Browse
Submit Data
Databases
API
Help

Dataset Information

0 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Effect of Vectashield-induced fluorescence quenching on conventional and super-resolution microscopy

ABSTRACT: In our upcoming publication "Effect of Vectashield-induced fluorescence quenching on conventional and super-resolution microscopy", we quantified dSTORM parameters of AF647- and AF(+)647-labelled cells in different imaging media - Vectashield (VS), 25% Vectashield (25% VS) and GLOX BME. Quantification was done for three targets: tubulin ?3 and neurofilament light chain (NfL) in ND7/23 cells and ?II spectrin in primary mouse cortical neurons. Here, we present raw dSTORM movies and corresponding molecule lists that were used for the analysis. Results of the analysis are shown in Figure 7 of the manuscript.

SUBMITTER: Ivana Niki?-Spiegel

PROVIDER: S-BIAD16 | bioimages | 2020 Apr

REPOSITORIES: bioimages

ACCESS DATA

Json Xml

Publications

Effect of Vectashield-induced fluorescence quenching on conventional and super-resolution microscopy.

Arsić Aleksandra A Stajković Nevena N Spiegel Rainer R Nikić-Spiegel Ivana I

Scientific reports 20200415 1

Finding the right combination of a fluorescent dye and a mounting medium is crucial for optimal microscopy of fixed samples. It was recently shown that Vectashield, one of the most commonly used mounting media for conventional microscopy, can also be applied to super-resolution direct stochastic optical reconstruction microscopy (dSTORM). dSTORM utilizes conventional dyes and starts with samples in a fluorescent "ON" state. This helps in identifying structures of interest. Subsequently, labelled ...[more]

PMID: 32296095

Similar Datasets

Example-Based Super-Resolution Fluorescence Microscopy.

Project description:Capturing biological dynamics with high spatiotemporal resolution demands the advancement in imaging technologies. Super-resolution fluorescence microscopy offers spatial resolution surpassing the diffraction limit to resolve near-molecular-level details. While various strategies have been reported to improve the temporal resolution of super-resolution imaging, all super-resolution techniques are still fundamentally limited by the trade-off associated with the longer image acquisition time that is needed to achieve higher spatial information. Here, we demonstrated an example-based, computational method that aims to obtain super-resolution images using conventional imaging without increasing the imaging time. With a low-resolution image input, the method provides an estimate of its super-resolution image based on an example database that contains super- and low-resolution image pairs of biological structures of interest. The computational imaging of cellular microtubules agrees approximately with the experimental super-resolution STORM results. This new approach may offer potential improvements in temporal resolution for experimental super-resolution fluorescence microscopy and provide a new path for large-data aided biomedical imaging.

| S-EPMC5913229 | biostudies-literature

Effect of Vectashield-induced fluorescence quenching on conventional and super-resolution microscopy.

Project description:Finding the right combination of a fluorescent dye and a mounting medium is crucial for optimal microscopy of fixed samples. It was recently shown that Vectashield, one of the most commonly used mounting media for conventional microscopy, can also be applied to super-resolution direct stochastic optical reconstruction microscopy (dSTORM). dSTORM utilizes conventional dyes and starts with samples in a fluorescent "ON" state. This helps in identifying structures of interest. Subsequently, labelled samples are induced into blinking, which is necessary for determining the position of single molecules and reconstruction of super-resolution images. This is only possible with certain fluorescent dyes and imaging buffers. One of the most widely used dyes for dSTORM, Alexa Fluor 647 (AF647), blinks in Vectashield. However, after preparing immunocytochemical samples in Vectashield, we noticed that the fluorescence intensity of AF647 is quenched. This is particularly evident for dimmer immunostainings, such as stainings of some components of neuronal cytoskeleton and axonal initial segment. Because structures of interest cannot be identified in quenched samples, loss of fluorescence intensity hinders imaging of AF647 in Vectashield. This has consequences for both conventional and dSTORM imaging. To overcome this, we provide: 1) a quantitative analysis of AF647 intensity in different imaging media, 2) a quantitative analysis of the suitability of Vectashield for dSTORM imaging of high and low-abundance AF647-labelled targets. Furthermore, for the first time, we quantitatively analyse the performance of Alexa Fluor Plus 647, a new variant of AF647-conjugated antibody, in dSTORM imaging.

| S-EPMC7160131 | biostudies-literature

Spatial covariance reconstructive (SCORE) super-resolution fluorescence microscopy.

Project description:Super-resolution fluorescence microscopy has become a powerful tool to resolve structural information that is not accessible to traditional diffraction-limited imaging techniques such as confocal microscopy. Stochastic optical reconstruction microscopy (STORM) and photoactivation localization microscopy (PALM) are promising super-resolution techniques due to their relative ease of implementation and instrumentation on standard microscopes. However, the application of STORM is critically limited by its long sampling time. Several recent works have been focused on improving the STORM imaging speed by making use of the information from emitters with overlapping point spread functions (PSF). In this work, we present a fast and efficient algorithm that takes into account the blinking statistics of independent fluorescence emitters. We achieve sub-diffraction lateral resolution of 100 nm from 5 to 7 seconds of imaging. Our method is insensitive to background and can be applied to different types of fluorescence sources, including but not limited to the organic dyes and quantum dots that we demonstrate in this work.

| S-EPMC4005777 | biostudies-literature

Super-resolution fluorescence microscopy by stepwise optical saturation.

Project description:Super-resolution fluorescence microscopy is an important tool in biomedical research for its ability to discern features smaller than the diffraction limit. However, due to its difficult implementation and high cost, the super-resolution microscopy is not feasible in many applications. In this paper, we propose and demonstrate a saturation-based super-resolution fluorescence microscopy technique that can be easily implemented and requires neither additional hardware nor complex post-processing. The method is based on the principle of stepwise optical saturation (SOS), where M steps of raw fluorescence images are linearly combined to generate an image with a [Formula: see text]-fold increase in resolution compared with conventional diffraction-limited images. For example, linearly combining (scaling and subtracting) two images obtained at regular powers extends the resolution by a factor of 1.4 beyond the diffraction limit. The resolution improvement in SOS microscopy is theoretically infinite but practically is limited by the signal-to-noise ratio. We perform simulations and experimentally demonstrate super-resolution microscopy with both one-photon (confocal) and multiphoton excitation fluorescence. We show that with the multiphoton modality, the SOS microscopy can provide super-resolution imaging deep in scattering samples.

| S-EPMC5905910 | biostudies-literature

Exchangeable HaloTag Ligands for Super-Resolution Fluorescence Microscopy.

Project description:The specific and covalent labeling of the protein HaloTag with fluorescent probes in living cells makes it a powerful tool for bioimaging. However, the irreversible attachment of the probe to HaloTag precludes imaging applications that require transient binding of the probe and comes with the risk of irreversible photobleaching. Here, we introduce exchangeable ligands for fluorescence labeling of HaloTag (xHTLs) that reversibly bind to HaloTag and that can be coupled to rhodamines of different colors. In stimulated emission depletion (STED) microscopy, probe exchange of xHTLs allows imaging with reduced photobleaching as compared to covalent HaloTag labeling. Transient binding of fluorogenic xHTLs to HaloTag fusion proteins enables points accumulation for imaging in nanoscale topography (PAINT) and MINFLUX microscopy. We furthermore introduce pairs of xHTLs and HaloTag mutants for dual-color PAINT and STED microscopy. xHTLs thus open up new possibilities in imaging across microscopy platforms for a widely used labeling approach.

| S-EPMC9912333 | biostudies-literature

Simultaneous spatiotemporal super-resolution and multi-parametric fluorescence microscopy.

Project description:Super-resolution microscopy and single molecule fluorescence spectroscopy require mutually exclusive experimental strategies optimizing either temporal or spatial resolution. To achieve both, we implement a GPU-supported, camera-based measurement strategy that highly resolves spatial structures (~100 nm), temporal dynamics (~2 ms), and molecular brightness from the exact same data set. Simultaneous super-resolution of spatial and temporal details leads to an improved precision in estimating the diffusion coefficient of the actin binding polypeptide Lifeact and corrects structural artefacts. Multi-parametric analysis of epidermal growth factor receptor (EGFR) and Lifeact suggests that the domain partitioning of EGFR is primarily determined by EGFR-membrane interactions, possibly sub-resolution clustering and inter-EGFR interactions but is largely independent of EGFR-actin interactions. These results demonstrate that pixel-wise cross-correlation of parameters obtained from different techniques on the same data set enables robust physicochemical parameter estimation and provides biological knowledge that cannot be obtained from sequential measurements.

| S-EPMC7979808 | biostudies-literature

Super-resolution vibrational microscopy by stimulated Raman excited fluorescence.

Project description:Inspired by the revolutionary impact of super-resolution fluorescence microscopy, super-resolution Raman imaging has been long pursued because of its much higher chemical specificity than the fluorescence counterpart. However, vibrational contrasts are intrinsically less sensitive compared with fluorescence, resulting in only mild resolution enhancement beyond the diffraction limit even with strong laser excitation power. As such, it is still a great challenge to achieve biocompatible super-resolution vibrational imaging in the optical far-field. In 2019 Stimulated Raman Excited Fluorescence (SREF) was discovered as an ultrasensitive vibrational spectroscopy that combines the high chemical specificity of Raman scattering and the superb sensitivity of fluorescence detection. Herein we developed a novel super-resolution vibrational imaging method by harnessing SREF as the contrast mechanism. We first identified the undesired role of anti-Stokes fluorescence background in preventing direct adoption of super-resolution fluorescence technique. We then devised a frequency-modulation (FM) strategy to remove the broadband backgrounds and achieved high-contrast SREF imaging. Assisted by newly synthesized SREF dyes, we realized multicolor FM-SREF imaging with nanometer spectral resolution. Finally, by integrating stimulated emission depletion (STED) with background-free FM-SREF, we accomplished high-contrast super-resolution vibrational imaging with STED-FM-SREF whose spatial resolution is only determined by the signal-to-noise ratio. In our proof-of-principle demonstration, more than two times of resolution improvement is achieved in biological systems with moderate laser excitation power, which shall be further refined with optimized instrumentation and imaging probes. With its super resolution, high sensitivity, vibrational contrast, and mild laser excitation power, STED-FM-SREF microscopy is envisioned to aid a wide variety of applications.

| S-EPMC8058038 | biostudies-literature

Single-shot super-resolution total internal reflection fluorescence microscopy.

Project description:We combined instant structured illumination microscopy (iSIM) with total internal reflection fluorescence microscopy (TIRFM) in an approach referred to as instant TIRF-SIM, thereby improving the lateral spatial resolution of TIRFM to 115 ± 13 nm without compromising speed, and enabling imaging frame rates up to 100 Hz over hundreds of time points. We applied instant TIRF-SIM to multiple live samples and achieved rapid, high-contrast super-resolution imaging close to the coverslip surface.

| S-EPMC7470603 | biostudies-literature

Multiple signal classification algorithm for super-resolution fluorescence microscopy.

Project description:Single-molecule localization techniques are restricted by long acquisition and computational times, or the need of special fluorophores or biologically toxic photochemical environments. Here we propose a statistical super-resolution technique of wide-field fluorescence microscopy we call the multiple signal classification algorithm which has several advantages. It provides resolution down to at least 50 nm, requires fewer frames and lower excitation power and works even at high fluorophore concentrations. Further, it works with any fluorophore that exhibits blinking on the timescale of the recording. The multiple signal classification algorithm shows comparable or better performance in comparison with single-molecule localization techniques and four contemporary statistical super-resolution methods for experiments of in vitro actin filaments and other independently acquired experimental data sets. We also demonstrate super-resolution at timescales of 245 ms (using 49 frames acquired at 200 frames per second) in samples of live-cell microtubules and live-cell actin filaments imaged without imaging buffers.

| S-EPMC5155148 | biostudies-literature

Towards correlative super-resolution fluorescence and electron cryo-microscopy.

Project description:Correlative light and electron microscopy (CLEM) has become a powerful tool in life sciences. Particularly cryo-CLEM, the combination of fluorescence cryo-microscopy (cryo-FM) permitting for non-invasive specific multi-colour labelling, with electron cryo-microscopy (cryo-EM) providing the undisturbed structural context at a resolution down to the Ångstrom range, has enabled a broad range of new biological applications. Imaging rare structures or events in crowded environments, such as inside a cell, requires specific fluorescence-based information for guiding cryo-EM data acquisition and/or to verify the identity of the structure of interest. Furthermore, cryo-CLEM can provide information about the arrangement of specific proteins in the wider structural context of their native nano-environment. However, a major obstacle of cryo-CLEM currently hindering many biological applications is the large resolution gap between cryo-FM (typically in the range of ∼400 nm) and cryo-EM (single nanometre to the Ångstrom range). Very recently, first proof of concept experiments demonstrated the feasibility of super-resolution cryo-FM imaging and the correlation with cryo-EM. This opened the door towards super-resolution cryo-CLEM, and thus towards direct correlation of structural details from both imaging modalities.

| S-EPMC5524168 | biostudies-literature

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data