Faculty Opinions recommendation of MINFLUX nanometer-scale 3D imaging and microsecond-range tracking on a common fluorescence microscope.

We present a light sheet fluorescence microscope dedicated to image “Organ-on-chip”-like biostructures in microfluidic chip. Based on a simple design, the setup is built around the chip and its environment to allow 3D imaging inside the chip in a microfluidic laboratory. The experimental setup, its optical characterization and first volumetric images are reported.

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A versatile tiling light sheet microscope for cleared tissues imaging

10.1101/829267 ◽

2019 ◽

Cited By ~ 1

Author(s):

Yanlu Chen ◽

Xiaoliang Li ◽

Dongdong Zhang ◽

Chunhui Wang ◽

Ruili Feng ◽

...

Keyword(s):

Spatial Resolution ◽

Phase Modulation ◽

3D Imaging ◽

Tissue Expansion ◽

Light Sheet ◽

Nanometer Scale ◽

Tissue Clearing ◽

Working Principle ◽

Tissue Size ◽

Imaging Speed

AbstractWe present a tiling light sheet microscope compatible with all tissue clearing methods for rapid multicolor 3D imaging of centimeter-scale cleared tissues with micron-scale (4×4×10 μm3) to nanometer-scale (70×70×200 nm3) spatial resolution. The microscope uses tiling light sheets to achieve a more advanced multicolor 3D imaging ability than conventional light sheet microscopes. The illumination light is phase modulated to adjust the 3D imaging ability of the microscope based on the tissue size and the desired spatial resolution and imaging speed, and also to keep the microscope aligned. The ability of the microscope to align via phase modulation alone also makes the microscope reliable and easy to operate. Here we describe the working principle and design of the microscope. We demonstrate its imaging ability by imaging various cleared tissues prepared by different tissue clearing and tissue expansion techniques.

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Development a flexible light‐sheet fluorescence microscope for high‐speed 3D imaging of calcium dynamics and 3D imaging of cellular microstructure

Journal of Biophotonics ◽

10.1002/jbio.201960239 ◽

2020 ◽

Vol 13 (6) ◽

Author(s):

Hugh Sparks ◽

Liuba Dvinskikh ◽

Jahn M. Firth ◽

Alice J. Francis ◽

Sian E. Harding ◽

...

Keyword(s):

High Speed ◽

3D Imaging ◽

Light Sheet ◽

Fluorescence Microscope ◽

Calcium Dynamics ◽

Cellular Microstructure

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MINFLUX nanometer-scale 3D imaging and microsecond-range tracking on a common fluorescence microscope

Nature Communications ◽

10.1038/s41467-021-21652-z ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Roman Schmidt ◽

Tobias Weihs ◽

Christian A. Wurm ◽

Isabelle Jansen ◽

Jasmin Rehman ◽

...

Keyword(s):

Fluorescence Microscopy ◽

3D Imaging ◽

Beam Steering ◽

Three Dimensions ◽

Nanometer Scale ◽

Custom Made ◽

Sample Position ◽

Localization Precision ◽

Spatio Temporal ◽

20 Nm

AbstractThe recently introduced minimal photon fluxes (MINFLUX) concept pushed the resolution of fluorescence microscopy to molecular dimensions. Initial demonstrations relied on custom made, specialized microscopes, raising the question of the method’s general availability. Here, we show that MINFLUX implemented with a standard microscope stand can attain 1–3 nm resolution in three dimensions, rendering fluorescence microscopy with molecule-scale resolution widely applicable. Advances, such as synchronized electro-optical and galvanometric beam steering and a stabilization that locks the sample position to sub-nanometer precision with respect to the stand, ensure nanometer-precise and accurate real-time localization of individually activated fluorophores. In our MINFLUX imaging of cell- and neurobiological samples, ~800 detected photons suffice to attain a localization precision of 2.2 nm, whereas ~2500 photons yield precisions <1 nm (standard deviation). We further demonstrate 3D imaging with localization precision of ~2.4 nm in the focal plane and ~1.9 nm along the optic axis. Localizing with a precision of <20 nm within ~100 µs, we establish this spatio-temporal resolution in single fluorophore tracking and apply it to the diffusion of single labeled lipids in lipid-bilayer model membranes.

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Design and calibration of an IVEM for general 3-dimensional imaging of non-diffracting materials

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100129838 ◽

1992 ◽

Vol 50 (2) ◽

pp. 1040-1041

Author(s):

Neil Rowlands ◽

Jeff Price ◽

Michael Kersker ◽

Seichi Suzuki ◽

Steve Young ◽

...

Keyword(s):

3D Imaging ◽

Tomographic Reconstruction ◽

Three Dimensional ◽

3 Dimensional ◽

3D Microstructure ◽

Image Translation ◽

Digital Correlation ◽

On Line ◽

Mechanical Stage ◽

Projection Angle

Three-dimensional (3D) microstructure visualization on the electron microscope requires that the sample be tilted to different positions to collect a series of projections. This tilting should be performed rapidly for on-line stereo viewing and precisely for off-line tomographic reconstruction. Usually a projection series is collected using mechanical stage tilt alone. The stereo pairs must be viewed off-line and the 60 to 120 tomographic projections must be aligned with fiduciary markers or digital correlation methods. The delay in viewing stereo pairs and the alignment problems in tomographic reconstruction could be eliminated or improved by tilting the beam if such tilt could be accomplished without image translation.A microscope capable of beam tilt with simultaneous image shift to eliminate tilt-induced translation has been investigated for 3D imaging of thick (1 μm) biologic specimens. By tilting the beam above and through the specimen and bringing it back below the specimen, a brightfield image with a projection angle corresponding to the beam tilt angle can be recorded (Fig. 1a).

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Analyzing reactions of single mechanoenzyme molecules by light microscopy

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100122538 ◽

1992 ◽

Vol 50 (1) ◽

pp. 426-427

Author(s):

Jeff Gelles

Keyword(s):

Image Processing ◽

Reaction Mechanisms ◽

Transient State ◽

Nanometer Scale ◽

Reaction Intermediates ◽

Enzyme Molecule ◽

Directed Movement ◽

Enzyme Molecules ◽

Individual Enzyme ◽

Single Reaction

Mechanoenzymes are enzymes which use a chemical reaction to power directed movement along biological polymer. Such enzymes include the cytoskeletal motors (e.g., myosins, dyneins, and kinesins) as well as nucleic acid polymerases and helicases. A single catalytic turnover of a mechanoenzyme moves the enzyme molecule along the polymer a distance on the order of 10−9 m We have developed light microscope and digital image processing methods to detect and measure nanometer-scale motions driven by single mechanoenzyme molecules. These techniques enable one to monitor the occurrence of single reaction steps and to measure the lifetimes of reaction intermediates in individual enzyme molecules. This information can be used to elucidate reaction mechanisms and determine microscopic rate constants. Such an approach circumvents difficulties encountered in the use of traditional transient-state kinetics techniques to examine mechanoenzyme reaction mechanisms.

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Scanning x-ray fluorescence microscopy and principal component analysis

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100133394 ◽

1992 ◽

Vol 50 (2) ◽

pp. 1752-1753

Author(s):

Brian Cross

Keyword(s):

Principal Component Analysis ◽

Fluorescence Microscopy ◽

Spatial Resolution ◽

High Speed ◽

Image Acquisition ◽

Principal Component ◽

Fluorescence Microscope ◽

Acquisition Rate ◽

X Ray ◽

Speed Up

A relatively new entry, in the field of microscopy, is the Scanning X-Ray Fluorescence Microscope (SXRFM). Using this type of instrument (e.g. Kevex Omicron X-ray Microprobe), one can obtain multiple elemental x-ray images, from the analysis of materials which show heterogeneity. The SXRFM obtains images by collimating an x-ray beam (e.g. 100 μm diameter), and then scanning the sample with a high-speed x-y stage. To speed up the image acquisition, data is acquired "on-the-fly" by slew-scanning the stage along the x-axis, like a TV or SEM scan. To reduce the overhead from "fly-back," the images can be acquired by bi-directional scanning of the x-axis. This results in very little overhead with the re-positioning of the sample stage. The image acquisition rate is dominated by the x-ray acquisition rate. Therefore, the total x-ray image acquisition rate, using the SXRFM, is very comparable to an SEM. Although the x-ray spatial resolution of the SXRFM is worse than an SEM (say 100 vs. 2 μm), there are several other advantages.

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