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1.
We used spectrally resolved fluorescence lifetime imaging (SLIM) to investigate the mitochondria staining dye rhodamine 123 and binding of DAPI to RNA and DNA in cells. Moreover, different components of the photosensitizer Photofrin were resolved in cell cultures by SLIM. To record lifetime images (tau-mapping) with spectral resolution we used a laser scanning microscope equipped with a spectrograph, a 16 channel multianode PMT, and multidimensional time-correlated single photon counting. A Ti:Saphir laser was used for excitation or alternatively a ps diode laser. With this system the time- and spectral-resolved fluorescence characteristics of different fluorophores were investigated in cell cultures. As an example, the mitochondria staining dye rhodamine I23 could be easily distinguished from DAPI, which binds to nucleic acids. Also different binding sites of DAPI could be discriminated. This was proved by the appearance of different lifetime components within different spectral channels. Moreover, we were able to detect monomeric and aggregated forms of Photofrin in cells. Different lifetimes could be attributed to the various compounds. In addition, a detailed analysis of the autofluorescence by SLIM could explain changes of mitochondrial metabolism during Photofrin-PDT.  相似文献   

2.
Development of cell-based therapy entails the use of different types of materials as support for cultured cells. Some of these materials are opaque. For a general microscope study of cell cultures prepared on transparent supports, Giemsa stain with bright field microscopy is useful. With opaque supports or scaffolds, epifluorescence microscopy is necessary. The method the authors describe uses eosin Y to stain cytoplasm and DAPI to stain nuclei under fluorescence microscopy. This method provides easy and fast fluorescent staining for a general morphological study of cultured cells on transparent or opaque supports.  相似文献   

3.
One of the most promising applications of encapsulated living cells is their use as protected transplanted tissue into the human body. A suitable system for the protection of living cells is the use of nano‐ or microcapsules of polyelectrolytes. These shells can be deposited easily on top of the cells by means of a layer‐by‐layer technique. An interesting feature of the capsules is the possibility to control their properties on a nanometre level, tuning their wall texture via the preparation conditions. Here we introduce a model system to test the protection ability of polyelectrolyte capsules. Common bakery yeast cells were encapsulated. They were coated with a fluorescently labelled shell at conditions known to guarantee cell survival, and the cell interior was stained with DAPI. The protozoan Paramecium primaurelia was incubated with this double‐stained living yeast and visualized by means of two‐photon excitation fluorescence microscopy. Cross‐sections of the dye‐stained material as well as autofluorescence of the fixed protozoan allowed us to follow the digestion of the coated yeast with time. Our investigation reveals that capsules prepared under these deposition conditions are permeable to lysosomal enzymes, leading to degradation of the yeast inside the intact capsules. Our preliminary results indicate the suitability of the introduced model as a test system of this permeability.  相似文献   

4.
Mitochondrial function plays an important role in the regulation of cellular life and death, including disease states. Disturbance in mitochondrial function and distribution can be accompanied by significant morphological alterations. Electron microscopy tomography (EMT) is a powerful technique to study the 3D structure of mitochondria, but the automatic detection and segmentation of mitochondria in EMT volumes has been challenging due to the presence of subcellular structures and imaging artifacts. Therefore, the interpretation, measurement and analysis of mitochondrial distribution and features have been time consuming, and development of specialized software tools is very important for high-throughput analyses needed to expedite the myriad studies on cellular events. Typically, mitochondrial EMT volumes are segmented manually using special software tools. Automatic contour extraction on large images with multiple mitochondria and many other subcellular structures is still an unaddressed problem. The purpose of this work is to develop computer algorithms to detect and segment both fully and partially seen mitochondria on electron microscopy images. The detection method relies on mitochondria's approximately elliptical shape and double membrane boundary. Initial detection results are first refined using active contours. Then, our seed point selection method automatically selects reliable seed points along the contour, and segmentation is finalized by automatically incorporating a live-wire graph search algorithm between these seed points. In our evaluations on four images containing multiple mitochondria, 52 ellipses are detected among which 42 are true and 10 are false detections. After false ellipses are eliminated manually, 14 out of 15 fully seen mitochondria and 4 out of 7 partially seen mitochondria are successfully detected. When compared with the segmentation of a trained reader, 91% Dice similarity coefficient was achieved with an average 4.9 nm boundary error.  相似文献   

5.
Veneous blood-citrate samples of patients with infectious mononucleosis were stained with DAPI, a newer fluorochrome (4',6-diamidino-2-phenylindole) to demonstrate the "large atypical lymphocytes" associated with this disease. As a result of the capacity of DAPI to stain DNA and certain acid mucopolysaccharides, a rapid detection of the pathognomonic white blood cells could be achieved. Most of them revealed the well known indented or lobulated nuclei and vacuolated (foamy) cytoplasm. Others exhibited kidney-shaped nuclei, often filled with yellow-fluorescent tiny granula. In our view, the rapid, specific and sensitive DAPI-technique for detecting pathognomonic blood cells in patients with infectious mononucleosis can be considered an improved microscopic method.  相似文献   

6.
The SAC8.5, a low-cost Peltier-cooled black and white 8-bit CCD camera for astronomy, was evaluated for its use in imaging microscopy. Two camera–microscope configurations were used: an epifluorescence microscope (Nikon Eclipse TE2000-U) and a bottom port laser scanning confocal microscope system (Zeiss LSCM 510 META). Main advantages of the CCD camera over the currently used photomultiplier detection in the scanning setup are fast image capturing, stable background, an improved signal-to-noise ratio and good linearity. Based on DAPI-labelled Chinese Hamster Ovarian cells, the signal-to-noise ratio was estimated to be 4 times higher with respect to the currently used confocal photomultiplier detector. A linear relationship between the fluorescence signal and the FITC-inulin concentrations ranging from 0.05 to 1.8 mg mL−1 could be established. With the SAC8.5 CCD camera and using DAPI, calcein-AM and propidium iodide we could also distinguish between viable, apoptotic and necrotic cells: exposure to CdCl2 caused necrosis in A6 cells. Additional examples include the observation of wire-like mitochondrial networks in Mito Tracker Green-loaded Madin–Darby canine kidney cells. Furthermore, it is straightforward to interface the SAC8.5 with automated shutters to prevent rapid fluorophore photobleaching via easy to use astrovideo software. In this study, we demonstrate that the SAC8.5 black and white CCD camera is an easy-to-implement and cost-conscious addition to quantitative fluorescence microfluorimetry on living tissues and is suitable for teaching laboratories.  相似文献   

7.
The rocks of the McMurdo Dry Valleys desert in Antarctica harbour endolithic communities of micro‐organisms such as lichens, fungi, cyanobacteria and bacteria. Establishing the physiological status and viability of these microbial colonies in their natural microhabitat has far‐reaching implications for understanding the microbial ecology of the harsh environment of this polar desert. Here we describe the use of confocal microscopy and a specific fluorescent probe (FUN‐1) to evaluate the metabolic activity of fungal cells. Application of confocal microscopy also served to identify living and dead bacteria or cyanobacteria using the fluorescent assay reagents Live/Dead SYTO 9 and propidium iodide or SYTOX Green, respectively. In addition, through the use of epifluorescence microscopy, live/dead bacteria and cyanobacteria could be detected by estimating fluorescence from their cell components provoked by simultaneously staining with nucleic acids stains such as DAPI and SYTOX Green.  相似文献   

8.
The native shape and intracellular distribution of newly synthesized DNA was visualized by correlative (light and electron) microscopy in ice embedded whole cells of Escherichia coli. For that purpose, the commercially available modified nucleoside triphosphate named BODIPY® FL‐14‐dUTP was enzymatically incorporated in vivo into the genome of E. coli mutant K12 strain, which cannot synthesize thymine. The successful incorporation of this thymidine analogue was confirmed first by fluorescence microscope, where the cells were stained in the typical for bodipy green color. Later the preselected labeled E. coli were observed by Hilbert Differential Transmission Electron Microscope (HDC TEM) and the distribution of elemental boron (contained in bodipy) was visualized at high‐resolution by an electron spectroscopic imaging (ESI) technique. The practical detection limit of boron was found to be around 5 ~ 10 mmol/kg in area of 0.1 μm2, which demonstrated that ESI is a suitable approach to study the cytochemistry and location of labeled nucleic fragments within the cytoplasmic chromosomal area. In addition, the fine cellular fibrous and chromosomal ultrastructures were revealed in situ by combing of phase‐plate HDC TEM and ESI. The obtained results conclude that the correlation between fluorescent microscopy with phase‐plate HDC TEM and ESI is a powerful approach to explore the structural and conformation dynamics of DNA replication machinery in frozen cells close to the living state.  相似文献   

9.
Virus aggregation is analyzed because of its potential use for both classifying viruses and understanding virus ecology and evolution. Virus aggregation is, however, problematic because aggregation causes loss of virions during processing for microscopy of any type. Thus, here we detect virus aggregation by fluorescence microscopy of wet-mounted dissections of dilute gel-supported plaques ( in situ fluorescence microscopy) of a test virus, the long-tail aggregating Bacillus thuringiensis bacteriophage, 0305φ8–36. Background fluorescence is reduced to nonproblematic levels by using the dye, DAPI (4',6-diamidino-2-phenylindole), to stain viral nucleic acid. In situ fluorescence microscopy reveals two in situ phases, one weakly fluorescent. Most bacteriophages partition to the weakly fluorescent phase. Aggregates of bacteriophage 0305φ8–36 are detected during fluorescence microscopy via the following. (1) Coordinated motion is found for visibly separate particles in solution; the motion is either thermally generated, fluid drift-induced or mechanical pressure-induced. (2) Aggregate dissociation is observed. Some of the larger aggregates are elastic and entangled with material of the weakly fluorescent phase. The larger aggregates sometimes sink at 1-g within specimens. In situ fluorescence microscopy rapidly distinguishes 0305φ8–36 from nonaggregating bacteriophages.  相似文献   

10.
Transit from the nuclear complex to the cytoplasm through the nuclear pore complex permits modification of mRNA, including processing such as splicing, capping, and polyadenylation, etc. At each of these events, mRNA interacts with various proteins to form mRNA‐protein complex. Visualizing the mRNA is crucial for understanding the mechanisms underlying mRNA processing and elucidating its structure and recent advances in mRNA imaging allow detection of real‐time mRNA localization in living cells. However, these techniques revealed only the location of mRNA but cannot visualize the conformation of mRNA‐protein complex in cells. On the other hand, transmission electron microscopy has been used to visualize the structure of the Balbiani ring‐derived large mRNA, but their observations were limited to the insect cells. In this study, we visualized the structure of mRNA‐protein complex in human culture cells by using immuno‐electron microscopy. Through immuno‐detection, an mRNA exon junction binding complex Y14, and its binding protien Upf2, different gold particle patterns were imaged with transmission electron microscopy and analyzed. Characteristic linear and stacked particle orientation were observed. Across the nuclear membrane, only linear aggregation pattern was observed, whereas the stacked aggregation pattern was detected in the cytoplasm. Our method is able to visualize mRNA‐conformation and applicable to many cell types, including mammalian cells, where genes can easily be manipulated.  相似文献   

11.
FUN‐1, a fluorescent vital dye, has been observed to form cylindrical intravacuolar structures within the vacuoles of metabolically active yeast cells. FUN‐1 staining, which begins as a diffuse pool of fluorescent cytoplasmic stain, uses an unknown endogenous biochemical processing mechanism to compact and form orange‐red cylindrical intravacuolar structures within the cell vacuole. In the clinical setting, FUN‐1 is primarily used for identification of fungal infection. FUN‐1 is utilized in the laboratory to distinguish between metabolically active and dead fungal cells. Although this stain is useful for distinguishing between live and dead fungal dead cells, few studies have utilized this chemical. This lack of use in the scientific community may be due to the requirement that cells are visualized directly after staining. Thus, it would be of interest to be able to stain cells and store them for later use. Our lab examined the longevity of cylindrical intravacuolar structures in two strains of Saccharomyces cerevisiae stained with FUN‐1 and stored at −20°C. We found that cylindrical intravacuolar structures could be reliably observed and imaged utilizing differential interference contrast microscopy and fluorescence microscopy for 21 days. We also observed that cells stained with FUN‐1 would resume propagation on yeast extract, peptone, dextrose (YPD) plates after being frozen at −20°C for 21 days. These modifications to the published procedure for FUN‐1 dye staining should allow for a more prevalent and less time sensitive use of this important biological tool.  相似文献   

12.
4', 6-diamidino-2-phenylindole dihydrochloride (DAPI) is a DNA dye widely used to mark and trace stem cells in therapy. We here studied the effect of DAPI staining on the behavior of mesenchymal stem cells cultured in either a control, non-osteogenic medium or in an osteogenic differentiation medium. In the control medium, the number of stem cells/field, as well as the number of fluorescent cells/field increased up to the sixth day in both control and DAPI-treated cultures. Afterwards, both the number of fluorescent cells and their fluorescence intensity decreased. Control cells were fusiform and with some long extensions that apparently linked them to neighboring cells, while DAPI-treated cells were mostly round cells with fine and short extensions. The trypan-blue exclusion method showed 99% cell viability in both groups, however, both alkaline phosphatase activity and the thiazolyl blue formazan assay (indicative of mitochondrial metabolism) gave significantly lower values in DAPI-marked cells. The mitochondrial mass, as indicated by specific staining and flow cytometry, showed no differences between groups. Mesenchymal stem cells gave origin to mineralized nodules in the osteogenic differentiation medium and there were not DAPI-marked cells on the ninth day of culture. Alkaline phosphatase activity, viability assay and number of cells/field and of mineralized nodules/field were similar in both groups. So, DAPI treatment did not change cell viability and proliferation during osteogenic differentiation of mesenchymal stem cells. However, since these cells loose DAPI marking after 9 days in osteogenic cultures suggests that DAPI may not be an effective marker for mesenchymal stem cells implanted in bone tissue for long periods.  相似文献   

13.
The study of distribution and quantitation of a fluorescent probe in living epithelia with the aid of an inverted microscope requires that individual cells can be analysed without optical interference from adjacent cells. This report describes the application of fluorescence microscopy and fluorometry to a recently developed in vitro culture system of renal epithelial cells. Epithelial cells derived from the mammalian renal cortical collecting tubule (CT) and the thick ascending loop of Henle (TAL) are cultivated as continuous monolayers in serum-free, hormone-supplemented media. A specific mitochondrial marker (DASPMI) is added to the medium and incorporated into the cytoplasm. The microscopic image reveals that the mitochondrial fluorescence distribution differs between CT and TAL cultures. The fluorometric quantitation shows a normally distributed histogram of medium-range intensity in TAL cell cultures while CT cultures exhibit a two-peak pattern of mitochondrial fluorescence distribution among epithelial cells.  相似文献   

14.
In this contribution, they have attempted to develop a labeling technique for in vivo imaging of functionally active plasmid DNA in cyanobacterial cells through its decoration with semiconductor quantum dots (Qdots) as fluorescent nanoprobes. For that purpose biotinylated plasmid slr2060 DNA was conjugated with Qdots‐streptavidine. The intact DNA was visualized in a single green color by light microscopy. These Qdots‐DNA conjugates were capable of expressing the acyltransferase enzyme. Qdots‐DNA conjugates and confocal microscope imaging technique were adopted to visualize the gene transport across the membrane of the live cyanobacteria cell in real time. Long‐term kinetic study enabled to reveal the steps of extracellular and intracellular microenvironment for plasmid transportation into the live cell. To confirm these processes a confocal microscope and indicator plate assay test were applied in tandem. In this contribution, Qdots‐labeled plasmid DNA was utilized for the first time for long‐term intracellular imaging studies in cyanobacteria species PCC6803. The results showed that the Qdots‐labeled plasmid DNA detection could be used as a powerful labeling technique for visualization of exogenous DNA entry and tracking into living cells by confocal microscopy. Microsc. Res. Tech. 79:447–452, 2016. © 2016 Wiley Periodicals, Inc.  相似文献   

15.
Objective: Although several methods have been used to detect the intracellular reactive oxygen species (ROS) generation, it is still difficult to determine where ROS generate from. This study aimed to demonstrate whether ROS generate from mitochondria during oxidative stress induced mitochondria damage in cardiac H9c2 cells by laser scanning confocal microscopy (LSCM). Methods: Cardiac H9c2 cells were exposed to H2O2 (1200μM) to induce mitochondrial oxidant damage. Mitochondrial membrane potential (ΔΨm) was measured by staining cells with tetramethylrhodamine ethyl ester (TMRE); ROS generation was measured by staining cells with dichlorodihydrofluorescein diacetate (H2DCFDA). Results: A rapid/transient ROS burst from mitochondria was induced in cardiac cells treated with H2O2 compared with the control group, suggesting that mitochondria are the main source of ROS induced by oxidative stress in H9c2 cells. Meanwhile, the TMRE fluorescence intensity of mitochondria which had produced a great deal of ROS decreased significantly, indicating that the burst of ROS induces the loss of ΔΨm. In addition, the structure of mitochondria was damaged seriously after ROS burst. However, we also demonstrated that the TMRE fluorescence intensity might be affected by H2DCFDA. Conclusions: Mitochondria are the main source of ROS induced by oxidative stress in H9c2 cells and these findings provide a new method to observe whether ROS generate from mitochondria by LSCM. However, these observations also suggested that it is inaccurate to test the fluorescence intensities of cells stained with two or more different fluorescent dyes which should be paid more attention to. Microsc. Res. Tech. 76:612–617, 2013. © 2013 Wiley Periodicals, Inc.  相似文献   

16.
The transforming growth factor β1 (TGF-β1) is a human cytokine which has been demonstrated to modulate cell surface integrin repertoire. In this work integrin expression in response to TGF-β1 stimulation has been investigated on the surface of human osteoblast-like cells. We used atomic force microscopy (AFM) and confocal laser scanning microscopy to assess integrin expression and to evaluate their distribution over the dorsal side of the plasma membrane. AFM probes have been covalently functionalised with monoclonal antibodies specific to the β1 integrin subunit. Force curves have been collected in order to obtain maps of the interaction between the immobilized antibody and the respective cell membrane receptors. Adhesion peaks have been automatically detected by means of an ad hoc developed data analysis software. The specificity of the detected interactions has been assessed by adding free antibody in the solution and monitoring the dramatic decrease in the recorded interactions. In addition, the effect of TGF-β1 treatment on both the fluorescence signal and the adhesion events has been tested. The level of expression of the β1 integrin subunit was enhanced by TGF-β1. As a further analysis, the adhesion force of the single living cells to the substrate was measured by laterally pushing the cell with the AFM tip and measuring the force necessary to displace it. The treatment with TGF-β1 resulted in a decrease of the cell/substrate adhesion force. Results obtained by AFM have been validated by confocal laser scanning microscopy thus demonstrating the high potential of the AFM technique for the investigation of cell surface receptors distribution and trafficking at the nanoscale.  相似文献   

17.
Confocal laser scanning microscopy (CLSM) offers improved depth discrimination and spatial resolution to the analysis of biologic samples. We demonstrate in this paper that such technology is valuable in examining DNA single-strand breaks in human cells. The single-cell-gel (SCG) assay is a new technique for measuring DNA strand breaks in individual cells. Cells embedded in lowmelting-point agarose are treated with varying concentrations of hydrogen peroxide to induce DNA strand breaks. Following cell lysis and alkaline electrophoresis, which enables single-stranded break detection, analysis of the resulting “comets” provides an accurate method of comparing changes in DNA migration patterns, which have been shown to reflect the DNA damage levels. A statistically significant difference (p < 0.01) in single-stranded DNA damage levels was detected in cells exposed to hydrogen peroxide concentrations as low as 10 nm for 2 min. LSM analysis of the SCG technique allows rapid, sensitive and reproducible quantitation of single-stranded breaks of cellular DNA.  相似文献   

18.
Bioluminescence from cells is so dim that bioluminescence microscopy is performed using an ultra low‐light imaging camera. Although the image sensor of such cameras has been greatly improved over time, such improvements have not been made commercially available for microscopes until now. Here, we customized the optical system of a microscope for bioluminescence imaging. As a result, bioluminescence images of cells could be captured with a conventional objective lens and colour imaging camera. As bioluminescence microscopy requires no excitation light, it lacks the photo‐toxicity associated with fluorescence imaging and permits the long‐term, nonlethal observation of living cells. Thus, bioluminescence microscopy would be a powerful tool in cellular biology that complements fluorescence microscopy.  相似文献   

19.
Multiphoton microscopy in life sciences   总被引:13,自引:1,他引:12  
Near infrared (NIR) multiphoton microscopy is becoming a novel optical tool of choice for fluorescence imaging with high spatial and temporal resolution, diagnostics, photochemistry and nanoprocessing within living cells and tissues. Three‐dimensional fluorescence imaging based on non‐resonant two‐photon or three‐photon fluorophor excitation requires light intensities in the range of MW cm?2 to GW cm?2, which can be derived by diffraction limited focusing of continuous wave and pulsed NIR laser radiation. NIR lasers can be employed as the excitation source for multifluorophor multiphoton excitation and hence multicolour imaging. In combination with fluorescence in situ hybridization (FISH), this novel approach can be used for multi‐gene detection (multiphoton multicolour FISH). Owing to the high NIR penetration depth, non‐invasive optical biopsies can be obtained from patients and ex vivo tissue by morphological and functional fluorescence imaging of endogenous fluorophores such as NAD(P)H, flavin, lipofuscin, porphyrins, collagen and elastin. Recent botanical applications of multiphoton microscopy include depth‐resolved imaging of pigments (chlorophyll) and green fluorescent proteins as well as non‐invasive fluorophore loading into single living plant cells. Non‐destructive fluorescence imaging with multiphoton microscopes is limited to an optical window. Above certain intensities, multiphoton laser microscopy leads to impaired cellular reproduction, formation of giant cells, oxidative stress and apoptosis‐like cell death. Major intracellular targets of photodamage in animal cells are mitochondria as well as the Golgi apparatus. The damage is most likely based on a two‐photon excitation process rather than a one‐photon or three‐photon event. Picosecond and femtosecond laser microscopes therefore provide approximately the same safe relative optical window for two‐photon vital cell studies. In labelled cells, additional phototoxic effects may occur via photodynamic action. This has been demonstrated for aminolevulinic acid‐induced protoporphyrin IX and other porphyrin sensitizers in cells. When the light intensity in NIR microscopes is increased to TW cm?2 levels, highly localized optical breakdown and plasma formation do occur. These femtosecond NIR laser microscopes can also be used as novel ultraprecise nanosurgical tools with cut sizes between 100 nm and 300 nm. Using the versatile nanoscalpel, intracellular dissection of chromosomes within living cells can be performed without perturbing the outer cell membrane. Moreover, cells remain alive. Non‐invasive NIR laser surgery within a living cell or within an organelle is therefore possible.  相似文献   

20.
Thymus-leukemia (TL) antigen is a class I molecule of the major histocompatibility complex that is expressed on the surface of mouse cortical thymocytes. Though not expected, it has been reported that TL antigen can be found on isolated mitochondria of TL+ cells. We used immuno-cryoultramicrotomy to look for TL on mitochondria in situ, thereby avoiding the plasma membrane contamination that occurs when isolating organelles. Establishing optimal fixation conditions was crucial, as mitochondrial structure was not preserved by the low concentrations of fixative needed for detection of antibody labeling. The plasma membranes of tissue culture and thymus cells were labeled well with anti-TL antibody and protein A-gold conjugate, while mitochondria within the cells were not labeled. Isolation of mitochondria on a one-step Ficoll gradient resulted in a purer organelle preparation than did isolation of mitochondria by centrifugation alone. Generally, mitochondria within this purer preparation were not labeled. Our data show that under conditions where contamination by plasma membrane is not a major concern, TL antigen cannot be detected on mitochondria.  相似文献   

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