Super‐resolution microscopy techniques can provide answers to still pending questions on prokaryotic organisms but are yet to be used at their full potential for this purpose. To address this, we evaluate the ability of the rhodamine‐like KK114 dye to label various types of bacteria, to enable imaging of fine structural details with stimulated emission depletion microscopy (STED). We assessed fluorescent labeling with KK114 for eleven Gram‐positive and Gram‐negative bacterial species and observed that this contrast agent binds to their cell membranes. Significant differences in the labeling outputs were noticed across the tested bacterial species, but importantly, KK114‐staining allowed the observation of subtle nanometric cell details in some cases. For example, a helix pattern resembling a cytoskeleton arrangement was detected in Bacillus subtilis. Furthermore, we found that KK114 easily penetrates the membrane of bacterial microorganism that lost their viability, which can be useful to discriminate between living and dead cells. 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
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
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
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 ).
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
Molecular bioswitches offer an invaluable asset in the shift from systemic to targeted treatments. Within the growing arsenal of switches are imaging probes that functionalize only in given locations or situations. Acetate esters are a common fluorescent example, known to activate upon interaction with esterases. Fluorescein diacetate (FDA) is one such fluorophore used in cell viability assays. These assays rely on the fact that the compound begins colorless and with no fluorescent signature whatsoever, and only after internalization into cells it is possible to detect a fluorescence signal. In this study, using fluorescence intensity (FI) and fluorescence lifetime (FLT) imaging, FDA is shown to be fluorescent even when unactivated. Furthermore, the FLT is shown to change with pH. Finally, the ability to image FDA in different environments simulated by tissue‐imitating phantoms is explored. Altogether, the ability of FDA to serve as a bioswitch when measured using FLT imaging microscopy (FLIM) is assessed. The combination of a spectrum of FDA activation and FLIM serves as a bioswitch, where biologically relevant stimulation can generate detectable and incremental variations. 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
Expansion microscopy is a super‐resolution method that allows expanding uniformly biological samples, by increasing the relative distances among fluorescent molecules labeling specific components. One of the main concerns in this approach regards the isotropic behavior at the nanoscale. The present study aims to determine the robustness of such a technique, quantifying the expansion parameters i.e. scale factor, isotropy, uniformity. Our focus is on the nuclear pore complex (NPC), as well‐known nanoscale component endowed of a preserved and symmetrical structure localized on the nuclear envelope. Here, we show that Nup153 is a good reporter to quantitatively address the isotropy of the expansion process. The quantitative analysis carried out on NPCs, at different spatial scales, allows concluding that expansion microscopy can be used at the nanoscale to measure subcellular features with an accuracy from 10 to 5 nm. Therefore, it is an excellent method for structural studies of macromolecular complexes. 相似文献
Visualizing fine neuronal structures deep inside strongly light‐scattering brain tissue remains a challenge in neuroscience. Recent nanoscopy techniques have reached the necessary resolution but often suffer from limited imaging depth, long imaging time or high light fluence requirements. Here, we present two‐photon super‐resolution patterned excitation reconstruction (2P‐SuPER) microscopy for 3‐dimensional imaging of dendritic spine dynamics at a maximum demonstrated imaging depth of 130 μm in living brain tissue with approximately 100 nm spatial resolution. We confirmed 2P‐SuPER resolution using fluorescence nanoparticle and quantum dot phantoms and imaged spiny neurons in acute brain slices. We induced hippocampal plasticity and showed that 2P‐SuPER can resolve increases in dendritic spine head sizes on CA1 pyramidal neurons following theta‐burst stimulation of Schaffer collateral axons. 2P‐SuPER further revealed nanoscopic increases in dendritic spine neck widths, a feature of synaptic plasticity that has not been thoroughly investigated due to the combined limit of resolution and penetration depth in existing imaging technologies. 相似文献
One of the main challenges for laser‐scanning microscopy of biological tissues with refractive heterogeneities is the degradation in spatial resolution that occurs as a result of beam steering and distortion. This challenge is particularly significant for dual‐axis confocal (DAC) microscopy, which achieves improved spatial‐filtering and optical‐sectioning performance over traditional confocal microscopy through off‐axis illumination and collection of light with low‐numerical aperture (NA) beams that must intersect precisely at their foci within tissues. DAC microscope image quality is sensitive to positional changes and distortions of these illumination‐ and collection‐beam foci. Previous studies have shown that Bessel beams display improved positional stability and beam quality than Gaussian beams when propagating through tissues with refractive heterogeneities, which suggests that Bessel‐beam illumination may enhance DAC microscopy of such tissues. Here, we utilize both Gaussian and Bessel illumination in a point‐scanned DAC microscope and quantify the resultant degradation in resolution when imaging within heterogeneous optical phantoms and fresh tissues. Results indicate that DAC microscopy with Bessel illumination exhibits reduced resolution degradation from microscopic tissue heterogeneities compared to DAC microscopy with conventional Gaussian illumination.
The advent of super‐resolution microscopy allowed for new insights into cellular and physiological processes of normal and diseased cells. In this study, we report for the first time on the super‐resolved DNA structure of buccal cells from patients with Alzheimer's disease (AD) versus age‐ and gender‐matched healthy, non‐caregiver controls. In this super‐resolution study cohort of 74 participants, buccal cells were collected and their spatial DNA organization in the nucleus examined by 3D Structured Illumination Microscopy (3D‐SIM). Quantitation of the super‐resolution DNA structure revealed that the nuclear super‐resolution DNA structure of individuals with AD significantly differs from that of their controls (p < 0.05) with an overall increase in the measured DNA‐free/poor spaces. This represents a significant increase in the interchromatin compartment. We also find that the DNA structure of AD significantly differs in mild, moderate, and severe disease with respect to the DNA‐containing and DNA‐free/poor spaces. We conclude that whole genome remodeling is a feature of buccal cells in AD. 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
Ex vivo confocal laser scanning microscopy (ex vivo CLSM) offers an innovative diagnostic approach through vertical scanning of skin samples with a resolution close to conventional histology. In addition, it enables fluorescence detection in tissues. We aimed to assess the applicability of ex vivo CLSM in the detection of vascular immune complexes in cutaneous vasculitis and to compare its diagnostic accuracy with direct immunofluorescence (DIF) microscopy. Eighty‐two sections of 49 vasculitis patients with relevant DIF microscopy findings were examined using ex vivo CLSM following staining with fluorescent‐labeled IgG, IgM, IgA, C3 and fibrinogen antibodies. DIF microscopy showed immunoreactivity of vessels with IgG, IgM, IgA, C3 and Fibrinogen in 2.0%, 49.9%, 12.2%, 59.2% and 44.9% of the patients, respectively. Ex vivo CLSM detected positive vessels with the same antibodies in 2.0%, 38.8%, 8.2%, 42.9% and 36.7% of the patients, respectively. The detection rate of positive superficial dermal vessels was significantly higher in DIF microscopy as compared to ex vivo CLSM (P < .05). Whereas, ex vivo CLSM identified positive deep dermal vessels more frequently compared to DIF microscopy. In conclusion, ex vivo CLSM could identify specific binding of the antibodies in vessels and showed a comparable performance to conventional DIF microscopy in diagnosing vasculitis. 相似文献
Confocal microscopy is an indispensable tool for biological imaging due to its high resolution and optical sectioning capability. However, its slow imaging speed and severe photobleaching have largely prevented further applications. Here, we present dual inclined beam line‐scanning (LS) confocal microscopy. The reduced excitation intensity of our imaging method enabled a 2‐fold longer observation time of fluorescence compared to traditional LS microscopy while maintaining a good sectioning capability and single‐molecule sensitivity. We characterized the performance of our method and applied it to subcellular imaging and three‐dimensional single‐molecule RNA imaging in mammalian cells. 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
Ex‐vivo confocal laser scanning microscopy (CLSM) is an emerging diagnostic tool allowing fast and easy microscopic tissue examination. The first generation of ex‐vivo devices have already shown promising results in the ex‐vivo evaluation of basal cell carcinoma compared to Mohs surgery. Nevertheless, for the diagnostics of pathological skin lesions the knowledge of normal skin features is essential. Therefore we examined 50 samples of healthy skin from various donor sites including head and neck (n = 25), trunk (n = 10), upper (n = 10) and lower extremities (n = 5) using a new generation ex‐vivo CLSM device offering three different laser wavelengths and compared the findings to the corresponding histological sections. In correlation with the histopathology we identified different layers of the epidermis, differentiated keratinocytes from melanocytes and described in detail skin appendages including hair follicle, sebaceous and sweat glands. Furthermore, structures of the dermis and subcutis were illustrated. Additionally, artefacts and pitfalls occurring with the use of ex‐vivo CLSM have been documented. The study offers an overview of the main ex‐vivo CLSM skin characteristics in comparison to the standard histological examination and helps to recognize and avoid common artefacts.
Anatomy of a hair follicle in the reflectance mode (RM) CLSM, fluorescence mode (FM) CLSM and in a routine hematoxylin‐eosin stained histological section (H). 相似文献
