Browse
Submit Data
Databases
API
Help

Dataset Information

17 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Subcellular Euclidean distance measurements with multicolor fluorescence localization imaging in cultured cells.

ABSTRACT: This protocol measures the 3D Euclidean distance (Δ_3D) between two/three fluorescently labeled kinetochore components in fixed samples using Kinetochore Delta software (KiDv1.0.1, MATLAB based). Overestimation of mean Δ_3D is corrected through a Bayesian algorithm, with Δ_EC distances reflecting the ensemble average positions of fluorophores within a kinetochore population. This package also enables kinetochore categorization, which can be used to sub-sample kinetochores and measure Δ_EC. Together, this allows the dynamic architecture of human kinetochores to be investigated (tested in hTERT-RPE1 cells). For complete details on the use and execution of this protocol, please refer to Roscioli et al. (2020).

SUBMITTER: Germanova TE

PROVIDER: S-EPMC8605398 | biostudies-literature |

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Similar Datasets

Nanoscale subcellular architecture revealed by multicolor three-dimensional salvaged fluorescence imaging.

Project description:Combining the molecular specificity of fluorescent probes with three-dimensional imaging at nanoscale resolution is critical for investigating the spatial organization and interactions of cellular organelles and protein complexes. We present a 4Pi single-molecule switching super-resolution microscope that enables ratiometric multicolor imaging of mammalian cells at 5-10-nm localization precision in three dimensions using 'salvaged fluorescence'. Imaging two or three fluorophores simultaneously, we show fluorescence images that resolve the highly convoluted Golgi apparatus and the close contacts between the endoplasmic reticulum and the plasma membrane, structures that have traditionally been the imaging realm of electron microscopy. The salvaged fluorescence approach is equally applicable in most single-objective microscopes.

| S-EPMC7028321 | biostudies-literature

Nanometer distance measurements between multicolor quantum dots.

Project description:Quantum dot dimers made of short double-stranded DNA molecules labeled with different color quantum dots at each end were imaged using multicolor stage-scanning confocal microscopy. This approach eliminates chromatic aberration and color registration issues usually encountered in other multicolor imaging techniques. We demonstrate nanometer accuracy in individual distance measurement by suppression of quantum dot blinking and thoroughly characterize the contribution of different effects to the variability observed between measurements. Our analysis opens the way to accurate structural studies of biomolecules and biomolecular complexes using multicolor quantum labeling.

| S-EPMC2748936 | biostudies-other

Spatial implications associated with using Euclidean distance measurements and geographic centroid imputation in health care research.

Project description:ObjectiveTo determine the effect of using Euclidean measurements and zip-code centroid geo-imputation versus more precise spatial analytical techniques in health care research.Data sourcesCommercially insured members from a southeastern managed care organization.Study designDistance from admitting inpatient facility to member's home and zip-code centroid (geographic placement) was compared using Euclidean straight-line and shortest-path drive distances (measurement technique).Data collectionAdministrative claims from October 2005 to September 2006.Principal findingsMeasurement technique had a greater impact on distance values compared with geographic placement. Drive distance from the geocoded address was highly correlated (r=0.99) with the Euclidean distance from the zip-code centroid.ConclusionsActual differences were relatively small. Researchers without capabilities to produce drive distance measurements and/or address geocoding techniques could rely on simple linear regressions to estimate correction factors with a high degree of confidence.

| S-EPMC2813451 | biostudies-literature

Ultraprecise single-molecule localization microscopy enables in situ distance measurements in intact cells.

Project description:Single-molecule localization microscopy (SMLM) has the potential to quantify the diversity in spatial arrangements of molecules in intact cells. However, this requires that the single-molecule emitters are localized with ultrahigh precision irrespective of the sample format and the length of the data acquisition. We advance SMLM to enable direct distance measurements between molecules in intact cells on the scale between 1 and 20 nm. Our actively stabilized microscope combines three-dimensional real-time drift corrections and achieves a stabilization of <1 nm and localization precision of ~1 nm. To demonstrate the biological applicability of the new microscope, we show a 4- to 7-nm difference in spatial separations between signaling T cell receptors and phosphatases (CD45) in active and resting T cells. In summary, by overcoming the major bottlenecks in SMLM imaging, it is possible to generate molecular images with nanometer accuracy and conduct distance measurements on the biological relevant length scales.

| S-EPMC7164934 | biostudies-literature

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

Project description: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.

| S-EPMC3353744 | biostudies-literature

Generating triangulated macromolecular surfaces by Euclidean Distance Transform.

Project description:Macromolecular surfaces are fundamental representations of their three-dimensional geometric shape. Accurate calculation of protein surfaces is of critical importance in the protein structural and functional studies including ligand-protein docking and virtual screening. In contrast to analytical or parametric representation of macromolecular surfaces, triangulated mesh surfaces have been proved to be easy to describe, visualize and manipulate by computer programs. Here, we develop a new algorithm of EDTSurf for generating three major macromolecular surfaces of van der Waals surface, solvent-accessible surface and molecular surface, using the technique of fast Euclidean Distance Transform (EDT). The triangulated surfaces are constructed directly from volumetric solids by a Vertex-Connected Marching Cube algorithm that forms triangles from grid points. Compared to the analytical result, the relative error of the surface calculations by EDTSurf is <2-4% depending on the grid resolution, which is 1.5-4 times lower than the methods in the literature; and yet, the algorithm is faster and costs less computer memory than the comparative methods. The improvements in both accuracy and speed of the macromolecular surface determination should make EDTSurf a useful tool for the detailed study of protein docking and structure predictions. Both source code and the executable program of EDTSurf are freely available at http://zhang.bioinformatics.ku.edu/EDTSurf.

| S-EPMC2779860 | biostudies-literature

Tumor Spheres Quantification with Smoothed Euclidean Distance Transform.

Project description:Tumor sphere quantification plays an important role in cancer research and drugs screening. Even though the number and size of tumor spheres can be found manually, this process is time-consuming, prone to making errors, and may not be viable when the number of images is very large. This manuscript presents a method for automated quantification of spheres with a novel segmentation technique. The segmentation method relies on initial watershed algorithm which detects the minima of the distance transform and finds a tumor sphere for each minimum. Due to the irregular edges of tumor spheres, the distance transform matrix has often more number of minima than the true number of spheres. This leads to the over segmentation problem. The proposed approach uses the smoothed form of the distance transform to effectively eliminate superfluous minima and then seeds the watershed algorithm with the remaining minima. The proposed method was validated over pancreatic tumor spheres images achieving high efficiency for tumor spheres quantification.

| S-EPMC6179360 | biostudies-literature

Blue-conversion of organic dyes produces artifacts in multicolor fluorescence imaging.

Project description:Multicolor fluorescence imaging is a powerful tool visualizing the spatiotemporal relationship among biomolecules. Here, we report that commonly employed organic dyes exhibit a blue-conversion phenomenon, which can produce severe multicolor image artifacts leading to false-positive colocalization by invading predefined spectral windows, as demonstrated in the case study using EGFR and Tensin2. These multicolor image artifacts become much critical in localization-based superresolution microscopy as the blue-converted dyes are photoactivatable. We provide a practical guideline for the use of organic dyes for multicolor imaging to prevent artifacts derived by blue-conversion.

| S-EPMC8246296 | biostudies-literature

Impact of emission anisotropy on fluorescence spectroscopy and FRET distance measurements.

Project description:The objective of this report is to provide a practical and improved method for estimating Förster resonance energy transfer distance measurement error due to unknown angles in the dipole orientation factor based on emission anisotropy measurements. We improve on the method of Dale et al. (1979), which has minor mistakes and is frequently interpreted in overly optimistic ways in the literature. To facilitate proper fluorescence intensity measurements, we also evaluated instrument parameters that could impact the measurement. The apparent fluorescence intensity of isotropic samples depends on the sample emission anisotropy, fluorometer geometry, and optical apertures. We separate parameters of the sample, and those of the cylindrically symmetric illumination source and detector in the equations describing results of unpolarized and polarized fluorescence intensity measurements. This approach greatly simplifies calculations compared with the more universal method of Axelrod (1989). We provide a full computational method for calculating the Förster resonance energy transfer distance error and present a graph describing distance error in the simplest case.

| S-EPMC2718156 | biostudies-literature

Multicolor miRNA fluorescence in situ hybridization for tumor differential diagnosis

Project description:We profiled and quantitated miRNAs in two skin tumors (Basal cell carcinoma and Merkel cell carcinoma) and identified tumor-specific miRNAs. We used these tumor-specific miRNAs to guide development of miRNA fluorescence in situ hybridization.

2013-06-28 | GSE34137 | GEO

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

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