Construction of an engineered yeast with glucose-inducible emission of green fluorescence from the cell surface

An engineered yeast with emission of fluorescence from the cell surface was constructed. Cell surface engineering was applied to display a visible reporter molecule, green fluorescent protein (GFP). A glucose-inducible promoter GAPDH as a model promoter was selected to control the expression of the reporter gene in response to environmental changes. The GFP gene was fused with the gene encoding the C-terminal half of α-agglutinin of Saccharomyces cerevisiae having a glycosylphosphatidylinositol anchor attachment signal sequence. A secretion signal sequence of the fungal glucoamylase precursor protein was connected to the N-terminal of GFP. This designed gene was integrated into the TRP1 locus of the chromosome of S. cerevisiae with homologous recombination. Fluorescence microscopy demonstrated that the transformant cells emitted green fluorescence derived from functionally expressed GFP involved in the fusion molecule. The surface display of GFP was further verified by immunofluorescence labeling with a polyclonal antibody (raised in rabbits) against GFP as the first antibody and Rhodamine Red-X-conjugated goat anti-rabbit IgG as the second antibody which cannot penetrate into the cell membrane. The display of GFP on the cell surface was confirmed using a confocal laser scanning microscope and by measuring fluorescence in each cell fraction obtained after the subcellular fractionation. As GFP was proved to be displayed as an active form on the cell surface, selection of promoters will endow yeast cells with abilities to respond to changes in environmental conditions, including nutrient concentrations in the media, through the emission of fluorescence. Received: 23 August 1999 / Received revision: 16 November 1999 / Accepted: 29 November 1999     

A new approach to the detection and statistical classification of Ca2+ sparks

The availability of high-speed, two-dimensional (2-D) confocal microscopes and the expanding armamentarium of fluorescent probes presents unprecedented opportunities and new challenges for studying the spatial and temporal dynamics of cellular processes. The need to remove subjectivity from the detection process, the difficulty of the human eye to detect subtle changes in fluorescence in these 2-D images, and the large volume of data produced by these confocal microscopes call for the need to develop algorithms to automatically mark the changes in fluorescence. These fluorescence signal changes are often subtle, so the statistical estimate of the likelihood that the detected signal is not noise is an integral part of the detection algorithm. This statistical estimation is fundamental to our new approach to detection; in earlier Ca(2+) spark detectors, this statistical assessment was incidental to detection. Importantly, the use of the statistical properties of the signal local to the spark, instead of over the whole image, reduces the false positive and false negative rates. We developed an automatic spark detection algorithm based on these principles and used it to detect sparks on an inhomogeneous background of transverse tubule-labeled rat ventricular cells. Because of the large region of the cell surveyed by the confocal microscope, we can detect a large enough number of sparks to measure the dynamic changes in spark frequency in individual cells. We also found, in contrast to earlier results, that cardiac sparks are spatially symmetric. This new approach puts the detection of fluorescent signals on a firm statistical foundation.     

Improved and high throughput quantitative measurements of weak GFP expression in transgenic plant materials

Green fluorescent proteins (GFPs) are widely used in tracing transgene expression and have been known as convenient and efficient markers for plant transformation. However, sometimes researchers are still puzzled by the weak fluorescence since it makes the observation of GFP signals and confirmation of transgenic plants difficult. In this investigation, we explored the possibility of enhancing the weak signals by changing the pH environment of detection and took microplate reader as a more effective instrument compared to traditional fluorescent microscope to detect the weak signals. It was found that the fluorescence intensity of enhanced GFP (EGFP) in transgenic plants can be increased 2–6 folds by altering the environmental pH, and the concentration of EGFP at a large scale (ranged from 20 ng/ml to 20 μg/ml) can be detected and quantified. It can exclude the influence of degradation fragment and hence facilitate later analysis; these advantages were further verified by comparing with western blotting and confocal microscopy. It was reliable and effective for the qualitative and quantitative analysis of transgenic plants and was more suitable for the detection of very weak fluorescent signals.     

Development and Evaluation of Chitosan-Coated Liposomes for Oral DNA Vaccine: The Improvement of Peyer’s Patch Targeting Using a Polyplex-Loaded Liposomes

The aim of this study was to develop chitosan-coated and polyplex-loaded liposomes (PLLs) containing DNA vaccine for Peyer’s patch targeting. Plain liposomes carrying plasmid pRc/CMV-HBs were prepared by the reverse-phase evaporation method. Chitosan coating was carried out by incubation of the liposomal suspensions with chitosan solution. Main lipid components of liposomes were phosphatidylcholine/cholesterol. Sodium deoxycholate and dicetyl phosphate were used as negative charge inducers. The zeta potentials of plain liposomes were strongly affected by the pH of the medium. Coating with chitosan variably increased the surface charges of the liposomes. To increase the zeta potential and stability of the liposome, chitosan was also used as a DNA condensing agent to form a polyplex. The PLLs were coated with chitosan solution. In vivo study of PLLs was carried out in comparison with chitosan-coated liposomes using plasmid encoding green fluorescence protein as a reporter. A single dose of plasmid equal to 100 μg was intragastrically inoculated into BALB/c mice. The expression of green fluorescence protein (GFP) was detected after 24 h using a confocal laser scanning microscope. The signal of GFP was obtained from positively charged chitosan-coated liposomes but found only at the upper part of duodenum. With chitosan-coated PLL540, the signal of GFP was found throughout the intestine. Chitosan-coated PLL demonstrated a higher potential to deliver the DNA to the distal intestine than the chitosan-coated liposomes due to the increase in permanent positive surface charges and the decreased enzymatic degradation.     

Video-Rate Confocal Microscopy for Single-Molecule Imaging in Live Cells and Superresolution Fluorescence Imaging

There is no confocal microscope optimized for single-molecule imaging in live cells and superresolution fluorescence imaging. By combining the swiftness of the line-scanning method and the high sensitivity of wide-field detection, we have developed a, to our knowledge, novel confocal fluorescence microscope with a good optical-sectioning capability (1.0 μm), fast frame rates (<33 fps), and superior fluorescence detection efficiency. Full compatibility of the microscope with conventional cell-imaging techniques allowed us to do single-molecule imaging with a great ease at arbitrary depths of live cells. With the new microscope, we monitored diffusion motion of fluorescently labeled cAMP receptors of Dictyostelium discoideum at both the basal and apical surfaces and obtained superresolution fluorescence images of microtubules of COS-7 cells at depths in the range 0–85 μm from the surface of a coverglass.     

A Highlights from MBoC Selection: Ultrafast superresolution fluorescence imaging with spinning disk confocal microscope optics

Most current superresolution (SR) microscope techniques surpass the diffraction limit at the expense of temporal resolution, compromising their applications to live-cell imaging. Here we describe a new SR fluorescence microscope based on confocal microscope optics, which we name the spinning disk superresolution microscope (SDSRM). Theoretically, the SDSRM is equivalent to a structured illumination microscope (SIM) and achieves a spatial resolution of 120 nm, double that of the diffraction limit of wide-field fluorescence microscopy. However, the SDSRM is 10 times faster than a conventional SIM because SR signals are recovered by optical demodulation through the stripe pattern of the disk. Therefore a single SR image requires only a single averaged image through the rotating disk. On the basis of this theory, we modified a commercial spinning disk confocal microscope. The improved resolution around 120 nm was confirmed with biological samples. The rapid dynamics of micro­tubules, mitochondria, lysosomes, and endosomes were observed with temporal resolutions of 30–100 frames/s. Because our method requires only small optical modifications, it will enable an easy upgrade from an existing spinning disk confocal to a SR microscope for live-cell imaging.     

Effects of Lipofectamine 2000/siRNA Complexes on Autophagy in Hepatoma Cells

Lipofectamine 2000 is commonly used for siRNA transfections. However, few studies have examined cellular responses to this delivery system. The purpose of this study is to evaluate the effect of siRNA transfection using Lipofectamine 2000 on cellular autophagy. Huh7.5 cells, stably transfected to express GFP–LC3, were treated with Lipofectamine 2000/negative control siRNA (NC siRNA) complexes. At different time points after treatment, cells were lysed and analyzed by immunoblotting and fluorescence spectroscopy. Cells were also observed using confocal microscopy. An increase of endogenous LC3 lipidation, GFP–LC3 fluorescence, and autophagosomal puncta was observed in cells treated with Lipofectamine 2000/NC siRNA complexes. The kinetics of the increase of GFP–LC3 fluorescence correlated with the concentration of NC siRNA transfected, where 50, 100, and 200 nM NC siRNA caused a significant increase at 72, 48, and 24 h, respectively, after transfection. A similar effect on the GFP–LC3 signal was also observed for cells treated with Lipofectamine 2000 complexed with two other NC siRNAs. The effects were also confirmed in another hepatoma cell line, H4IIE, by immunoblotting. Lipofectamine 2000-mediated transport of NC siRNAs led to an increase of autophagosomes in a dose- and time-dependent manner. Thus, this effect on cells should be taken into consideration when using this approach for intracellular delivery of siRNA.     

Frequency division multiplexed multichannel high-speed fluorescence confocal microscope

In this article, we report a new type of fluorescence confocal microscope: frequency division multiplexed multichannel fluorescence confocal microscope, in which we encode the spatial location information into the frequency domain. In this microscope, the exciting laser beam is first split into multiple beams and each beam is modulated at a different frequency. These multiple beams are focused at different locations of the target to form multiple focal points, which further generate multiple fluorescent emission spots. The fluorescent emissions from different focal points are also modulated at different frequencies, because the exciting beams are modulated at different frequencies (or difference carrier frequency). Then, all the fluorescent emissions (modulated at different frequencies) are collected together and detected by a highly sensitive, large-dynamic-range photomultiplier tube. By demodulating the detected signal (i.e., via the Fourier transform), we can distinguish the fluorescent light emitted from the different locations by the corresponding carrier frequencies. The major advantage of this unique fluorescence confocal microscope is that it not only has a high sensitivity because of the use of photomultiplier tube but also can get multiple-point data simultaneously, which is crucial to study the dynamic behavior of many biological process. As an initial step, to verify the feasibility of the proposed multichannel confocal microscope, we have developed a two-channel confocal fluorescence microscope and applied it to study the dynamic behavior of the changes of the calcium ion concentration during the single cardiac myocyte contraction. Our preliminary experimental results demonstrated that we could indeed realize multichannel confocal fluorescence microscopy by utilizing the frequency division multiplexed microscope, which could become an effective tool to study the dynamic behavior of many biological processes.     

Fluorescence and confocal laser scanning microscopy imaging of elastic fibers in hematoxylin-eosin stained sections

 We have studied the possibility of associating fluorescence microscopy and hematoxylin-eosin staining for the identification of elastic fibers in elastin-rich tissues. Elastic fibers and elastic laminae were consistently identified by the proposed procedure, which revealed itself to be easy and useful for the determination of such structures and their distribution. The fluorescence properties of stained elastic fibers are due to eosin staining as revealed by fluorescence analysis of the dye in solution, with no or only minor contribution by the elastin auto-fluorescence. The main advantage of this technique resides in the possibility of studying the distribution of elastic fibers in file material without further sectioning and staining. The use of the confocal laser scanning microscope greatly improved the resolution and selectivity of imaging elastic fibers in different tissues. The determination of the three-dimensional distribution and structure of elastic fiber and laminae using the confocal laser scanning microscope was evaluated and also produced excellent results. Accepted: 28 August 1996     

isoSTED microscopy with water-immersion lenses and background reduction

Fluorescence microscopy is an excellent tool to gain knowledge on cellular structures and biochemical processes. Stimulated emission depletion (STED) microscopy provides a resolution in the range of a few 10 nm at relatively fast data acquisition. As cellular structures can be oriented in any direction, it is of great benefit if the microscope exhibits an isotropic resolution. Here, we present an isoSTED microscope that utilizes water-immersion objective lenses and enables imaging of cellular structures with an isotropic resolution of better than 60 nm in living samples at room temperature and without CO₂ supply or another pH control. This corresponds to a reduction of the focal volume by far more than two orders of magnitude as compared to confocal microscopy. The imaging speed is in the range of 0.8 s/μm³. Because fluorescence signal can only be detected from a diffraction-limited volume, a background signal is inevitably observed at resolutions well beyond the diffraction limit. Therefore, we additionally present a method that allows us to identify this unspecific background signal and to remove it from the image.     

Highly sensitive analytical method for aluminum movement in soybean root through lumogallion staining

We present highly sensitive aluminum detection method in root using fluorescent lumogallion. Roots treated with 100 μM AlCl₃ including 0.2 mM CaCl₂ (pH 4.5) were stained for 60 min with 10 μM lumogallion fluorescence solution and fluorescence from aluminum complex in root was observed under confocal laser microscope. There was a good correlation between the intensity of fluorescence and aluminum content. When the amount of aluminum lost during each step in staining process was measured, it was found that about 10% of aluminum was lost only at staining stage. Through lumogallion staining method, aluminum accumulation especially at an early stage of aluminum treatment in root was shown. At the beginning (2 hr), aluminum began to be accumulated in root cap. After 4 hr treatment, the aluminum distribution was spread to about 3 mm from root apex in the root cap and outer cortex. When aluminum was found in the outer cortex in 3–5 mm from the root apex, the viability was tended to be decreased in the same area (6 hr). At the same time, aluminum amount in meristem was increased. However the comparison of lumogallion staining method with that of morin, which has been widely used to detect aluminum in root, the sensitivity of lumogallion method was found to be much higher.     

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     

Display of adenoregulin with a novel <Emphasis Type="Italic">Pichia pastoris</Emphasis> cell surface display system

Two Pichia pastoris cell surface display vectors were constructed. The vectors consisted of the flocculation functional domain of Flo 1p with its own secretion signal sequence or the α-factor secretion signal sequence, a polyhistidine (6×His) tag for detection, an enterokinase recognition site, and the insertion sites for target proteins. Adenoregulin (ADR) is a 33-amino-acid antimicrobial peptide isolated from Phyllomedusa bicolor skin. The ADR was expressed and displayed on the Pichia pastoris KM71 cell surface with the system reported. The displayed recombinant ADR fusion protein was detected by fluorescence microscopy and confocal laser scanning microscopy (CLSM). The antimicrobial activity of the recombinant adenoregulin was detected after proteolytic cleavage of the fusion protein on cell surface. The validity of the Pichia pastoris cell surface display vectors was proved by the displayed ADR.     

Osmotic water permeability measurements using confocal laser scanning microscopy

 We have developed a method for measurement of plasma membrane water permeability (P _f) in intact cells using laser scanning confocal microscopy. The method is based on confocal recording of the fluorescence intensity emitted by calcein-loaded adherent cells during osmotic shock. P _f is calculated as a function of the time constant in the fluorescence intensity change, the cell surface-to-volume ratio and the fractional content of the osmotically active cell volume. The method has been applied to the measurement of water permeability in MDCK cells. The cells behaved as linear osmometers in the interval from 100 to 350 mosM. About 57% of the total cell volume was found to be osmotically inactive. Water movement across the plasma membrane in intact MDCK cells was highly temperature dependent. HgCl₂ had no effect on water permeability, while amphotericin B and DMSO significantly increased P _f values. The water permeability in MDCK cells transfected with aquaporin 2 was an order of magnitude higher than in the intact MDCK cell line. The water permeability of the nuclear membrane in both cell lines was found to be unlimited. Thus the intranuclear fluid belongs to the osmotically active portion of the cell. We conclude that the use of confocal microscopy provides a sensitive and reproducible method for measurement of water permeability in different types of adherent cells and potentially for coverslip-attached tissue preparations. Received: 12 June 1999 / Revised version: 21 February 2000 / Accepted: 25 February 2000     

Comparative FISH analysis of numerical chromosome 7 abnormalities in 5-μm and 15-μm paraffin-embedded tissue sections from prostatic carcinoma

 Interphase fluorescence in situ hybridization (FISH) was performed on 15-μm-thick paraffin sections from prostatic carcinomas using a chromosome 7-specific α-satellite DNA probe. A confocal laser scanning microscope (CLSM) was used for optical sectioning of the thick sections and reconstruction of 3D images. The number of FISH signals was determined by a gallery of optical sections evaluating only complete nuclei. To investiate the influence of section thickness and truncation and nuclei on scoring results, we compared the FISH data from 15-μm sections with signal counts obtained from 5-μm sections. The latter were evaluated by conventional fluorescence microscopy in the same tumor regions previously defined and marked on the slides. After statistical analysis of spot frequencies in tumor and non-tumorous cells (χ² test), we transferred the signal frequencies into a cytogenetic classification (−7, +7, polysomy 7). Based on this classification, most cases showed more than one chromosome 7 aberration type. Trisomy 7 (+7) became apparent in 15-μm-thick sections in all 19 tumors, polysomy 7 (>3 spots) in 18/19 cases, and monosomy 7 (−7) in 13/19 cases. In 5-μm sections, however, trisomy 7 and polysomy 7 were found in only 7/19 and 13/19 cases, respectively, and monosomy 7 in 7/19 cases. When comparing the classification results of tumor cells of the same tumor regions originating either from 5-μm or 15-μm sections, the following discrepancies were noted: in 15-μm sections exclusively, in 12/19 tumors, trisomy 7 was found; in 6/19 cases, polysomy 7; in 8/19 cases, monosomy 7. The high proportion of cases with tumor nuclei expressing only one hybridization signal of chromosome 7 in 15-μm sections could be confirmed as monosomy 7 in five selected cases by double-hybridization using centromere-specific probes for chromosomes 7 and 12. These results demonstrate that numerical chromosome 7 aberrations are more frequently observed in thick (15-μm) paraffin-embedded tissue sections by evaluating only complete nuclei. The use of routine sections (5-μm) for interphase cytogenetic analyses is compromised by a remarkable underestimation of the real chromosome copy numbers. Accepted: 7 November 1996     

Image correlation spectroscopy. II. Optimization for ultrasensitive detection of preexisting platelet-derived growth factor-beta receptor oligomers on intact cells.

Previously we introduced image correlation spectroscopy (ICS) as an imaging analog of fluorescence correlation spectroscopy (FCS). Implementation of ICS with image collection via a standard fluorescence confocal microscope and computer-based autocorrelation analysis was shown to facilitate measurements of absolute number densities and determination of changes in aggregation state for fluorescently labeled macromolecules. In the present work we illustrate how to use ICS to quantify the aggregation state of immunolabeled plasma membrane receptors in an intact cellular milieu, taking into account background fluorescence. We introduce methods that enable us to completely remove white noise contributions from autocorrelation measurements for individual images and illustrate how to perform background corrections for autofluorescence and nonspecific fluorescence on cell population means obtained via ICS. The utilization of photon counting confocal imaging with ICS analysis in combination with the background correction techniques outlined enabled us to achieve very low detection limits with standard immunolabeling methods on normal, nontransformed human fibroblasts (AG1523) expressing relatively low numbers of platelet-derived growth factor-beta (PDGF-beta) receptors. Specifically, we determined that the PDGF-beta receptors were preaggregated as tetramers on average with a mean surface density of 2.3 clusters micrometer(-2) after immunolabeling at 4 degreesC. These measurements, which show preclustering of PDGF-beta receptors on the surface of normal human fibroblasts, contradict a fundamental assumption of the ligand-induced dimerization model for signal transduction and provide support for an alternative model that posits signal transduction from within preexisting receptor aggregates.     

Confined detection volume of fluorescence correlation spectroscopy by bare fiber probes

A fiber-tip-based near-field fluorescence correlation spectroscopy (FCS) has been developed for confining the detection volume to sub-diffraction-limited dimensions. This near-field FCS is based on near-field illumination by coupling a scanning near-field optical microscope (SNOM) to a conventional confocal FCS. Single-molecule FCS analysis at 100 nM Rhodamine 6G has been achieved by using bare chemically etched, tapered fiber tips. The detection volume under control of the SNOM system has been reduced over one order of magnitude compared to that of the conventional confocal FCS. Related factors influencing the near-field FCS performance are investigated and discussed in detail. In this proof-of-principle study, the preliminary experimental results suggest that the fiber-tip-based near-field FCS might be a good alternative to realize localized analysis at the single-molecule level.     

Subcellular localization of PS1 based on PS1/GFP fusing protein

Mutations in presenilin 1 (PS1) gene are closely associated with the early onset of familial Alzheimer’s disease (EOFAD). The fusion genes, GFPPS1 (recombinant plasmid pEGFP-C1-PS1) and PS1-GFP (recombinant plasmid pEGFP-N2-PS1) were constructed to study the subcellular localization of PS1 holoprotein. Recombinant plasmids were transiently transfected into two cell lines, HEK293 and CHO, respectively, using the green fluorescence from GFP (green fluorescence protein) as the PS1 localization signal. Then, we observed green fluorescence with a SPOT II (Olympus, BH2) and CONFOCAL microscope (Olympus, FV300) under 488 nm. The results show that PS1 located on the nuclear envelope. A few can be found on the cellular membrane and in the cytosol in a non-homogeneous distribution. __________ Translated from China Biotechnology, 2006, 26(6): 17-22 [译自: 中 国生物工程杂志]     

Cholesterol modulation of nicotinic acetylcholine receptor surface mobility

Nicotinic acetylcholine receptor (AChR) function and distribution are quite sensitive to cholesterol (Chol) levels in the plasma membrane (reviewed by Barrantes in J Neurochem 103 (suppl 1):72–80, 2007). Here we combined confocal fluorescence recovery after photobleaching (FRAP) and confocal fluorescence correlation spectroscopy (FCS) to examine the mobility of the AChR and its dependence on Chol content at the cell surface of a mammalian cell line. Plasma membrane AChR exhibited limited mobility and only ~55% of the fluorescence was recovered within 10 min after photobleaching. Depletion of membrane Chol by methyl-β-cyclodextrin strongly affected the mobility of the AChR at the plasma membrane; the fraction of mobile AChR fell from 55 to 20% in Chol-depleted cells, whereas Chol enrichment by methyl-β-cyclodextrin-Chol treatment did not reduce receptor mobility at the cell surface. Actin depolymerization caused by latrunculin A partially restored receptor mobility in Chol-depleted cells. In agreement with the FRAP data, scanning FCS experiments showed that the diffusion coefficient of the AChR was about 30% lower upon Chol depletion. Taken together, these results suggest that membrane Chol modulates AChR mobility at the plasma membrane through a Chol-dependent mechanism sensitive to cortical actin.