Browse
Submit Data
Databases
API
Help

Dataset Information

28 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Multicolor super-resolution fluorescence imaging via multi-parameter fluorophore detection.

ABSTRACT: Understanding the complexity of the cellular environment will benefit from the ability to unambiguously resolve multiple cellular components, simultaneously and with nanometer-scale spatial resolution. Multicolor super-resolution fluorescence microscopy techniques have been developed to achieve this goal, yet challenges remain in terms of the number of targets that can be simultaneously imaged and the crosstalk between color channels. Herein, we demonstrate multicolor stochastic optical reconstruction microscopy (STORM) based on a multi-parameter detection strategy, which uses both the fluorescence activation wavelength and the emission color to discriminate between photo-activatable fluorescent probes. First, we obtained two-color super-resolution images using the near-infrared cyanine dye Alexa 750 in conjunction with a red cyanine dye Alexa 647, and quantified color crosstalk levels and image registration accuracy. Combinatorial pairing of these two switchable dyes with fluorophores which enhance photo-activation enabled multi-parameter detection of six different probes. Using this approach, we obtained six-color super-resolution fluorescence images of a model sample. The combination of multiple fluorescence detection parameters for improved fluorophore discrimination promises to substantially enhance our ability to visualize multiple cellular targets with sub-diffraction-limit resolution.

SUBMITTER: Bates M

PROVIDER: S-EPMC3353744 | biostudies-literature |

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Similar Datasets

Multicolor super-resolution DNA imaging for genetic analysis.

Project description:Many types of cancer and neurodegenerative diseases are caused by abnormalities and variations in the genome. We have designed a high-resolution imaging technique with high throughput and low cost for determining structural variations of genes related to genetic diseases. We initially mapped all seven nicking sites of Nb.BbvCI endonuclease enzyme on lambda DNA. Then we resolved densely labeled patterns of 107 nicking sites on human BAC DNA that is digested by Nb.BsmI and Nb.BbvCI endonuclease enzymes. This high density resulted in several dyes being closer together than the diffraction limit. Overall, detailed DNA nicking sites mapping with 100 bp resolution was achieved, which has the potential to reveal information about genetic variance and to facilitate medical diagnosis of several genetic diseases.

| S-EPMC3880789 | biostudies-literature

Multicolor super-resolution imaging with photo-switchable fluorescent probes.

Project description:Recent advances in far-field optical nanoscopy have enabled fluorescence imaging with a spatial resolution of 20 to 50 nanometers. Multicolor super-resolution imaging, however, remains a challenging task. Here, we introduce a family of photo-switchable fluorescent probes and demonstrate multicolor stochastic optical reconstruction microscopy (STORM). Each probe consists of a photo-switchable "reporter" fluorophore that can be cycled between fluorescent and dark states, and an "activator" that facilitates photo-activation of the reporter. Combinatorial pairing of reporters and activators allows the creation of probes with many distinct colors. Iterative, color-specific activation of sparse subsets of these probes allows their localization with nanometer accuracy, enabling the construction of a super-resolution STORM image. Using this approach, we demonstrate multicolor imaging of DNA model samples and mammalian cells with 20- to 30-nanometer resolution. This technique will facilitate direct visualization of molecular interactions at the nanometer scale.

| S-EPMC2633025 | biostudies-literature

Resolving Multi-Asperity Contacts at the Nanoscale through Super-Resolution Fluorescence Imaging.

Project description:Contact mechanics, spanning nanometer to tectonic scales, faces long-standing challenges arising from multiscale random roughness, which hinders experimental validation of theories. Understanding multi-asperity rough contacts is vital for addressing catastrophic consequences of these contacts failing such as earthquakes and for diverse technological applications. To visualize such contacts, we introduce a super-resolution microscopy method utilizing spontaneous millisecond ON/OFF fluorescence blinking of contact-sensitive molecular rotor molecules immobilized on a glass coverslip. This technique achieves ∼55 nm lateral imaging resolution for rough poly(methyl methacrylate) and glass spheres on glass contacts. For soft polymer spheres due to large plastic deformation, the resolution improvement does not significantly affect the area of real contact. However, for hard glass spheres, the real contact area is found to be 2.4 times smaller than that found by diffraction-limited imaging. This study highlights, through direct visualization, the impact of material stiffness on the nanoscale structure within the area of real contact.

| S-EPMC10895690 | biostudies-literature

Super-resolution multicolor fluorescence microscopy enabled by an apochromatic super-oscillatory lens with extended depth-of-focus.

Project description:Planar super-oscillatory lens (SOL), a far-field subwavelength-focusing diffractive device, holds great potential for achieving sub-diffraction-limit imaging at multiple wavelengths. However, conventional SOL devices suffer from a numerical-aperture-related intrinsic tradeoff among the depth of focus (DoF), chromatic dispersion and focusing spot size. Here, we apply a multi-objective genetic algorithm (GA) optimization approach to design an apochromatic binary-phase SOL having a prolonged DoF, customized working distance (WD), minimized main-lobe size, and suppressed side-lobe intensity. Experimental implementation demonstrates simultaneous focusing of blue, green and red light beams into an optical needle of ~0.5λ in diameter and DOF > 10λ at WD = 428 μm. By integrating this SOL device with a commercial fluorescence microscope, we perform, for the first time, three-dimensional super-resolution multicolor fluorescence imaging of the "unseen" fine structures of neurons. The present study provides not only a practical route to far-field multicolor super-resolution imaging but also a viable approach for constructing imaging systems avoiding complex sample positioning and unfavorable photobleaching.

| S-EPMC10444772 | biostudies-literature

Three-dimensional super-resolution fluorescence imaging of DNA.

Project description:Recent advances in fluorescence super-resolution microscopy are providing important insights into details of cellular structures. To acquire three dimensional (3D) super-resolution images of DNA, we combined binding activated localization microscopy (BALM) using fluorescent double-stranded DNA intercalators and optical astigmatism. We quantitatively establish the advantage of bis- over mono-intercalators before demonstrating the approach by visualizing single DNA molecules stretched between microspheres at various heights. Finally, the approach is applied to the more complex environment of intact and damaged metaphase chromosomes, unravelling their structural features.

| S-EPMC7385144 | 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

Multicolor fluorescence fluctuation spectroscopy in living cells via spectral detection.

Project description:Signaling pathways in biological systems rely on specific interactions between multiple biomolecules. Fluorescence fluctuation spectroscopy provides a powerful toolbox to quantify such interactions directly in living cells. Cross-correlation analysis of spectrally separated fluctuations provides information about intermolecular interactions but is usually limited to two fluorophore species. Here, we present scanning fluorescence spectral correlation spectroscopy (SFSCS), a versatile approach that can be implemented on commercial confocal microscopes, allowing the investigation of interactions between multiple protein species at the plasma membrane. We demonstrate that SFSCS enables cross-talk-free cross-correlation, diffusion, and oligomerization analysis of up to four protein species labeled with strongly overlapping fluorophores. As an example, we investigate the interactions of influenza A virus (IAV) matrix protein 2 with two cellular host factors simultaneously. We furthermore apply raster spectral image correlation spectroscopy for the simultaneous analysis of up to four species and determine the stoichiometry of ternary IAV polymerase complexes in the cell nucleus.

| S-EPMC8545396 | biostudies-literature

Imaging HIV-1 RNA dimerization in cells by multicolor super-resolution and fluctuation microscopies.

Project description:Dimerization is a unique and vital characteristic of retroviral genomes. It is commonly accepted that genomic RNA (gRNA) must be dimeric at the plasma membrane of the infected cells to be packaged during virus assembly. However, where, when and how HIV-1 gRNA find each other and dimerize in the cell are long-standing questions that cannot be answered using conventional approaches. Here, we combine two state-of-the-art, multicolor RNA labeling strategies with two single-molecule microscopy technologies to address these questions. We used 3D-super-resolution structured illumination microscopy to analyze and quantify the spatial gRNA association throughout the cell and monitored the dynamics of RNA-RNA complexes in living-cells by cross-correlation fluctuation analysis. These sensitive and complementary approaches, combined with trans-complementation experiments, reveal for the first time the presence of interacting gRNA in the cytosol, a challenging observation due to the low frequency of these events and their dilution among the bulk of other RNAs, and allow the determination of the subcellular orchestration of the HIV-1 dimerization process.

| S-EPMC5027490 | biostudies-literature

Multi-dimensional super-resolution imaging enables surface hydrophobicity mapping.

Project description:Super-resolution microscopy allows biological systems to be studied at the nanoscale, but has been restricted to providing only positional information. Here, we show that it is possible to perform multi-dimensional super-resolution imaging to determine both the position and the environmental properties of single-molecule fluorescent emitters. The method presented here exploits the solvatochromic and fluorogenic properties of nile red to extract both the emission spectrum and the position of each dye molecule simultaneously enabling mapping of the hydrophobicity of biological structures. We validated this by studying synthetic lipid vesicles of known composition. We then applied both to super-resolve the hydrophobicity of amyloid aggregates implicated in neurodegenerative diseases, and the hydrophobic changes in mammalian cell membranes. Our technique is easily implemented by inserting a transmission diffraction grating into the optical path of a localization-based super-resolution microscope, enabling all the information to be extracted simultaneously from a single image plane.

| S-EPMC5155161 | biostudies-literature

Fluorescence lifetime DNA-PAINT for multiplexed super-resolution imaging of cells.

Project description:DNA point accumulation for imaging in nanoscale topography (DNA-PAINT) is a powerful super-resolution technique highly suitable for multi-target (multiplexing) bio-imaging. However, multiplexed imaging of cells is still challenging due to the dense and sticky environment inside a cell. Here, we combine fluorescence lifetime imaging microscopy (FLIM) with DNA-PAINT and use the lifetime information as a multiplexing parameter for targets identification. In contrast to Exchange-PAINT, fluorescence lifetime PAINT (FL-PAINT) can image multiple targets simultaneously and does not require any fluid exchange, thus leaving the sample undisturbed and making the use of flow chambers/microfluidic systems unnecessary. We demonstrate the potential of FL-PAINT by simultaneous imaging of up to three targets in a cell using both wide-field FLIM and 3D time-resolved confocal laser scanning microscopy (CLSM). FL-PAINT can be readily combined with other existing techniques of multiplexed imaging and is therefore a perfect candidate for high-throughput multi-target bio-imaging.

| S-EPMC8752799 | 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