Analysis of cellular processes with microscopic bright field defocused imaging has the advantage of low phototoxicity and minimal sample preparation. However bright field images lack the contrast and nuclei reporting available with florescent approaches and therefore present a challenge to methods that segment and track the live cells. Moreover, such methods must be robust to systemic and random noise, variability in experimental configuration, and the multiple unknowns in the biological system under study.
A new method called maximal-information is introduced that applies a non-parametric information theoretic approach to segment bright field defocused images. The method utilizes a combinatorial optimization strategy to select specific defocused images from each image stack such that set complexity, a Kolmogorov complexity measure, is maximized. Differences among these selected images are then applied to initialize and guide a level set based segmentation algorithm. The performance of the method is compared with a recent approach that uses a fixed defocused image selection strategy over an image data set of embryonic kidney cells (HEK 293T) from multiple experiments. Results demonstrate that the adaptive maximal-information approach significantly improves precision and recall of segmentation over the diversity of data sets.
Integrating combinatorial optimization with non-parametric Kolmogorov complexity has been shown to be effective in extracting information from microscopic bright field defocused images. The approach is application independent and has the potential to be effective in processing a diversity of noisy and redundant high throughput biological data.
We present a very simple procedure yielding high-contrast images of adherent, confluent cells such as human neuroblastoma (SH-EP) cells by ordinary bright-field microscopy. Cells are illuminated through a color filter and a pinhole aperture placed between the condenser and the cell culture surface. Refraction by each cell body generates a sharp, bright spot when the image is defocused. The technique allows robust, automatic cell counting from a single bright-field image in a wide range of focal positions; it does this via free, readily available image-analysis tools. Contrast may be enhanced by swelling cell bodies by brief incubation in PBS. The procedure was benchmarked against manual counting and automated counting of fluorescently labeled cell nuclei.. Counts from day-old and freshly seeded plates were compared in a range of densities, from sparse to densely overgrown. On average bright-field images produced the same counts as fluorescent images, with less than 5% error. This method will allow routine cell counting using a plain bright-field microscope, absent cell-line modification or cell staining.
Brightfield; microscopy; fluorescence; cell culture; counting; pinhole; monochomatic; PBS; defocusing
Confocal microscopy analysis of fluorescence and morphology is becoming the standard tool in cell biology and molecular imaging. Accurate quantification algorithms are required to enhance the understanding of different biological phenomena. We present a novel approach based on image-segmentation of multi-cellular regions in bright field images demonstrating enhanced quantitative analyses and better understanding of cell motility. We present MultiCellSeg, a segmentation algorithm to separate between multi-cellular and background regions for bright field images, which is based on classification of local patches within an image: a cascade of Support Vector Machines (SVMs) is applied using basic image features. Post processing includes additional classification and graph-cut segmentation to reclassify erroneous regions and refine the segmentation. This approach leads to a parameter-free and robust algorithm. Comparison to an alternative algorithm on wound healing assay images demonstrates its superiority. The proposed approach was used to evaluate common cell migration models such as wound healing and scatter assay. It was applied to quantify the acceleration effect of Hepatocyte growth factor/scatter factor (HGF/SF) on healing rate in a time lapse confocal microscopy wound healing assay and demonstrated that the healing rate is linear in both treated and untreated cells, and that HGF/SF accelerates the healing rate by approximately two-fold. A novel fully automated, accurate, zero-parameters method to classify and score scatter-assay images was developed and demonstrated that multi-cellular texture is an excellent descriptor to measure HGF/SF-induced cell scattering. We show that exploitation of textural information from differential interference contrast (DIC) images on the multi-cellular level can prove beneficial for the analyses of wound healing and scatter assays. The proposed approach is generic and can be used alone or alongside traditional fluorescence single-cell processing to perform objective, accurate quantitative analyses for various biological applications.
We describe a technique based on moment-analysis for the measurement of the average number of molecules and brightness in each pixel in fluorescence microscopy images. The average brightness of the particle is obtained from the ratio of the variance to the average intensity at each pixel. To obtain the average number of fluctuating particles, we divide the average intensity at one pixel by the brightness. This analysis can be used in a wide range of concentrations. In cells, the intensity at any given pixel may be due to bright immobile structures, dim fast diffusing particles, and to autofluorescence or scattering. The total variance is given by the variance of each of the above components in addition to the variance due to detector noise. Assuming that all sources of variance are independent, the total variance is the sum of the variances of the individual components. The variance due to the particles fluctuating in the observation volume is proportional to the square of the particle brightness while the variance of the immobile fraction, the autofluorescence, scattering, and that of the detector is proportional to the intensity of these components. Only the fluctuations that depend on the square of the brightness (the mobile particles) will have a ratio of the variance to the intensity >1. Furthermore, changing the fluorescence intensity by increasing the illumination power, distinguishes between these possible contributions. We show maps of molecular brightness and number of cell migration proteins obtained using a two-photon scanning microscope operating with a photon-counting detector. These brightness maps reveal binding dynamics at the focal adhesions with pixel resolution and provide a picture of the binding and unbinding process in which dim molecules attach to the adhesions or large molecular aggregates dissociate from adhesion.
In recent years, high-throughput microscopy has emerged as a powerful tool to analyze cellular dynamics in an unprecedentedly high resolved manner. The amount of data that is generated, for example in long-term time-lapse microscopy experiments, requires automated methods for processing and analysis. Available software frameworks are well suited for high-throughput processing of fluorescence images, but they often do not perform well on bright field image data that varies considerably between laboratories, setups, and even single experiments.
In this contribution, we present a fully automated image processing pipeline that is able to robustly segment and analyze cells with ellipsoid morphology from bright field microscopy in a high-throughput, yet time efficient manner. The pipeline comprises two steps: (i) Image acquisition is adjusted to obtain optimal bright field image quality for automatic processing. (ii) A concatenation of fast performing image processing algorithms robustly identifies single cells in each image. We applied the method to a time-lapse movie consisting of ∼315,000 images of differentiating hematopoietic stem cells over 6 days. We evaluated the accuracy of our method by comparing the number of identified cells with manual counts. Our method is able to segment images with varying cell density and different cell types without parameter adjustment and clearly outperforms a standard approach. By computing population doubling times, we were able to identify three growth phases in the stem cell population throughout the whole movie, and validated our result with cell cycle times from single cell tracking.
Our method allows fully automated processing and analysis of high-throughput bright field microscopy data. The robustness of cell detection and fast computation time will support the analysis of high-content screening experiments, on-line analysis of time-lapse experiments as well as development of methods to automatically track single-cell genealogies.
Nosema locustae, a protozoan parasite of grasshoppers, is used as a bioinsecticide. In the present study, the persistence of N. locustae spores in soil and the interaction of these spores with the indigenous soil microflora were examined with various forms of microscopy and staining. Fluorescence microscopy was found to be better than phase-contrast or bright-field microscopy for detecting and viewing spores in soil. Fluorescein isothiocyanate was a better fluorescent stain than acridine orange or fluorescein diacetate; water-soluble aniline blue did not stain spores. The eight bright-field microscopy stains tested (phenolic erythrosin, phenolic rose bengal, malachite green, crystal violet, safranin, Congo red, methyl red, and eosin B) were not satisfactory, as spore staining characteristics were either poor or masked by overstained soil debris. A procedure was developed which allowed spores to be extracted from soil with a peptone-phosphate buffer, recovered on a membrane filter, and stained with fluorescein isothiocyanate for microscopic counting. This procedure was used to assess the persistence of N. locustae spores in field and laboratory soils. The number of N. locustae spores in a laboratory model soil system persisted at a high level for over 8 weeks when the soil was incubated at 5°C but exhibited a 1,000-fold decrease after 1 week of incubation at 27°C. Persistence was related to the temperature-dependent activity of the indigenous soil microflora, which, on the basis of microscopic observations, appeared to prey on N. locustae spores. N. locustae spores were detected in an N. locustae-treated field soil at a low level consistent with the level for laboratory soil incubated at 27°C, and they persisted at this level for over 2 months. No spores were detected on vegetation from this field or in the soil from an adjacent, nontreated control field. N. locustae-like spores were also detected in soil from nontreated fields supporting large grasshopper populations.
This unit describes a method for quantifying various cellular features (e.g., volume, total and subcellular fluorescence localization) from sets of microscope images of individual cells. It includes procedures for tracking cells over time. One purposefully defocused transmission image (sometimes referred to as bright-field or BF) is acquired to segment the image and locate each cell. Fluorescent images (one for each of the color channels to be analyzed) are then acquired by conventional wide-field epifluorescence or confocal microscopy. This method uses the image processing capabilities of Cell-ID (Gordon et al., 2007, as updated here) and data analysis by the statistical programming framework R (R-Development-Team, 2008), which we have supplemented with a package of routines for analyzing Cell-ID output. Both Cell-ID and the analysis package are open-source.
image processing; fluorescence microscopy; Cell-ID; R
A light-diffraction microscope was modified to allow sequential viewing of the microorganisms in a soil smear by transmitted, reflected, and reflected-polarized incandescent light and by reflected ultraviolet light. Observations were also made by conventional incandescent and ultraviolet transmitted-light microscopy. All results for the various forms of bright-field microscopy with stained and unstained soils were in agreement, but they differed from the results obtained for two types of ultraviolet-fluorescence microscopy. The latter proved to be nonspecific for in situ soil microorganisms. Capsule-like areas were noted surrounding many of the resident microbial cells of soil when viewed by the various forms of bright-field microscopy. These areas could not be stained or removed by a variety of treatments, but they apparently often did take up stain after in situ soil growth had been initiated. It was concluded that these areas are not capsules but may represent a structural component of nonmultiplying microbial cells in soil.
The applications of multiphoton microscopy for deep tissue imaging in basic and clinical research are ever increasing, supplementing confocal imaging of the surface layers of cells in tissue. However, imaging living tissue is made difficult by the light scattering properties of the tissue, and this is extraordinarily apparent in the mouse mammary gland which contains a stroma filled with fat cells surrounding the ductal epithelium. Whole mount mammary glands stained with Carmine Alum are easily archived for later reference and readily viewed using bright field microscopy to observe branching architecture of the ductal network. Here, we report on the advantages of multiphoton imaging of whole mount mammary glands. Chief among them is that optical sectioning of the terminal end bud (TEB) and ductal epithelium allows the appreciation of abnormalities in structure that are very difficult to ascertain using either bright field imaging of the stained gland or the conventional approach of hematoxylin and eosin staining of fixed and paraffin-embedded sections. A second advantage is the detail afforded by second harmonic generation (SHG) in which collagen fiber orientation and abundance can be observed.
GFP-mouse mammary glands were imaged live or after whole mount preparation using a Zeiss LSM510/META/NLO multiphoton microscope with the purpose of obtaining high resolution images with 3D content, and evaluating any structural alterations induced by whole mount preparation. We describe a simple means for using a commercial confocal/ multiphoton microscope equipped with a Ti-Sapphire laser to simultaneously image Carmine Alum fluorescence and collagen fiber networks by SHG with laser excitation set to 860 nm. Identical terminal end buds (TEBs) were compared before and after fixation, staining, and whole mount preparation and structure of collagen networks and TEB morphologies were determined. Flexibility in excitation and emission filters was explored using the META detector for spectral emission scanning. Backward scattered or reflected SHG (SHG-B) was detected using a conventional confocal detector with maximum aperture and forward scattered or transmitted SHG (SHG-F) detected using a non-descanned detector.
We show here that the developing mammary gland is encased in a thin but dense layer of collagen fibers. Sparse collagen layers are also interspersed between stromal layers of fat cells surrounding TEBs. At the margins, TEBs approach the outer collagen layer but do not penetrate it. Abnormal mammary glands from an HAI-1 transgenic FVB mouse model were found to contain TEBs with abnormal pockets of cells forming extra lumens and zones of continuous lateral bud formation interspersed with sparse collagen fibers.
Parameters influencing live imaging and imaging of fixed unstained and Carmine Alum stained whole mounts were evaluated. Artifacts induced by light scattering of GFP and Carmine Alum signals from epithelial cells were identified in live tissue as primarily due to fat cells and in whole mount tissue as due to dense Carmine Alum staining of epithelium. Carmine Alum autofluorescence was detected at excitation wavelengths from 750 to 950 nm with a peak of emission at 623 nm (~602-656 nm). Images of Carmine Alum fluorescence differed dramatically at emission wavelengths of 565–615 nm versus 650–710 nm. In the latter, a mostly epithelial (nuclear) visualization of Carmine Alum predominates. Autofluorescence with a peak emission of 495 nm was derived from the fixed and processed tissue itself as it was present in the unstained whole mount. Contribution of autofluorescence to the image decreases with increasing laser excitation wavelengths. SHG-B versus SHG-F signals revealed collagen fibers and could be found within single fibers, or in different fibers within the same layer. These differences presumably reflected different states of collagen fiber maturation. Loss of SHG signals from layer to layer could be ascribed to artifacts rendered by light scattering from the dense TEB structures, and unless bandpass emissions were selected, contained unfiltered non-SHG fluorescence and autofluorescent emissions. Flexibility in imaging can be increased using spectral emission imaging to optimize emission bandwidths and to separate SHG-B, GFP, and Carmine Alum signals, although conventional filters were also useful.
Collagen fibril arrangement and TEB structure is well preserved during the whole mount procedure and light scattering is reduced dramatically by extracting fat resulting in improved 3D structure, particularly for SHG signals originating from collagen. In addition to providing a bright signal, Carmine Alum stained whole mount slides can be imaged retrospectively such as performed for the HAI-1 mouse gland revealing new aspects of abnormal TEB morphology. These studies demonstrated the intimate contact, but relatively sparse abundance of collagen fibrils adjacent to normal and abnormal TEBS in the developing mammary gland and the ability to obtain these high resolution details subject to the discussed limitations. Our studies demonstrated that the TEB architecture is essentially unchanged after processing.
Giemsa staining of thick blood smears remains the "gold standard" for detecting malaria. However, this method is not very good for diagnosing low-level infections. A method for the simultaneous staining of Plasmodium-parasitized culture and blood smears for both bright field and fluorescence was developed and its ability to improve detection efficiency tested.
A total of 22 nucleic acid-specific fluorescent dyes were tested for their ability to provide easily observable staining of Plasmodium falciparum-parasitized red blood cells following Giemsa staining.
Of the 14 dyes that demonstrated intense fluorescence staining, only SYBR Green 1, YOYO-1 and ethidum homodimer-2 could be detected using fluorescent microscopy, when cells were first stained with Giemsa. Giemsa staining was not effective when applied after the fluorescent dyes. SYBR Green 1 provided the best staining in the presence of Giemsa, as a very high percentage of the parasitized cells were simultaneously stained. When blood films were screened using fluorescence microscopy the parasites were more readily detectable due to the sharp contrast between the dark background and the specific, bright fluorescence produced by the parasites.
The dual staining method reported here allows fluorescence staining, which enhances the reader's ability to detect parasites under low parasitaemia conditions, coupled with the ability to examine the same cell under bright field conditions to detect the characteristic morphology of Plasmodium species that is observed with Giemsa staining.
Automated microscopy to detect Mycobacterium tuberculosis in sputum smear slides would enable laboratories in countries with a high tuberculosis burden to cope efficiently with large numbers of smears. Focusing is a core component of automated microscopy, and successful autofocusing depends on selection of an appropriate focus algorithm for a specific task. We examined autofocusing algorithms for bright-field microscopy of Ziehl–Neelsen stained sputum smears. Six focus measures, defined in the spatial domain, were examined with respect to accuracy, execution time, range, full width at half maximum of the peak and the presence of local maxima. Curve fitting around an estimate of the focal plane was found to produce good results and is therefore an acceptable strategy to reduce the number of images captured for focusing and the processing time. Vollath's F4 measure performed best for full z-stacks, with a mean difference of 0.27 μm between manually and automatically determined focal positions, whereas it is jointly ranked best with the Brenner gradient for curve fitting.
Autofocusing; automated microscopy; focus measure; tuberculosis; Ziehl-Neelsen
The goals of this study were to create cryo-imaging methods to quantify characteristics (size, dispersal, and blood vessel density) of mouse orthotopic models of glioblastoma multiforme (GBM) and to enable studies of tumor biology, targeted imaging agents, and theranostic nanoparticles.
Green fluorescent protein-labeled, human glioma LN-229 cells were implanted into mouse brain. At 20–38 days, cryo-imaging gave whole brain, 4-GB, 3D microscopic images of bright field anatomy, including vasculature, and fluorescent tumor. Image analysis/visualization methods were developed.
Vessel visualization and segmentation methods successfully enabled analyses. The main tumor mass volume, the number of dispersed clusters, the number of cells/cluster, and the percent dispersed volume all increase with age of the tumor. Histograms of dispersal distance give a mean and median of 63 and 56 μm, respectively, averaged over all brains. Dispersal distance tends to increase with age of the tumors. Dispersal tends to occur along blood vessels. Blood vessel density did not appear to increase in and around the tumor with this cell line.
Cryo-imaging and software allow, for the first time, 3D, whole brain, microscopic characterization of a tumor from a particular cell line. LN-229 exhibits considerable dispersal along blood vessels, a characteristic of human tumors that limits treatment success.
Glioblastoma multiforme; GBM; Migration; Dispersal; Invasion; Blood vessel detection; 3D region growing; Cryo-imaging; LN-229
Many modern super-resolution imaging methods based on single-molecule fluorescence require the conversion of a dark fluorogen into a bright emitter to control emitter concentration. We have synthesized and characterized a nitro-aryl fluorogen which can be converted by a nitroreductase enzyme into a bright push–pull red-emitting fluorophore. Synthesis of model compounds and optical spectroscopy identify a hydroxyl-amino derivative as the product fluorophore, which is bright and detectable on the single-molecule level for fluorogens attached to a surface. Solution kinetic analysis shows Michaelis–Menten rate dependence upon both NADH and the fluorogen concentrations as expected. The generation of low concentrations of single-molecule emitters by enzymatic turnovers is used to extract subdiffraction information about localizations of both fluorophores and nitroreductase enzymes in cells. Enzymatic Turnover Activated Localization Microscopy (ETALM) is a complementary mechanism to photoactivation and blinking for controlling the emission of single molecules to image beyond the diffraction limit.
Studies of stochasticity in gene expression typically make use of fluorescent protein reporters, which permit the measurement of expression levels within individual cells by fluorescence microscopy. Analysis of such microscopy images is almost invariably based on a segmentation algorithm, where the image of a cell or cluster is analyzed mathematically to delineate individual cell boundaries. However segmentation can be ineffective for studying bacterial cells or clusters, especially at lower magnification, where outlines of individual cells are poorly resolved. Here we demonstrate an alternative method for analysing such images without segmentation. The method employs a comparison between the pixel brightness in phase contrast vs. fluorescence microscopy images. By fitting the correlation between phase contrast and fluorescence intensity to a physical model, we obtain well-defined estimates for the different expression levels of gene expression that are present in the cell or cluster. The method reveals the boundaries of the individual cells, even if the source images lack the resolution to show these boundaries clearly.
Stochasticity; GFP; phase contrast; single cell; segmentation; image analysis
A technique is described for the determination of bacterial numbers and the spectrum of actively metabolizing cells on the same microscopic preparation by a combined autoradiography/epifluorescence microscopy technique. Natural bacterial populations incubated with [3H]glucose were filtered onto 0.2-μm Nuclepore polycarbonate membranes. The filters were cut into quarters and fixed on the surface of glass slides, coated with NTB-2 nuclear track emulsion (Kodak), and exposed to the radiation. After processing, the autoradiographs were stained with acridine orange. A combination of overstaining on the slightly alkaline side and gradual destaining on the acid side of neutrality gave the best results. Epifluorescence microscopy revealed bright-orange fluorescent cells with dark-silver grains associated against a greenish-to-grayish background. Based on the standardization curves, detection of actually metabolizing cells was optimal when cells were incubated with 1 to 5 μCi of [3H]glucose per ml of sample for 4 h and the autoradiographs were exposed to NTB-2 emulsion at 7°C for 3 days. In water samples taken immediately above sandy sediments at beaches of the Kiel Fjord and the Kiel Bight (Baltic Sea, FRG), between 2.3 and 56.2% (average, 31.3%) of the total number of bacteria were actually metabolizing cells. Spearman rank correlation analysis revealed significant interrelationships between the number of active bacteria and the actual uptake rate of glucose.
A novel nucleic acid stain, SYBR Gold, was used to stain marine viral particles in various types of samples. Viral particles stained with SYBR Gold yielded bright and stable fluorescent signals that could be detected by a cooled charge-coupled device camera or by flow cytometry. The fluorescent signal strength of SYBR Gold-stained viruses was about twice that of SYBR Green I-stained viruses. Digital images of SYBR Gold-stained viral particles were processed to enumerate the concentration of viral particles by using digital image analysis software. Estimates of viral concentration based on digitized images were 1.3 times higher than those based on direct counting by epifluorescence microscopy. Direct epifluorescence counts of SYBR Gold-stained viral particles were in turn about 1.34 times higher than those estimated by the transmission electron microscope method. Bacteriophage lysates stained with SYBR Gold formed a distinct population in flow cytometric signatures. Flow cytometric analysis revealed at least four viral subpopulations for a Lake Erie sample and two subpopulations for a Georgia coastal sample. Flow cytometry-based viral counts for various types of samples averaged 1.1 times higher than direct epifluorescence microscopic counts. The potential application of digital image analysis and flow cytometry for rapid and accurate measurement of viral abundance in aquatic environments is discussed.
Fluorescence microscopy has become a principle methodology in the field of developmental biology. Recent technological advances have led to the design of high-speed and high-resolution confocal and multiphoton microscopes that enable researchers to obtain three- and four-dimensional information in living cells and whole embryos. Paralleling this progress, the development of stable and bright vital fluorescent probes has revolutionized the ability to track individual cells in vitro and in vivo and to visualize intercellular and subcellular molecular interactions in real time. Combining imaging modalities and labeling techniques that are increasingly unobtrusive to cell and whole animal function, our understanding of how proteins interact, tissues take form, and organs synchronize to create a functioning animal is reaching a whole new level.
EGF, epidermal growth factor; FP, fluorescent protein; FRET, Forster or fluorescence resonance energy transfer; GFP, green fluorescent protein; LSM, laser scanning microscopy; MB, molecular beacons; QD, quantum dot
Many biological specimens, such as living cells and their intracellular components, often exhibit very little amplitude contrast, making it difficult for conventional bright field microscopes to distinguish them from their surroundings. To overcome this problem phase contrast techniques such as Zernike, Normarsky and dark-field microscopies have been developed to improve specimen visibility without chemically or physically altering them by the process of staining. These techniques have proven to be invaluable tools for studying living cells and furthering scientific understanding of fundamental cellular processes such as mitosis. However a drawback of these techniques is that direct quantitative phase imaging is not possible. Quantitative phase imaging is important because it enables determination of either the refractive index or optical thickness variations from the measured optical path length with sub-wavelength accuracy.
Digital holography is an emergent phase contrast technique that offers an excellent approach in obtaining both qualitative and quantitative phase information from the hologram. A CCD camera is used to record a hologram onto a computer and numerical methods are subsequently applied to reconstruct the hologram to enable direct access to both phase and amplitude information. Another attractive feature of digital holography is the ability to focus on multiple focal planes from a single hologram, emulating the focusing control of a conventional microscope.
A modified Mach-Zender off-axis setup in transmission is used to record and reconstruct a number of holographic amplitude and phase images of cellular and sub-cellular features.
Both cellular and sub-cellular features are imaged with sub-micron, diffraction-limited resolution. Movies of holographic amplitude and phase images of living microbes and cells are created from a series of holograms and reconstructed with numerically adjustable focus, so that the moving object can be accurately tracked with a reconstruction rate of 300ms for each hologram. The holographic movies show paramecium swimming among other microbes as well as displaying some of their intracellular processes. A time lapse movie is also shown for fibroblast cells in the process of migration.
Digital holography and movies of digital holography are seen to be useful new tools for visualization of dynamic processes in biological microscopy. Phase imaging digital holography is a promising technique in terms of the lack of coherent noise and the precision with which the optical thickness of a sample can be profiled, which can lead to images with an axial resolution of a few nanometres.
Cytological studies of fluorescent proteins are rapidly yielding insights into chromatin structure and dynamics. Here we describe the production and cytological characterization of new transgenic maize lines expressing a fluorescent histone fusion protein, H2B-mCherry. The transgene is expressed under the control of the maize ubiquitin1 promoter, including its first exon and intron. Polymerase chain reaction–based genotyping and root-tip microscopy showed that most of the lines carrying the transgene also expressed it, producing bright uniform staining of nuclei. Further, plants showing expression in root tips at the seedling stage also showed expression during meiosis, late in the life cycle. Detailed high-resolution three-dimensional imaging of cells and nuclei from various somatic and meiotic cell types showed that H2B-mCherry produced remarkably clear images of chromatin and chromosome fiber morphology, as seen in somatic, male meiotic prophase, and early microgametophyte cells. H2B-mCherry also yielded distinct nucleolus staining and was shown to be compatible with fluorescence in situ hybridization. We found several instances where H2B-mCherry was superior to DAPI as a generalized chromatin stain. Our study establishes these histone H2B-mCherry lines as new biological reagents for visualizing chromatin structure, chromosome morphology, and nuclear dynamics in fixed and living cells in a model plant genetic system.
Obtaining information on the genetic capabilities and phylogenetic affinities of individual prokaryotic cells within natural communities is a high priority in the fields of microbial ecology, microbial biogeochemistry, and applied microbiology, among others. A method for prokaryotic in situ PCR (PI-PCR), a technique which will allow single cells within complex mixtures to be identified and characterized genetically, is presented here. The method involves amplification of specific nuclei acid sequences inside intact prokaryotic cells followed by color or fluorescence detection of the localized PCR product via bright-field or epifluorescence microscopy. Prokaryotic DNA and mRNA were both used successfully as targets for PI-PCR. We demonstrate the use of PI-PCR to identify nahA-positive cells in mixtures of bacterial isolates and in model marine bacterial communities.
The development of transgenic mouse lines that selectively label a subset of neurons provides unique opportunities to study detailed neuronal morphology and morphological changes under experimental conditions. In the present study, a mouse line in which a small number of retinal ganglion cells (RGCs) express yellow fluorescent protein (YFP) under control of the Thy-1 promoter was used (Feng et al., 2000). We characterized the number, distribution by retinal region and eccentricity of YFP-labeled RGCs using fluorescence microscopy and StereoInvestigator software (MicroBrightField, VT, USA). Then, we captured images of 4–6 YFP-expressing RGCs from each of 8 retinal regions by confocal microscopy, producing 3-dimensional and flattened data sets. A new semi-automated method to quantify the soma size, dendritic length and dendritic arbor complexity was developed using MetaMorph software (Molecular Devices, PA, USA). Our results show that YFP is expressed in 0.2% of all RGCs. Expression of YFP was not significantly different in central versus peripheral retina, but there were higher number of YFP expressing RGCs in the temporal quadrant than in the nasal. By confocal-based analysis, 58% of RGCs expressing YFP did so at a high level, with the remainder distributed in decreasing levels of brightness. Variability in detailed morphometric parameters was as great between two fellow retinas as in retinas from different mice. The analytic methods developed for this selective YFP expressing RGC model permit quantitative comparisons of parameters relevant to neuronal injury.
mouse; retina; ganglion cell; glaucoma; optic nerve; neuropathy; yellow fluorescent protein
Halogenoquinolones are potent and widely used antimicrobials blocking microbial DNA synthesis. However, they induce adverse photoresponses through the absorption of UV light, including phototoxicity and photocarcinogenicity. The phototoxic responses may be the result of photosensitization of singlet oxygen, production of free radicals and/or other reactive species resulting from photodehalogenation. Here, we report the use of laser scanning confocal microscopy to detect and to follow the fluorescence changes of one monohalogenated and three di-halogenated quinolones in live human epidermal keratinocyte cells during in situ irradiation by confocal laser in real time. Fluorescence image analysis and co-staining with the LysoTracker probe showed that lysosomes are a preferential site of drug localization and phototransformations. As the lysosomal environment is relatively acidic, we also determined how low pH may affect the dehalogenation and concomitant fluorescence. With continued UV irradiation, fluorescence increased in the photoproducts from BAY y3118 and clinafloxacin, whereas it decreased for lomefloxacin and moxifloxacin. Our images not only help to localize these phototoxic agents in the cell, but also provide means for dynamic monitoring of their phototransformations in the cellular environment.
Determinations of the number of microorganisms in lake water samples with the bright-field light microscope were performed using conventional counting chambers. Determinations with the fluorescence microscope were carried out after staining the organisms with acridine orange and filtering them onto Nuclepore filters. For transmission electron microscopy, a water sample was concentrated by centrifugation. The pellet was solidifed in agar, fixed, dehydrated, embedded in Epon, and cut into thin sections. The number and area of organism profiles per unit area of the sections were determined. The number of organisms per unit volume of the pellet was then calculated using stereological formulae. The corresponding number in the lake water was obtained from the ratio of volume of solidified pellet/volume of water sample. Control experiments with pure cultures of bacteria and algae showed good agreement between light and electron microscopic counts. This was also true for most lake water samples, but the electron microscopic preparations from some samples contained small vibrio-like bodies and ill-defined structures that made a precise comparison more difficult. Bacteria and small blue-green and green algae could not always be differentiated with the light microscope, but this was easily done by electron microscopy. Our results show that transmission electron microscopy can be used for checking light microscopic counts of microorganisms in lake water.
Mosaicing of confocal images enables observation of nuclear morphology in large areas of tissue. An application of interest is rapid detection of basal cell carcinomas (BCCs) in skin excisions during Mohs surgery. A mosaic is currently created in less than 9 min, whereas preparing frozen histology requires 20 to 45 min for an excision. In reflectance mosaics, using acetic acid as a contrast agent to brighten nuclei, large and densely nucleated BCC tumors were detectable in fields of view of 12 × 12 mm (which is equivalent to a 2×-magnified view as required by Mohs surgeons). However, small and sparsely nucleated tumors remained undetectable. Their diminutive size within the large field of view resulted in weak light backscatter and contrast relative to the bright surrounding normal dermis. In fluorescence, a nuclear-specific contrast agent may be used and light emission collected specifically from nuclei but almost none from the dermis. Acridine orange of concentration 1 mM stains nuclei in 20 s with high specificity and strongly enhances nuclear-to-dermis contrast of BCCs. Comparison of fluorescence mosaics to histology shows that both large and small tumors are detectable. The results demonstrate the feasibility of confocal mosaicing microscopy toward rapid surgical pathology to potentially expedite and guide surgery.
confocal microscopy; mosaicing; surgical pathology; basal cell carcinomas (BCCs); Mohs surgery; acridine orange
Bright field imaging of biological samples stained with antibodies and/or special stains provides a rapid protocol for visualizing various macromolecules. However, this method of sample staining and imaging is rarely employed for direct quantitative analysis due to variations in sample fixations, ambiguities introduced by color composition and the limited dynamic range of imaging instruments. We demonstrate that, through the decomposition of color signals, staining can be scored on a cell-by-cell basis. We have applied our method to fibroblasts grown from histologically normal breast tissue biopsies obtained from two distinct populations. Initially, nuclear regions are segmented through conversion of color images into gray scale, and detection of dark elliptic features. Subsequently, the strength of staining is quantified by a color decomposition model that is optimized by a graph cut algorithm. In rare cases where nuclear signal is significantly altered as a result of sample preparation, nuclear segmentation can be validated and corrected. Finally, segmented stained patterns are associated with each nuclear region following region-based tessellation. Compared to classical non-negative matrix factorization, proposed method: (i) improves color decomposition, (ii) has a better noise immunity, (iii) is more invariant to initial conditions and (iv) has a superior computing performance.