Cooperative 4Pi excitation and detection yields sevenfold sharper optical sections in live-cell microscopy

Although the addition of just the excitation light field at the focus, or of just the fluorescence field at the detector is sufficient for a three- to fivefold resolution increase in 4Pi-fluorescence microscopy, substantial improvements of its optical properties are achieved by exploiting both effects simultaneously. They encompass not only an additional expansion of the optical bandwidth, but also an amplified transfer of the newly gained spatial frequencies to the image. Here we report on the realization and the imaging properties of this 4Pi microscopy mode of type C that also is the far-field microscope with the hitherto largest aperture. We show that in conjunction with two-photon excitation, the resulting optical transfer function displays a sevenfold improvement of axial three-dimensional resolution over confocal microscopy in aqueous samples, and more importantly, a marked transfer of all frequencies within its inner region of support. The latter is present also without the confocal pinhole. Thus, linear image deconvolution is possible both for confocalized and nonconfocalized live-cell 4Pi imaging. Realized in a state-of-the-art scanning microscope, this approach enables robust three-dimensional imaging of fixed and live cells at approximately 80 nm axial resolution.     

High-speed high-resolution imaging of intercellular immune synapses using optical tweezers

Imaging in any plane other than horizontal in a microscope typically requires a reconstruction from multiple optical slices that significantly decreases the spatial and temporal resolution that can be achieved. This can limit the precision with which molecular events can be detected, for example, at intercellular contacts. This has been a major issue for the imaging of immune synapses between live cells, which has generally required the reconstruction of en face intercellular synapses, yielding spatial resolution significantly above the diffraction limit and updating at only a few frames per minute. Strategies to address this issue have usually involved using artificial activating substrates such as antibody-coated slides or supported planar lipid bilayers, but synapses with these surrogate stimuli may not wholly resemble immune synapses between two cells. Here, we combine optical tweezers and confocal microscopy to realize generally applicable, high-speed, high-resolution imaging of almost any arbitrary plane of interest. Applied to imaging immune synapses in live-cell conjugates, this has enabled the characterization of complex behavior of highly dynamic clusters of T cell receptors at the T cell/antigen-presenting cell intercellular immune synapse and revealed the presence of numerous, highly dynamic long receptor-rich filopodial structures within inhibitory Natural Killer cell immune synapses.     

Super-Resolution Imaging Strategies for Cell Biologists Using a Spinning Disk Microscope

In this study we use a spinning disk confocal microscope (SD) to generate super-resolution images of multiple cellular features from any plane in the cell. We obtain super-resolution images by using stochastic intensity fluctuations of biological probes, combining Photoactivation Light-Microscopy (PALM)/Stochastic Optical Reconstruction Microscopy (STORM) methodologies. We compared different image analysis algorithms for processing super-resolution data to identify the most suitable for analysis of particular cell structures. SOFI was chosen for X and Y and was able to achieve a resolution of ca. 80 nm; however higher resolution was possible >30 nm, dependant on the super-resolution image analysis algorithm used. Our method uses low laser power and fluorescent probes which are available either commercially or through the scientific community, and therefore it is gentle enough for biological imaging. Through comparative studies with structured illumination microscopy (SIM) and widefield epifluorescence imaging we identified that our methodology was advantageous for imaging cellular structures which are not immediately at the cell-substrate interface, which include the nuclear architecture and mitochondria. We have shown that it was possible to obtain two coloured images, which highlights the potential this technique has for high-content screening, imaging of multiple epitopes and live cell imaging.     

Imaging Dendritic Spines of Rat Primary Hippocampal Neurons using Structured Illumination Microscopy

Dendritic spines are protrusions emerging from the dendrite of a neuron and represent the primary postsynaptic targets of excitatory inputs in the brain. Technological advances have identified these structures as key elements in neuron connectivity and synaptic plasticity. The quantitative analysis of spine morphology using light microscopy remains an essential problem due to technical limitations associated with light''s intrinsic refraction limit. Dendritic spines can be readily identified by confocal laser-scanning fluorescence microscopy. However, measuring subtle changes in the shape and size of spines is difficult because spine dimensions other than length are usually smaller than conventional optical resolution fixed by light microscopy''s theoretical resolution limit of 200 nm.Several recently developed super resolution techniques have been used to image cellular structures smaller than the 200 nm, including dendritic spines. These techniques are based on classical far-field operations and therefore allow the use of existing sample preparation methods and to image beyond the surface of a specimen. Described here is a working protocol to apply super resolution structured illumination microscopy (SIM) to the imaging of dendritic spines in primary hippocampal neuron cultures. Possible applications of SIM overlap with those of confocal microscopy. However, the two techniques present different applicability. SIM offers higher effective lateral resolution, while confocal microscopy, due to the usage of a physical pinhole, achieves resolution improvement at the expense of removal of out of focus light. In this protocol, primary neurons are cultured on glass coverslips using a standard protocol, transfected with DNA plasmids encoding fluorescent proteins and imaged using SIM. The whole protocol described herein takes approximately 2 weeks, because dendritic spines are imaged after 16-17 days in vitro, when dendritic development is optimal. After completion of the protocol, dendritic spines can be reconstructed in 3D from series of SIM image stacks using specialized software.     

Programmable illumination and high-speed, multi-wavelength, confocal microscopy using a digital micromirror

Confocal microscopy is routinely used for high-resolution fluorescence imaging of biological specimens. Most standard confocal systems scan a laser across a specimen and collect emitted light passing through a single pinhole to produce an optical section of the sample. Sequential scanning on a point-by-point basis limits the speed of image acquisition and even the fastest commercial instruments struggle to resolve the temporal dynamics of rapid cellular events such as calcium signals. Various approaches have been introduced that increase the speed of confocal imaging. Nipkov disk microscopes, for example, use arrays of pinholes or slits on a spinning disk to achieve parallel scanning which significantly increases the speed of acquisition. Here we report the development of a microscope module that utilises a digital micromirror device as a spatial light modulator to provide programmable confocal optical sectioning with a single camera, at high spatial and axial resolution at speeds limited by the frame rate of the camera. The digital micromirror acts as a solid state Nipkov disk but with the added ability to change the pinholes size and separation and to control the light intensity on a mirror-by-mirror basis. The use of an arrangement of concave and convex mirrors in the emission pathway instead of lenses overcomes the astigmatism inherent with DMD devices, increases light collection efficiency and ensures image collection is achromatic so that images are perfectly aligned at different wavelengths. Combined with non-laser light sources, this allows low cost, high-speed, multi-wavelength image acquisition without the need for complex wavelength-dependent image alignment. The micromirror can also be used for programmable illumination allowing spatially defined photoactivation of fluorescent proteins. We demonstrate the use of this system for high-speed calcium imaging using both a single wavelength calcium indicator and a genetically encoded, ratiometric, calcium sensor.     

Real-time nanoscopy by using blinking enhanced quantum dots

Superresolution optical microscopy (nanoscopy) is of current interest in many biological fields. Superresolution optical fluctuation imaging, which utilizes higher-order cumulant of fluorescence temporal fluctuations, is an excellent method for nanoscopy, as it requires neither complicated optics nor illuminations. However, it does need an impractical number of images for real-time observation. Here, we achieved real-time nanoscopy by modifying superresolution optical fluctuation imaging and enhancing the fluctuation of quantum dots. Our developed quantum dots have higher blinking than commercially available ones. The fluctuation of the blinking improved the resolution when using a variance calculation for each pixel instead of a cumulant calculation. This enabled us to obtain microscopic images with 90-nm and 80-ms spatial-temporal resolution by using a conventional fluorescence microscope without any optics or devices.     

Two-Photon Excitation STED Microscopy in Two Colors in Acute Brain Slices

Many cellular structures and organelles are too small to be properly resolved by conventional light microscopy. This is particularly true for dendritic spines and glial processes, which are very small, dynamic, and embedded in dense tissue, making it difficult to image them under realistic experimental conditions. Two-photon microscopy is currently the method of choice for imaging in thick living tissue preparations, both in acute brain slices and in vivo. However, the spatial resolution of a two-photon microscope, which is limited to ∼350 nm by the diffraction of light, is not sufficient for resolving many important details of neural morphology, such as the width of spine necks or thin glial processes. Recently developed superresolution approaches, such as stimulated emission depletion microscopy, have set new standards of optical resolution in imaging living tissue. However, the important goal of superresolution imaging with significant subdiffraction resolution has not yet been accomplished in acute brain slices. To overcome this limitation, we have developed a new microscope based on two-photon excitation and pulsed stimulated emission depletion microscopy, which provides unprecedented spatial resolution and excellent experimental access in acute brain slices using a long-working distance objective. The new microscope improves on the spatial resolution of a regular two-photon microscope by a factor of four to six, and it is compatible with time-lapse and simultaneous two-color superresolution imaging in living cells. We demonstrate the potential of this nanoscopy approach for brain slice physiology by imaging the morphology of dendritic spines and microglial cells well below the surface of acute brain slices.     

Virtual slit scanning microscopy

We present a novel slit scanning confocal microscope with a CCD camera image sensor and a virtual slit aperture for descanning that can be adjusted during post-processing. A very efficient data structure and mathematical criteria for aligning the virtual aperture guarantee the ease of use. We further introduce a method to reduce the anisotropic lateral resolution of slit scanning microscopes. System performance is evaluated against a spinning disk confocal microscope on identical specimens. The virtual slit scanning microscope works as the spinning disk type and outperforms on thick specimens.     

Distinct modulated pupil function system for real-time imaging of living cells

Optical microscopy is one of the most contributive tools for cell biology in the past decades. Many microscopic techniques with various functions have been developed to date, i.e., phase contrast microscopy, differential interference contrast (DIC) microscopy, confocal microscopy, two photon microscopy, superresolution microscopy, etc. However, person who is in charge of an experiment has to select one of the several microscopic techniques to achieve an experimental goal, which makes the biological assay time-consuming and expensive. To solve this problem, we have developed a microscopic system with various functions in one instrument based on the optical Fourier transformation with a lens system for detection while focusing on applicability and user-friendliness for biology. The present instrument can arbitrarily modulate the pupil function with a micro mirror array on the Fourier plane of the optical pathway for detection. We named the present instrument DiMPS (Distinct optical Modulated Pupil function System). The DiMPS is compatible with conventional fluorescent probes and illumination equipment, and gives us a Fourier-filtered image, a pseudo-relief image, and a deep focus depth. Furthermore, DiMPS achieved a resolution enhancement (pseudo-superresolution) of 110 nm through the subtraction of two images whose pupil functions are independently modulated. In maximum, the spatial and temporal resolution was improved to 120 nm and 2 ms, respectively. Since the DiMPS is based on relay optics, it can be easily combined with another microscopic instrument such as confocal microscope, and provides a method for multi-color pseudo-superresolution. Thus, the DiMPS shows great promise as a flexible optical microscopy technique in biological research fields.     

Live-Cell Superresolution Imaging by Pulsed STED Two-Photon Excitation Microscopy

Two-photon laser scanning microscopy (2PLSM) allows fluorescence imaging in thick biological samples where absorption and scattering typically degrade resolution and signal collection of one-photon imaging approaches. The spatial resolution of conventional 2PLSM is limited by diffraction, and the near-infrared wavelengths used for excitation in 2PLSM preclude the accurate imaging of many small subcellular compartments of neurons. Stimulated emission depletion (STED) microscopy is a superresolution imaging modality that overcomes the resolution limit imposed by diffraction and allows fluorescence imaging of nanoscale features. Here, we describe the design and operation of a superresolution two-photon microscope using pulsed excitation and STED lasers. We examine the depth dependence of STED imaging in acute tissue slices and find enhancement of 2P resolution ranging from approximately fivefold at 20 μm to approximately twofold at 90-μm deep. The depth dependence of resolution is found to be consistent with the depth dependence of depletion efficiency, suggesting resolution is limited by STED laser propagation through turbid tissue. Finally, we achieve live imaging of dendritic spines with 60-nm resolution and demonstrate that our technique allows accurate quantification of neuronal morphology up to 30-μm deep in living brain tissue.     

Confocal microscopy: a tool to visualise differentiation of stem cells into cardiomyocytes

Confocal microscopy offers important advantages compared to conventional epifluorescence microscopy. It works as an "optical microtome" leading to a accurate image resolution of a defined focal plane. Furthermore, the addition of a Nipkow disk on the confocal microscope greatly accelerates the image acquisition, up to 30 frames per second. Nevertheless, the software-assisted mathematical restoration of images acquired using a wide-field microscope allows to get images with a resolution similar to the one obtained in confocal microscopy. These imaging technologies allowed us to monitor on line cardiac differentiation of murine embryonic stem (ES) cells within 3D structures called embryoid bodies. The high rate acquisition of images using the confocal microscope equipped with a Nipkow disk allows to monitor calcium spiking in differentiating cardiomyocytes within embryoid bodies.     

Slicing embryos gently with laser light sheets

Light sheet microscopy is an easy to implement and extremely powerful alternative to established fluorescence imaging techniques such as laser scanning confocal, multi-photon and spinning disk microscopy. By illuminating the sample only with a thin slice of light, photo-bleaching is reduced to a minimum, making light sheet microscopy ideal for non-destructive imaging of fragile samples over extended periods of time. Millimeter-sized samples can be imaged rapidly with high resolution and high depth penetration. A large variety of instruments have been developed and optimized for a number of different samples: Bessel beams form thin light sheets for single cells, and selective plane illumination microscopy (SPIM) offers multi-view acquisition to image entire embryos with isotropic resolution. This review explains how light sheet microscopy involves a conceptually new microscope design and how it changes modern imaging in biology.     

Sequential Superresolution Imaging of Multiple Targets Using a Single Fluorophore

Fluorescence superresolution (SR) microscopy, or fluorescence nanoscopy, provides nanometer scale detail of cellular structures and allows for imaging of biological processes at the molecular level. Specific SR imaging methods, such as localization-based imaging, rely on stochastic transitions between on (fluorescent) and off (dark) states of fluorophores. Imaging multiple cellular structures using multi-color imaging is complicated and limited by the differing properties of various organic dyes including their fluorescent state duty cycle, photons per switching event, number of fluorescent cycles before irreversible photobleaching, and overall sensitivity to buffer conditions. In addition, multiple color imaging requires consideration of multiple optical paths or chromatic aberration that can lead to differential aberrations that are important at the nanometer scale. Here, we report a method for sequential labeling and imaging that allows for SR imaging of multiple targets using a single fluorophore with negligible cross-talk between images. Using brightfield image correlation to register and overlay multiple image acquisitions with ~10 nm overlay precision in the x-y imaging plane, we have exploited the optimal properties of AlexaFluor647 for dSTORM to image four distinct cellular proteins. We also visualize the changes in co-localization of the epidermal growth factor (EGF) receptor and clathrin upon EGF addition that are consistent with clathrin-mediated endocytosis. These results are the first to demonstrate sequential SR (s-SR) imaging using direct stochastic reconstruction microscopy (dSTORM), and this method for sequential imaging can be applied to any superresolution technique.     

Conical diffraction illumination opens the way for low phototoxicity super-resolution imaging

We present a new technology for super-resolution fluorescence imaging, based on conical diffraction. Conical diffraction is a linear, singular phenomenon, taking place when a laser beam is diffracted through a biaxial crystal. We use conical diffraction in a thin biaxial crystal to generate illumination patterns that are more compact than the classical Gaussian beam, and use them to generate a super-resolution imaging modality.

While there already exist several super-resolution modalities, our technology (biaxial super-resolution: BSR) is distinguished by the unique combination of several performance features. Using BSR super-resolution data are achieved using low light illumination significantly less than required for classical confocal imaging, which makes BSR ideal for live-cell, long-term time-lapse super-resolution imaging. Furthermore, no specific sample preparation is required, and any fluorophore can be used. Perhaps most exciting, improved resolution BSR-imaging resolution enhancement can be achieved with any type of objective no matter the magnification, numerical aperture, working distance, or the absence or presence of immersion medium.

In this article, we present the first implementation of BSR modality on a commercial confocal microscope. We acquire and analyze validation data, showing high quality super-resolved images of biological objects, and demonstrate the wide applicability of the technology. We report live-cell super-resolution imaging over a long period, and show that the light dose required for super-resolution imaging is far below the threshold likely to generate phototoxicity.     

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.     

A mercury arc lamp-based multi-color confocal real time imaging system for cellular structure and function

Multi-point scanning confocal microscopy using a Nipkow disk enables the acquisition of fluorescent images with high spatial and temporal resolutions. Like other single-point scanning confocal systems that use Galvano meter mirrors, a commercially available Nipkow spinning disk confocal unit, Yokogawa CSU10, requires lasers as the excitation light source. The choice of fluorescent dyes is strongly restricted, however, because only a limited number of laser lines can be introduced into a single confocal system. To overcome this problem, we developed an illumination system in which light from a mercury arc lamp is scrambled to make homogeneous light by passing it through a multi-mode optical fiber. This illumination system provides incoherent light with continuous wavelengths, enabling the observation of a wide range of fluorophores. Using this optical system, we demonstrate both the high-speed imaging (up to 100 Hz) of intracellular Ca(2+) propagation, and the multi-color imaging of Ca(2+) and PKC-gamma dynamics in living cells.     

Confocal scanning optical microscopy and three-dimensional imaging

Summary— Confocal scanning optical microscopy has significant advantages over conventional fluorescence microscopy: it rejects the out-of-locus light and provides a greater resolution than the wide-field microscope. In laser scanning optical microscopy, the specimen is scanned by a diffraction-limited spot of laser light and the fluorescence emission (or the reflected light) is focused onto a photodetector. The imaged point is then digitized, stored into the memory of a computer and displayed at the appropriate spatial position on a graphic device as a part of a two-dimensional image. Thus, confocal scanning optical microscopy allows accurate non-invasive optical sectioning and further three-dimensional reconstruction of biological specimens. Here we review the recent technological aspects of the principles and uses of the confocal microscope, and we introduce the different methods of three-dimensional imaging.     

Control and manipulation of pathogens with an optical trap for live cell imaging of intercellular interactions

The application of live cell imaging allows direct visualization of the dynamic interactions between cells of the immune system. Some preliminary observations challenge long-held beliefs about immune responses to microorganisms; however, the lack of spatial and temporal control between the phagocytic cell and microbe has rendered focused observations into the initial interactions of host response to pathogens difficult. This paper outlines a method that advances live cell imaging by integrating a spinning disk confocal microscope with an optical trap, also known as an optical tweezer, in order to provide exquisite spatial and temporal control of pathogenic organisms and place them in proximity to host cells, as determined by the operator. Polymeric beads and live, pathogenic organisms (Candida albicans and Aspergillus fumigatus) were optically trapped using non-destructive forces and moved adjacent to living cells, which subsequently phagocytosed the trapped particle. High resolution, transmitted light and fluorescence-based movies established the ability to observe early events of phagocytosis in living cells. To demonstrate the broad applicability of this method to immunological studies, anti-CD3 polymeric beads were also trapped and manipulated to form synapses with T cells in vivo, and time-lapse imaging of synapse formation was also obtained. By providing a method to exert fine control of live pathogens with respect to immune cells, cellular interactions can be captured by fluorescence microscopy with minimal perturbation to cells and can yield powerful insight into early responses of innate and adaptive immunity.     

基于激光共聚焦同步双扫描技术的LC3脱乙酰化修饰调控自噬的机理研究

激光共聚焦同步双扫描(simultaneous,SIM)技术在常规扫描单元的基础上,引入一个同步扫描单元(SIM scanner),该技术独立控制了两个激光束,一个用于激光光刺激,另一个用于同步成像。本实验中,采用激光共聚焦同步双扫描系统的405 nm和488 nm激光分别对细胞的特定部位进行刺激和同步成像,实时检测了LC3复合物的形成,记录并分析了乙酰化前后LC3的光动力学变化过程,证实了LC3的脱乙酰化修饰是自噬性降解所必须的,本实验体系为激光共聚焦双扫描技术的推广提供了一个很好的平台。SIM技术的应用,解决了刺激过程无法成像的问题,为漂白后荧光恢复(fluorescence recovery after photobleaching,FRAP)、漂白后荧光损失(fluorescence loss in photobleaching,FLIP)和光诱导激活等研究提供了最佳的解决方案,可作为光刺激的一种实验模式在很多实验设计中进行延伸应用。     

Single-Molecule and Superresolution Imaging in Live Bacteria Cells

Single-molecule imaging enables biophysical measurements devoid of ensemble averaging, gives enhanced spatial resolution beyond the diffraction limit, and permits superresolution reconstructions. Here, single-molecule and superresolution imaging are applied to the study of proteins in live Caulobacter crescentus cells to illustrate the power of these methods in bacterial imaging. Based on these techniques, the diffusion coefficient and dynamics of the histidine protein kinase PleC, the localization behavior of the polar protein PopZ, and the treadmilling behavior and protein superstructure of the structural protein MreB are investigated with sub-40-nm spatial resolution, all in live cells.Since its advent 20 years ago, single-molecule fluorescence imaging has given rise to a host of exciting experiments (Ambrose and Moerner 1991). Beyond enabling fundamental investigations of the physics of emissive molecules, one main advantage of this technique is its use in biologically relevant, live-cell experiments. Optical fluorescence microscopy is an important instrument for cell biology, as light can be used to noninvasively probe a sample with relatively small perturbation of the specimen, enabling dynamical observation of the motions of internal structures in living cells. Single-molecule epifluorescence microscopy extends these capabilities by achieving nanometer-scale resolution, taking advantage of the fact that one can precisely characterize the point spread function (PSF) of a microscope, allowing the center of a distribution, and thus the exact position of an emitter, to be localized with accuracy much better than the diffraction limit itself. This localization accuracy improves beyond the diffraction limit roughly as one over the square root of the number of detected photons (Thompson et al. 2002). Detecting 100 photons from a single, isolated molecule can therefore improve the resolution of an optical measurement from the ∼250-nm diffraction limit down to 25 nm.Single-molecule imaging has been used in the investigation of a number of live-cell samples. In 2000, the lateral heterogeneity of the plasma membrane was investigated by tracing the motion of single dye-labeled lipids in native human airway smooth muscle (HASM) cells (Schütz et al. 2000), and epidermal growth factor (EGF) receptor signaling was explored with a fluorescent protein fusion and a labeled ligand (Sako et al. 2000). Single fluorophore-labeled molecules have subsequently been used in many ways (Moerner 2003), for instance to investigate the effect of varying cholesterol concentration on the mobility of proteins in the plasma membrane of Chinese hamster ovary (CHO) cells (Vrljic et al. 2002; Vrljic et al. 2005) and to explore the real-time dynamic behavior of cell-penetrating-peptide (CPP) molecular transporters on the plasma membrane of CHO cells (Lee et al. 2008). Furthermore, in 2001, Harms et al. characterized the emission of fluorescent proteins in biocompatible environments and noted that the yellow fluorescent protein EYFP was well-suited to single-molecule imaging in cells (Harms et al. 2001). Such fluorescent proteins can be genetically encoded as tags for native proteins in cells; these fusions have been used in many live-cell single-molecule experiments.More recently, single-molecule epifluorescence microscopy has been used to probe the inner workings of live bacteria. The small size of prokaryotic cells makes the optical diffraction limit particularly noticeable, which has stimulated the push toward superlocalization and superresolution to overcome this obstacle. As a result, the nascent field of bacterial structural biology has benefited greatly from single-molecule investigations of proteins in live cells. The overall shapes of such cells can be seen in a standard light microscope, but those interested in probing subcellular details, such as protein structure and localization, have typically had to resort to in vitro characterization combined with extrapolation to the cellular environment, as well as to indirect methods such as biochemical assays. Although cryo-electron microscopy can provide extremely high spatial resolution, fixation or plunge-freezing is essential, and methods for identifying specific proteins out of many are still lacking. As a consequence, bacterial cell biology is an area of study ripe for investigation with direct, noninvasive optical methods of probing position, coupling and structure, with resolution below the standard diffraction limit.Several groups have extended single-molecule imaging techniques to live bacterial samples. In 2004, single PleC proteins were visualized in Caulobacter crescentus cells (Deich et al. 2004), and the behavior of this system is described in more detail later. More recently, Xie and coauthors have used single-molecule fluorescence techniques to study DNA-binding proteins, mRNA, and membrane proteins to provide much insight into the mechanisms of bacterial gene expression; these efforts have been documented in a recent review (Xie et al. 2008). As well, Conley et al. used covalently linked Cy3-Cy5-thiol switchable fluorophores to illuminate the stalks of C. crescentus cells with high resolution (Conley et al. 2008). In this article, we focus on the application of single-molecule imaging and single-molecule-based superresolution imaging to investigate the localization, movement, and structure of three important proteins, PleC, PopZ, and MreB, in live C. crescentus cells.