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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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Optical microscopy uses optic principles to provide detailed images of samples. Antonie van Leeuwenhoek designed the first compound optical microscope in the 17th century to visualize blood cells, bacteria, and yeast cells. In 1830, Joseph Jackson Lister created an essentially modern light microscope. The 20th century saw the development of microscopes with enhanced magnification and resolution.
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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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Related Experiment Video

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Cornea characterization using a combined multiphoton microscopy and optical coherence tomography system.

Tom Lai1, Shuo Tang1

  • 1Department of Electrical and Computer Engineering, University of British Columbia, 2332 Main Mall, Vancouver, BC, V6T 1Z4, Canada.

Biomedical Optics Express
|May 31, 2014
PubMed
Summary

A new multimodal imaging system combining multiphoton microscopy and optical coherence tomography reveals species-specific corneal collagen structures and quantifies refractive index (RI) and thickness for comprehensive characterization.

Keywords:
(110.4500) Optical coherence tomography(180.4315) Nonlinear microscopy

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Area of Science:

  • Ophthalmology
  • Biomedical Imaging
  • Biophysics

Background:

  • Corneal structure and optical properties are crucial for vision.
  • Existing imaging techniques have limitations in simultaneously visualizing morphology and quantifying optical properties.

Purpose of the Study:

  • To develop and validate a multimodal imaging system for comprehensive corneal characterization.
  • To compare corneal morphology and optical properties across different species.

Main Methods:

  • Combined multiphoton microscopy and optical coherence tomography.
  • Visualized corneal layers (epithelium, stroma, Bowman's layer, Descemet's membrane).
  • Quantified refractive index (RI) and thickness of epithelial and stromal layers.

Main Results:

  • Identified species-specific stromal collagen patterns (fibrous in piscine/human, interwoven in murine/porcine/bovine).
  • Observed collagen micro-folds in human corneas causing light attenuation.
  • Epithelium consistently showed a slightly higher RI than the stroma across species.
  • Provided quantitative RI and thickness data for murine, piscine, human, porcine, and bovine corneas.

Conclusions:

  • The multimodal system effectively visualizes corneal morphology and quantifies optical properties.
  • Significant species-dependent variations in corneal structure and RI exist.
  • This system offers a powerful tool for detailed corneal analysis and research.