characteristics. These same differences can also be observed between sectors isolated from one parental culture. Isolated sectors varied from each other and the parental culture from which they were derived in morphology, carbon assimilation, fermentation reaction, intracellular reduction tests, and nutritional requirements.These variable characteristics may be due to an extremely flexible adaptive mechanism or a multinucleate condition in which the nuclear types are not always identical to each other.     

Visualization of mismatch repair complexes using fluorescence microscopy

DNA mismatch repair (MMR) is a surveillance mechanism present in most living organisms, which repairs errors introduced by DNA polymerases. Importantly, loss of MMR function due to inactivating mutations and/or epigenetic silencing results in the accumulation of mutations and as consequence increased cancer susceptibility, as observed in Lynch syndrome patients.During the past decades important progress has been made in the MMR field resulting in the identification and characterization of essential MMR components, culminating in the in vitro reconstitution of 5′ and 3′ nick-directed MMR. However, several mechanistic aspects of the MMR reaction remain not fully understood, therefore alternative approaches and further investigations are needed.Recently, the use of imaging techniques and, more specifically, visualization of MMR components in living cells, has broadened our mechanistic understanding of the repair reaction providing more detailed information about the spatio-temporal organization of MMR in vivo. In this review we would like to comment on mechanistic aspects of the MMR reaction in light of these and other recent findings. Moreover, we will discuss the current limitations and provide future perspectives regarding imaging of mismatch repair components in diverse organisms.     

Tools and techniques to measure mitophagy using fluorescence microscopy

Mitophagy is a specialized form of autophagy that removes damaged mitochondria, thereby maintaining efficient cellular metabolism and reducing cellular stress caused by aberrant oxidative bursts. Deficits in mitophagy underlie several diseases, and a substantial body of research has elucidated key steps in the pathways that lead to and execute autophagic clearance of mitochondria. Many of these studies employ fluorescence microscopy to visualize mitochondrial morphology, mass, and functional state. Studies in this area also examine colocalization/recruitment of accessory factors, components of the autophagic machinery and signaling molecules to mitochondria. In this review, we provide a brief summary of the current understanding about the processes involved in mitophagy followed by a discussion of probes commonly employed and important considerations of the methodologies to study and analyze mitophagy using fluorescence microscopy. Representative data, where appropriate, are provided to highlight the use of key probes to monitor mitophagy. The review will conclude with a consideration of new possibilities for mitophagy research and a discussion of recently developed technologies for this emerging area of cell biology.     

Detection of rat ceruloplasmin receptors using fluorescence microscopy and microdensitometry

Rat ceruloplasmin (rCp) has been labeled with the fluorophores fluorescein and rhodamine by using the isothiocyanate derivatives (FITC, RBITC). High p-phenylenediamine oxidase activity of the resulting conjugates was observed (70-90% of native activity). Polyacrylamide gel electrophoresis showed fluorescein-labeled rCp (FITC-rCp) had an increased mobility, while rhodamine-labeled rCp (RBITC-rCp) showed no increase in mobility when compared to native rCp. RBITC-rCp was tested as a probe for ceruloplasmin receptors on rat erythrocytes using fluorescence microscopy to detect membrane binding. Film negatives of blood smears exposed under identical conditions were analyzed by microdensitometry to give relative optical densities for the amount of RBITC-rCp bound per unit area of the plasma membrane. With this technique, binding of rCp was observed to be saturable, reversible, and specific. Competition of the protein ligands superoxide dismutase, catalase, and unlabeled rCp against RBITC-rCp already bound on erythrocytes showed that only rCp could displace the bound RBITC-rCp with 32% specific binding being observed. Low levels of membrane binding were seen after the erythrocytes had been trypsin treated. Platelet binding was also detected and it was saturable, reversible, and trypsin sensitive. However, only 20% of the bound RBITC-rCp could be displaced by rCp. These studies demonstrate a versatile technique for detection and localization of Cp receptors.     

Characterization of single-cell electroporation by using patch-clamp and fluorescence microscopy

Electroporation of single NG108-15 cells with carbon-fiber microelectrodes was characterized by patch-clamp recordings and fluorescence microscopy. To minimize adverse capacitive charging effects, the patch-clamp pipette was sealed on the cell at a 90(o) angle with respect to the microelectrodes where the applied potential reaches a minimum. From transmembrane current responses, we determined the electric field strengths necessary for ion-permeable pore formation and investigated the kinetics of pore opening and closing as well as pore open times. From both patch-clamp and fluorescence microscopy experiments, the threshold transmembrane potentials for dielectric breakdown of NG108-15 cells, using 1-ms rectangular waveform pulses, was approximately 250 mV. The electroporation pulse preceded pore formation, and analyte entry into the cells was dictated by concentration, and membrane resting potential driving forces. By stepwise moving a cell out of the focused field while measuring the transmembrane current response during a supramaximal pulse, we show that cells at a distance of approximately 30 microm from the focused field were not permeabilized.     

Genistein effects on growth and cell cycle ofCandida albicans

Microbial virulence is generally considered to be multifactorial with infection resulting from the sum of several globally regulated virulence factors. Estrogen may serve as a signal for global virulence induction in Candida albicans. Nonsteroidal estrogens and estrogen receptor antagonists may therefore have interesting effects on yeast and their virulence factors. Growth of C. albicans was monitored by viable plate counts at timed intervals after inoculation into yeast nitrogen broth plus glucose. To determine if increased growth of yeast in the presence of estradiol was due to tyrosine kinase-mediated signaling, we measured growth in the presence of genistein, estradiol or genistein plus estradiol and compared these conditions to controls, which were not supplemented with either compound. Unexpectedly, genistein stimulated growth of C. albicans. In addition, genistein was found to increase the rate of germination (possibly reflecting release from G(0) into G(1) cell cycle phase) and also increased Hsp90 expression, demonstrated by a dot blot technique which employed a commercial primary antibody detected with chemiluminescence with horseradish peroxidase-labeled secondary antibody. These biological effects may be attributable to genistein's activity as a phytoestrogen. In contrast, nafoxidine suppressed growth of Candida and mildly diminished Hsp90 expression. This study raises the possibility of receptor cross-talk between estrogen and isoflavinoid compounds, and antiestrogens which may affect the same signaling system, though separate targets for each compound were not ruled out.     

Differentiation of Candida stellatoidea from C. albicans and C. tropicalis by temperature-dependent growth responses on defined media

C. stellatoidea differs from both C. albicans and C. tropicalis in its i) much greater growth differential on minimal and amino acid enriched media and ii) unique inability to grow on minimal medium containing glycerol as carbon source at 37C. The relative responses to amino acid enrichment occur on media containing either fermentative or oxidative carbon sources, at 25C or 37C. Under any given conditions of carbon source and temperature, different assortments of individual amino acids are stimulatory for each of the three species. All assortments include one or more members of the glutamic acid family. However, sulfur amino acids stimulate only C. stellatoidea on all three carbon sources. On minimal-glycerol medium, wild type strains of C. stellatoidea grow prototrophically at 25C but are auxotrophic for amino acids at 37C; the particular auxotrophies expressed vary from strain to strain. Slow growing, mycelial mutants, prototrophic on glycerol at 37C arise spontaneously in wild type strains at frequencies indicating nuclear gene mutation. Such mutants can be induced by both transition and frame shift mutagens. The implications of these observations for the taxonomic relationships between the three Candida species and for identification of C. stellatoidea in particular are discussed.     

Morphological study of bacteria of the respiratory system using fluorescence microscopy of Papanicolaou-stained smears with special regard to the identification of Mycobacteria sp.

In Papanicolaou-stained smears certain structures such as nucleoli, Pneumocystis carinii , Charcot-Leyden crystals, bacteria and fungi show a brilliant fluorescence. the morphological characteristics of microorganisms which can be detected by this system, especially mycobacteria, are described. This screening method offers the possibility of providing the clinician with a provisional diagnosis within hours. Proof of the nature of the organisms should be obtained by culture.     

Antagonistic effects of Bacillus natto and Streptococcus faecalis on growth of Candida albicans.

The growth-inhibitory effects of Bacillus natto and Streptococcus faecalis on Canida albicans were investigated. When inoculated into the filtrate of a long-term culture of B. natto strain BN (BN), a stock culture of C. albicans RIMD 0301020 lost its viability completely, whereas C. albicans RIMD 0301011, a fresh isolate from a clinical source, did not. In continuous flow (CF) culture the growth of both strains of C. albicans was suppressed by mixed cultivation with BN. On the other hand, in classical batch culture BN did not suppress the growth of C. albicans. S. faecalis BIO-4R, a multi-drug resistant strain, was also antagonistic to C. albicans RIMD 0301011 but symbiotic with BN in CF culture. These findings suggest that BN in concert with S. faecalis BIO-4R may inhibit the growth of C. albicans in the intestinal tract.     

An electron microscopy study of wall expansion during Candida albicans yeast and mycelial growth using concanavalin A-ferritin labelling of mannoproteins

Depending upon growth temperature, Candida albicans can exhibit two different morphologies, a budding yeast or a mycelium. By studying the distribution of concanavalin A-ferritin particles on the cell wall surface during bud and germ tube formation, we have elucidated the way cell wall extension occurs. Both processes initially require the localized lysis of the wall in order to allow the incorporation of the newly synthesized material. Later on, the cell wall behaves as an elastic structure, allowing extension by an intosusception process and, as a consequence, cell growth.Abbreviation Con A concanavalin A