The fluorescence robot imaging technology multi-epitope-ligand-cartography/toponome imaging system has revolutionized the field of proteomics/functional genomics, because it enables the investigator to locate and decipher functional protein networks, the toponome, consisting of hundreds of different proteins in a single cell or tissue section. The technology has been proven to solve key problems in biology and therapy research. It has uncovered a new cellular transdifferentiation mechanism of vascular cells giving rise to myogenic cells in situ and in vivo; a finding that has led to efficient cell therapy models of muscle disorders, and discovered a new target protein in sporadic amyotrophic lateral sclerosis by hierarchical protein network analysis, a finding that has been confirmed by a mouse knockout model. A lead target protein in tumor cells that controls cell polarization as a mechanism that is fundamental for migration and metastasis formation has also been uncovered, and new functional territories in the CNS defined by high-dimensional synaptic protein clusters have been unveiled. The technology can be effectively interlocked with genomics and proteomics to optimize time-to-market and the overall attrition rate of new drugs. This review outlines major proofs of principle with an emphasis on neurotoponomics. 相似文献
In fluctuation‐based optical nanoscopy, investigating high‐density labeled subcellular structures with high fidelity has been a significant challenge. In this study, based on super‐resolution radial fluctuation (SRRF) microscopy, the joint tagging (JT) strategy is employed to enable fast high‐density nanoscopic imaging and tracking. In fixed cell experiment, multiple types of quantum dots with distinguishable fluorescence spectra are jointly tagged to subcellular microtubules. In each spectral channel, the decrease in labeling density guarantees the high‐fidelity super‐resolution reconstruction using SRRF microscopy. Subsequently, the combination of all spectral channels achieves high‐density super‐resolution imaging of subcellular microtubules with a resolution of ~62 nm using JT assisted SRRF technique. In the live‐cell experiment, 3‐channel JT is utilized to track the dynamic motions of high‐density toxin‐induced lipid clusters for 1 minute, achieving the simultaneous tracking of many individual toxin‐induced lipid clusters spatially distributed significantly below the optical diffraction limit in living cells. 相似文献
Based on multicolor quantum dots (QDs) labeling, the joint tagging assisted super‐resolution radial fluctuation (JT‐SRRF) nanoscopy achieves high‐fidelity super‐resolution imaging of subcellular microtubules and fast live‐cell parallel tracking of cholera toxin subunit B (CTB) induced lipid clusters spatially distributed below the optical diffraction limit. This method paves the way for fast high‐density parallel tracking, which is especially beneficial for the investigation of the intensive dynamics in live‐cell applications. Further details can be found in the article by Zhiping Zeng, Jing Ma, Peng Xi, and Canhua Xu ( e201800020 ).
Super‐resolution microscopy (SRM) has had a substantial impact on the biological sciences due to its ability to observe tiny objects less than 200 nm in size. Stimulated emission depletion (STED) microscopy represents a major category of these SRM techniques that can achieve diffraction‐unlimited resolution based on a purely optical modulation of fluorescence behaviors. Here, we investigated how the laser beams affect fluorescence lifetime in both confocal and STED imaging modes. The results showed that with increasing illumination time, the fluorescence lifetime in two kinds of fluorescent microspheres had an obvious change in STED imaging mode, compared with that in confocal imaging mode. As a result, the reduction of saturation intensity induced by the increase of fluorescence lifetime can improve the STED imaging resolution at the same depletion power. The phenomenon was also observed in Star635P‐labeled human Nup153 in fixed HeLa cells, which can be treated as a reference for the synthesis of fluorescent labels with the sensitivity to the surrounding environment for resolution improvement in STED nanoscopy. 相似文献
Label‐free optical nano‐imaging of dendritic structures and intracellular granules in biological cells is demonstrated using a bright and homogeneous nanometric light source. The optical nanometric light source is excited using a focused electron beam. A zinc oxide (ZnO) luminescent thin film was fabricated by atomic layer deposition (ALD) to produce the nanoscale light source. The ZnO film formed by ALD emitted the bright, homogeneous light, unlike that deposited by another method. The dendritic structures of label‐free macrophage receptor with collagenous structure‐expressing CHO cells were clearly visualized below the diffraction limit. The inner fiber structure was observed with 120 nm spatial resolution. Because the bright homogeneous emission from the ZnO film suppresses the background noise, the signal‐to‐noise ratio (SNR) for the imaging results was greater than 10. The ALD method helps achieve an electron beam excitation assisted microscope with high spatial resolution and high SNR.
The replication of HIV‐1, like that of all viruses, is intimately connected with cellular structures and pathways. For many years, bulk biochemical and cell biological methods were the main approaches employed to investigate interactions between HIV‐1 and its host cell. However, during the past decade advancements in fluorescence imaging technologies opened new possibilities for the direct visualization of individual steps occurring throughout the viral replication cycle. Electron microscopy (EM) methods, which have traditionally been employed for the study of viruses, are complemented by fluorescence microscopy (FM) techniques that allow us to follow the dynamics of virus–cell interaction. Subdiffraction fluorescence microscopy, as well as correlative EM/FM approaches, are narrowing the fundamental gap between the high structural resolution provided by EM and the high temporal resolution and throughput accomplished by FM. The application of modern microscopy to the study of HIV‐1–host cell interactions has provided insights into the biology of the virus which could not easily, or not at all, have been gained by other methods. Here, we review how modern fluorescence imaging techniques enhanced our knowledge of the dynamic and structural changes involved in HIV‐1 particle formation. 相似文献
Dendritic spines arise as small protrusions from the dendritic shaft of various types of neuron and receive inputs from excitatory axons. Ever since dendritic spines were first described in the nineteenth century, questions about their function have spawned many hypotheses. In this review, we introduce understanding of the structural and biochemical properties of dendritic spines with emphasis on components studied with imaging methods. We then explore advances in in vivo imaging methods that are allowing spine activity to be studied in living tissue, from super-resolution techniques to calcium imaging. Finally, we review studies on spine structure and function in vivo. These new results shed light on the development, integration properties and plasticity of spines. 相似文献
Localization‐based super‐resolution microscopy relies on the detection of individual molecules cycling between fluorescent and non‐fluorescent states. These transitions are commonly regulated by high‐intensity illumination, imposing constrains to imaging hardware and producing sample photodamage. Here, we propose single‐molecule self‐quenching as a mechanism to generate spontaneous photoswitching. To demonstrate this principle, we developed a new class of DNA‐based open‐source super‐resolution probes named super‐beacons, with photoswitching kinetics that can be tuned structurally, thermally and chemically. The potential of these probes for live‐cell compatible super‐resolution microscopy without high‐illumination or toxic imaging buffers is revealed by imaging interferon inducible transmembrane proteins (IFITMs) at sub‐100 nm resolutions. 相似文献
STED (stimulated emission depletion) microscopy is one of the most promising super‐resolution fluorescence microscopies,due to its fast imaging and ultra‐high resolution. In this paper, we present a dual‐color STED microscope with a single laser source. Polarization beam splitters are used to separate the output from a supercontinuum laser source into four laser beams, including two excitation beams (488, 635 nm) and two depletion beams (592, 775 nm). These four laser beams are then used to build a low cost dual‐color STED system to achieve a spatial resolution of 75 nm in cell samples. 相似文献
For both fundamental study of biological processes and early diagnosis of diseases, information about nanoscale changes in tissue and cell structure is crucial. Nowadays, almost all currently known nanoscopy methods rely upon the contrast created by fluorescent stains attached to the object or molecule of interest. This causes limitations due to the impact of the label on the object and its environment, as well as its applicability in vivo, particularly in humans. In this paper, a new label‐free approach to visualize small structure with nano‐sensitivity to structural alterations is introduced. Numerically synthesized profiles of the axial spatial frequencies are used to probe the structure within areas whose size can be beyond the diffraction resolution limit. Thereafter, nanoscale structural alterations within such areas can be visualized and objects, including biological ones, can be investigated with sub‐wavelength resolution, in vivo, in their natural environment. Some preliminary results, including numerical simulations and experiments, which demonstrate the nano‐sensitivity and super‐resolution ability of our approach, are presented. 相似文献
Light‐sheet fluorescence microscopy (LSFM) allows volumetric live imaging at high‐speed and with low photo‐toxicity. Various LSFM modalities are commercially available, but their size and cost limit their access by the research community. A new method, termed sub‐voxel‐resolving (SVR) light‐sheet add‐on microscopy (SLAM), is presented to enable fast, resolution‐enhanced light‐sheet fluorescence imaging from a conventional wide‐field microscope. This method contains two components: a miniature add‐on device to regular wide‐field microscopes, which contains a horizontal laser light‐sheet illumination path to confine fluorophore excitation at the vicinity of the focal plane for optical sectioning; an off‐axis scanning strategy and a SVR algorithm that utilizes sub‐voxel spatial shifts to reconstruct the image volume that results in a twofold increase in resolution. SLAM method has been applied to observe the muscle activity change of crawling C. elegans, the heartbeat of developing zebrafish embryo, and the neural anatomy of cleared mouse brains, at high spatiotemporal resolution. It provides an efficient and cost‐effective solution to convert the vast number of in‐service microscopes for fast 3D live imaging with voxel‐super‐resolved capability. 相似文献
Optical‐resolution photoacoustic microscopy (OR‐PAM), which has been widely used and studied as a noninvasive and in vivo imaging technique, can yield high‐resolution and absorption contrast images. Recently, metallic nanoparticles and dyes, such as gold nanoparticles, methylene blue, and indocyanine green, have been used as contrast agents of OR‐PAM. This study demonstrates real‐time functional OR‐PAM images with high‐speed alternating illumination at 2 wavelengths. To generate 2 wavelengths, second harmonic generation at 532 nm with an LBO crystal and a pump wavelength of 1064 nm is applied at a pulse repetition rate of 300 kHz. For alternating illumination, an electro‐optical modulator is used as an optical switch. Therefore, the A‐line rate for the functional image is 150 kHz, which is half of the laser repetition rate. To enable fast signal processing and real‐time displays, parallel signal processing using a graphics processing unit (GPU) is performed. OR‐PAM images of the distribution of blood vessels and gold nanorods in a BALB/c‐nude mouse's ear can be simultaneously obtained with 500 × 500 pixels and real‐time display at 0.49 fps. 相似文献
Light‐sheet fluorescence microscopy (LSFM) is a powerful technique that can provide high‐resolution images of biological samples. Therefore, this technique offers significant improvement for three‐dimensional (3D) imaging of living cells. However, producing high‐resolution 3D images of a single cell or biological tissues, normally requires high acquisition rate of focal planes, which means a large amount of sample sections. Consequently, it consumes a vast amount of processing time and memory, especially when studying real‐time processes inside living cells. We describe an approach to minimize data acquisition by interpolation between planes using a phase retrieval algorithm. We demonstrate this approach on LSFM data sets and show reconstruction of intermediate sections of the sparse samples. Since this method diminishes the required amount of acquisition focal planes, it also reduces acquisition time of samples as well. Our suggested method has proven to reconstruct unacquired intermediate planes from diluted data sets up to 10× fold. The reconstructed planes were found correlated to the original preacquired samples (control group) with correlation coefficient of up to 90%. Given the findings, this procedure appears to be a powerful method for inquiring and analyzing biological samples. 相似文献
Super-resolution microscopy has become an increasingly popular and robust tool across the life sciences to study minute cellular structures and processes. However, with the increasing number of available super-resolution techniques has come an increased complexity and burden of choice in planning imaging experiments. Choosing the right super-resolution technique to answer a given biological question is vital for understanding and interpreting biological relevance. This is an often-neglected and complex task that should take into account well-defined criteria (e.g., sample type, structure size, imaging requirements). Trade-offs in different imaging capabilities are inevitable; thus, many researchers still find it challenging to select the most suitable technique that will best answer their biological question. This review aims to provide an overview and clarify the concepts underlying the most commonly available super-resolution techniques as well as guide researchers through all aspects that should be considered before opting for a given technique. 相似文献
TIRF and STORM microscopy are super‐resolving fluorescence imaging modalities for which current implementations on standard microscopes can present significant complexity and cost. We present a straightforward and low‐cost approach to implement STORM and TIRF taking advantage of multimode optical fibres and multimode diode lasers to provide the required excitation light. Combined with open source software and relatively simple protocols to prepare samples for STORM, including the use of Vectashield for non‐TIRF imaging, this approach enables TIRF and STORM imaging of cells labelled with appropriate dyes or expressing suitable fluorescent proteins to become widely accessible at low cost.
Either modulated illumination or temporal fluctuation analysis can assist super‐resolution techniques in overcoming the diffraction limit of conventional optical microscopy. As they are not contradictory to each other, an effective combination of spatial and temporal super‐resolution mechanisms would further improve the resolution of fluorescent images. Here, a super‐resolution imaging method called fluctuation‐enhanced Airyscan technology (FEAST) is proposed, which achieves ~40 nm lateral imaging resolution and is useful for a range of fluorescent proteins and organic dyes. It was demonstrated not only to obtain different subcellular super‐resolution images, but also to improve the accuracy of counting the average human epidermal growth factor receptor 2 (HER2) copy number for diagnosis in breast cancer. Furthermore, the combination of FEAST and sample expansion microscopy (Ex‐FEAST) improves the lateral resolution to ~26 nm. 相似文献
Recently developed super‐resolution microscopy techniques are changing our understanding of lipid rafts and membrane organisation in general. The lipid raft hypothesis postulates that cholesterol can drive the formation of ordered domains within the plasma membrane of cells, which may serve as platforms for cell signalling and membrane trafficking. There is now a wealth of evidence for these domains. However, their study has hitherto been hampered by the resolution limit of optical microscopy, making the definition of their properties problematic and contentious. New microscopy techniques circumvent the resolution limit and, for the first time, allow the fluorescence imaging of structures on length scales below 200 nm. This review describes such techniques, particularly as applied to the study of membrane organisation, synthesising newly emerging facets of lipid raft biology into a state‐of‐the art model. Editor's suggested further reading in BioEssays: Super‐resolution imaging prompts re‐thinking of cell biology mechanisms Abstract and Quantitative analysis of photoactivated localization microscopy (PALM) datasets using pair‐correlation analysis Abstract 相似文献
Opioid receptors are important pharmacological targets for the management of numerous medical conditions (eg, severe pain), but they are also the gateway to the development of deleterious side effects (eg, opiate addiction). Opioid receptor signaling cascades are well characterized. However, quantitative information regarding their lateral dynamics and nanoscale organization in the plasma membrane remains limited. Since these dynamic properties are important determinants of receptor function, it is crucial to define them. Herein, the nanoscale lateral dynamics and spatial organization of kappa opioid receptor (KOP), wild type mu opioid receptor (MOPwt), and its naturally occurring isoform (MOPN40D) were quantitatively characterized using fluorescence correlation spectroscopy and photoactivated localization microscopy. Obtained results, supported by ensemble‐averaged Monte Carlo simulations, indicate that these opioid receptors dynamically partition into different domains. In particular, significant exclusion from GM1 ganglioside‐enriched domains and partial association with cholesterol‐enriched domains was observed. Nanodomain size, receptor population density and the fraction of receptors residing outside of nanodomains were receptor‐specific. KOP‐containing domains were the largest and most densely populated, with the smallest fraction of molecules residing outside of nanodomains. The opposite was true for MOPN40D. Moreover, cholesterol depletion dynamically regulated the partitioning of KOP and MOPwt, whereas this effect was not observed for MOPN40D. 相似文献
