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Related Concept Videos

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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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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 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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Related Experiment Video

Updated: Nov 4, 2025

Dissection and 2-Photon Imaging of Peripheral Lymph Nodes in Mice
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Two-Photon Fluorescence Imaging.

Fan Feng1, Heng Mao2, Aimin Wang3

  • 1Center for Bioinformatics, School of Life Sciences, Peking University, Beijing, China.

Advances in Experimental Medicine and Biology
|May 30, 2021
PubMed
Summary
This summary is machine-generated.

Two-photon fluorescence imaging enables deep tissue visualization of cell dynamics in vivo. This advanced microscopy technique offers high-resolution, high-contrast imaging for neuroscience research.

Keywords:
Miniature two-photon microscopePan-neuronal imagingTwo-photon imaging

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In vivo Imaging of Biological Tissues with Combined Two-Photon Fluorescence and Stimulated Raman Scattering Microscopy
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Area of Science:

  • Neuroscience
  • Biomedical Imaging
  • Cell Biology

Background:

  • Two-photon fluorescence imaging is crucial for observing cellular dynamics within intact tissues.
  • It is particularly well-suited for imaging neuronal activity in neuroscience research.
  • The technique offers inherent optical sectioning due to nonlinear two-photon absorption.

Purpose of the Study:

  • To highlight the capabilities of two-photon fluorescence imaging for in vivo studies.
  • To emphasize its advantages for neuroscience research and deep tissue penetration.
  • To discuss its ongoing development for advanced biological applications.

Main Methods:

  • Utilizes nonlinear two-photon absorption for inherent optical sectioning.
  • Employs longer wavelength excitation light for reduced scattering and absorption by tissue.
  • Leverages efficient illumination for high signal contrast and signal-to-noise ratio.

Main Results:

  • Achieves high signal contrast and signal-to-noise ratio in two-photon images.
  • Enables deeper tissue penetration compared to one-photon imaging.
  • Facilitates detailed observation of cellular dynamics in vivo.

Conclusions:

  • Two-photon fluorescence imaging is a powerful tool for in vivo cellular dynamics and neuronal activity.
  • Its properties allow for deeper, clearer imaging in biological tissues.
  • Rapid advancements are expanding its applications in high-speed, high-resolution, and long-term live imaging.