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Related Concept Videos

Confocal Fluorescence Microscopy01:16

Confocal Fluorescence Microscopy

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,...
Imaging Biological Samples with Optical Microscopy01:18

Imaging Biological Samples with Optical Microscopy

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.
In optical microscopy, the specimen to be viewed is placed on a glass slide and clipped on the stage...

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Near Simultaneous Laser Scanning Confocal and Atomic Force Microscopy (Conpokal) on Live Cells
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Near Simultaneous Laser Scanning Confocal and Atomic Force Microscopy (Conpokal) on Live Cells

Published on: August 11, 2020

Measurement in the confocal microscope.

Guy Cox1

  • 1Australian Centre for Microscopy & Microanalysis, University of Sydney, Sydney, NSW, Australia.

Methods in Molecular Biology (Clifton, N.J.)
|September 21, 2013
PubMed
Summary
This summary is machine-generated.

Accurate microscope measurements, including surface profiles and lengths, are achievable with proper alignment and settings. Advanced techniques like fluorescence lifetime imaging (FLIM) enable precise molecular interaction studies.

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Area of Science:

  • Microscopy and Image Analysis
  • Biophysics
  • Molecular Biology

Background:

  • Accurate quantitative measurements using microscopes depend critically on proper instrument alignment and signal level settings (gain/PMT voltage, black level) to prevent data loss.
  • While basic measurements like surface profiles, depths, lengths, and 2D areas are relatively straightforward with standard image analysis software, more complex analyses like volume and 3D surface area require specialized software or stereological techniques.

Purpose of the Study:

  • To outline the methodologies and considerations for various quantitative measurements using microscopy.
  • To highlight the capabilities and requirements for advanced techniques such as fluorescence intensity, ratiometric measurements, and Förster Resonance Energy Transfer (FRET) analysis.

Main Methods:

  • Standard image analysis software for length, 2D area, surface profiles, and relative depth measurements.
  • 3D analysis software or stereological techniques for volume and 3D surface area calculations.
  • Fluorescence Lifetime Imaging Microscopy (FLIM) for exploring chemical environments, multi-labeling, and FRET measurements; intensity-based methods with corrections for bleed-through and cross-talk or acceptor bleaching as alternatives for FRET without FLIM hardware.

Main Results:

  • Surface profile and relative depth measurements can exceed the microscope's depth resolution.
  • Measuring labeled object thickness is less accurate than other dimensional measurements.
  • FLIM provides detailed insights into molecular interactions at the nanometer scale and is the preferred method for FRET when available.

Conclusions:

  • Careful calibration and appropriate method selection are crucial for accurate fluorescence intensity and ratiometric measurements.
  • FLIM offers superior capabilities for studying molecular interactions and chemical environments compared to traditional intensity-based methods.
  • While basic measurements are accessible, advanced quantitative analyses in microscopy necessitate specialized tools and techniques for reliable results.