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.     

Image analysis of particle dispersions in microscopy images of cryo-sectioned sausages

Two feature extraction methods, the three-dimensional (3-D) local box-counting method and the area distribution method, are presented to describe the fat dispersion pattern on digital microscopy images of cryo-sectioned sausages. Both methods calculate whole arrays of variables for each microscopy image. The 3-D box-counting method calculates scale dependent (local) dimensions. This is in contrast to common fractal methods, which are univariate. Principal component analysis (PCA) was used to show that different sausages yield different fat dispersion patterns. Partial least square regression (PLS) shows that there is a correlation between the variables gained with both methods and the fat content.     

Inherent bias in correlation averaged images

The correlation averaging algorithm frequently used to enhance micrographs of repeating structures contains an inherent bias that favours images with larger pixel values or positive noise levels. This bias not only skews the composite image toward higher pixel values, but also distorts the image by increasing the value of high-valued pixels more than that of low-valued pixels. These errors are especially important in scanning probe microscopy images where the pixel value reflects a distinct height. A similar algorithm that uses a structure function in place of the correlation function eliminates this bias.     

Image analysis for denoising full-field frequency-domain fluorescence lifetime images

Video-rate fluorescence lifetime-resolved imaging microscopy (FLIM) is a quantitative imaging technique for measuring dynamic processes in biological specimens. FLIM offers valuable information in addition to simple fluorescence intensity imaging; for instance, the fluorescence lifetime is sensitive to the microenvironment of the fluorophore allowing reliable differentiation between concentration differences and dynamic quenching. Homodyne FLIM is a full-field frequency-domain technique for imaging fluorescence lifetimes at every pixel of a fluorescence image simultaneously. If a single modulation frequency is used, video-rate image acquisition is possible. Homodyne FLIM uses a gain-modulated image intensified charge-coupled device (ICCD) detector, which unfortunately is a major contribution to the noise of the measurement. Here we introduce image analysis for denoising homodyne FLIM data. The denoising routine is fast, improves the extraction of the fluorescence lifetime value(s) and increases the sensitivity and fluorescence lifetime resolving power of the FLIM instrument. The spatial resolution (especially the high spatial frequencies not related to noise) of the FLIM image is preserved, because the denoising routine does not blur or smooth the image. By eliminating the random noise known to be specific to photon noise and from the intensifier amplification, the fidelity of the spatial resolution is improved. The polar plot projection, a rapid FLIM analysis method, is used to demonstrate the effectiveness of the denoising routine with exemplary data from both physical and complex biological samples. We also suggest broader impacts of the image analysis for other fluorescence microscopy techniques (e.g. super-resolution imaging).     

Streak time-lapse video microscopy: analysis of protoplasmic motility and cell division in Tradescantia stamen hair cells

We have developed a video time-lapse analogue of a streak camera using a digital image processing technique, and have used it to analyse dynamic processes in plant cells. The same image area is selected from a succession of frames in a video tape recording. The successive images are stored in an image memory, rearranged, and displayed in a single frame according to the time course. Each image area is rectangular, with selectable length and width: the closest analogy to a streak camera is obtained when one of its sides is a single pixel. The composite image displays sequential changes in the distribution of subcellular components in the selected area. The method was shown to be useful for monitoring details of cytoplasmic streaming and organelle movement, observing the time course of chemical fixation, and also for studying kinetochore movement, shortening of chromatids and cell plate formation during mitosis and cytokinesis. Cessation and resumption of cytoplasmic movement during the cell division cycle were also clearly localized outside the nuclear region in apical cells of Tradescantia stamen hairs.     

Real-time quantitative elemental analysis and mapping: microchemical imaging in cell physiology

Recent advances in widely available microcomputers have made the acquisition and processing of digital quantitative X-ray maps of one to several cells readily feasible. Here we describe a system which uses a graphics-based microcomputer to acquire spectrally filtered X-ray elemental image maps that are fitted to standards, to display the image in real time, and to correct the post-acquisition image map with regard to specimen drift. Both high-resolution quantitative energy-dispersive X-ray images of freeze-dried cyrosections and low-dose quantitative bright-field images of frozen-hydrated sections can be acquired to obtain element and water content from the same intracellular regions. The software programs developed, together with the associated hardware, also allow static probe acquisition of data from selected cell regions with spectral processing and quantification performed on-line in real time. In addition, the unified design of the software program provides for off-line processing and analysing by several investigators at microcomputers remote from the microscope. The overall experimental strategy employs computer-aided imaging, combined with static probes, as an essential interactive tool of investigation for biological analysis. This type of microchemical microscopy facilitates studies in cell physiology and pathophysiology which focus on mechanisms of ionic (elemental) compartmentation, i.e. structure-function correlation at cellular and subcellular levels; it allows investigation of intracellular concentration gradients, of the heterogeneity of cell responses to stimuli, of certain fast physiological events in vivo at ultrastructural resolution, and of events occurring with low incidence or involving cell-to-cell interactions.     

Three-dimensional analysis of cell nucleus structures visualized by confocal scanning laser microscopy

Studies of the three-dimensional (3-D) organization of cell nuclei are becoming increasingly important for the understanding of basic cellular events such as growth and differentiation. Modern methods of molecular biology, including in situ hybridization and immunofluorescence, allow the visualization of specific nuclear structures and the study of spatial arrangements of chromosome domains in interphase nuclei. Specific methods for labelling nuclear structures are used to develop computerized techniques for the automated analysis of the 3-D organization of cell nuclei. For this purpose, a coordinate system suitable for the analysis of tri-axial ellipsoidal nuclei is determined. High-resolution 3-D images are obtained using confocal scanning laser microscopy. The results demonstrate that with these methods it is possible to recognize the distribution of visualized structures and to obtain useful information regarding the 3-D organization of the nuclear structure of different cell systems.     

Probing confined fields with single molecules and vice versa

Single dye molecules are used as local probes to map the spatial distribution of the squared electric field components in the focus of a high numerical aperture lens. Simulated field distributions are quantitatively verified by experimentally obtained fluorescence excitation maps. We show that annular illumination can be used to engineer the field distribution in the focus at a dielectric/air interface such that electric field components in all directions acquire comparable magnitudes. The 3D orientation of molecular absorption dipoles can be determined by comparing measured to simulated image patterns. The presence of longitudinal electric field components in a focus is of particular interest in tip-enhanced scanning near-field optical microscopy.     

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.     

Fluorescence photobleaching-based image standardization for fluorescence microscopy

A method is presented for the standardization of images acquired with fluorescence microscopy, based on the knowledge of spatial distributions proportional to the microscope's absolute excitation intensity and fluorescence detection efficiency distributions over the image field. These distributions are determined using a thin fluorescent test layer, employed under practically mono-exponential photobleaching conditions. It is demonstrated that these distributions can be used for (i) the quantitative evaluation of differences between both the excitation intensity and the fluorescence detection efficiency of different fluorescence microscopes and (ii) the standardization of images acquired with different microscopes, permitting the deduction of quantitative relationships between images obtained under different imaging conditions.     

Quantification of colocalization and cross-talk based on spectral angles

Common methods for quantification of colocalization in fluorescence microscopy typically require cross-talk free images or images where cross-talk has been eliminated by image processing, as they are based on intensity thresholding. Quantification of colocalization includes not only calculating a global measure of the degree of colocalization within an image, but also a classification of each image pixel as showing colocalized signals or not. In this paper, we present a novel, automated method for quantification of colocalization and classification of image pixels. The method, referred to as SpecDec, is based on an algorithm for spectral decomposition of multispectral data borrowed from the field of remote sensing. Pixels are classified based on hue rather than intensity. The hue distribution is presented as a histogram created by a series of steps that compensate for the quantization noise always present in digital image data, and classification rules are thereafter based on the shape of the angle histogram. Detection of colocalized signals is thus only dependent on the hue, making it possible to classify also low-intensity objects, and decoupling image segmentation from detection of colocalization. Cross-talk will show up as shifts of the peaks of the histogram, and thus a shift of the classification rules, making the method essentially insensitive to cross-talk. The method can also be used to quantify and compensate for cross-talk, independent of the microscope hardware.     

An overview of state‐of‐the‐art image restoration in electron microscopy

In Life Science research, electron microscopy (EM) is an essential tool for morphological analysis at the subcellular level as it allows for visualization at nanometer resolution. However, electron micrographs contain image degradations such as noise and blur caused by electromagnetic interference, electron counting errors, magnetic lens imperfections, electron diffraction, etc. These imperfections in raw image quality are inevitable and hamper subsequent image analysis and visualization. In an effort to mitigate these artefacts, many electron microscopy image restoration algorithms have been proposed in the last years. Most of these methods rely on generic assumptions on the image or degradations and are therefore outperformed by advanced methods that are based on more accurate models. Ideally, a method will accurately model the specific degradations that fit the physical acquisition settings. In this overview paper, we discuss different electron microscopy image degradation solutions and demonstrate that dedicated artefact regularisation results in higher quality restoration and is applicable through recently developed probabilistic methods.     

A novel approach for correlative light electron microscopy analysis

Correlative light and electron microscopy (CLEM) is a multimodal technique of increasing utilization in functional, biochemical, and molecular biology. CLEM attempts to combine multidimensional information from the complementary fluorescence light microscopy (FLM) and electron microscopy (EM) techniques to bridge the various resolution gaps. Within this approach the very same cell/structure/event observed at level can be analyzed as well by FLM and EM. Unfortunately, these studies turned out to be extremely time consuming and are not suitable for statistical relevant data. Here, we describe a new CLEM method based on a robust specimen preparation protocol, optimized for cryosections (Tokuyasu method) and on an innovative image processing toolbox for a novel type of multimodal analysis. Main advantages obtained using the proposed CLEM method are: (1) hundred times more cells/structures/events that can be correlated in each single microscopy session; (2) three‐dimensional correlation between FLM and EM, obtained by means of ribbons of serial cryosections and electron tomography microscopy (ETM); (3) high rate of success for each CLEM experiment, obtained implementing protection of samples from physical damage and from loss of fluorescence; (4) compatibility with the classical immunogold and immunofluorescence labeling techniques. This method has been successfully validated for the correlative analysis of Russel Bodies subcellular compartments. Microsc. Res. Tech., 2010. © 2009 Wiley‐Liss, Inc.     

Algorithms for automated characterization of cell populations in thick specimens from 3-D confocal fluorescence microscopy data

Methods are presented for the automated, quantitative and three-dimensional (3-D) analysis of cell populations in thick, essentially intact tissue sections while maintaining intercell spatial relationships. This analysis replaces current manual methods which are tedious and subjective. The thick sample is imaged in three dimensions using a confocal scanning laser microscope. The stack of optical slices is processed by a 3-D segmentation algorithm that separates touching and overlapping structures using localization constraints. Adaptive data reduction is used to achieve computational efficiency. A hierarchical cluster analysis algorithm is used automatically to characterize the cell population by a variety of cell features. It allows automatic detection and characterization of patterns such as the 3-D spatial clustering of cells, and the relative distributions of cells of various sizes. It also permits the detection of structures that are much smaller, larger, brighter, darker, or differently shaped than the rest of the population. The overall method is demonstrated for a set of rat brain tissue sections that were labelled for tyrosine hydroxylase using fluorescein-conjugated antibodies. The automated system was verified by comparison with computer-assisted manual counts from the same image fields.     

Image processing techniques for 3-D chromosome analysis

3-D karyotype analysis is developing rapidly due to the availability of confocal microscopes and CCD video cameras, and the development of 3-D processing techniques. Here, image enhancement and visualization techniques specifically designed for 3-D karyotype analysis are described. To facilitate a good comparison between the different techniques, the same 3-D image, obtained with a confocal scanning laser microscope (CSLM), of a mitotic prophase nucleus of a root-tip cell of Crepis capillaris was used throughout. Besides well-known stereoscopic presentation, another means of improving depth perception is shown, i.e. a solid modelling algorithm, which simulates the process of fluorescence. An interactive routine to dissect objects in the image is presented as an alternative for automated segmentation algorithms, which cannot be applied to closely apposed or merging objects. As an example of a convenient way to reduce the vast amount of data (2 Mbyte per image), a partly automated 3-D cursor is presented in detail. This cursor is used to trace the central axes of chromosomes and record them as strings of Cartesian coordinates. The advantages of a computer graphics display, which facilitates real-time rotation and hence is a powerful tool in studying 3-D features of chromosomes, are also shown.     

Three-dimensional imaging of corneal cells using in vivo confocal microscopy

Confocal microscopy is a unique and powerful imaging paradigm which allows optical sectioning through intact tissue. Real-time tandem scanning confocal microscopy has previously been used to generate high-magnification two-dimensional (2-D) images of cells in living organ systems. Inherent problems with movement, however, have prevented the in vivo acquisition of complete 3-D datasets. The development of a new objective lens, used in combination with specialized real-time image acquisition procedures, has allowed sequential serial sections to be obtained in vivo from the rabbit cornea for the first time. These sections can be digitially registered and stacked on the computer to provide a 3-D reconstruction of the corneal cells. This technique should serve as a useful method for studying 3-D structures and analysing 4-D phenomena at the cellular level in living animals. Three-dimensional images of a stromal nerve in normal rabbit cornea and of fibroblasts within a rabbit corneal wound are presented as examples of current capabilities.     

激光扫描共聚焦显微镜技术的发展及应用

激光扫描共聚焦显微术是先进的分子和细胞生物学研究技术。它在荧光显微镜成像的基础上加装激光扫描装置,结合数据化图像处理技术,采集组织和细胞内荧光标记图像。在亚细胞水平观察钙等离子水平的变化,并结合电生理等技术观察细胞生理活动与细胞形态及运动变化的相互关系。由于它的应用范围较广泛,已成为形态学、分子细胞生物学、神经科学和药理学等研究领域中很重要的研究技术。     

Image division technique in pre-acquisition analysis of information content for automated microscopy

This article presents a method that allows for reliable automated image acquisition of specimens with high information content in light microscopy with emphasis on fluorescence microscopy applications. Automated microscopy typically relies on autofocusing used for the analysis of information content behaviour along the z-axis within each field of view. However, in the case of a field of view containing more objects that do not lie precisely in one z-plane, traditional autofocusing methods fail due to their principle of operation. We avoid this issue by reducing the original problem to a set of simple and performable tasks: we divide the field of view into a small number of tiles and process each of them individually. The obtained results enable discovering z-planes with rich information content that remain hidden during global analysis of the whole field of view. Our approach therefore outperforms other acquisition methods including the manual one. A large part of the contribution is oriented towards practical application.     

Virtual electron microscopy in cell biology

Virtual microscopy of histological glass slides can emulate conventional light microscopy. Up till now, such a digital simulation does not exist for ultrathin electron microscopic slides. Because of the relative inaccessibility of electron microscopy, evaluation of subcellular structures by (bio)medical students is performed with the aid of photographic prints. In this article, the generation and evaluation of virtual electron microscopic slides is discussed. A T‐lymphoblastic cell was used as an example. Electron microscopic pictures were taken at two magnifications (25,000 and 50,000), processed in an analogue or digital way and stitched to reconstruct the image of the total cell. This image is viewed with a webviewer equipped with pan and zoom functions. The possibility of distinguishing the trilaminar structure of cellular membranes was the requisite. Virtual images obtained at an original magnification of 25,000, scanned at a resolution of 800 ppi could compete with pictures developed directly from negatives obtained by electron microscopy. It is possible to navigate and zoom into details in a way emulating electron microscopy. Virtual electron microscopy is innovative and offers new perspectives to interpret cytological pictures and to teach cell biology in an interactive and unique way. Microsc. Res. Tech., 2010. © 2010 Wiley‐Liss, Inc.