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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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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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Fluholoscopy-Compact and Simple Platform Combining Fluorescence and Holographic Microscopy.

David Alonso1, Javier Garcia1, Vicente Micó1

  • 1Departamento de Óptica y de Optometría y Ciencias de la Visión, Facultad de Física, Universidad de Valencia, C/Doctor Moliner 50, 46100 Burjassot, Spain.

Biosensors
|February 25, 2023
PubMed
Summary

This study introduces a simple, cost-effective microscope platform for simultaneous fluorescence and quantitative phase imaging. This dual-mode imaging enables comprehensive analysis of biological samples in a single snapshot.

Keywords:
Gabor holographyfluorescence imagingmultimodal imagingmultiplexed microscopyquantitative phase imaging

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Area of Science:

  • Biomedical optics
  • Microscopy
  • Imaging technologies

Background:

  • Combining imaging modalities enhances sample analysis by revealing complementary properties.
  • Existing multimodal imaging platforms can be complex and costly.

Purpose of the Study:

  • To develop a simple, cost-effective, and compact microscope for simultaneous fluorescence and quantitative phase imaging.
  • To demonstrate the platform's capability for single-snapshot, dual-mode imaging.

Main Methods:

  • A single illumination wavelength excites fluorescence and provides coherent light for phase imaging.
  • A bandpass filter separates the two imaging paths.
  • Two digital cameras capture simultaneous fluorescence and phase images.

Main Results:

  • The platform successfully integrates fluorescence and quantitative phase imaging.
  • Independent calibration and analysis of each modality were performed.
  • Experimental validation was conducted on static and dynamic biological samples.

Conclusions:

  • The developed common-path dual-mode imaging platform is effective for simultaneous fluorescence and quantitative phase imaging.
  • This technology offers a cost-effective solution for advanced biomedical imaging.
  • The platform's versatility is demonstrated across various static and dynamic samples.