In situ detection of rhodococci associated with activated sludge foams

Genus-specific 16S rRNA targeted oligonucleotide probes, Rco1 and Rco2, were designed and used to detect rhodococci in activated sludge foam samples by confocal laser scanning microscopy. Pure cultures were used to find the optimal hybridisation conditions which were determined by comparing the mean fluorescent intensities of target and non-target cells from images captured using a confocal laser scanning microscope (CLSM). The combination of fluorescent in situ hybridisation with rRNA-targeted oligonucleotide probes and confocal laser scanning microscopy provides an effective way of detecting rhodococci in environmental samples.     

激光扫描共聚焦显微镜荧光探针的选择和应用

激光扫描共聚焦显微镜是检测生物荧光信号的最新技术手段。不仅广泛用于荧光定性、定量测量,还可用于活细胞动态荧光监测、组织细胞断层扫描、三维图象重建、共聚焦图象分析、荧光光漂白恢复、激光显微切割手术等。本文拟就激光扫描共聚焦显微镜常用的检测内容及其相关荧光探针的选择和应用做一简单的介绍。     

钙荧光探剂的研究及其在生命科学中的应用

钙荧光探剂测量活细胞胞浆游离Ｃａ２＋浓度的方法在钙研究中已成为一种越来越重要的技术。特别是由于新的一代荧光探剂的合成和激光共聚焦显微镜的发展,使其应用更加广泛。由于国内使用这种技术的实验室逐渐增多,本文将系统介绍钙荧光探剂的发展、测量原理和方法、新的常用钙荧光探剂的比较及其在生命科学中的应用。     

Changes in retention behavior of fluorescently labeled proteins during ion-exchange chromatography caused by different protein surface labeling positions

Confocal laser scanning microscopy (CLSM) is a method allowing in situ visualization of protein transport in porous chromatography resins. CLSM requires labeling a protein with a fluorescent probe. Recent work has shown that conjugation of the protein with fluorescent probes can lead to significant changes in the retention time of the protein-dye conjugate with respect to the unlabeled protein. In this study, we show that common labeling procedures result in a heterogeneous mixture of different variants and that attachment location of the fluorescent probe on the protein surface can have a strong effect on the retention of protein-dye conjugate. Lysozyme was labeled with Cy5 and BODIPY-FL succinimidyl esters, followed by chromatographic separation of the different lysozyme-dye conjugates and subsequent determination of the label position using MALDI-TOF-MS. Finally, homogenously labeled lysozyme-dye conjugates were used in CLSM experimentation and compared to published results arising from heterogeneously labeled feedstocks. The results confirm that the attachment location of the fluorescent probe has a strong effect on chromatographic retention behavior. When addressing the binding affinities of the different labeled protein fractions, it was found that native lysozyme was able to displace lysozyme-dye conjugates when the fluorescent label was attached to lysine-33, but not when attached to lysine-97. Finally, it could be shown that when superimposing the single profiles of the three major fractions obtained during a labeling procedure a qualitative picture of the net profile is obtained.     

Confocal imaging of in situ natural microbial communities and their extracellular polymeric secretions using Nanoplast resin

A novel method using excision and fixation in Nanoplast, a hydrophilic embedding resin, allows confocal imaging of natural microbial communities and their extracellular polymeric secretions (EPS) while in situ. Prestaining with fluorescent probes permits the observation of specific cellular and extracellular components. Marine stromatolite sediments were examined using this method. Optical sectioning using confocal laser scanning microscopy (CLSM) permitted high-resolution imaging through sediments. Delicate arrangements of the EPS that are associated with sedimentary microbial biofilms were imaged using a fluorescein isothiocyanate (FITC)-labeled lectin (concanavalin-A) probe. Close microspatial associations of heterotrophic bacteria cells and autotrophic cyanobacteria cells were also observed. The nanoplast resin produces no detectable autofluorescence. Further coupling of multi-photon scanning laser microscopy (2P-LSM) with a conventional single photon CLSM allowed concurrent imaging of DAPI-labeled microbial cells, FITC-labeled EPS and autofluorescent carbonate sand grains. The multi-photon infrared laser permits deep (approximately 1 mm) penetration of samples and the excitation of DAPI, which normally requires UV-excitation with minimal disturbance to samples. The unique combination of Nanoplast with fluorescent probes, CLSM and 2P-LSM allows for the preservation and imaging of natural microbial communities in their in situ state, a method easily adapted for examinations of other microbial systems.     

Fluorescent in situ hybridisation on tissue sections: a quantitative approach with confocal laser scanning microscopy

The use of fluorescent detection methods in association with digital microscopy technologies is an innovative approach for tissue localisation of messenger RNA. The success of such methods relies on the tissue preservation, local availability of the probe and on the existence of high resolution tridimensional analysis systems. Cryostatic sections, mild denaturation, short oligonucleotide probes (20mer) and confocal laser scanning microscopy allow the fulfillment of all these conditions avoiding photobleaching and tissue autofluorescence. In this paper, we describe in detail a method for in situ hybridisation set up with digoxigenin-coupled oligonucleotide complementary to beta-actin mRNA as a probe and an anti-hapten fluorescent antibody as second step for detecting specific hybridisation. Fluorescence was analysed by means of a confocal laser scanning microscope (CLSM) that provides images with low out-of-focus blurring also with relatively low numerical aperture (NA) objectives. We propose also an easy method to perform semi-quantitative thresholding analysis which allows to discriminate between background and specific signal.     

Measurement of mitochondrial pH in situ

In this article, we describe the advantages and disadvantages of procedures for monitoring mitochondrial pH in situ using optical microscopic techniques. The first method employs the combination of the fluorescent pH-sensitive indicator carboxy-SNARF and laser scanning confocal microscopy. Manipulation of the loading and post-loading conditions enables relatively specific accumulation of carboxy-SNARF into mitochondria. With the use of a mitochondrial-specific marker, mitochondrial pH can be accurately monitored. More recently, mitochondrial-targeted, pH-sensitive probes have been used to monitor mitochondrial pH. In particular, mitochondrial targeting of the yellow fluorescent protein (YFP) mutant of green fluorescent protein (GFP) combines the advantages of specific mitochondrial localization, high-fluorophore quantum yield, and extinction coefficient with an appropriate pKa for measuring mitochondrial pH. The use of dual-excitation ratiometry with mitochondrially targeted YFP increases the dynamic range of mitochondrial pH measurements and corrects for differences in the amount of expression of mitochondrially targeted YFP at the level of individual mitochondria.     

Enzymatic permeabilization of the thecate dinoflagellate Alexandrium minutum (Dinophyceae) yields detection of intracellularly associated bacteria via catalyzed reporter deposition-fluorescence in situ hybridization

The enzymatic permeabilization procedure described here allows the detection of intracellular bacteria in the thecate dinoflagellate Alexandrium minutum by using catalyzed reporter deposition-fluorescence in situ hybridization. The combined use of propidium iodide and calcofluor for confocal laser scanning microscopy, together with general and specific fluorescent bacterial probes, demonstrated the intracellular presence of bacteria, including members of the phylum Bacteroidetes.     

Phloem mobility of fluorescent xenobiotics in Arabidopsis in relation to their physicochemical properties

The transport of a range of fluorescent probes within the rootphloem of Arabidopsis thaliana has been imaged using the confocallaser scanning microscope. The phloem mobility of these probesand their subsequent subcellular distribution in the cells ofthe ‘unloading zone’, close to the root tip, arediscussed in relation to a structure-activity relations (SAR)model. This is a generalized model describing the interactionof low molecular weight xenobiotics with living cells, basedon the physicochemical properties of the former. The work demonstratesthat the model can be used to predict the phloem mobility ofxenobiotics, but only partly predicts the subcellular distributionof phloem-mobile probes following unloading. The potential useof phloem-mobile fluorescent probes as physiological indicatorsis discussed. Key words: Arabidopsis, confocal laser scanning microscopy (CLSM), fluorescent probes, phloem transport, xenobiotic transport     

In Situ Microspatial Imaging Using Two-Photon and Confocal Laser Scanning Microscopy of Bacteria and Extracellular Polymeric Secretions (EPS) Within Marine Stromatolites

The combination of a hydrophilic embedding resin, Nanoplast, with fluorescent probes, and subsequent imaging using two-photon and confocal laser scanning microscopy (2P-LSM and CLSM) has allowed in imaging of the in situ microspatial arrangements of microbial cells and their extracellular polymeric secretion (EPS) within marine stromatolites. Optical sectioning by 2P-LSM and CLSM allowed imaging of endolithic cyanobacteria cells, Solentia sp., seen within carbonate sand grains. 2P-LSM allowed very clear imaging with a high resolution of bacteria using DAPI, which normally require UV excitation and reduced photo-bleaching of fluorescent probes.     

激光扫描共聚焦显微镜在蛋白质晶体生长研究中的应用

激光扫描共聚焦显微镜与普通光学显微镜相比,其分辨率高,同时具有可对样品进行非侵入性无损伤断层扫描,以及对样品形貌进行三维成建等特点,因此,可作为研究晶体生长强有利的工具。本文介绍了其在定量测量晶体的个数,重组三维图像以获得晶体生长的过程信息及测定晶体生长台阶动态变化等方面的应用。还对激光扫描共聚焦显微镜在晶体生长研究的其它方面应用前景作了展望。     

Confocal microscopy of fertilization-induced calcium dynamics in sea urchin eggs.

Although confocal microscopy has typically been utilized in studies of fixed specimens, its potential for exploring dynamic processes in living cells is rapidly being realized. In this report, confocal laser scanning microscopy is used to analyze the calcium wave that occurs following fertilization in living sea urchin eggs microinjected with the calcium-sensitive fluorescent probes fluo-3 or calcium green. Time-lapse recordings of optical sections depicting calcium dynamics within the eggs are also subjected to volumetric reconstructions. Such analyses indicate that (1) cytoplasmic free calcium levels become elevated throughout the fertilized egg, (2) fertilization also causes the egg nucleus to undergo a transient increase in free calcium, and (3) normal cleavage can be obtained following time-lapse imaging of the calcium waves.     

Confocal laser scanning microscopy

Many technological advancements of the past decade have contributed to improvements in the photon efficiency of the confocal laser scanning microscope (CLSM). The resolution of images from the new generation of CLSMs is approaching that achieved by the microscope itself because of continued development in digital imaging methods, laser technology and the availability of brighter and more photostable fluorescent probes. Such advances have made possible novel experimental approaches for multiple label fluorescence, live cell imaging and multidimensional microscopy.     

Babesia bovis: subcellular localization of host erythrocyte membrane components during their asexual growth

In the present study, the subcellular localization of the host red blood cell (RBC) membrane components, the alpha2-3-linked sialic acid (SA) residues and the lipid bilayer, was observed during the asexual growth of Babesia bovis using two erythrocyte probes, the SA-specific lectin (MALII) and the lipophilic fluorescent (PKH2) probes, respectively. In confocal laser scanning microscopy with MALII, the SA residues on the surface of parasitized RBCs appeared to accumulate into the intracellular parasites as the parasites matured as well as to remain on the surface of extracellular parasites. Furthermore, when PKH2-labeled RBCs were infected with B. bovis, PKH2 signals were also observed around both the intracellular and the extracellular parasites, similarly to the results of MALII. These results indicated that the components derived from the host erythrocyte membrane are incorporated into the intracellular parasites during their asexual growth within the parasitized RBC, suggesting the possible formation of a parasitophorous vacuole-based network or a parasite surface coat.     

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

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

Anti-Fading Agents for Confocal Immunofluorescence: Colocalization of Nuclear Polypeptides

We evaluated the performance of four anti-fading agents during acquisition of multiple optical sections near the widest diameter of Drosophila accessory gland nuclei using indirect immunofluorescence and confocal laser scanning microscopy. Two commercially available agents, Vectashield® and SlowFade® showed anti-fading properties that alleviated fluorochrome fading associated with the acquisition of multiple fluorescent optical Z-series from a single specimen by a confocal laser scanning system. Using these reagents, we were able to colocalize polypeptides through immunostained whole Drosophila nuclei.     

Preparation of chromosome spreads for electron (TEM,SEM, STEM), light and confocal microscopy

In the past, ultrastructural studies on chromosome morphology have been carried out using light microscopy, scanning electron microscopy and transmission electron microscopy of whole mounted or sectioned samples. Until now, however, it has not been possible to use all of these techniques on the same specimen. In this paper we describe a specimen preparation method that allows one to study the same chromosomes by transmission, scanning-transmission and scanning electron microscopy, as well as by standard light microscopy and confocal microscopy. Chromosome plates are obtained on a carbon coated glass slide. The carbon film carrying the chromosomes is then transferred to electron microscopy grids, subjected to various treatments and observed. The results show a consistent morphological correspondence between the different methods. This method could be very useful and important because it makes possible a direct comparison between the various techniques used in chromosome studies such as banding, in situ hybridization, fluorescent probe localization, ultrastructural analysis, and colloidal gold cytochemical reactionsAbbreviations CLSM confocal laser scanning microscope - EM electron microscopy - kV kilovolt(s) - LM light microscope - SEM scanning electron microscope - STEM scanning-transmission electron microscope - TEM transmission electron microscope     

Visualizing two-component protein diffusion in porous adsorbents by confocal scanning laser microscopy.

The use of confocal scanning laser microscopy (CSLM) has recently been described for the visualization of intraparticle protein profiles during single-protein finite bath uptake experiments. By coupling of fluorescent molecules to proteins the penetration of porous media by labeled macromolecules could be detected by scanning single adsorbent particles for fluorescence emission after laser excitation. Thus the internal protein distribution profile, which is a central element in modeling of protein transport in porous adsorbents, became experimentally accessible. Results from the simultaneous visualization of two proteins by this technology are shown here. The use of two different fluorescent dyes for protein labeling and two independent detectors in the CSLM allowed for the first time ever the direct observation of a two-component diffusion process within a porous stationary phase. The finite bath uptake of human immunoglobulin G (hIgG) and bovine serum albumin (BSA) to two different ion exchange adsorbents (SP Sepharose Fast Flow and Source 30S) and to an affinity adsorbent (Protein A Sepharose) was measured using Cy5 and Oregon Green as labels. Single adsorbent particles were scanned for intensity distribution of fluorescence emission from the two fluorophors. The intraparticle profiles obtained from the confocal images were translated into a relative protein concentration thus allowing the calculation of protein uptake kinetics from direct measurement in the stationary phase. The confocal technique may prove to be a very powerful means of data generation for modeling of multi-component mass transfer phenomena in protein adsorption.     

Mapping of Heavy Metal Ion Sorption to Cell-Extracellular Polymeric Substance-Mineral Aggregates by Using Metal-Selective Fluorescent Probes and Confocal Laser Scanning Microscopy

Biofilms, organic matter, iron/aluminum oxides, and clay minerals bind toxic heavy metal ions and control their fate and bioavailability in the environment. The spatial relationship of metal ions to biomacromolecules such as extracellular polymeric substances (EPS) in biofilms with microbial cells and biogenic minerals is complex and occurs at the micro- and submicrometer scale. Here, we review the application of highly selective and sensitive metal fluorescent probes for confocal laser scanning microscopy (CLSM) that were originally developed for use in life sciences and propose their suitability as a powerful tool for mapping heavy metals in environmental biofilms and cell-EPS-mineral aggregates (CEMAs). The benefit of using metal fluorescent dyes in combination with CLSM imaging over other techniques such as electron microscopy is that environmental samples can be analyzed in their natural hydrated state, avoiding artifacts such as aggregation from drying that is necessary for analytical electron microscopy. In this minireview, we present data for a group of sensitive fluorescent probes highly specific for Fe³⁺, Cu²⁺, Zn²⁺, and Hg²⁺, illustrating the potential of their application in environmental science. We evaluate their application in combination with other fluorescent probes that label constituents of CEMAs such as DNA or polysaccharides and provide selection guidelines for potential combinations of fluorescent probes. Correlation analysis of spatially resolved heavy metal distributions with EPS and biogenic minerals in their natural, hydrated state will further our understanding of the behavior of metals in environmental systems since it allows for identifying bonding sites in complex, heterogeneous systems.     

Simultaneous force and fluorescence measurements of a protein that forms a bond between a living bacterium and a solid surface

All microbial biofilms are initiated through direct physical contact between a bacterium and a solid surface, a step that is controlled by inter- and intramolecular forces. Atomic force microscopy and confocal laser scanning microscopy were used simultaneously to observe the formation of a bond between a fluorescent chimeric protein on the surface of a living Escherichia coli bacterium and a solid substrate in situ. The chimera was composed of a portion of outer membrane protein A (OmpA) fused to the cyan-fluorescent protein AmCyan. Sucrose gradient centrifugation and fluorescent confocal slices through bacteria demonstrated that the chimeric protein was targeted and anchored to the external cell surface. The wormlike chain theory predicted that this protein should exhibit a nonlinear force-extension "signature" consistent with the sequential unraveling of the AmCyan and OmpA domains. Experimentally measured force-extension curves revealed a unique pair of "sawtooth" features that were present when a bond formed between a silicon nitride surface (atomic force microscopy tip) and E. coli cells expressing the OmpA-AmCyan protein. The observed sawtooth pair closely matched the wormlike chain model prediction for the mechanical unfolding of the AmCyan and OmpA substructures in series. These sawteeth disappeared from the measured force-extension curves when cells were treated with proteinase K. Furthermore, these unique sawteeth were absent for a mutant stain of E. coli incapable of expressing the AmCyan protein on its outer surface. Together, these data show that specific proteins exhibit unique force signatures characteristic of the bond that is formed between a living bacterium and another surface.