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.     

Comparison of the axial resolution of practical Nipkow-disk confocal fluorescence microscopy with that of multifocal multiphoton microscopy: theory and experiment

We compare the axial sectioning capability of multifocal confocal and multifocal multiphoton microscopy in theory and in experiment, with particular emphasis on the background arising from the cross‐talk between adjacent imaging channels. We demonstrate that a time‐multiplexed non‐linear excitation microscope exhibits significantly less background and therefore a superior axial resolution as compared to a multifocal single‐photon confocal system. The background becomes irrelevant for thin (< 15 µm) and sparse fluorescent samples, in which case the confocal parallelized system exhibits similar or slightly better sectioning behaviour due to its shorter excitation wavelength. Theoretical and experimental axial responses of practically implemented microscopes are given.     

Axial resolution of confocal fluorescence microscopy

A simple analytic expression is given for the axial resolution of a confocal fluorescence microscope. The expression, which is based on the spatial frequency cut-off criterion of resolution, is valid for high aperture optics and arbitrary fluorescence wavelength.     

An efficient algorithm for measurement and correction of chromatic aberrations in fluorescence microscopy

Even the best optical microscopes available on the market exhibit chromatic aberrations to some extent. In some types of study, chromatic aberrations of current optics cannot be neglected and a software correction is highly desirable. This paper describes a novel method of chromatic aberration measurement and software correction using sub-resolution bead imaging and computer image analysis. The method is quick, precise and enables the determination of both longitudinal and lateral chromatic aberrations. Correction function can be computed in about half an hour, including image acquisition. Using this approach, chromatic aberrations can be reduced to 10–20 nm laterally and 10–60 nm axially depending on the type of optical set-up. The method is especially suitable for fluorescence microscopy, where a limited number of wavelengths are observed.     

A method for personal expertise-independent evaluation of image resolution in scanning electron microscopy

The two conventional methods currently employed for the evaluation of image resolution in scanning electron microscopy are the gap method and a fast Fourier transform (FFT) method. These can be highly dependent on personal expertise on the distinction between signal information and noise contained in a micrograph. Hence, the present paper proposes an alternative method (referred to as a contrast-to-gradient (CG) method) that can determine the image resolution of a micrograph without requiring personal expertise on the judgment of noise. The image resolution in the CG method is defined as a weighted harmonic mean of the local resolution, which is proportional to the quotient of the threshold contrast divided by the local gradient. The local gradient is calculated from the quadratic function that best fits the local pixel intensities over 5 x 5 pixels. It has been shown that the CG method, compared with the FFT method, has a broader range of applications for various types of images, such as low-contrast, noise-containing, filter-processed, highly directional, and quasi-periodic feature images.     

Surpassing the lateral resolution limit by a factor of two using structured illumination microscopy

Lateral resolution that exceeds the classical diffraction limit by a factor of two is achieved by using spatially structured illumination in a wide-field fluorescence microscope. The sample is illuminated with a series of excitation light patterns, which cause normally inaccessible high-resolution information to be encoded into the observed image. The recorded images are linearly processed to extract the new information and produce a reconstruction with twice the normal resolution. Unlike confocal microscopy, the resolution improvement is achieved with no need to discard any of the emission light. The method produces images of strikingly increased clarity compared to both conventional and confocal microscopes.     

Contrast,resolution, pixelation,dynamic range and signal-to-noise ratio: fundamental limits to resolution in fluorescence light microscopy

In a perfect optical system numerical aperture and wavelength determine resolution. In a real optical system, however, the number of photons collected from a specimen determines the contrast and this limits the resolution. Contrast is affected by the number of picture elements per unit area, the number of photons and the aberrations present in every optical system. The concept of contrast vs. distance functions is used to compare the resolution achievable in confocal and wide-field fluorescence microscopes and the effect of a further reduction of the observable volume. In conclusio: (a) real optical systems will never be able to achieve the theoretical resolution, (b) wide-field fluorescence microscopy will often provide a better resolution than confocal fluorescence microscopy, (c) decreasing the observed volume does not necessarily increase the resolution and (d) using multiple fluorophores can improve the accuracy with which distances are measured. Some numbers for typical situations are provided.     

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.     

Axial super resolution topography of focal adhesion by confocal microscopy

The protein organization within focal adhesions has been studied by state‐of‐the‐art super resolution methods because of its thin structure, well below diffraction limit. However, to achieve high axial resolution, most of the current approaches rely on either sophisticated optics or diligent sample preparation, limiting their application. In this report we present a phasor‐based method that can be applied to fluorescent samples to determine the precise axial position of proteins using a conventional confocal microscope. We demonstrate that with about 4,000 photon counts collected along a z‐scan, axial localization precision close to 10 nm is achievable. We show that, with within 10 nm, the axial location of paxillin, FAK, and talin is similar at focal adhesion sites, while F‐actin shows a sharp increase in height towards the cell center. We further demonstrated the live imaging capability of this method. With the advantage of simple data acquisition and no special instrument requirement, this approach could have wide dissemination and application potentials. Microsc. Res. Tech., 76:1070–1078, 2013. © 2013 Wiley Periodicals, Inc.     

激光共焦扫描显微镜及其应用

介绍了共焦激光显微镜的基本光路、成像原理、关键技术及应用。     

Super‐resolution optical microscopy based on scannable cantilever‐combined microsphere

We report an ingenious method of super‐resolution optical microscopy utilizing scannable cantilever‐combined microsphere. By scanning the microsphere over the sample surface in a cantilever‐combined microsphere‐sample contact state, super‐resolution images can be acquired at arbitrary sample regions through near‐field information collection by the microsphere. In addition, such a state can effectively reduce the possibility of breaking the cantilever and damaging the microsphere or sample surface. This work has developed a new method and technique of sub‐diffraction‐limit optical microscopy, and can be practically applied in various fields of micro/nanoscopy. Microsc. Res. Tech. 78:1128–1132, 2015. © 2015 Wiley Periodicals, Inc.     

Differential phase contrast in scanning optical microscopy

High-quality high-resolution transmission and reflection images produced using a scanning optical microscope and the split-detector technique are presented. These images exhibit differential phase contrast, the method avoiding some drawbacks of the usual Nomarski DIC arrangement. Imaging is treated theoretically and compared with the Nomarski method.     

Three-dimensional optical microscopy using tilted views

The resolution of an optical microscope is considerably less in the direction of the optical axis (z) than in the x–y plane. This is true of conventional or confocal microscopes. To alleviate this problem we used multiple tilted views to supply the ‘missing data’ and thus increase the resolution in z. A special tilting stage was constructed which allowed specimens to be rotated through large angles. The relative, translation, rotation and z-spacing between data sets were determined by a novel Wiener/phase cross-correlation function. Once brought to a common coordinate system the data sets can be combined by Fourier space techniques similar to those used in X-ray crystallography. We applied this technique to metaphase chromosomes from intact embryos of Drosophila melanogaster. As determined from significant intensity in the Fourier transform, the resolution of the final reconstruction was about 0?25 μm in x and y, and 0?4 μm in z.     

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.     

On-line image processing in high resolution electron microscopy

On-line processing and analysis of high resolution electron microscope (HREM) images has been implemented using a software controlled image processor based on a commercial digital video framestore. It directly digitizes the output from the low light TV camera on the HREM and applies any processing necessary. The conditions under which images from amorphous specimens are being obtained can be established on-line. The fast fourier transforms (FFTs) of 256 times 256 pixels produced in 15 s with the system off-line are comparable to those from diffraction analysis of the same data on a laser optical bench. The precision of analysis of defocus, aberrations and astigmatism on-line is discussed, together with possible approaches to routine analysis of crystalline specimens and for interactive control.     

Scanning optical fluorescence microscopy

A scanning optical fluorescence microscope is described which possesses several advantages over a conventional fluorescence microscope. These include improved resolution, a reduction in background- and auto-fluorescence, an increase in the available fluorescence spectrum and simple modification for automated fluorescence studies. Experimental results are included.     

Multimodal and non‐linear optical microscopy applications in reproductive biology

A plethora of optical techniques is currently available to obtain non‐destructive, contactless, real time information with subcellular spatial resolution to observe cell processes. Each technique has its own unique features for imaging and for obtaining certain biological information. However none of the available techniques can be of universal use. For a comprehensive investigation of biological specimens and events, one needs to use a combination of bioimaging methods, often at the same time. Some modern confocal/multiphoton microscopes provide simultaneous fluorescence, fluorescence lifetime imaging, and four‐dimensional imaging. Some of them can also easily be adapted for harmonic generation imaging, and to permit cell manipulation technique. In this work we present a multimodal optical workstation that extends a commercially available confocal microscope to include nonlinear/multiphoton microscopy and optical manipulation/stimulation tools. The nonlinear microscopy capabilities were added to the commercial confocal microscope by exploiting all the flexibility offered by the manufacturer. The various capabilities of this workstation as applied directly to reproductive biology are discussed. Microsc. Res. Tech. 79:567–582, 2016. © 2016 Wiley Periodicals, Inc.     

Rapid histologic diagnosis using quick fluorescence staining and tissue confocal microscopy

With the development of advanced and minimally invasive surgical techniques, and in view of the functional and cosmetic aspects, the need for rapid and accurate diagnosis during surgery is increasing. This study was conducted to develop a tissue diagnosis method using confocal microscopy after simple tissue staining that does not require freezing and slicing. At present, fluorescence staining with confocal microscopy is not generalized for real‐time diagnosis during surgery. In this paper, we propose a fluorescence staining method using Hoechst 33342 and Eosin that does not require tissue freezing and slicing. The proposed method can be used as part of a rapid tissue diagnosis method that is suitable for use in the operating room, although further research is required before it can be applied in clinical practice.     

Out‐of‐focus background subtraction for fast structured illumination super‐resolution microscopy of optically thick samples

We propose a structured illumination microscopy method to combine super resolution and optical sectioning in three‐dimensional (3D) samples that allows the use of two‐dimensional (2D) data processing. Indeed, obtaining super‐resolution images of thick samples is a difficult task if low spatial frequencies are present in the in‐focus section of the sample, as these frequencies have to be distinguished from the out‐of‐focus background. A rigorous treatment would require a 3D reconstruction of the whole sample using a 3D point spread function and a 3D stack of structured illumination data. The number of raw images required, 15 per optical section in this case, limits the rate at which high‐resolution images can be obtained. We show that by a succession of two different treatments of structured illumination data we can estimate the contrast of the illumination pattern and remove the out‐of‐focus content from the raw images. After this cleaning step, we can obtain super‐resolution images of optical sections in thick samples using a two‐beam harmonic illumination pattern and a limited number of raw images. This two‐step processing makes it possible to obtain super resolved optical sections in thick samples as fast as if the sample was two‐dimensional.     

Quantitative microscopy reveals 3D organization and kinetics of endocytosis in rat hepatocytes

In order to demonstrate the power of quantitative microscopy, the endocytic apparatus of rat hepatocytes was reexamined using in situ liver and short term cultured hepatocyte couplets that were allowed to internalize endocytic markers for various time intervals. Correlative confocal light and electron microscopy demonstrate a tubulovesicular reticulum representing the endocytic apparatus. Volume and membrane area account for 2% of cell volume and 30% plasma membrane surface. Colocalization analysis demonstrated that pathway-specific ligands and fluid-phase markers enter EEA1-positive vesicles, the early endosomal compartment, immediately after internalization. These vesicles are translocated rapidly from basolateral to perinuclear and apical locations. Ligands are sorted within 5 min to their respective pathways. Sequential colocalization of an asialoglycoprotein-pulse with rab7 and lamp3 demonstrates that early endosomes change into or fuse with late endosomes and lysosomes. Alternatively, markers are sequestered into the common endosome consisting of rab11-positive, long tubules that originate from early endosomes and show an affinity for the transcytotic marker pIgA and its receptor. This compartment mediates transcytosis by delivering the receptor-ligand complex to the subapical compartment, a set of apical, rab11-positive vesicles, which are connected to the tubular reticulum. We conclude that vesicular traffic between preexisting compartments, maturation or fusion of endocytic organelles, and transport in tubules act in concert and together mediate transport between compartments of a tubulovesicular endocytic apparatus. In addition, we show that quantitative microscopy using high resolution data sets can detect and characterize kinetics of various parameters thus adding a dynamic component to 3D information.