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Immunofluorescence Microscopy

Immunofluorescence Microscopy

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.

Immunogold Electron Microscopy

Immunogold Electron Microscopy

Immunoelectron microscopy utilizes immunogold labeling of endogenous proteins with specific antibodies to detect and localize these proteins in cells and tissues. The procedure provides insights into the distribution and quantification of protein under different stimulation conditions offering clues about their functions. Conjugating highly electron-dense gold particles with primary or secondary antibodies allow antigen detection on and within cells, with high resolution and specificity.

Fixation and Sectioning

Fixation and Sectioning

Two basic types of preparation are used to visualize specimens with a light microscope: wet mounts and fixed specimens.
The simplest type of preparation is the wet mount, in which the specimen is placed in a drop of liquid on the slide. A liquid specimen can be directly deposited on the slide using a dropper. Solid specimens, such as skin scraping, can be placed on the slide before adding a drop of liquid to prepare the wet mount. Sometimes the liquid is simply water, but stains are often added...

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Related Experiment Video

Updated: Nov 1, 2025

Author Spotlight: Enhanced Multiplex Immunofluorescent Microscopy Protocol for Neuroscience Research

Author Spotlight: Enhanced Multiplex Immunofluorescent Microscopy Protocol for Neuroscience Research

Published on: June 21, 2024

Chemical Sectioning for Immunofluorescence Imaging.

Ruixi Chen^1,2, Hongfu Zhou^1,2, Anan Li^1,2

¹Britton Chance Center for Biomedical Photonics, Wuhan National Laboratory for Optoelectronics-Huazhong University of Science and Technology, Wuhan, Hubei 430074, China.

Analytical Chemistry

|June 17, 2021

Summary

This summary is machine-generated.

Immunofluorescence chemical sectioning (IF-CS) enables clear imaging of tissues labeled with common dyes by controlling fluorescence. This new method overcomes limitations of existing techniques for detailed subcellular studies.

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Area of Science:

Biomedical Imaging
Cell Biology
Microscopy

Background:

Conventional imaging methods face trade-offs between speed, resolution, and specimen volume.
Chemical sectioning (CS) improves imaging by selectively activating superficial fluorescence but is limited to pH-sensitive fluorescent proteins.
Existing CS methods cannot be used with commonly employed commercial fluorescent dyes in immunolabeling.

Purpose of the Study:

To develop a novel chemical sectioning (CS) method for immunofluorescence (IF) imaging.
To enable CS imaging for samples labeled with frequently used commercial fluorescent dyes, such as Alexa dyes.
To overcome the limitations of conventional imaging and existing CS techniques for high-resolution tissue analysis.

Main Methods:

Development of immunofluorescence chemical sectioning (IF-CS) utilizing complexation reactions to control Alexa dye fluorescence.
Application of IF-CS to wide-field block-face imaging systems.
Demonstration of 3D submicron-resolution imaging of large immunolabeled tissues.

Main Results:

IF-CS successfully enabled chemical sectioning for tissues labeled with Alexa dyes.
The method eliminated out-of-focus interference in wide-field immunofluorescence imaging.
High-resolution 3D imaging of large immunolabeled tissues was achieved, supporting multicolor imaging.

Conclusions:

IF-CS provides a versatile solution for imaging immunolabeled tissues, overcoming previous limitations.
This technique allows for detailed submicron-resolution 3D imaging of large tissue samples.
IF-CS is expected to significantly advance systematic studies of subcellular architectures in intact biological systems.