Method for localization of sialic acid on cell surface and cell interior in peripheral blood: a pilot study

This article presents a method for identification and localization of cell surface and intracellular sialoglycoconjugates of peripheral blood cells. To reveal cell surface conjugates, a sample of peripheral blood was incubated with lectin after centrifugation and rinsing. For intracellular localization in leukocytes, RBCs were lysed and the membranes were permeabilized prior to cytochemical reaction. Fluorescein isothiocyanate conjugated lectins were used for visualization in fluorescence microscope. All lectins bound specifically to the surface of erythrocytes. Confocal microscopy showed surface and intracellular labeling of permeabilized leukocytes. A part of the signal in eosinophils originated from binding of anionic fluorophore to cationic granular proteins.     

Application of the Mesolens for subcellular resolution imaging of intact larval and whole adult Drosophila

In a previous paper, we showed a new giant lens called the Mesolens and presented performance data and images from whole fixed and intact fluorescently‐stained 12.5‐day old mouse embryos. Here, we show that using the Mesolens we can image an entire Drosophila larva or adult fly in confocal epifluorescence and show subcellular detail in all tissues. By taking several hundreds of optical sections through the entire volume of the specimen, we show cells and nuclear details within the gut, brain, salivary glands and reproductive system that normally require dissection for study. Organs are imaged in situ in correct 3D arrangement. Imaginal discs are imaged in mature larvae and it proved possible to image pachytene chromosomes in cells within ovarian follicles in intact female flies. Methods for fixing, staining and clearing are given.     

Automatic measurement of compression wood cell attributes in fluorescence microscopy images

This paper presents a new automated method for analyzing compression wood fibers in fluorescence microscopy. Abnormal wood known as compression wood is present in almost every softwood tree harvested. Compression wood fibers show a different cell wall morphology and chemistry compared to normal wood fibers, and their mechanical and physical characteristics are considered detrimental for both construction wood and pulp and paper purposes. Currently there is the need for improved methodologies for characterization of lignin distribution in wood cell walls, such as from compression wood fibers, that will allow for a better understanding of fiber mechanical properties. Traditionally, analysis of fluorescence microscopy images of fiber cross-sections has been done manually, which is time consuming and subjective. Here, we present an automatic method, using digital image analysis, that detects and delineates softwood fibers in fluorescence microscopy images, dividing them into cell lumen, normal and highly lignified areas. It also quantifies the different areas, as well as measures cell wall thickness. The method is evaluated by comparing the automatic with a manual delineation. While the boundaries between the various fiber wall regions are detected using the automatic method with precision similar to inter and intra expert variability, the position of the boundary between lumen and the cell wall has a systematic shift that can be corrected. Our method allows for transverse structural characterization of compression wood fibers, which may allow for improved understanding of the micro-mechanical modeling of wood and pulp fibers.     

In vivo spatio-temporal visualization of the human skin by real-time confocal microscopy

A modified tandem scanning confocal microscope is used to obtain in vivo images of the human skin in real time. Three-dimensional and temporal visualizations are demonstrated with volume reconstruction and blood flow images. Two image processing methods based on Fourier transform and logarithmic processing are presented. Their applications in noise removal of the scanning disk lines and of the heterogeneity of light are illustrated.     

A guided tour into subcellular colocalization analysis in light microscopy

It is generally accepted that the functional compartmentalization of eukaryotic cells is reflected by the differential occurrence of proteins in their compartments. The location and physiological function of a protein are closely related; local information of a protein is thus crucial to understanding its role in biological processes. The visualization of proteins residing on intracellular structures by fluorescence microscopy has become a routine approach in cell biology and is increasingly used to assess their colocalization with well‐characterized markers. However, image‐analysis methods for colocalization studies are a field of contention and enigma. We have therefore undertaken to review the most currently used colocalization analysis methods, introducing the basic optical concepts important for image acquisition and subsequent analysis. We provide a summary of practical tips for image acquisition and treatment that should precede proper colocalization analysis. Furthermore, we discuss the application and feasibility of colocalization tools for various biological colocalization situations and discuss their respective strengths and weaknesses. We have created a novel toolbox for subcellular colocalization analysis under ImageJ, named JACoP, that integrates current global statistic methods and a novel object‐based approach.     

Automatic real-time three-dimensional cell tracking by fluorescence microscopy

Live cell imaging has become an indispensable technique for cell biologists. However, when grown on coverslip glass used for live cell imaging many cultured cells move even during relatively short observation times and focus can drift as a result of mechanical instabilities and/or temperature fluctuations. Time‐lapse imaging therefore requires constant adjustment of the imaging field and focus position to keep the cell of interest centred in the imaged volume. We show here that this limitation can be overcome by tracking cells in a fully automated way using the mass centre of cellular fluorescence. Combined with automated multiple location revisiting, this method dramatically increases the throughput of high‐resolution live cell imaging experiments.     

Automatic particle detection in microscopy using temporal correlations

One of the fundamental problems in the analysis of single particle tracking data is the detection of individual particle positions from microscopy images. Distinguishing true particles from noise with a minimum of false positives and false negatives is an important step that will have substantial impact on all further analysis of the data. A common approach is to obtain a plausible set of particles from a larger set of candidate particles by filtering using manually selected threshold values for intensity, size, shape, and other parameters describing a particle. This introduces subjectivity into the analysis and hinders reproducibility. In this paper, we introduce a method for automatic selection of these threshold values based on maximizing temporal correlations in particle count time series. We use Markov Chain Monte Carlo to find the threshold values corresponding to the maximum correlation, and we study several experimental data sets to assess the performance of the method in practice by comparing manually selected threshold values from several independent experts with automatically selected threshold values. We conclude that the method produces useful results, reducing subjectivity and the need for manual intervention, a great benefit being its easy integratability into many already existing particle detection algorithms. Microsc. Res. Tech., 76:997–1006, 2013. © 2013 Wiley Periodicals, Inc.     

Reduction of cross-talk between fluorescent labels in scanning laser microscopy

By using dual detectors in combination with a dichroic filter, it is possible to record simultaneously the distribution of two fluorescent labels in a specimen. It is often difficult, however, to obtain a good separation, i.e. each detector will generally detect light from more than one fluorophore. In such cases it is desirable to find image-processing methods to improve the separation. A simple method is to form a linear combination of the recorded images. In this paper we investigate the necessary prerequisites for this method to be successful, and we also investigate to what extent these are fulfilled in some practical cases. In this context the spectral properties of the fluorophores turn out to be of crucial importance. Even when the necessary prerequisites are not strictly fulfilled, a considerable improvement in image quality can, nevertheless, be obtained.     

Review of free software tools for image analysis of fluorescence cell micrographs

An increasing number of free software tools have been made available for the evaluation of fluorescence cell micrographs. The main users are biologists and related life scientists with no or little knowledge of image processing. In this review, we give an overview of available tools and guidelines about which tools the users should use to segment fluorescence micrographs. We selected 15 free tools and divided them into stand‐alone, Matlab‐based, ImageJ‐based, free demo versions of commercial tools and data sharing tools. The review consists of two parts: First, we developed a criteria catalogue and rated the tools regarding structural requirements, functionality (flexibility, segmentation and image processing filters) and usability (documentation, data management, usability and visualization). Second, we performed an image processing case study with four representative fluorescence micrograph segmentation tasks with figure‐ground and cell separation. The tools display a wide range of functionality and usability. In the image processing case study, we were able to perform figure‐ground separation in all micrographs using mainly thresholding. Cell separation was not possible with most of the tools, because cell separation methods are provided only by a subset of the tools and are difficult to parametrize and to use. Most important is that the usability matches the functionality of a tool. To be usable, specialized tools with less functionality need to fulfill less usability criteria, whereas multipurpose tools need a well‐structured menu and intuitive graphical user interface.     

Morphometrical study of plant vacuolar dynamics in single cells using three-dimensional reconstruction from optical sections

In higher plants, vacuoles increase their volumes in accordance with cell enlargement and occupy most of the cell volume. However, quantitative analyses of vacuolar contributions during changes in cell morphology have been hampered by the inadequacies and frequent artifacts associated with current three-dimensional (3-D) reconstruction methods of images derived from light microscopy. To overcome the limitations of quantifying 3-D structures, we have introduced 3-D morphometrics into light microscopy, adopting a contour-based approach for which we have developed an interpolation method. Using this software, named REANT, the morphological and morphometrical changes in protoplasts and vacuoles during plasmolysis could be investigated. We employed the tobacco (Nicotiana tabacum) BY-2 cell line No.7, expressing a GFP-AtVam3p fusion protein, BY-GV7, using GFP as a marker of vacuolar membranes (VMs). By vital staining of the plasma membrane (PM) of cells, we simultaneously obtained optical sections of both the PM and VM. We, therefore, reconstructed the 3-D structures of protoplasts and vacuoles before and after plasmolysis. We were able to identify the appearance of elliptical structures of VMs in the vacuolar lumen, and to determine that they were derived from cytoplasmic strands. From the 3-D structures, the volumes and surface areas were measured at the single cell level. The shrinkage of vacuoles accounted for most of the decrease in protoplast volume, while the surface area of the vacuoles remained mostly unchanged. These morphometrical analyses suggest that the elliptical structures are reservoirs for excess VMs that result from the response to rapid decreases in vacuolar and protoplast volumes.     

An automatic system for in vitro cell migration studies

This paper describes a system for in vitro cell migration analysis. Adult neural stem/progenitor cells are studied using time-lapse bright-field microscopy and thereafter stained immunohistochemically to find and distinguish undifferentiated glial progenitor cells and cells having differentiated into type-1 or type-2 astrocytes. The cells are automatically segmented and tracked through the time-lapse sequence. An extension to the Chan-Vese Level Set segmentation algorithm, including two new terms for specialized growing and pruning, made it possible to resolve clustered cells, and reduced the tracking error by 65%. We used a custom-built manual correction module to form a ground truth used as a reference for tracked cells that could be identified from the fluorescence staining. On average, the tracks were correct 95% of the time, using our new segmentation. The tracking, or association of segmented cells, was performed using a 2-state Hidden Markov Model describing the random behaviour of the cells. By re-estimating the motion model to conform with the segmented data we managed to reduce the number of tracking parameters to essentially only one. Upon characterization of the cell migration by the HMM state occupation function, it was found that glial progenitor cells were moving randomly 2/3 of the time, while the type-2 astrocytes showed a directed movement 2/3 of the time. This finding indicates possibilities for cell-type specific identification and cell sorting of live cells based on specific movement patterns in individual cell populations, which would have valuable applications in neurobiological research.     

In vivo determination of fibril orientation in plant cell walls with polarization CSLM

Congo Red fluorescence is used to detect cellulose in the wall of plant cells. The orientation of the cellulose fibrils is determined by using polarized light for excitation. The absorption characteristics of Congo Red make this approach a method of choice for applications with any standard confocal scanning laser microscope (CSLM). The semiquantitative character of CSLM observations combined with the non-toxicity of the stain allow a very fast and reliable assessment of cellulose orientation in the wall of living plant cells.     

Theoretical versus experimental resolution in optical microscopy

The aim of this article is to compare experimental resolution under different conditions with theoretical resolution predicted using electromagnetic diffraction theory. Imaging properties of fluorescent beads of three different diameters (0.1 microm, 0.2 microm, and 0.5 microm) as well as imaging properties of four different fluorescence-stained DNA targets (ABL gene, BCR gene, centromere 6, and centromere 17) are studied. It is shown how the dependence of the resolution on object size varies with wavelength (520 nm versus 580 nm), type of microscopy (wide-field, confocal using Nipkow disk, confocal laser scanning) and basic image processing steps (median and gaussian filters). Furthermore, specimen influence on the resolution was studied (the influence of embedding medium, coverglass thickness, and depth below the coverglass). Both lateral and axial resolutions are presented. The results clearly show that real objects are far from being points and that experimental resolution is often much worse than the theoretical one. Although the article concentrates on fluorescence imaging using high NA objectives, similar dependence can also be expected for other optical arrangements.     

Automated noninvasive epithelial cell counting in phase contrast microscopy images with automated parameter selection

Cell counting is commonly used to determine proliferation rates in cell cultures and for adherent cells it is often a ‘destructive’ process requiring disruption of the cell monolayer resulting in the inability to follow cell growth longitudinally. This process is time consuming and utilises significant resource. In this study a relatively inexpensive, rapid and widely applicable phase contrast microscopy‐based technique has been developed that emulates the contrast changes taking place when bright field microscope images of epithelial cell cultures are defocused. Processing of the resulting images produces an image that can be segmented using a global threshold; the number of cells is then deduced from the number of segmented regions and these cell counts can be used to generate growth curves. The parameters of this method were tuned using the discrete mereotopological relations between ground truth and processed images. Cell count accuracy was improved using linear discriminant analysis to identify spurious noise regions for removal. The proposed cell counting technique was validated by comparing the results with a manual count of cells in images, and subsequently applied to generate growth curves for oral keratinocyte cultures supplemented with a range of concentrations of foetal calf serum. The approach developed has broad applicability and utility for researchers with standard laboratory imaging equipment.     

Three-dimensional computer reconstruction of large tissue volumes based on composing series of high-resolution confocal images by GlueMRC and LinkMRC software

Computer-based visualization of large tissue volumes with high resolution based on composing series of high-resolution confocal images is presented. GlueMRC and LinkMRC programs are introduced, implementing composition of overlapping series of optical sections captured by a confocal microscope, registration and subsequent composition of successive confocal stacks. Both programs are using an interactive approach in combination with automatic algorithms for image registration. Further, the method for obtaining surface renderings of microscopical structure under study is described. For this purpose, structure contours visible in the sections are interactively digitized using a Colon plug-in module running in Ellipse environment. Then the coordinates of the contours are processed by special modules in the graphic programming environment IRIS Explorer and the structure surface is rendered. The method is shown on the 3-D reconstruction of the capillary bed of human placental villi and chick embryonic gut and its vascular bed.     

Comparison of three-dimensional imaging properties between two-photon and single-photon fluorescence microscopy

The imaging performance in single-photon (1-p) and two-photon (2-p) fluorescence microscopy is described. Both confocal and conventional systems are compared in terms of the three-dimensional (3-D) point spread function and the 3-D optical transfer function. Images of fluorescent sharp edges and layers are modelled, giving resolution in transverse and axial directions. A comparison of the imaging properties is also given for a 4Pi confocal system. Confocal 2-p 4Pi fluorescence microscopy gives the best axial resolution in the sense that its 3-D optical transfer function has the strongest response along the axial direction.     

Automated assessment of keratocyte density in clinical confocal microscopy of the corneal stroma

Cell density in the corneal stroma is typically determined by counting the number of bright objects, presumably keratocyte nuclei, in images from clinical confocal microscopy. We present a program that identifies bright objects and counts those that most likely represent cells. Selection variables were determined from 125 normal corneas with cell densities that had been assessed manually. The program was tested on 17 corneas of patients before and at several intervals to 5 years after laser in situ keratomileusis (LASIK) surgery. In these corneas, which showed a decrease in cell density after surgery, the program identified cells as well as human observers did.     

Cosmetic assessment of the human hair by confocal microscopy

The optical sectioning property of the confocal microscope offers a breakthrough from the classic observation of the hair in a scanning electron microscope (SEM). Confocal microscopy requires minimal sampling preparation, and the hair can be observed in its natural environment with less damage than by other microscopic methods such as SEM. While used in the reflection mode, the true morphology of the cuticle and the various exogenous deposits at the surface can be identified and quantified. This relatively noninvasive, nondestructive technique is routinely used by us to monitor the efficiency of cleansing shampoos, to assess the homogeneity of layering polymers, and to evaluate the changes they induce in the optical properties of the hair surface in terms of opacity, transparency, and brilliancy. A second important field of investigation uses the fluorescence channel which reveals the internal structure of the hair. Fluorescent probes (rhodamine and its derivatives) demonstrate the routes of penetration and outline the geometry of cortical cells and of the medulla according to their lipophilic or hydrophilic properties. A volume rendering of a hair cylinder provides a better understanding of the interrelationships between cuticle cells, cortical cells, and the medullar channel. This recent technology is becoming an invaluable tool for the cosmetic assessment of the hair.     

Novel methods for the quantification of changes in actin organization in chondrocytes using fluorescent imaging and linear profiling

We present three novel reproducible methodologies for the quantification of changes in actin organization from microscope images. Striation and integrative analysis were devised for the investigation of trans-cellular filaments and F-actin localization, respectively, in response to physiological or mechanical actin-modulatory conditions. Additionally, the Parker-Qusous (PQ) formula was developed as a measure of total quantity of F-actin, independent of cell volume changes, whereby fluorescence intensity was divided by the cube root of cell volume, squared. Values obtained were quantified in Mauricean Units (Mu; pixel/μm(3)). Upon isolation, there was a 49% decrease in total F-actin fluorescence from 1.91 ± 0.16 pixel/μm(3) (Mu) to 0.95 ± 0.55 Mu, whereas upon culture, an apparent increase in total fluorescence was deemed insignificant due to an increase in average cell volume, with a rise, however, in striation units (StU) from 1 ± 1 to 5 ± 1 StU/cell, and a decrease in percentage cortical fluorescence to 30.45% ± 1.52% (P = 7.8 × 10(-5)). Freshly isolated chondrocytes exhibited a decrease in total F-actin fluorescence to 0.61 ± 0.05 Mu and 0.32 ± 0.02 Mu, 10 min posthypertonic and hypotonic challenges, respectively. Regulatory volume decrease was inhibited in the presence of REV5901 with maintenance of actin levels at 1.15 Mu. Following mechanical impact in situ, there was a reduction in total F-actin fluorescence to 0.95 ± 0.08 Mu and 0.74 ± 0.06 Mu under isotonic and hypotonic conditions, respectively, but not under hypertonic conditions. We report simple methodologies for quantification of changes in actin organization, which will further our understanding of the role of actin in various cellular stress responses. These techniques can be applied to better quantify changes in localization of various proteins using fluorescent labeling.