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Related Concept Videos

Super-resolution Fluorescence Microscopy01:37

Super-resolution Fluorescence Microscopy

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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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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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Fluorescence and Phosphorescence: Instrumentation01:25

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Fluorometers and spectrofluorometers are two types of instruments used for measuring molecular fluorescence. These instruments differ in how they select excitation and emission wavelengths and the type of light sources they utilize. Fluorometers use absorption interference filters to choose excitation and emission wavelengths. The excitation source in a fluorometer is typically a low-pressure mercury vapor lamp that emits intense lines distributed throughout the ultraviolet and visible regions.
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Total Internal Reflection Fluorescence Microscopy01:05

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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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Updated: May 29, 2025

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Merging Vibrational Spectroscopy with Fluorescence Microscopy: Combining the Best of Two Worlds.

Naixin Qian1, Hanqing Xiong2, Lu Wei3

  • 1Department of Chemistry, Columbia University, New York, NY, USA; email: qn2120@columbia.edu, wm2256@columbia.edu.

Annual Review of Physical Chemistry
|February 3, 2025
PubMed
Summary

This review explores vibrational-encoded fluorescence microscopy, merging vibrational and fluorescence spectroscopy for enhanced chemical specificity and detection sensitivity. New techniques enable single-molecule vibrational spectroscopy and superresolution imaging.

Keywords:
double-resonance spectroscopyfluorescence microscopynonlinear spectroscopyoptical imagingsingle-molecule spectroscopyvibrational spectroscopyvibrational-encoded fluorescence microscopy

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Area of Science:

  • Molecular Spectroscopy
  • Nonlinear Optical Spectroscopy
  • Microscopy

Background:

  • Vibrational spectroscopy offers chemical specificity, while fluorescence spectroscopy provides high sensitivity.
  • Historically, these two fields were separate, each lacking the other's advantages.
  • Novel nonlinear optical techniques now merge these spectroscopies.

Purpose of the Study:

  • To review the emerging field of vibrational-encoded fluorescence microscopy.
  • To summarize key technical developments and applications.
  • To discuss future prospects in this interdisciplinary area.

Main Methods:

  • Development of double-resonance nonlinear optical spectroscopy.
  • Combining Raman or infrared (IR) spectroscopy with fluorescence microscopy.
  • Exploring time and frequency domains, and spectroscopy versus microscopy.

Main Results:

  • Achieved single-molecule vibrational spectroscopy at room temperature without plasmonics.
  • Demonstrated superresolution vibrational imaging beyond the diffraction limit.
  • Successfully merged chemical specificity with high detection sensitivity and spatial resolution.

Conclusions:

  • Vibrational-encoded fluorescence microscopy represents a significant advancement in molecular spectroscopy.
  • This technique offers unprecedented capabilities for chemical analysis and imaging.
  • The field holds great promise for future research and applications.