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Immunofluorescence Microscopy01:12

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.
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Updated: Jul 15, 2026

A Custom Multiphoton Microscopy Platform for Live Imaging of Mouse Cornea and Conjunctiva
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Published on: May 17, 2020

Multiphoton fluorescence and second harmonic generation microscopy for imaging infectious keratitis.

Hsin-Yuan Tan1, Yen Sun, Wen Lo

  • 1Chang Gung University, College of Medicine, Chang Gung Memorial Hospital, Department of Ophthalmology, Tao Yuan, Taiwan.

Journal of Biomedical Optics
|May 5, 2007
PubMed
Summary

Multiphoton microscopy visualizes human corneal infections ex-vivo by imaging cellular and collagen changes. This technique identifies structural alterations without histology, aiding potential clinical diagnosis of corneal diseases.

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Area of Science:

  • Ophthalmology
  • Biomedical Imaging
  • Microscopy

Background:

  • Corneal infections cause significant structural changes.
  • Accurate visualization is crucial for diagnosis and treatment.
  • Current diagnostic methods may require invasive procedures.

Purpose of the Study:

  • To demonstrate multiphoton fluorescence and second harmonic generation (SHG) microscopy for ex-vivo human corneal visualization.
  • To assess the capability of these imaging techniques in identifying infectious processes.
  • To evaluate the potential for clinical application in diagnosing corneal infections.

Main Methods:

  • Utilized multiphoton fluorescence microscopy to visualize cellular components.
  • Employed second harmonic generation (SHG) microscopy to image collagenous structures.
  • Applied these techniques for ex-vivo analysis of human corneal samples with infectious processes.

Main Results:

  • Multiphoton microscopy effectively visualized structural alterations in corneal cells and collagen.
  • Fluorescence imaging identified cellular changes, while SHG imaging revealed collagenous component alterations.
  • Pathogens exhibiting fluorescence were detectable within turbid corneal specimens.
  • The technique successfully identified structural alterations without the need for histological processing.

Conclusions:

  • Multiphoton microscopy is effective for ex-vivo visualization of corneal infections.
  • The combined fluorescence and SHG imaging modalities provide comprehensive structural information.
  • Further development could lead to a valuable clinical diagnostic and monitoring tool for corneal infections.