Optical imaging of nanoscale cellular structures

Visualization of subcellular structures and their temporal evolution is of utmost importance to understand a vast range of biological processes. Optical microscopy is the method of choice for imaging live cells and tissues; it is minimally invasive, so processes can be observed over extended periods of time without generating artifacts due to intense light irradiation. The use of fluorescence microscopy is advantageous because biomolecules or supramolecular structures of interest can be labeled specifically with fluorophores, so the images reveal information on processes involving only the labeled molecules. The key restriction of optical microscopy is its moderate resolution, which is limited to about half the wavelength of light (～200 nm) due to fundamental physical laws governing wave optics. Consequently, molecular processes taking place at spatial scales between 1 and 100 nm cannot be studied by regular optical microscopy. In recent years, however, a variety of super-resolution fluorescence microscopy techniques have been developed that circumvent the resolution limitation. Here, we present a brief overview of these techniques and their application to cellular biophysics.     

Microscopy basics and the study of actin–actin-binding protein interactions

Actin is a multifunctional eukaryotic protein with a globular monomer form that polymerizes into a thin, linear microfilament in cells. Through interactions with various actin-binding proteins (ABPs), actin plays an active role in many cellular processes, such as cell motility and structure. Microscopy techniques are powerful tools for determining the role and mechanism of actin–ABP interactions in these processes. In this article, we describe the basic concepts of fluorescent speckle microscopy, total internal reflection fluorescence microscopy, atomic force microscopy, and cryoelectron microscopy and review recent studies that utilize these techniques to visualize the binding of actin with ABPs.     

Light sheet microscopes: Novel imaging toolbox for visualizing life’s processes

Capturing dynamic processes in live samples is a nontrivial task in biological imaging. Although fluorescence provides high specificity and contrast compared to other light microscopy techniques, the photophysical principles of this method can have a harmful effect on the sample. Current advances in light sheet microscopy have created a novel imaging toolbox that allows for rapid acquisition of high-resolution fluorescent images with minimal perturbation of the processes of interest. Each unique design has its own advantages and limitations. In this review, we describe several cutting edge light sheet microscopes and their optimal applications.     

Fluorescence approaches for determining protein conformations, interactions and mechanisms at membranes

Processes that occur at membranes are essential for the viability of every cell, but such processes are the least well understood at the molecular level. The complex nature and physical properties of the molecular components involved, as well as the requirement for two separated aqueous compartments, restrict the experimental approaches that can be successfully applied to examine the structure, conformational changes and interactions of the membrane-bound proteins that accomplish these processes. In particular, to accurately elucidate the molecular mechanisms that effect and regulate such processes, one must use experimental approaches that do not disrupt the structural integrity or functionality of the protein-membrane complexes being examined. To best accomplish this goal, especially when large multicomponent complexes and native membranes are involved, the optimal experimental approach to use is most often fluorescence spectroscopy. Using multiple independent fluorescence techniques, one can determine structural information in real time and in intact membranes under native conditions that cannot be obtained by crystallography, electron microscopy and NMR techniques, among others. Furthermore, fluorescence techniques provide a comprehensive range of information, from kinetic to thermodynamic, about the assembly, structure, function and regulation of membrane-bound proteins and complexes. This article describes the use of various fluorescence techniques to characterize different aspects of proteins bound to or embedded in membranes.     

Extending the knowledge in histochemistry and cell biology

Central to modern Histochemistry and Cell Biology stands the need for visualization of cellular and molecular processes. In the past several years, a variety of techniques has been achieved bridging traditional light microscopy, fluorescence microscopy and electron microscopy with powerful software-based post-processing and computer modeling. Researchers now have various tools available to investigate problems of interest from bird’s- up to worm’s-eye of view, focusing on tissues, cells, proteins or finally single molecules. Applications of new approaches in combination with well-established traditional techniques of mRNA, DNA or protein analysis have led to enlightening and prudent studies which have paved the way toward a better understanding of not only physiological but also pathological processes in the field of cell biology. This review is intended to summarize articles standing for the progress made in “histo-biochemical” techniques and their manifold applications.     

Monitoring and quantifying dynamic physiological processes in live neurons using fluorescence recovery after photobleaching

The direct visualization of subcellular dynamic processes is often hampered by limitations in the resolving power achievable with conventional microscopy techniques. Fluorescence recovery after photobleaching has emerged as a highly informative approach to address this challenge, permitting the quantitative measurement of the movement of small organelles and proteins in living functioning cells, and offering detailed insights into fundamental cellular phenomena of physiological importance. In recent years, its implementation has benefited from the increasing availability of confocal microscopy systems and of powerful labeling techniques based on genetically encoded fluorescent proteins or other chemical markers. In this review, we present fluorescence recovery after photobleaching and related techniques in the context of contemporary neurobiological research and discuss quantitative and semi‐quantitative approaches to their interpretation.     

Super-resolution Imaging of the Cytokinetic Z Ring in Live Bacteria Using Fast 3D-Structured Illumination Microscopy (f3D-SIM)

Imaging of biological samples using fluorescence microscopy has advanced substantially with new technologies to overcome the resolution barrier of the diffraction of light allowing super-resolution of live samples. There are currently three main types of super-resolution techniques – stimulated emission depletion (STED), single-molecule localization microscopy (including techniques such as PALM, STORM, and GDSIM), and structured illumination microscopy (SIM). While STED and single-molecule localization techniques show the largest increases in resolution, they have been slower to offer increased speeds of image acquisition. Three-dimensional SIM (3D-SIM) is a wide-field fluorescence microscopy technique that offers a number of advantages over both single-molecule localization and STED. Resolution is improved, with typical lateral and axial resolutions of 110 and 280 nm, respectively and depth of sampling of up to 30 µm from the coverslip, allowing for imaging of whole cells. Recent advancements (fast 3D-SIM) in the technology increasing the capture rate of raw images allows for fast capture of biological processes occurring in seconds, while significantly reducing photo-toxicity and photobleaching. Here we describe the use of one such method to image bacterial cells harboring the fluorescently-labelled cytokinetic FtsZ protein to show how cells are analyzed and the type of unique information that this technique can provide.     

Imaging adhesion and signaling dynamics in Xenopus laevis growth cones

Xenopus laevis provides a robust model system to study cellular signaling and downstream processes during development both in vitro and in vivo. Intracellular signals must function within highly restricted spatial and temporal domains to activate specific downstream targets and cellular processes. Combining the versatility of developing Xenopus neurons with advances in fluorescent protein biosensors and imaging technologies has allowed many dynamic cellular processes to be visualized. This review will focus on the techniques we use to visualize and measure cell signaling, motility and adhesion by quantitative fluorescence microscopy in vitro and in vivo.     

Fluorescence microscopy

Although fluorescence microscopy permeates all of cell and molecular biology, most biologists have little experience with the underlying photophysical phenomena. Understanding the principles underlying fluorescence microscopy is useful when attempting to solve imaging problems. Additionally, fluorescence microscopy is in a state of rapid evolution, with new techniques, probes and equipment appearing almost daily. Familiarity with fluorescence is a prerequisite for taking advantage of many of these developments. This review attempts to provide a framework for understanding excitation of and emission by fluorophores, the way fluorescence microscopes work, and some of the ways fluorescence can be optimized.     

Combining multiple fluorescence imaging techniques in biology: when one microscope is not enough

While fluorescence microscopy has proven to be an exceedingly useful tool in bioscience, it is difficult to offer simultaneous high resolution, fast speed, large volume, and good biocompatibility in a single imaging technique. Thus, when determining the image data required to quantitatively test a complex biological hypothesis, it often becomes evident that multiple imaging techniques are necessary. Recent years have seen an explosion in development of novel fluorescence microscopy techniques, each of which features a unique suite of capabilities. In this Technical Perspective, we highlight recent studies to illustrate the benefits, and often the necessity, of combining multiple fluorescence microscopy modalities. We provide guidance in choosing optimal technique combinations to effectively address a biological question. Ultimately, we aim to promote a more well-rounded approach in designing fluorescence microscopy experiments, leading to more robust quantitative insight.     

Functional studies in living animals using multiphoton microscopy

In vivo microscopy is a powerful method for studying fundamental issues of physiology and pathophysiology. The recent development of multiphoton fluorescence microscopy has extended the reach of in vivo microscopy, supporting high-resolution imaging deep into the tissues and organs of living animals. As compared with other in vivo imaging techniques, multiphoton microscopy is uniquely capable of providing a window into cellular and subcellular processes in the context of the intact, functioning animal. In addition, the ability to collect multiple colors of fluorescence from the same sample makes in vivo microscopy uniquely capable of characterizing up to three parameters from the same volume, supporting powerful correlative analyses. Since its invention in 1990, multiphoton microscopy has been increasingly applied to numerous areas of medical investigation, providing invaluable insights into cell physiology and pathology. However, researchers have only begun to realize the true potential of this powerful technology as it has proliferated beyond the laboratories of a relatively few pioneers. In this article we present an overview of the advantages and limitations of multiphoton microscopy as applied to in vivo imaging. We also review specific examples of the application of in vivo multiphoton microscopy to studies of physiology and pathology in a variety of organs including the brain, skin, skeletal muscle, tumors, immune cells, and visceral organs.     

Image analysis in fluorescence microscopy: bacterial dynamics as a case study

Fluorescence microscopy is the primary tool for studying complex processes inside individual living cells. Technical advances in both molecular biology and microscopy have made it possible to image cells from many genetic and environmental backgrounds. These images contain a vast amount of information, which is often hidden behind various sources of noise, convoluted with other information and stochastic in nature. Accessing the desired biological information therefore requires new tools of computational image analysis and modeling. Here, we review some of the recent advances in computational analysis of images obtained from fluorescence microscopy, focusing on bacterial systems. We emphasize techniques that are readily available to molecular and cell biologists but also point out examples where problem-specific image analyses are necessary. Thus, image analysis is not only a toolkit to be applied to new images but also an integral part of the design and implementation of a microscopy experiment.     

Real-time imaging of single synaptic vesicles in live neurons

Recent advances in fluorescence microscopy have provided researchers with powerful new tools to visualize cellular processes occurring in real time, giving researchers an unprecedented opportunity to address many biological questions that were previously inaccessible. With respect to neurobiology, these real-time imaging techniques have deepened our understanding of molecular and cellular processes, including the movement and dynamics of single proteins and organelles in living cells. In this review, we summarize recent advances in the field of real-time imaging of single synaptic vesicles in live neurons.     

Visualizing recycling of synaptic vesicle proteins

The use of modern techniques (in particular, novel fluorescence markers of a few molecular participants of the exo-and endocytotic processes, including pH-sensitive agents, immuno-electron and laser-scanning microscopy) allows experimenters to visualize different stages of recycling of synaptic vesicle proteins. Neirofiziologiya/Neurophysiology, Vol. 39, Nos. 4/5, pp. 350–351, July–October, 2007.     

Multilayer mounting enables long-term imaging of zebrafish development in a light sheet microscope

Light sheet microscopy techniques, such as selective plane illumination microscopy (SPIM), are ideally suited for time-lapse imaging of developmental processes lasting several hours to a few days. The success of this promising technology has mainly been limited by the lack of suitable techniques for mounting fragile samples. Embedding zebrafish embryos in agarose, which is common in conventional confocal microscopy, has resulted in severe growth defects and unreliable results. In this study, we systematically quantified the viability and mobility of zebrafish embryos mounted under more suitable conditions. We found that tubes made of fluorinated ethylene propylene (FEP) filled with low concentrations of agarose or methylcellulose provided an optimal balance between sufficient confinement of the living embryo in a physiological environment over 3 days and optical clarity suitable for fluorescence imaging. We also compared the effect of different concentrations of Tricaine on the development of zebrafish and provide guidelines for its optimal use depending on the application. Our results will make light sheet microscopy techniques applicable to more fields of developmental biology, in particular the multiview long-term imaging of zebrafish embryos and other small organisms. Furthermore, the refinement of sample preparation for in toto and in vivo imaging will promote other emerging optical imaging techniques, such as optical projection tomography (OPT).     

Beyond the diffraction limit: far-field fluorescence imaging with ultrahigh resolution

Fluorescence microscopy is an important and extensively utilised tool for imaging biological systems. However, the image resolution that can be obtained has a limit as defined through the laws of diffraction. Demand for improved resolution has stimulated research into developing methods to image beyond the diffraction limit based on far-field fluorescence microscopy techniques. Rapid progress is being made in this area of science with methods emerging that enable fluorescence imaging in the far-field to possess a resolution well beyond the diffraction limit. This review outlines developments in far-field fluorescence methods which enable ultrahigh resolution imaging and application of these techniques to biology. Future possible trends and directions in far-field fluorescence imaging with ultrahigh resolution are also outlined.     

Hypothesis testing via integrated computer modeling and digital fluorescence microscopy

Computational modeling has the potential to add an entirely new approach to hypothesis testing in yeast cell biology. Here, we present a method for seamless integration of computational modeling with quantitative digital fluorescence microscopy. This integration is accomplished by developing computational models based on hypotheses for underlying cellular processes that may give rise to experimentally observed fluorescent protein localization patterns. Simulated fluorescence images are generated from the computational models of underlying cellular processes via a "model-convolution" process. These simulated images can then be directly compared to experimental fluorescence images in order to test the model. This method provides a framework for rigorous hypothesis testing in yeast cell biology via integrated mathematical modeling and digital fluorescence microscopy.     

Single-molecule analysis of DNA replication in Xenopus egg extracts

The recent advent in single-molecule imaging and manipulation methods has made a significant impact on the understanding of molecular mechanisms underlying many essential cellular processes. Single-molecule techniques such as electron microscopy and DNA fiber assays have been employed to study the duplication of genome in eukaryotes. Here, we describe a single-molecule assay that allows replication of DNA attached to the functionalized surface of a microfluidic flow cell in a soluble Xenopus leavis egg extract replication system and subsequent visualization of replication products via fluorescence microscopy. We also explain a method for detection of replication proteins, through fluorescently labeled antibodies, on partially replicated DNA immobilized at both ends to the surface.     

Optical sectioning microscopy

Confocal scanning microscopy, a form of optical sectioning microscopy, has radically transformed optical imaging in biology. These devices provide a powerful means to eliminate from images the background caused by out-of-focus light and scatter. Confocal techniques can also improve the resolution of a light microscope image beyond what is achievable with widefield fluorescence microscopy. The quality of the images obtained, however, depends on the user's familiarity with the optical and fluorescence concepts that underlie this approach. We describe the core concepts of confocal microscopes and important variables that adversely affect confocal images. We also discuss data-processing methods for confocal microscopy and computational optical sectioning techniques that can perform optical sectioning without a confocal microscope.