scholarly journals Multifocal imaging for precise, label-free tracking of fast biological processes in 3D

2020 ◽  
Author(s):  
Jan N. Hansen ◽  
An Gong ◽  
Dagmar Wachten ◽  
René Pascal ◽  
Alex Turpin ◽  
...  
Keyword(s):  
Spatial Resolution ◽  
High Speed ◽  
Reconstruction Algorithm ◽  
Cost Effective ◽  
Human Sperm ◽  
Dark Field ◽  
Biological Processes ◽  
Label Free ◽  
Flagellar Beat ◽  
Dark Field Microscopy

AbstractMany biological processes happen on a nano- to millimeter scale and within milliseconds. Established methods such as confocal microscopy are suitable for precise 3D recordings but lack the temporal or spatial resolution to resolve fast 3D processes and require labeled samples. Multifocal imaging (MFI) allows high-speed 3D imaging but suffers from the compromise between spatial resolution and field-of-view (FOV), requiring bright fluorescent labels and limiting its application. Here, we present a new approach for high-resolution, label-free, high-speed MFI, based on dark-field microscopy and operative over large volumes. We introduce a 3D reconstruction algorithm that increases resolution and depth of the sampled volume without compromising speed and FOV. This allowed us to characterize the flagellar beat of human sperm and surrounding fluid flow with a precision below the Abbe limit, in a large volume, and at high speed. Our MFI concept is cost-effective, can be easily built, and does not rely on object labeling, making it broadly applicable.

scholarly journals Multifocal imaging for precise, label-free tracking of fast biological processes in 3D

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Jan N. Hansen ◽  
An Gong ◽  
Dagmar Wachten ◽  
René Pascal ◽  
Alex Turpin ◽  
...  
Keyword(s):  
Spatial Resolution ◽  
High Speed ◽  
Reconstruction Algorithm ◽  
Cost Effective ◽  
Large Field ◽  
Biological Processes ◽  
Label Free ◽  
Flagellar Beat ◽  
Sampling Volume ◽  
High Depth

AbstractMany biological processes happen on a nano- to millimeter scale and within milliseconds. Established methods such as confocal microscopy are suitable for precise 3D recordings but lack the temporal or spatial resolution to resolve fast 3D processes and require labeled samples. Multifocal imaging (MFI) allows high-speed 3D imaging but is limited by the compromise between high spatial resolution and large field-of-view (FOV), and the requirement for bright fluorescent labels. Here, we provide an open-source 3D reconstruction algorithm for multi-focal images that allows using MFI for fast, precise, label-free tracking spherical and filamentous structures in a large FOV and across a high depth. We characterize fluid flow and flagellar beating of human and sea urchin sperm with a z-precision of 0.15 µm, in a volume of 240 × 260 × 21 µm, and at high speed (500 Hz). The sampling volume allowed to follow sperm trajectories while simultaneously recording their flagellar beat. Our MFI concept is cost-effective, can be easily implemented, and does not rely on object labeling, which renders it broadly applicable.

scholarly journals Label-Free Single-Particle Imaging of the Influenza Virus by Objective-Type Total Internal Reflection Dark-Field Microscopy

PLoS ONE ◽  
2012 ◽  
Vol 7 (11) ◽  
pp. e49208 ◽  
Author(s):  
Sawako Enoki ◽  
Ryota Iino ◽  
Nobuhiro Morone ◽  
Kunihiro Kaihatsu ◽  
Shouichi Sakakihara ◽  
...  
Keyword(s):  
Influenza Virus ◽  
Single Particle ◽  
Total Internal Reflection ◽  
Dark Field ◽  
Internal Reflection ◽  
Label Free ◽  
Dark Field Microscopy ◽  
Particle Imaging ◽  
Single Particle Imaging ◽  
Objective Type

Label-free hyperspectral dark-field microscopy for quantitative scatter imaging

2017 ◽  
Author(s):  
Philip Cheney ◽  
David McClatchy ◽  
Stephen Kanick ◽  
Paul Lemaillet ◽  
David Allen ◽  
...  
Keyword(s):  
Dark Field ◽  
Label Free ◽  
Dark Field Microscopy

scholarly journals Label-free high-speed wide-field imaging of single microtubules using interference reflection microscopy

2018 ◽  
Author(s):  
Mohammed Mahamdeh ◽  
Steve Simmert ◽  
Anna Luchniak ◽  
Erik Schäeffer ◽  
Jonathon Howard
Keyword(s):  
High Speed ◽  
Signal To Noise Ratio ◽  
Dark Field ◽  
Fluorescent Labeling ◽  
Wide Field ◽  
Differential Interference Contrast ◽  
Label Free ◽  
High Speed Imaging ◽  
Highly Sensitive

SummaryWhen studying microtubules in vitro, label free imaging of single microtubules is necessary when the quantity of purified tubulin is too low for efficient fluorescent labeling or there is concern that labelling will disrupt its function. Commonly used techniques for observing unlabeled microtubules, such as video enhanced differential interference contrast, dark-field and more recently laser-based interferometric scattering microscopy, suffer from a number of drawbacks. The contrast of differential interference contrast images depends on the orientation of the microtubules, dark-field is highly sensitive to impurities and optical misalignments, and interferometric scattering has a limited field of view. In addition, all of these techniques require costly optical components such as Nomarski prisms, dark-field condensers, lasers and laser scanners. Here we show that single microtubules can be imaged at high speed and with high contrast using interference reflection microscopy without the aforementioned drawbacks. Interference reflection microscopy is simple to implement, requiring only the incorporation of a 50/50 mirror instead of a dichroic in a fluorescence microscope, and with appropriate microscope settings has similar signal-to-noise ratio to differential interference contrast and fluorescence. We demonstrated the utility of interference reflection microscopy by high speed imaging and tracking of dynamic microtubules at 100 frames per second. In conclusion, the image quality of interference reflection microscopy is similar to or exceeds that of all other techniques and, with minimal microscope modification, can be used to study the dynamics of unlabeled microtubules.

Elastic Scattering in Electron Microscopy

1973 ◽  
Vol 31 ◽  
pp. 252-253
Author(s):  
J. Langmore ◽  
M. Isaacson ◽  
J. Wall ◽  
A. V. Crewe
Keyword(s):  
Electron Microscopy ◽  
Elastic Scattering ◽  
Cross Section ◽  
Quantum Noise ◽  
Dark Field ◽  
Elastic Cross Section ◽  
Biological Molecules ◽  
Dark Field Microscopy ◽  
Field Systems ◽  
Effects Of Radiation

High resolution dark field microscopy is becoming an important tool for the investigation of unstained and specifically stained biological molecules. Of primary consideration to the microscopist is the interpretation of image Intensities and the effects of radiation damage to the specimen. Ignoring inelastic scattering, the image intensity is directly related to the collected elastic scattering cross section, σɳ, which is the product of the total elastic cross section, σ and the eficiency of the microscope system at imaging these electrons, η. The number of potentially bond damaging events resulting from the beam exposure required to reduce the effect of quantum noise in the image to a given level is proportional to 1/η. We wish to compare η in three dark field systems.

Simulated Dark-Field Electron Micrographs of Point Defects and Organometallics

1975 ◽  
Vol 33 ◽  
pp. 200-201
Author(s):  
William Krakow
Keyword(s):  
Electron Micrographs ◽  
Point Defects ◽  
Structure Determination ◽  
Atomic Structure ◽  
Image Interpretation ◽  
Dark Field ◽  
Field Electron ◽  
Dark Field Microscopy ◽  
Dark Field Electron

Tilted beam dark-field microscopy has been applied to atomic structure determination in perfect crystals, several synthesized molecules with heavy atcm markers and in the study of displaced atoms in crystals. Interpretation of this information in terms of atom positions and atom correlations is not straightforward. Therefore, calculated dark-field images can be an invaluable aid in image interpretation.

Scanning x-ray fluorescence microscopy and principal component analysis

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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