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Related Concept Videos

Two-Dimensional Microscopy in Microbiology01:29

Two-Dimensional Microscopy in Microbiology

Two-dimensional (2D) microscopy encompasses a range of optical techniques that capture images within a single focal plane, offering detailed representations of microscopic structures. These techniques are essential in biological and medical research, enabling the visualization of cellular and subcellular structures with different levels of contrast and specificity.There are several major types of 2D microscopy, each with strengths and applications.Bright-Field MicroscopyBright-field microscopy...
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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,...
Total Internal Reflection Fluorescence Microscopy01:05

Total Internal Reflection Fluorescence Microscopy

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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Conducting Multiple Imaging Modes with One Fluorescence Microscope
08:32

Conducting Multiple Imaging Modes with One Fluorescence Microscope

Published on: October 28, 2018

Dual objective fluorescence microscopy for single molecule imaging applications.

Sripad Ram1, Prashant Prabhat, E Sally Ward

  • 1Department of Immunology, University of Texas Southwestern Medical Center, Dallas, TX, 75390.

Proceedings of Spie--The International Society for Optical Engineering
|August 21, 2012
PubMed
Summary
This summary is machine-generated.

This study introduces a dual objective imaging configuration for enhanced single molecule fluorescence microscopy. This setup improves photon collection efficiency, leading to more accurate single molecule localization in cellular studies.

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Last Updated: May 19, 2026

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Published on: October 28, 2018

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Area of Science:

  • Biophysics
  • Optical Microscopy
  • Cell Biology

Background:

  • Fluorescence microscopy is crucial for studying cellular processes.
  • Single molecule imaging offers advantages over bulk experiments by eliminating ensemble averaging.
  • Detecting sufficient photons is essential for identifying and quantitatively analyzing single molecules.

Purpose of the Study:

  • To demonstrate a dual objective imaging configuration for single molecule studies.
  • To evaluate the photon collection efficiency of this novel configuration.
  • To improve the accuracy of single molecule localization.

Main Methods:

  • Utilizing a dual objective imaging setup with opposing inverted and upright objective lenses.
  • Comparing photon collection efficiency against a regular microscope under identical illumination.
  • Developing analytical tools for estimating 2D molecular locations and their accuracy.

Main Results:

  • The dual objective configuration significantly enhances photon collection efficiency compared to conventional microscopes.
  • This increased efficiency enables more precise localization of single molecules.
  • Analytical tools were successfully applied to estimate molecular positions and their accuracy.

Conclusions:

  • The dual objective imaging configuration is a valuable advancement for single molecule fluorescence microscopy.
  • Improved photon collection leads to superior accuracy in single molecule localization.
  • This technique provides enhanced capabilities for quantitative analysis in cell biology.