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Related Concept Videos

Confocal Fluorescence Microscopy01:16

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Confocal microscopy is an advanced microscopic technique. The prime advantage of the confocal microscope over other microscopy techniques is its ability to block the out-of-focus light from the illuminated samples using pinholes. It is widely used with fluorescence optics to obtain high-resolution, sharp contrast images. Unlike optical microscopes, confocal microscopes use a focused beam of light laser to scan the entire sample surface at different z-planes. These microscopes are, therefore,...
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Total internal reflection fluorescence microscopy or TIRF is an advanced microscopic technique used to visualize fluorophores in samples close to a solid surface with a higher refractive index, such as a glass coverslip. TIRF only allows fluorophores in proximity to the solid surface to be excited. When light from a medium with a lower refractive index (such as air) hits the glass coverslip at a critical angle, the light undergoes total internal reflection stead of passing through the glass.
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Super-resolution fluorescence microscopy (SRFM) provides a better resolution than conventional fluorescence microscopy by reducing the point spread function (PSF). PSF is the light intensity distribution from a point that causes it to appear blurred. Due to PSF, each fluorescing point appears bigger than its actual size, and it is the PSF interference of nearby fluorophores that causes the blurred image. Various approaches to achieving higher resolution through SRFM have recently been...
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Imaging Biological Samples with Optical Microscopy01:18

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Optical microscopy uses optic principles to provide detailed images of samples. Antonie van Leeuwenhoek designed the first compound optical microscope in the 17th century to visualize blood cells, bacteria, and yeast cells. In 1830, Joseph Jackson Lister created an essentially modern light microscope. The 20th century saw the development of microscopes with enhanced magnification and resolution.
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Transmission electron microscopy (TEM) can be used to determine the 3D structure of biological samples with the help of techniques such as electron microscope tomography and single-particle reconstruction. While single-particle reconstruction can examine macromolecules and macromolecular complexes in vitro conditions only, tomography permits the study of cell components or small cells in vivo.
Electron Tomography
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Phase-Contrast Microscopes
In-phase-contrast microscopes, interference between light directly passing through a cell and light refracted by cellular components is used to create high-contrast, high-resolution images without staining. It is the oldest and simplest type of microscope that creates an image by altering the wavelengths of light rays passing through the specimen. Altered wavelength paths are created using an annular stop in the condenser. The annular stop produces a hollow cone of...
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Related Experiment Video

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Workflow Using a Cryogenic Coincident Fluorescence, Electron, and Ion Beam Microscope for Targeted Milling of Cells
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Fluorescence micro-optical sectioning tomography using acousto-optical deflector-based confocal scheme.

Xiaoli Qi1, Tao Yang1, Longhui Li1

  • 1Britton Chance Center for Biomedical Photonics, Huazhong University of Science and Technology, Wuhan National Laboratory for Optoelectronics, 1037 Luoyu Road, Wuhan 430074, China; Huazhong University of Science and Technology, Department of Biomedical Engineering, Key Laboratory of Biomedical Photonics of Ministry of Education, 1037 Luoyu Road, Wuhan 430074, China.

Neurophotonics
|January 22, 2016
PubMed
Summary
This summary is machine-generated.

This study optimizes a fluorescence micro-optical sectioning tomography system for brain-wide neural circuit imaging. Combining mechanical and optical sectioning enhances signal-to-background noise ratio for clearer neural wiring visualization.

Keywords:
acousto-optical deflectorbrain-wide imagingconfocal imagingfluorescence microscopy

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Area of Science:

  • Neuroscience
  • Optical Imaging
  • Biotechnology

Background:

  • Fluorescent labeling enables detailed study of neural circuits.
  • Previous development of fluorescence micro-optical sectioning tomography (fMOST) for whole-mouse brain imaging.
  • Need for optimizing imaging parameters for high-resolution neural circuit mapping.

Purpose of the Study:

  • To analyze optical considerations for an acousto-optical deflector (AOD) scanner-based confocal detection scheme in fMOST.
  • To improve signal-to-background noise ratio for brain-wide neural wiring visualization.
  • To optimize simultaneous mechanical and optical sectioning for enhanced background suppression.

Main Methods:

  • Utilized an acousto-optical deflector (AOD) scanner-based confocal detection scheme.
  • Analyzed the influence of confocal detection parameters on imaging.
  • Investigated the impact of imaging site during sectioning and AOD fast scan mode.
  • Evaluated signal-to-background noise ratio under different optical and mechanical sectioning conditions.

Main Results:

  • Characterized the influence of confocal detection on image quality.
  • Determined the effect of imaging site during sectioning on signal-to-background noise ratio.
  • Quantified the impact of AOD fast scan mode on imaging performance.
  • Demonstrated that combining mechanical and optical sectioning maximizes background suppression.

Conclusions:

  • Optimized optical and mechanical sectioning strategies are crucial for high-quality fMOST.
  • AOD scanner-based confocal detection can be effectively integrated into fMOST systems.
  • The developed system enables clearer visualization of brain-wide neural wiring for functional studies.