Browse
Submit Data
Databases
API
Help

Dataset Information

25 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Three-dimensional, tomographic super-resolution fluorescence imaging of serially sectioned thick samples.

ABSTRACT: Three-dimensional fluorescence imaging of thick tissue samples with near-molecular resolution remains a fundamental challenge in the life sciences. To tackle this, we developed tomoSTORM, an approach combining single-molecule localization-based super-resolution microscopy with array tomography of structurally intact brain tissue. Consecutive sections organized in a ribbon were serially imaged with a lateral resolution of 28 nm and an axial resolution of 40 nm in tissue volumes of up to 50 µm×50 µm×2.5 µm. Using targeted expression of membrane bound (m)GFP and immunohistochemistry at the calyx of Held, a model synapse for central glutamatergic neurotransmission, we delineated the course of the membrane and fine-structure of mitochondria. This method allows multiplexed super-resolution imaging in large tissue volumes with a resolution three orders of magnitude better than confocal microscopy.

SUBMITTER: Nanguneri S

PROVIDER: S-EPMC3360663 | biostudies-literature | 2012

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Publications

Three-dimensional, tomographic super-resolution fluorescence imaging of serially sectioned thick samples.

Nanguneri Siddharth S Flottmann Benjamin B Horstmann Heinz H Heilemann Mike M Kuner Thomas T

PloS one 20120525 5

Three-dimensional fluorescence imaging of thick tissue samples with near-molecular resolution remains a fundamental challenge in the life sciences. To tackle this, we developed tomoSTORM, an approach combining single-molecule localization-based super-resolution microscopy with array tomography of structurally intact brain tissue. Consecutive sections organized in a ribbon were serially imaged with a lateral resolution of 28 nm and an axial resolution of 40 nm in tissue volumes of up to 50 µm×50 ...[more]

PMID: 22662272

Similar Datasets

Multilayer three-dimensional super resolution imaging of thick biological samples.

Project description:Recent advances in optical microscopy have enabled biological imaging beyond the diffraction limit at nanometer resolution. A general feature of most of the techniques based on photoactivated localization microscopy (PALM) or stochastic optical reconstruction microscopy (STORM) has been the use of thin biological samples in combination with total internal reflection, thus limiting the imaging depth to a fraction of an optical wavelength. However, to study whole cells or organelles that are typically up to 15 microm deep into the cell, the extension of these methods to a three-dimensional (3D) super resolution technique is required. Here, we report an advance in optical microscopy that enables imaging of protein distributions in cells with a lateral localization precision better than 50 nm at multiple imaging planes deep in biological samples. The approach is based on combining the lateral super resolution provided by PALM with two-photon temporal focusing that provides optical sectioning. We have generated super-resolution images over an axial range of approximately 10 microm in both mitochondrially labeled fixed cells, and in the membranes of living S2 Drosophila cells.

| S-EPMC2603430 | biostudies-literature

Three-dimensional super-resolution fluorescence imaging of DNA.

Project description:Recent advances in fluorescence super-resolution microscopy are providing important insights into details of cellular structures. To acquire three dimensional (3D) super-resolution images of DNA, we combined binding activated localization microscopy (BALM) using fluorescent double-stranded DNA intercalators and optical astigmatism. We quantitatively establish the advantage of bis- over mono-intercalators before demonstrating the approach by visualizing single DNA molecules stretched between microspheres at various heights. Finally, the approach is applied to the more complex environment of intact and damaged metaphase chromosomes, unravelling their structural features.

| S-EPMC7385144 | biostudies-literature

Compressive three-dimensional super-resolution microscopy with speckle-saturated fluorescence excitation.

Project description:Nonlinear structured illumination microscopy (nSIM) is an effective approach for super-resolution wide-field fluorescence microscopy with a theoretically unlimited resolution. In nSIM, carefully designed, highly-contrasted illumination patterns are combined with the saturation of an optical transition to enable sub-diffraction imaging. While the technique proved useful for two-dimensional imaging, extending it to three-dimensions is challenging due to the fading of organic fluorophores under intense cycling conditions. Here, we present a compressed sensing approach that allows 3D sub-diffraction nSIM of cultured cells by saturating fluorescence excitation. Exploiting the natural orthogonality of speckles at different axial planes, 3D probing of the sample is achieved by a single two-dimensional scan. Fluorescence contrast under saturated excitation is ensured by the inherent high density of intensity minima associated with optical vortices in polarized speckle patterns. Compressed speckle microscopy is thus a simple approach that enables 3D super-resolved nSIM imaging with potentially considerably reduced acquisition time and photobleaching.

| S-EPMC6430798 | biostudies-literature

Three-dimensional super-resolution longitudinal magnetization spot arrays.

Project description:We demonstrate an all-optical strategy for realizing spherical three-dimensional (3D) super-resolution (??3/22) spot arrays of pure longitudinal magnetization by exploiting a 4? optical microscopic setup with two high numerical aperture (NA) objective lenses, which focus and interfere two modulated vectorial beams. Multiple phase filters (MPFs) are designed via an analytical approach derived from the vectorial Debye diffraction theory to modulate the two circularly polarized beams. The system is tailored to constructively interfere the longitudinal magnetization components, while simultaneously destructively interfering the azimuthal ones. As a result, the magnetization field is not only purely longitudinal but also super-resolved in all three dimensions. Furthermore, the MPFs can be designed analytically to control the number and locations of the super-resolved magnetization spots to produce both uniform and nonuniform arrays in a 3D volume. Thus, an all-optical control of all the properties of light-induced magnetization spot arrays has been demonstrated for the first time. These results open up broad applications in magnetic-optical devices such as confocal and multifocal magnetic resonance microscopy, 3D ultrahigh-density magneto-optic memory, and light-induced magneto-lithography.

| S-EPMC6062314 | biostudies-other

Super-resolution fluorescence-assisted diffraction computational tomography reveals the three-dimensional landscape of the cellular organelle interactome.

Project description:The emergence of super-resolution (SR) fluorescence microscopy has rejuvenated the search for new cellular sub-structures. However, SR fluorescence microscopy achieves high contrast at the expense of a holistic view of the interacting partners and surrounding environment. Thus, we developed SR fluorescence-assisted diffraction computational tomography (SR-FACT), which combines label-free three-dimensional optical diffraction tomography (ODT) with two-dimensional fluorescence Hessian structured illumination microscopy. The ODT module is capable of resolving the mitochondria, lipid droplets, the nuclear membrane, chromosomes, the tubular endoplasmic reticulum, and lysosomes. Using dual-mode correlated live-cell imaging for a prolonged period of time, we observed novel subcellular structures named dark-vacuole bodies, the majority of which originate from densely populated perinuclear regions, and intensively interact with organelles such as the mitochondria and the nuclear membrane before ultimately collapsing into the plasma membrane. This work demonstrates the unique capabilities of SR-FACT, which suggests its wide applicability in cell biology in general.

| S-EPMC6987131 | biostudies-literature

Fast, three-dimensional super-resolution imaging of live cells.

Project description:We report super-resolution fluorescence imaging of live cells with high spatiotemporal resolution using stochastic optical reconstruction microscopy (STORM). By labeling proteins either directly or via SNAP tags with photoswitchable dyes, we obtained two-dimensional (2D) and 3D super-resolution images of living cells, using clathrin-coated pits and the transferrin cargo as model systems. Bright, fast-switching probes enabled us to achieve 2D imaging at spatial resolutions of ?25 nm and temporal resolutions as fast as 0.5 s. We also demonstrated live-cell 3D super-resolution imaging. We obtained 3D spatial resolution of ?30 nm in the lateral direction and ?50 nm in the axial direction at time resolutions as fast as 1-2 s with several independent snapshots. Using photoswitchable dyes with distinct emission wavelengths, we also demonstrated two-color 3D super-resolution imaging in live cells. These imaging capabilities open a new window for characterizing cellular structures in living cells at the ultrastructural level.

| S-EPMC3137767 | biostudies-literature

Epi-mode tomographic quantitative phase imaging in thick scattering samples.

Project description:Quantitative phase imaging (QPI) is an important tool in biomedicine that allows for the microscopic investigation of live cells and other thin, transparent samples. Importantly, this technology yields access to the cellular and sub-cellular structure and activity at nanometer scales without labels or dyes. Despite this unparalleled ability, QPI's restriction to relatively thin samples severely hinders its versatility and overall utility in biomedicine. Here we overcome this significant limitation of QPI to enable the same rich level of quantitative detail in thick scattering samples. We achieve this by first illuminating the sample in an epi-mode configuration and using multiple scattering within the sample-a hindrance to conventional transmission imaging used in QPI-as a source of transmissive illumination from within. Second, we quantify phase via deconvolution by modeling the transfer function of the system based on the ensemble average angular distribution of light illuminating the sample at the focal plane. This technique packages the quantitative, real-time sub-cellular imaging capabilities of QPI into a flexible configuration, opening the door for truly non-invasive, label-free, tomographic quantitative phase imaging of unaltered thick, scattering specimens. Images of controlled scattering phantoms, blood in collection bags, cerebral organoids and freshly excised whole mouse brains are presented to validate the approach.

| S-EPMC6640824 | biostudies-literature

Three-dimensional super-resolution imaging by stochastic optical reconstruction microscopy.

Project description:Recent advances in far-field fluorescence microscopy have led to substantial improvements in image resolution, achieving a near-molecular resolution of 20 to 30 nanometers in the two lateral dimensions. Three-dimensional (3D) nanoscale-resolution imaging, however, remains a challenge. We demonstrated 3D stochastic optical reconstruction microscopy (STORM) by using optical astigmatism to determine both axial and lateral positions of individual fluorophores with nanometer accuracy. Iterative, stochastic activation of photoswitchable probes enables high-precision 3D localization of each probe, and thus the construction of a 3D image, without scanning the sample. Using this approach, we achieved an image resolution of 20 to 30 nanometers in the lateral dimensions and 50 to 60 nanometers in the axial dimension. This development allowed us to resolve the 3D morphology of nanoscopic cellular structures.

| S-EPMC2633023 | biostudies-literature

Live-cell multiplane three-dimensional super-resolution optical fluctuation imaging.

Project description:Super-resolution optical fluctuation imaging (SOFI) provides an elegant way of overcoming the diffraction limit in all three spatial dimensions by computing higher-order cumulants of image sequences of blinking fluorophores acquired with a classical widefield microscope. Previously, three-dimensional (3D) SOFI has been demonstrated by sequential imaging of multiple depth positions. Here we introduce a multiplexed imaging scheme for the simultaneous acquisition of multiple focal planes. Using 3D cross-cumulants, we show that the depth sampling can be increased. The simultaneous acquisition of multiple focal planes significantly reduces the acquisition time and thus the photobleaching. We demonstrate multiplane 3D SOFI by imaging fluorescently labelled cells over an imaged volume of up to 65 × 65 × 3.5 ?m(3) without depth scanning. In particular, we image the 3D network of mitochondria in fixed C2C12 cells immunostained with Alexa 647 fluorophores and the 3D vimentin structure in living Hela cells expressing the fluorescent protein Dreiklang.

| S-EPMC4284648 | biostudies-literature

Three-dimensional super-resolution protein localization correlated with vitrified cellular context.

Project description:We demonstrate the use of cryogenic super-resolution correlative light and electron microscopy (csCLEM) to precisely determine the spatial relationship between proteins and their native cellular structures. Several fluorescent proteins (FPs) were found to be photoswitchable and emitted far more photons under our cryogenic imaging condition, resulting in higher localization precision which is comparable to ambient super-resolution imaging. Vitrified specimens were prepared by high pressure freezing and cryo-sectioning to maintain a near-native state with better fluorescence preservation. A 2-3-fold improvement of resolution over the recent reports was achieved due to the photon budget performance of screening out Dronpa and optimized imaging conditions, even with thin sections which is at a disadvantage when calculate the structure resolution from label density. We extended csCLEM to mammalian cells by introducing cryo-sectioning and observed good correlation of a mitochondrial protein with the mitochondrial outer membrane at nanometer resolution in three dimensions.

| S-EPMC4604464 | 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