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Related Concept Videos

Super-resolution Fluorescence Microscopy01:37

Super-resolution Fluorescence Microscopy

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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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Two-Dimensional Microscopy in Microbiology01:29

Two-Dimensional Microscopy in Microbiology

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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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Immunofluorescence Microscopy01:12

Immunofluorescence Microscopy

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A fluorescence microscope uses fluorescent chromophores called fluorochromes, which can absorb energy from a light source and then emit this energy as visible light. Fluorochromes include naturally fluorescent substances (such as chlorophylls) and fluorescent stains that are added to the specimen to create contrast. Dyes such as Texas red and FITC are examples of fluorochromes. Other examples include the nucleic acid dyes 4’,6’-diamidino-2-phenylindole (DAPI), and acridine orange.
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Confocal Fluorescence Microscopy01:16

Confocal Fluorescence Microscopy

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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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Fluorescence and Phosphorescence: Instrumentation01:25

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Fluorometers and spectrofluorometers are two types of instruments used for measuring molecular fluorescence. These instruments differ in how they select excitation and emission wavelengths and the type of light sources they utilize. Fluorometers use absorption interference filters to choose excitation and emission wavelengths. The excitation source in a fluorometer is typically a low-pressure mercury vapor lamp that emits intense lines distributed throughout the ultraviolet and visible regions.
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Total Internal Reflection Fluorescence Microscopy01:05

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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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Conducting Multiple Imaging Modes with One Fluorescence Microscope
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Conducting Multiple Imaging Modes with One Fluorescence Microscope

Published on: October 28, 2018

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Fluorescence microscopy.

Michael J Sanderson1, Ian Smith2, Ian Parker2

  • 1Department of Microbiology and Physiology Systems, University of Massachusetts Medical School, Worcester, Massachusetts 01655;

Cold Spring Harbor Protocols
|October 3, 2014
PubMed
Summary
This summary is machine-generated.

This review covers advanced fluorescence microscopy techniques for cell physiology studies. It details laser scanning confocal, two-photon, and super-resolution microscopy, highlighting their image formation principles and applications.

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Area of Science:

  • Cell Biology
  • Microscopy Techniques

Background:

  • Fluorescence microscopy is essential for monitoring cell physiology.
  • Microscope design advances aim to improve image contrast and spatial resolution.

Purpose of the Study:

  • To outline the basics of wide-field microscopy.
  • To emphasize selection, advantages, and correct use of advanced microscopy.
  • To review image formation principles, capabilities, and limitations.

Main Methods:

  • Discussion of wide-field microscopy principles.
  • Emphasis on laser scanning confocal, two-photon, scanning disk confocal, total internal reflection, and super-resolution microscopy.
  • Review of image formation principles for each technique.

Main Results:

  • Provides an overview of various fluorescence microscopy techniques.
  • Explains the operational principles and applications of advanced microscopes.
  • Highlights the capabilities and limitations of different imaging modalities.

Conclusions:

  • Understanding different microscopy techniques is crucial for effective cell physiology research.
  • Proper selection and use of advanced microscopes enhance biological insights.
  • Knowledge of image formation aids in appreciating microscope constraints and potential.