Browse
Submit Data
Databases
API
Help

Dataset Information

15 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Simultaneous multiplane in vivo nonlinear microscopy using spectral encoding.

ABSTRACT: Conventional point-by-point imaging schemes for laser scanning microscopy limit acquisition speeds, particularly when imaging three-dimensional volumes. We report a novel approach that achieves parallelization of multiple fields of view through the use of spectral encoding. By focusing two or more beams of different wavelengths at different positions within a suitable tissue, fluorescence or second/third harmonic generation emissions from these regions can be uniquely separated. We demonstrate that this approach can allow simultaneous in vivo imaging of fluorescence in two planes within the living rodent cortex, and of second harmonic generation in fresh tissue.

SUBMITTER: Grosberg LE

PROVIDER: S-EPMC3708965 | biostudies-literature | 2012 Jul

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Publications

Simultaneous multiplane in vivo nonlinear microscopy using spectral encoding.

Grosberg Lauren E LE Chen Brenda R BR Hillman Elizabeth M C EM

Optics letters 20120701 14

Conventional point-by-point imaging schemes for laser scanning microscopy limit acquisition speeds, particularly when imaging three-dimensional volumes. We report a novel approach that achieves parallelization of multiple fields of view through the use of spectral encoding. By focusing two or more beams of different wavelengths at different positions within a suitable tissue, fluorescence or second/third harmonic generation emissions from these regions can be uniquely separated. We demonstrate t ...[more]

PMID: 22825194

Similar Datasets

Simultaneous multiplane imaging with reverberation two-photon microscopy.

Project description:Multiphoton microscopy has gained enormous popularity because of its unique capacity to provide high-resolution images from deep within scattering tissue. Here, we demonstrate video-rate multiplane imaging with two-photon microscopy by performing near-instantaneous axial scanning while maintaining three-dimensional micrometer-scale resolution. Our technique, termed reverberation microscopy, enables the monitoring of neuronal populations over large depth ranges and can be implemented as a simple add-on to a conventional design.

| S-EPMC8590882 | biostudies-literature

Assessment of breast pathologies using nonlinear microscopy.

Project description:Rapid intraoperative assessment of breast excision specimens is clinically important because up to 40% of patients undergoing breast-conserving cancer surgery require reexcision for positive or close margins. We demonstrate nonlinear microscopy (NLM) for the assessment of benign and malignant breast pathologies in fresh surgical specimens. A total of 179 specimens from 50 patients was imaged with NLM using rapid extrinsic nuclear staining with acridine orange and intrinsic second harmonic contrast generation from collagen. Imaging was performed on fresh, intact specimens without the need for fixation, embedding, and sectioning required for conventional histopathology. A visualization method to aid pathological interpretation is presented that maps NLM contrast from two-photon fluorescence and second harmonic signals to features closely resembling histopathology using hematoxylin and eosin staining. Mosaicking is used to overcome trade-offs between resolution and field of view, enabling imaging of subcellular features over square-centimeter specimens. After NLM examination, specimens were processed for standard paraffin-embedded histology using a protocol that coregistered histological sections to NLM images for paired assessment. Blinded NLM reading by three pathologists achieved 95.4% sensitivity and 93.3% specificity, compared with paraffin-embedded histology, for identifying invasive cancer and ductal carcinoma in situ versus benign breast tissue. Interobserver agreement was ? = 0.88 for NLM and ? = 0.89 for histology. These results show that NLM achieves high diagnostic accuracy, can be rapidly performed on unfixed specimens, and is a promising method for intraoperative margin assessment.

| S-EPMC4217415 | biostudies-literature

Simultaneous imaging of GFP, CFP and collagen in tumors in vivo using multiphoton microscopy.

Project description:BackgroundThe development of multiphoton laser scanning microscopy has greatly facilitated the imaging of living tissues. However, the use of genetically encoded fluorescent proteins to distinguish different cell types in living animals has not been described at single cell resolution using multiphoton microscopy.ResultsHere we describe a method for the simultaneous imaging, by multiphoton microscopy, of Green Fluorescent Protein, Cyan Fluorescent Protein and collagen in vivo in living tumors. This novel method enables: 1) the simultaneous visualization of overall cell shape and sub-cellular structures such as the plasma membrane or proteins of interest in cells inside living animals, 2) direct comparison of the behavior of single cells from different cell lines in the same microenvironment in vivo.ConclusionUsing this multi-fluor, multiphoton technique, we demonstrate that motility and metastatic differences between carcinoma cells of differing metastatic potential can be imaged in the same animal simultaneously at sub-cellular resolution.

| S-EPMC1159039 | biostudies-literature

SMAC: Simultaneous Mapping and Clustering Using Spectral Decompositions.

Project description:We introduce a principled approach for simultaneous mapping and clustering (SMAC) for establishing consistent maps across heterogeneous object collections (e.g., 2D images or 3D shapes). Our approach takes as input a heterogeneous object collection and a set of maps computed between some pairs of objects, and outputs a homogeneous object clustering together with a new set of maps possessing optimal intra- and inter-cluster consistency. Our approach is based on the spectral decomposition of a data matrix storing all pairwise maps in its blocks. We additionally provide tight theoretical guarantees for the accuracy of SMAC under established noise models. We also demonstrate the usefulness of our approach on synthetic and real datasets.

| S-EPMC7394310 | biostudies-literature

Readily Accessible Multiplane Microscopy: 3D Tracking the HIV-1 Genome in Living Cells.

Project description:Human immunodeficiency virus (HIV)-1 infection and the associated disease AIDS are a major cause of human death worldwide with no vaccine or cure available. The trafficking of HIV-1 RNAs from sites of synthesis in the nucleus, through the cytoplasm, to sites of assembly at the plasma membrane are critical steps in HIV-1 viral replication, but are not well characterized. Here we present a broadly accessible microscopy method that captures multiple focal planes simultaneously, which allows us to image the trafficking of HIV-1 genomic RNAs with high precision. This method utilizes a customization of a commercial multichannel emission splitter that enables high-resolution 3D imaging with single-macromolecule sensitivity. We show with high temporal and spatial resolution that HIV-1 genomic RNAs are most mobile in the cytosol, and undergo confined mobility at sites along the nuclear envelope and in the nucleus and nucleolus. These provide important insights regarding the mechanism by which the HIV-1 RNA genome is transported to the sites of assembly of nascent virions.

| S-EPMC4729623 | biostudies-literature

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

Project description: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.

| S-EPMC5789017 | biostudies-literature

Optical Tweezers with Integrated Multiplane Microscopy (OpTIMuM): a new tool for 3D microrheology.

Project description:We introduce a novel 3D microrheology system that combines for the first time Optical Tweezers with Integrated Multiplane Microscopy (OpTIMuM). The system allows the 3D tracking of an optically trapped bead, with ~ 20 nm accuracy along the optical axis. This is achieved without the need for a high precision z-stage, separate calibration sample, nor a priori knowledge of either the bead size or the optical properties of the suspending medium. Instead, we have developed a simple yet effective in situ spatial calibration method using image sharpness and exploiting the fact we image at multiple planes simultaneously. These features make OpTIMuM an ideal system for microrheology measurements, and we corroborate the effectiveness of this novel microrheology tool by measuring the viscosity of water in three dimensions, simultaneously.

| S-EPMC7946888 | biostudies-literature

Robust blind spectral unmixing for fluorescence microscopy using unsupervised learning.

Project description:Due to the overlapping emission spectra of fluorophores, fluorescence microscopy images often have bleed-through problems, leading to a false positive detection. This problem is almost unavoidable when the samples are labeled with three or more fluorophores, and the situation is complicated even further when imaged under a multiphoton microscope. Several methods have been developed and commonly used by biologists for fluorescence microscopy spectral unmixing, such as linear unmixing, non-negative matrix factorization, deconvolution, and principal component analysis. However, they either require pre-knowledge of emission spectra or restrict the number of fluorophores to be the same as detection channels, which highly limits the real-world applications of those spectral unmixing methods. In this paper, we developed a robust and flexible spectral unmixing method: Learning Unsupervised Means of Spectra (LUMoS), which uses an unsupervised machine learning clustering method to learn individual fluorophores' spectral signatures from mixed images, and blindly separate channels without restrictions on the number of fluorophores that can be imaged. This method highly expands the hardware capability of two-photon microscopy to simultaneously image more fluorophores than is possible with instrumentation alone. Experimental and simulated results demonstrated the robustness of LUMoS in multi-channel separations of two-photon microscopy images. We also extended the application of this method to background/autofluorescence removal and colocalization analysis. Lastly, we integrated this tool into ImageJ to offer an easy to use spectral unmixing tool for fluorescence imaging. LUMoS allows us to gain a higher spectral resolution and obtain a cleaner image without the need to upgrade the imaging hardware capabilities.

| S-EPMC6886781 | biostudies-literature

Phototoxic effects of nonlinear optical microscopy on cell cycle, oxidative states, and gene expression

Project description:Nonlinear optical imaging modalities, such as stimulated Raman scattering (SRS) microscopy, use pulsed-laser excitation with high peak intensity that can perturb the native state of cells. In this study, we used bulk RNA sequencing, quantitative measurement of cell proliferation, and fluorescent measurement of the generation of reactive oxygen species (ROS) to assess phototoxic effects of near-IR pulsed laser radiation, at different time scales, for laser excitation settings relevant to SRS imaging. We define a range of laser excitation settings for which there was no significant change in mouse Neuro2A cells after laser exposure. This study provides guidance for imaging parameters that minimize photo-induced perturbations in SRS microscopy to ensure accurate interpretation of experiments with time-lapse imaging or with paired measurements of imaging and sequencing on the same cells.

2022-03-06 | GSE197666 | GEO

Simultaneous detection of glutathione and lactate using spectral editing at 3 T.

Project description:Two spectral editing techniques for the simultaneous detection of glutathione (GSH) and lactate (Lac) in the human brain at 3 T are described and evaluated. These methods, 'sMEGA' (sinc-MEscher and GArwood) and 'DEW' (Double Editing With), were optimized to detect GSH and Lac simultaneously at 3 T using density-matrix simulations and validation in phantoms. Simulations to test for co-edited metabolites within the detected GSH region of the spectrum were also performed. In vivo data were acquired in the midline parietal region of seven subjects using both methods, and compared with conventional MEGA-PRESS (MEscher and GArwood-Point RESolved Spectroscopy) acquisitions of GSH and Lac. Simulations and phantom experiments showed that sMEGA and DEW had a high editing efficiency for both GSH and Lac. In the phantom, the editing efficiency of GSH was >88% relative to a conventional GSH MEGA-PRESS acquisition, whereas, for Lac, the editing efficiency was >95% relative to a conventional Lac MEGA-PRESS acquisition. Simulations also showed that the editing efficiency of both methods was comparable with separate MEGA-PRESS acquisitions of the same metabolites. In addition, simulations and in vivo spectra showed that, at a TE of 140 ms, there was a partial overlap between creatine (Cr) and GSH peaks, and that N-acetyl aspartate/N-acetyl aspartyl glutamate (NAA/NAAG) were sufficiently resolved from GSH. In vivo measurements showed that both sMEGA and DEW edited GSH and Lac reliably with the same editing efficiency as conventional MEGA-PRESS acquisitions of the same metabolites, with measured GSH integrals of 2.23 ± 0.51, 2.31 ± 0.38, 2.38 ± 0.53 and measured Lac integrals of 1.72 ± 0.67, 1.55 ± 0.35 and 1.53 ± 0.54 for MEGA-PRESS, DEW and sMEGA, respectively. Simultaneous detection of GSH and Lac using sMEGA and DEW is possible at 3 T with high editing efficiency.

| S-EPMC5685873 | biostudies-literature

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