Browse
Submit Data
Databases
API
Help

Dataset Information

42 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Ultrahigh-speed point scanning two-photon microscopy using high dynamic range silicon photomultipliers.

ABSTRACT: Conventional two-photon microscopes use photomultiplier tubes, which enable high sensitivity but can detect relatively few photons per second, forcing longer pixel integration times and limiting maximum imaging rates. We introduce novel detection electronics using silicon photomultipliers that greatly extend dynamic range, enabling more than an order of magnitude increased photon detection rate as compared to state-of-the-art photomultiplier tubes. We demonstrate that this capability can dramatically improve both imaging rates and signal-to-noise ratio (SNR) in two-photon microscopy using human surgical specimens. Finally, to enable wider use of more advanced detection technology, we have formed the OpenSiPM project, which aims to provide open source detector designs for high-speed two-photon and confocal microscopy.

SUBMITTER: Ching-Roa VD

PROVIDER: S-EPMC7933192 | biostudies-literature | 2021 Mar

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Publications

Ultrahigh-speed point scanning two-photon microscopy using high dynamic range silicon photomultipliers.

Ching-Roa Vincent D VD Olson Eben M EM Ibrahim Sherrif F SF Torres Richard R Giacomelli Michael G MG

Scientific reports 20210304 1

Conventional two-photon microscopes use photomultiplier tubes, which enable high sensitivity but can detect relatively few photons per second, forcing longer pixel integration times and limiting maximum imaging rates. We introduce novel detection electronics using silicon photomultipliers that greatly extend dynamic range, enabling more than an order of magnitude increased photon detection rate as compared to state-of-the-art photomultiplier tubes. We demonstrate that this capability can dramati ...[more]

PMID: 33664354

Similar Datasets

Neuronal imaging with ultrahigh dynamic range multiphoton microscopy.

Project description:Multiphoton microscopes are hampered by limited dynamic range, preventing weak sample features from being detected in the presence of strong features, or preventing the capture of unpredictable bursts in sample strength. We present a digital electronic add-on technique that vastly improves the dynamic range of a multiphoton microscope while limiting potential photodamage. The add-on provides real-time negative feedback to regulate the laser power delivered to the sample, and a log representation of the sample strength to accommodate ultrahigh dynamic range without loss of information. No microscope hardware modifications are required, making the technique readily compatible with commercial instruments. Benefits are shown in both structural and in-vivo functional mouse brain imaging applications.

| S-EPMC5517475 | biostudies-literature

Real-time high dynamic range laser scanning microscopy.

Project description:In conventional confocal/multiphoton fluorescence microscopy, images are typically acquired under ideal settings and after extensive optimization of parameters for a given structure or feature, often resulting in information loss from other image attributes. To overcome the problem of selective data display, we developed a new method that extends the imaging dynamic range in optical microscopy and improves the signal-to-noise ratio. Here we demonstrate how real-time and sequential high dynamic range microscopy facilitates automated three-dimensional neural segmentation. We address reconstruction and segmentation performance on samples with different size, anatomy and complexity. Finally, in vivo real-time high dynamic range imaging is also demonstrated, making the technique particularly relevant for longitudinal imaging in the presence of physiological motion and/or for quantification of in vivo fast tracer kinetics during functional imaging.

| S-EPMC4821995 | biostudies-literature

High-speed two-photon microscopy with adaptive line-excitation.

Project description:We present a two-photon fluorescence microscope designed for high-speed imaging of neural activity at cellular resolution. Our microscope uses an adaptive sampling scheme with line illumination. Instead of building images pixel by pixel via scanning a diffraction-limited spot across the sample, our scheme only illuminates the regions of interest (i.e., neuronal cell bodies) and samples a large area of them in a single measurement. Such a scheme significantly increases the imaging speed and reduces the overall laser power on the brain tissue. Using this approach, we performed high-speed imaging of the neuronal activity in mouse cortex in vivo. Our method provides a sampling strategy in laser-scanning two-photon microscopy and will be powerful for high-throughput imaging of neural activity.

| S-EPMC11601119 | biostudies-literature

Throughput-Speed Product Augmentation for Scanning Fiber-Optic Two-Photon Endomicroscopy.

Project description:Compactness, among several others, is one unique and very attractive feature of a scanning fiber-optic two-photon endomicroscope. To increase the scanning area and the total number of resolvable pixels (i.e., the imaging throughput), it typically requires a longer cantilever which, however, leads to a much undesired, reduced scanning speed (and thus imaging frame rate). Herein we introduce a new design strategy for a fiber-optic scanning endomicroscope, where the overall numerical aperture (NA) or beam focusing power is distributed over two stages: 1) a mode-field focuser engineered at the tip of a double-clad fiber (DCF) cantilever to pre-amplify the single-mode core NA, and 2) a micro objective of a lower magnification (i.e., ∼ 2× in this design) to achieve final tight beam focusing. This new design enables either an ~9-fold increase in imaging area (throughput) or an ~3-fold improvement in imaging frame rate when compared to traditional fiber-optic endomicroscope designs. The performance of an as-designed endomicroscope of an enhanced throughput-speed product was demonstrated by two representative applications: (1) high-resolution imaging of an internal organ (i.e., mouse kidney) in vivo over a large field of view without using any fluorescent contrast agents, and (2) real-time neural imaging by visualizing dendritic calcium dynamics in vivo with sub-second temporal resolution in GCaMP6m-expressing mouse brain. This cascaded NA amplification strategy is universal and can be readily adapted to other types of fiber-optic scanners in compact linear or nonlinear endomicroscopes.

| S-EPMC7773217 | biostudies-literature

Two-photon image-scanning microscopy with SPAD array and blind image reconstruction.

Project description:Two-photon excitation (2PE) laser scanning microscopy is the imaging modality of choice when one desires to work with thick biological samples. However, its spatial resolution is poor, below confocal laser scanning microscopy. Here, we propose a straightforward implementation of 2PE image scanning microscopy (2PE-ISM) that, by leveraging our recently introduced single-photon avalanche diode (SPAD) array detector and a novel blind image reconstruction method, is shown to enhance the effective resolution, as well as the overall image quality of 2PE microscopy. With our adaptive pixel reassignment procedure ?1.6 times resolution increase is maintained deep into thick semi-transparent samples. The integration of Fourier ring correlation based semi-blind deconvolution is shown to further enhance the effective resolution by a factor of ?2 - and automatic background correction is shown to boost the image quality especially in noisy images. Most importantly, our 2PE-ISM implementation requires no calibration measurements or other input from the user, which is an important aspect in terms of day-to-day usability of the technique.

| S-EPMC7316014 | biostudies-literature

Visible-wavelength two-photon excitation microscopy with multifocus scanning for volumetric live-cell imaging.

Project description:Two-photon excitation microscopy is one of the key techniques used to observe three-dimensional (3-D) structures in biological samples. We utilized a visible-wavelength laser beam for two-photon excitation in a multifocus confocal scanning system to improve the spatial resolution and image contrast in 3-D live-cell imaging. Experimental and numerical analyses revealed that the axial resolution has improved for a wide range of pinhole sizes used for confocal detection. We observed the 3-D movements of the Golgi bodies in living HeLa cells with an imaging speed of 2 s per volume. We also confirmed that the time-lapse observation up to 8 min did not cause significant cell damage in two-photon excitation experiments using wavelengths in the visible light range. These results demonstrate that multifocus, two-photon excitation microscopy with the use of a visible wavelength can constitute a simple technique for 3-D visualization of living cells with high spatial resolution and image contrast.

| S-EPMC7008499 | biostudies-literature

Changes in cortical microvasculature during misery perfusion measured by two-photon laser scanning microscopy.

Project description:This study aimed to examine the cortical microvessel diameter response to hypercapnia in misery perfusion using two-photon laser scanning microscopy (TPLSM). We evaluated whether the vascular response to hypercapnia could represent the cerebrovascular reserve. Cerebral blood flow (CBF) during normocapnia and hypercapnia was measured by laser-Doppler flowmetry through cranial windows in awake C57/BL6 mice before and at 1, 7, 14, and 28 days after unilateral common carotid artery occlusion (UCCAO). Diameters of the cortical microvessels during normocapnia and hypercapnia were also measured by TPLSM. Cerebral blood flow and the vascular response to hypercapnia were decreased after UCCAO. Before UCCAO, vasodilation during hypercapnia was found primarily in arterioles (22.9%±3.5%). At 14 days after UCCAO, arterioles, capillaries, and venules were autoregulatorily dilated by 79.5%±19.7%, 57.2%±32.3%, and 32.0%±10.8%, respectively. At the same time, the diameter response to hypercapnia in arterioles was significantly decreased to 1.9%±1.5%. A significant negative correlation was observed between autoregulatory vasodilation and the diameter response to hypercapnia in arterioles. Our findings indicate that arterioles play main roles in both autoregulatory vasodilation and hypercapnic vasodilation, and that the vascular response to hypercapnia can be used to estimate the cerebrovascular reserve.

| S-EPMC4126097 | biostudies-literature

Supraresolution imaging in brain slices using stimulated-emission depletion two-photon laser scanning microscopy.

Project description:Two-photon laser scanning microscopy (2PLSM) has allowed unprecedented fluorescence imaging of neuronal structure and function within neural tissue. However, the resolution of this approach is poor compared to that of conventional confocal microscopy. Here, we demonstrate supraresolution 2PLSM within brain slices. Imaging beyond the diffraction limit is accomplished by using near-infrared (NIR) lasers for both pulsed two-photon excitation and continuous wave stimulated emission depletion (STED). Furthermore, we demonstrate that Alexa Fluor 594, a bright fluorophore commonly used for both live cell and fixed tissue fluorescence imaging, is suitable for STED 2PLSM. STED 2PLSM supraresolution microscopy achieves approximately 3-fold improvement in resolution in the radial direction over conventional 2PLSM, revealing greater detail in the structure of dendritic spines located approximately 100 microns below the surface of brain slices. Further improvements in resolution are theoretically achievable, suggesting that STED 2PLSM will permit nanoscale imaging of neuronal structures located in relatively intact brain tissue.

| S-EPMC2756148 | biostudies-literature

Longitudinal imaging of the taste bud in vivo with two-photon laser scanning microscopy.

Project description:Taste bud cells in the tongue transduce taste information from chemicals in food and transmit this information to gustatory neurons in the geniculate ganglion that innervate taste buds. The peripheral taste system is a dynamic environment where taste bud cells are continuously replaced, but further understanding of this phenomenon has been limited by the inability to directly observe this process. To overcome this challenge, we combined chronic in vivo two-photon laser scanning microscopy with genetic labeling of gustatory neurons and taste buds to observe how cells within the taste bud change over time. This method expands the investigative possibilities beyond those offered by fixed-tissue methods. This method permits direct observation of taste bud cell entry, cell differentiation, cell loss, and arbor plasticity. We demonstrate that a few stains/dyes can be used to observe nuclei and organelles in the taste bud in vivo. We also describe a workflow for reconstructing composite z-stacks with grayscale data of both cells and arbors using ImageJ, Neurolucida 360, and Neurolucida Explorer software. Together, the methodology and software options for analyses presented here provide a novel approach for longitudinally observing taste bud cells and arbors in the taste bud in vivo.

| S-EPMC11642993 | biostudies-literature

In Vivo Neurodynamics Mapping via High-Speed Two-Photon Fluorescence Lifetime Volumetric Projection Microscopy.

Project description:Monitoring the morphological and biochemical information of neurons and glial cells at high temporal resolution in three-dimensional (3D) volumes of in vivo is pivotal for understanding their structure and function, and quantifying the brain microenvironment. Conventional two-photon fluorescence lifetime volumetric imaging speed faces the acquisition speed challenges of slow serial focal tomographic scanning, complex post-processing procedures for lifetime images, and inherent trade-offs among contrast, signal-to-noise ratio, and speed. This study presents a two-photon fluorescence lifetime volumetric projection microscopy using an axially elongated Bessel focus and instant frequency-domain fluorescence lifetime technique, and integrating with a convolutional network to enhance the imaging speed for in vivo neurodynamics mapping. The proposed method is validated by monitoring intracellular Ca2+ concentration throughout whole volume, tracking microglia movement and microenvironmental changes following thermal injury in the zebrafish brain, analyzing structural and functional variations of gap junctions in astrocyte networks, and measuring the Ca2+ concentration in neurons in mouse brains. This innovative methodology enables quantitative in vivo visualization of neurodynamics and the cellular processes and interactions in the brain.

| S-EPMC11831470 | 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