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Related Concept Videos

Confocal Fluorescence Microscopy01:16

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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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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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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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Multifaceted mirror array illuminator for fluorescence excitation-scanning spectral imaging microscopy.

Marina Parker1,2, Samuel A Mayes1,2, Craig M Browning1,2

  • 1University of South Alabama, Department of Chemical and Biomolecular Engineering, Mobile, Alabama, United States.

Journal of Biomedical Optics
|February 10, 2023
PubMed
Summary

Hyperspectral imaging fluorescence excitation-scanning (HIFEX) microscopy uses an array of LEDs to rapidly acquire spectral data. This novel technique improves speed and discrimination for fluorescence microscopy applications.

Keywords:
excitation scanningfluorescence microscopyhyperspectral imaginglight emitting diodespectral imagingspectroscopy

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Area of Science:

  • Optical microscopy
  • Spectroscopy
  • Biophotonics

Background:

  • Hyperspectral imaging (HSI) in fluorescence microscopy enables multiplexed imaging and autofluorescence removal.
  • Current HSI systems face trade-offs like reduced speed and resolution due to spectral data acquisition.
  • Signal loss can occur in emission-based spectral discrimination methods.

Purpose of the Study:

  • To develop an optical technique for hyperspectral imaging fluorescence excitation-scanning (HIFEX) microscopy.
  • To mitigate trade-offs associated with traditional HSI fluorescence microscopy.
  • To decrease complexity in the emission light path by scanning the excitation spectrum.

Main Methods:

  • Designed an array of wavelength-dependent light emitting diodes (LEDs).
  • Developed a unique beam combining system utilizing a multifurcated mirror.
  • Modeled and optimized the system using optical ray trace simulations, built a prototype, and calibrated it.

Main Results:

  • The HIFEX system demonstrated discrimination between at least six fluorescent labels and autofluorescence.
  • Achieved decreased wavelength switching times compared to mechanically tuned filters.
  • Feasibility testing included imaging of multilabel slide preparations.

Conclusions:

  • LED-based HIFEX microscopy offers a promising approach for fluorescence imaging.
  • The technique may enhance performance for time-dependent and photosensitive assays.
  • Reduced complexity and improved speed are key advantages for advanced microscopy.