Browse
Submit Data
Databases
API
Help

Dataset Information

0 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Multiplane and Spectrally-Resolved Single Molecule Localization Microscopy with Industrial Grade CMOS cameras.

ABSTRACT: This work explores the use of industrial grade CMOS cameras for single molecule localization microscopy (SMLM). We show that industrial grade CMOS cameras approach the performance of scientific grade CMOS cameras at a fraction of the cost. This makes it more economically feasible to construct high-performance imaging systems with multiple cameras that are capable of a diversity of applications. In particular we demonstrate the use of industrial CMOS cameras for biplane, multiplane and spectrally resolved SMLM. We also provide open-source software for simultaneous control of multiple CMOS cameras and for the reduction of the movies that are acquired to super-resolution images.

SUBMITTER: Babcock HP

PROVIDER: S-EPMC5789017 | biostudies-literature | 2018 Jan

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Publications

Multiplane and Spectrally-Resolved Single Molecule Localization Microscopy with Industrial Grade CMOS cameras.

Babcock Hazen P HP

Scientific reports 20180129 1

This work explores the use of industrial grade CMOS cameras for single molecule localization microscopy (SMLM). We show that industrial grade CMOS cameras approach the performance of scientific grade CMOS cameras at a fraction of the cost. This makes it more economically feasible to construct high-performance imaging systems with multiple cameras that are capable of a diversity of applications. In particular we demonstrate the use of industrial CMOS cameras for biplane, multiplane and spectrally ...[more]

PMID: 29379074

Similar Datasets

Enabling Spectrally Resolved Single-Molecule Localization Microscopy at High Emitter Densities.

Project description:Single-molecule localization microscopy (SMLM) is a powerful super-resolution technique for elucidating structure and dynamics in the life- and material sciences. Simultaneously acquiring spectral information (spectrally resolved SMLM, sSMLM) has been hampered by several challenges: an increased complexity of the optical detection pathway, lower accessible emitter densities, and compromised spatio-spectral resolution. Here we present a single-component, low-cost implementation of sSMLM that addresses these challenges. Using a low-dispersion transmission grating positioned close to the image plane, the +1stdiffraction order is minimally elongated and is analyzed using existing single-molecule localization algorithms. The distance between the 0th and 1st order provides accurate information on the spectral properties of individual emitters. This method enables a 5-fold higher emitter density while discriminating between fluorophores whose peak emissions are less than 15 nm apart. Our approach can find widespread use in single-molecule applications that rely on distinguishing spectrally different fluorophores under low photon conditions.

| S-EPMC9650776 | biostudies-literature

Characterization of an industry-grade CMOS camera well suited for single molecule localization microscopy - high performance super-resolution at low cost.

Project description:Many commercial as well as custom-built fluorescence microscopes use scientific-grade cameras that represent a substantial share of the instrument's cost. This holds particularly true for super-resolution localization microscopy where high demands are placed especially on the detector with respect to sensitivity, noise, and also image acquisition speed. Here, we present and carefully characterize an industry-grade CMOS camera as a cost-efficient alternative to commonly used scientific cameras. Direct experimental comparison of these two detector types shows widely similar performance for imaging by single molecule localization microscopy (SMLM). Furthermore, high image acquisition speeds are demonstrated for the CMOS detector by ultra-fast SMLM imaging.

| S-EPMC5663701 | biostudies-other

Correcting Artifacts in Single Molecule Localization Microscopy Analysis Arising from Pixel Quantum Efficiency Differences in sCMOS Cameras.

Project description:Optimal analysis of single molecule localization microscopy (SMLM) data acquired with a scientific Complementary Metal-Oxide-Semiconductor (sCMOS) camera relies on statistical compensation for its pixel-dependent gain, offset and readout noise. In this work we show that it is also necessary to compensate for differences in the relative quantum efficiency (RQE) of each pixel. We found differences in RQE on the order of 4% in our tested sCMOS sensors. These differences were large enough to have a noticeable effect on analysis algorithm results, as seen both in simulations and biological imaging data. We discuss how the RQE differences manifest themselves in the analysis results and present the modifications to the Poisson maximum likelihood estimation (MLE) sCMOS analysis algorithm that are needed to correct for the RQE differences.

| S-EPMC6889274 | biostudies-literature

Single-molecule localization microscopy.

Project description:Single-molecule localization microscopy (SMLM) describes a family of powerful imaging techniques that dramatically improve spatial resolution over standard, diffraction-limited microscopy techniques and can image biological structures at the molecular scale. In SMLM, individual fluorescent molecules are computationally localized from diffraction-limited image sequences and the localizations are used to generate a super-resolution image or a time course of super-resolution images, or to define molecular trajectories. In this Primer, we introduce the basic principles of SMLM techniques before describing the main experimental considerations when performing SMLM, including fluorescent labelling, sample preparation, hardware requirements and image acquisition in fixed and live cells. We then explain how low-resolution image sequences are computationally processed to reconstruct super-resolution images and/or extract quantitative information, and highlight a selection of biological discoveries enabled by SMLM and closely related methods. We discuss some of the main limitations and potential artefacts of SMLM, as well as ways to alleviate them. Finally, we present an outlook on advanced techniques and promising new developments in the fast-evolving field of SMLM. We hope that this Primer will be a useful reference for both newcomers and practitioners of SMLM.

| S-EPMC9160414 | biostudies-literature

Line-scanning microscopy for time-gated and spectrally resolved fluorescence imaging.

Project description:Laser-scanning fluorescence microscopy for efficient acquisition of time-gated and spectrally resolved fluorescence images was developed based on line illumination of the laser beam and detection of the fluorescence image through a slit. In this optical arrangement, the fluorescence image was obtained by scanning only one axis perpendicular to the excitation line, and the acquisition time was significantly reduced compared with conventional laser-scanning confocal microscopy. A multidimensional fluorescence dataset consisting of fluorescence intensities as a function of x-position, y-position, fluorescence wavelength, and delay time after photoexcitation was analyzed and decomposed based on the parallel factor analysis model. The performance of the line-scanning microscopy was examined by applying it to the analysis of one of the plant defense responses, accumulation of antimicrobial compounds of phytoalexin in oat (Avena sativa), induced by the elicitor treatment.

| S-EPMC2577744 | biostudies-other

Instant three-color multiplane fluorescence microscopy.

Project description:One of the most widely used microscopy techniques in biology and medicine is fluorescence microscopy, offering high specificity in labeling as well as maximal sensitivity. For live-cell imaging, the ideal fluorescence microscope should offer high spatial resolution, fast image acquisition, three-dimensional sectioning, and multicolor detection. However, most existing fluorescence microscopes have to compromise between these different requirements. Here, we present a multiplane, multicolor wide-field microscope that uses a dedicated beam splitter for recording volumetric data in eight focal planes and for three emission colors with frame rates of hundreds of volumes per second. We demonstrate the efficiency and performance of our system by three-dimensional imaging of multiply labeled fixed and living cells. The use of commercially available components makes our proposed microscope straightforward for implementation, thus promising for widely used applications.

| S-EPMC9680778 | biostudies-literature

Measuring spectrally-resolved information transfer.

Project description:Information transfer, measured by transfer entropy, is a key component of distributed computation. It is therefore important to understand the pattern of information transfer in order to unravel the distributed computational algorithms of a system. Since in many natural systems distributed computation is thought to rely on rhythmic processes a frequency resolved measure of information transfer is highly desirable. Here, we present a novel algorithm, and its efficient implementation, to identify separately frequencies sending and receiving information in a network. Our approach relies on the invertible maximum overlap discrete wavelet transform (MODWT) for the creation of surrogate data in the computation of transfer entropy and entirely avoids filtering of the original signals. The approach thereby avoids well-known problems due to phase shifts or the ineffectiveness of filtering in the information theoretic setting. We also show that measuring frequency-resolved information transfer is a partial information decomposition problem that cannot be fully resolved to date and discuss the implications of this issue. Last, we evaluate the performance of our algorithm on simulated data and apply it to human magnetoencephalography (MEG) recordings and to local field potential recordings in the ferret. In human MEG we demonstrate top-down information flow in temporal cortex from very high frequencies (above 100Hz) to both similarly high frequencies and to frequencies around 20Hz, i.e. a complex spectral configuration of cortical information transmission that has not been described before. In the ferret we show that the prefrontal cortex sends information at low frequencies (4-8 Hz) to early visual cortex (V1), while V1 receives the information at high frequencies (> 125 Hz).

| S-EPMC7793276 | biostudies-literature

Quantitative, spectrally-resolved intraoperative fluorescence imaging.

Project description:Intraoperative visual fluorescence imaging (vFI) has emerged as a promising aid to surgical guidance, but does not fully exploit the potential of the fluorescent agents that are currently available. Here, we introduce a quantitative fluorescence imaging (qFI) approach that converts spectrally-resolved data into images of absolute fluorophore concentration pixel-by-pixel across the surgical field of view (FOV). The resulting estimates are linear, accurate, and precise relative to true values, and spectral decomposition of multiple fluorophores is also achieved. Experiments with protoporphyrin IX in a glioma rodent model demonstrate in vivo quantitative and spectrally-resolved fluorescence imaging of infiltrating tumor margins for the first time. Moreover, we present images from human surgery which detect residual tumor not evident with state-of-the-art vFI. The wide-field qFI technique has broad implications for intraoperative surgical guidance because it provides near real-time quantitative assessment of multiple fluorescent biomarkers across the operative field.

| S-EPMC3497712 | biostudies-literature

Switchable Fluorophores for Single-Molecule Localization Microscopy.

Project description:The past decade has witnessed an explosion in the use of super-resolution fluorescence microscopy methods in biology and other fields. Single-molecule localization microscopy (SMLM) is one of the most widespread of these methods and owes its success in large part to the ability to control the on-off state of fluorophores through various chemical, photochemical, or binding-unbinding mechanisms. We provide here a comprehensive overview of switchable fluorophores in SMLM including a detailed review of all major classes of SMLM fluorophores, and we also address strategies for labeling specimens, considerations for multichannel and live-cell imaging, potential pitfalls, and areas for future development.

| S-EPMC6476321 | biostudies-literature

Symmetrically dispersed spectroscopic single-molecule localization microscopy.

Project description:Spectroscopic single-molecule localization microscopy (sSMLM) was used to achieve simultaneous imaging and spectral analysis of single molecules for the first time. Current sSMLM fundamentally suffers from a reduced photon budget because the photons from individual stochastic emissions are divided into spatial and spectral channels. Therefore, both spatial localization and spectral analysis only use a portion of the total photons, leading to reduced precisions in both channels. To improve the spatial and spectral precisions, we present symmetrically dispersed sSMLM, or SDsSMLM, to fully utilize all photons from individual stochastic emissions in both spatial and spectral channels. SDsSMLM achieved 10-nm spatial and 0.8-nm spectral precisions at a total photon budget of 1000. Compared with the existing sSMLM using a 1:3 splitting ratio between spatial and spectral channels, SDsSMLM improved the spatial and spectral precisions by 42% and 10%, respectively, under the same photon budget. We also demonstrated multicolour imaging of fixed cells and three-dimensional single-particle tracking using SDsSMLM. SDsSMLM enables more precise spectroscopic single-molecule analysis in broader cell biology and material science applications.

| S-EPMC7248114 | 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