Browse
Submit Data
Databases
API
Help

Dataset Information

44 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

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

ABSTRACT: 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.

SUBMITTER: Guo M

PROVIDER: S-EPMC7470603 | biostudies-literature | 2018 Jun

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Publications

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

Guo Min M Chandris Panagiotis P Giannini John Paul JP Trexler Adam J AJ Fischer Robert R Chen Jiji J Vishwasrao Harshad D HD Rey-Suarez Ivan I Wu Yicong Y Wu Xufeng X Waterman Clare M CM Patterson George H GH Upadhyaya Arpita A Taraska Justin W JW Shroff Hari H

Nature methods 20180507 6

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. ...[more]

PMID: 29735999

Similar Datasets

Single-shot, shadowless total internal reflection fluorescence microscopy via annular fiber bundle.

Project description:We demonstrate a method of generating instantaneous and uniform total internal reflection fluorescence (TIRF) excitation by using an annular fiber bundle and spatially incoherent light sources. We show the flexibility of our method in that it can generate TIRF excitation with either a laser light source or an LED of different wavelengths, and facilitate switching between TIRF and epi illumination. In this report we detail the design of the fiber bundle, then demonstrate the performance via single-molecule imaging in the presence of high background and high throughput, and uniform TIRF imaging of cells over a large field of view. Our versatile method will enable quantitative shadowless TIRF imaging.

| S-EPMC8323474 | biostudies-literature

Measurement of single macromolecule orientation by total internal reflection fluorescence polarization microscopy.

Project description:A new approach is presented for measuring the three-dimensional orientation of individual macromolecules using single molecule fluorescence polarization (SMFP) microscopy. The technique uses the unique polarizations of evanescent waves generated by total internal reflection to excite the dipole moment of individual fluorophores. To evaluate the new SMFP technique, single molecule orientation measurements from sparsely labeled F-actin are compared to ensemble-averaged orientation data from similarly prepared densely labeled F-actin. Standard deviations of the SMFP measurements taken at 40 ms time intervals indicate that the uncertainty for individual measurements of axial and azimuthal angles is approximately 10 degrees at 40 ms time resolution. Comparison with ensemble data shows there are no substantial systematic errors associated with the single molecule measurements. In addition to evaluating the technique, the data also provide a new measurement of the torsional rigidity of F-actin. These measurements support the smaller of two values of the torsional rigidity of F-actin previously reported.

| S-EPMC1366610 | biostudies-literature

Total internal reflection fluorescence microscopy imaging-guided confocal single-molecule fluorescence spectroscopy.

Project description:We have developed an integrated spectroscopy system combining total internal reflection fluorescence microscopy imaging with confocal single-molecule fluorescence spectroscopy for two-dimensional interfaces. This spectroscopy approach is capable of both multiple molecules simultaneously sampling and in situ confocal fluorescence dynamics analyses of individual molecules of interest. We have demonstrated the calibration with fluorescent microspheres, and carried out single-molecule spectroscopy measurements. This integrated single-molecule spectroscopy is powerful in studies of single molecule dynamics at interfaces of biological and chemical systems.

| S-EPMC3277604 | biostudies-other

Total internal reflection fluorescence (TIRF) microscopy of Chlamydomonas flagella.

Project description:The eukaryotic flagellum is host to a variety of dynamic behaviors, including flagellar beating, the motility of glycoproteins in the flagellar membrane, and intraflagellar transport (IFT), the bidirectional traffic of protein particles between the flagellar base and tip. IFT is of particular interest, as it plays integral roles in flagellar length control, cell signaling, development, and human disease. However, our ability to understand dynamic flagellar processes such as IFT is limited in large part by the fidelity with which we can image these behaviors in living cells. This chapter introduces the application of total internal reflection fluorescence (TIRF) microscopy to visualize the flagella of Chlamydomonas reinhardtii. The advantages and challenges of TIRF are discussed in comparison to confocal and differential interference contrast techniques. This chapter also reviews current IFT insights gleaned from TIRF microscopy of Chlamydomonas and provides an outlook on the future of the technique, with particular emphasis on combining TIRF with other emerging imaging technologies.

| S-EPMC3686088 | biostudies-literature

Two forms of single-vesicle astrocyte exocytosis imaged with total internal reflection fluorescence microscopy.

Project description:Transmitters such as glutamate and ATP are released from brain astrocytes. Several pathways for their release have been proposed, including exocytosis. In the present study we sought to measure exocytosis from astrocytes with single vesicle imaging methods using synaptopHlourin (SpH) as an optical reporter. We imaged single SpH-laden vesicles with total internal reflection fluorescence (TIRF) microscopy. We observed spontaneous, as well as evoked, single-vesicle exocytosis events. Analysis of the kinetics and spatial spread associated with these events indicated two discernible forms of single vesicle exocytosis. One form, constituting approximately 40% of the spontaneous events, was akin to kiss-and-run vesicle fusion and captured a mobile proton buffer from the extracellular medium. The other form seems to represent full vesicle fusion, constitutes approximately 60% of the spontaneous events, and is associated with complete mixing of the vesicle and plasma membranes. Activation of calcium-mobilizing receptors on the astrocyte surface selected between the different forms of exocytosis. These data provide evidence for two forms of simultaneously occurring single-vesicle exocytosis events in astrocytes, and also suggest that SpH imaging and TIRF microscopy is useful to study the mechanisms of astrocyte transmitter release.

| S-EPMC1820734 | biostudies-literature

Quantifying F-actin patches in single melanoma cells using total-internal reflection fluorescence microscopy

Project description:Total-internal reflection fluorescence (TIRF) microscope is a unique technique for selective excitation of only those fluorophore molecules in a cellular environment, which are located at the sub-diffraction axial distance of a cell’s contact-area. Despite this prominent feature of the TIRF microscope, making quantitative use of this technique has been a challenge, since the excitation intensity strongly depends on the axial position of a fluorophore molecule. Here, we present an easy-implemented data analysis method to quantitatively characterize the fluorescent signal, without considering the intensity-value. We use F-actin patches in single-melanoma cells as an example and define two quantities of elongation and surface density for F-actin patches at the contact-area of a melanoma cell. The elongation parameter can evaluate the dispersion of F-actin patches at the contact-area of a cell and is useful to classify the attaching, spreading, and expanding stages of a cell. Following that, we present the profile of the surface density of F-actin patches as a quantity to probe the spatio-temporal distribution of the F-actin patches at the contact-area of a cell. The data analysis methods that are proposed here will also be applicable in the image analysis of the other advanced optical microscopic methods.

| S-EPMC9678867 | biostudies-literature

Fast high-resolution 3D total internal reflection fluorescence microscopy by incidence angle scanning and azimuthal averaging.

Project description:Total internal reflection fluorescence microscopy (TIRFM) is the method of choice to visualize a variety of cellular processes in particular events localized near the plasma membrane of live adherent cells. This imaging technique not relying on particular fluorescent probes provides a high sectioning capability. It is, however, restricted to a single plane. We present here a method based on a versatile design enabling fast multiwavelength azimuthal averaging and incidence angles scanning to computationally reconstruct 3D images sequences. We achieve unprecedented 50-nm axial resolution over a range of 800 nm above the coverslip. We apply this imaging modality to obtain structural and dynamical information about 3D actin architectures. We also temporally decipher distinct Rab11a-dependent exocytosis events in 3D at a rate of seven stacks per second.

| S-EPMC4260613 | biostudies-literature

Total internal reflection fluorescence microscopy of intraflagellar transport in Tetrahymena thermophila.

Project description:Live imaging has become a powerful tool in studies of ciliary proteins. Tetrahymena thermophila is an established ciliated model with well-developed genetic and biochemical approaches, but its large size, complex shape, and the large number of short and overlapping cilia, have made live imaging of ciliary proteins challenging. Here we describe a method that combines paralysis of cilia by nickel ions and total internal reflection microscopy for live imaging of fluorescent proteins inside cilia of Tetrahymena. Using this method, we quantitatively documented the intraflagellar transport in Tetrahymena.

| S-EPMC5301313 | biostudies-literature

Using total internal reflection fluorescence microscopy to visualize rhodopsin-containing cells.

Project description:Sunlight is captured and converted to chemical energy in illuminated environments. Although (bacterio)chlorophyll-based photosystems have been characterized in detail, retinal-based photosystems, rhodopsins, have only recently been identified as important mediators of light energy capture and conversion. Recent estimates suggest that up to 70% of cells in some environments harbor rhodopsins. However, because rhodopsin autofluorescence is low-comparable to that of carotenoids and significantly less than that of (bacterio)chlorophylls-these estimates are based on metagenomic sequence data, not direct observation. We report here the use of ultrasensitive total internal reflection fluorescence (TIRF) microscopy to distinguish between unpigmented, carotenoid-producing, and rhodopsin-expressing bacteria. Escherichia coli cells were engineered to produce lycopene, β-carotene, or retinal. A gene encoding an uncharacterized rhodopsin, actinorhodopsin, was cloned into retinal-producing E. coli. The production of correctly folded and membrane-incorporated actinorhodopsin was confirmed via development of pink color in E. coli and SDS-PAGE. Cells expressing carotenoids or actinorhodopsin were imaged by TIRF microscopy. The 561-nm excitation laser specifically illuminated rhodopsin-containing cells, allowing them to be differentiated from unpigmented and carotenoid-containing cells. Furthermore, water samples collected from the Delaware River were shown by PCR to have rhodopsin-containing organisms and were examined by TIRF microscopy. Individual microorganisms that fluoresced under illumination from the 561-nm laser were identified. These results verify the sensitivity of the TIRF microscopy method for visualizing and distinguishing between different molecules with low autofluorescence, making it useful for analyzing natural samples.

| S-EPMC4407218 | biostudies-literature

Imaging with total internal reflection fluorescence microscopy for the cell biologist.

Project description:Total internal reflection fluorescence (TIRF) microscopy can be used in a wide range of cell biological applications, and is particularly well suited to analysis of the localization and dynamics of molecules and events near the plasma membrane. The TIRF excitation field decreases exponentially with distance from the cover slip on which cells are grown. This means that fluorophores close to the cover slip (e.g. within ~100 nm) are selectively illuminated, highlighting events that occur within this region. The advantages of using TIRF include the ability to obtain high-contrast images of fluorophores near the plasma membrane, very low background from the bulk of the cell, reduced cellular photodamage and rapid exposure times. In this Commentary, we discuss the applications of TIRF to the study of cell biology, the physical basis of TIRF, experimental setup and troubleshooting.

| S-EPMC2964103 | 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