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Related Concept Videos

Super-resolution Fluorescence Microscopy01:37

Super-resolution Fluorescence Microscopy

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 developed.
Total Internal Reflection Fluorescence Microscopy01:05

Total Internal Reflection Fluorescence Microscopy

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

Updated: May 14, 2026

Conducting Multiple Imaging Modes with One Fluorescence Microscope
08:32

Conducting Multiple Imaging Modes with One Fluorescence Microscope

Published on: October 28, 2018

Single-molecule imaging and spectroscopy using fluorescence and surface-enhanced Raman scattering.

M Ishikawa1, Y Maruyama, J-Y Ye

  • 1Joint Research Center for Atom Technology (JRCAT), 1-1-4 Higashi, Tsukuba, Ibaraki, 305-0046 Japan.

Journal of Biological Physics
|January 25, 2013
PubMed
Summary

Researchers developed new methods for detecting single nucleic acid bases using violet light fluorescence and surface-enhanced Raman scattering. This breakthrough enables precise identification of individual molecules for advanced biological studies.

Keywords:
Klenow fragmentTNP-ATPadeninefluorescencesilver particlessingle molecule imagingsurface-enhanced Raman scattering

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In vivo Imaging of Biological Tissues with Combined Two-Photon Fluorescence and Stimulated Raman Scattering Microscopy
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Last Updated: May 14, 2026

Conducting Multiple Imaging Modes with One Fluorescence Microscope
08:32

Conducting Multiple Imaging Modes with One Fluorescence Microscope

Published on: October 28, 2018

In vivo Imaging of Biological Tissues with Combined Two-Photon Fluorescence and Stimulated Raman Scattering Microscopy
09:06

In vivo Imaging of Biological Tissues with Combined Two-Photon Fluorescence and Stimulated Raman Scattering Microscopy

Published on: December 20, 2021

Area of Science:

  • Biophysics
  • Spectroscopy
  • Molecular Biology

Background:

  • Single-molecule detection is crucial for understanding biological processes.
  • Current methods for analyzing nucleic acid bases at the single-molecule level have limitations.

Purpose of the Study:

  • To explore the feasibility of detecting and identifying fluorescent analogs of nucleic acid bases at the single-molecule level.
  • To extend single-molecule fluorescence imaging and time-resolved fluorometry to the violet-excitation regime.
  • To implement Raman imaging and spectroscopy for single-molecule nucleic acid base identification.

Main Methods:

  • Utilized violet-excitation single-molecule fluorescence imaging and time-resolved fluorometry.
  • Observed fluorescent spots from single complexes of nucleotide analogs and DNA polymerase I Klenow fragment.
  • Employed surface-enhanced Raman scattering (SERS) spectroscopy of adenine molecules on silver colloidal nanoparticles.

Main Results:

  • Successfully detected fluorescent spots from single nucleotide analog-DNA polymerase complexes using violet excitation.
  • Observed intermittent on-and-off blinking behavior in the SERS spectra of single adenine molecules.
  • Demonstrated that blinking provides substantial evidence for single adenine molecule detection.

Conclusions:

  • Violet-excitation fluorescence techniques are feasible for single-molecule detection of nucleotide analogs.
  • SERS spectroscopy, evidenced by blinking, is a viable method for identifying individual nucleic acid bases.
  • These advancements offer new possibilities for high-resolution molecular analysis in biology.