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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.
Confocal Fluorescence Microscopy01:16

Confocal Fluorescence Microscopy

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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Related Experiment Video

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Single Molecule Fluorescence Microscopy on Planar Supported Bilayers
20:00

Single Molecule Fluorescence Microscopy on Planar Supported Bilayers

Published on: October 31, 2015

Spatial filter based bessel-like beam for improved penetration depth imaging in fluorescence microscopy.

Subhajit B Purnapatra1, Sampa Bera, Partha Pratim Mondal

  • 1Nanobioimaging Laboratory, Department of Instrumentation and Applied Physics, Indian Institute of Science, Bangalore 560012, India.

Scientific Reports
|September 27, 2012
PubMed
Summary

This study introduces a novel Bessel-like beam and orthogonal detection system for deep fluorescence imaging. The system achieves 650 µm penetration depth, enabling detailed visualization in thick specimens and advancing nano-particle tracking.

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Area of Science:

  • Biophysics
  • Optical Imaging
  • Microscopy

Background:

  • Deep tissue imaging is limited by scattering, distortion, and noise.
  • Current methods struggle to visualize biological phenomena at depths beyond hundreds of microns.

Purpose of the Study:

  • To develop an advanced imaging system for deep penetration fluorescence microscopy.
  • To overcome the limitations of conventional imaging techniques in thick biological samples.

Main Methods:

  • Utilized a Bessel-like penetrating diffractionless beam generated by engineering the excitation objective's back-aperture.
  • Implemented an orthogonal detection system allowing continuous scanning via detection point spread function (PSF) translation.
  • Tested the system on fluorescent polymer-tagged-CaCO₃ particles and yeast cells within a tissue-like gel matrix.

Main Results:

  • Achieved a penetration depth of up to 650 µm in a tissue-like gel matrix.
  • Demonstrated effective imaging of nano-particles and cellular structures at significant depths.
  • Maintained image quality despite depth-induced physical effects like scattering and noise.

Conclusions:

  • The Bessel-like beam and orthogonal detection system significantly enhance deep fluorescence imaging capabilities.
  • This approach is highly beneficial for applications such as deep nano-particle tracking in thick tissues.
  • The developed system represents a substantial advancement for fluorescence microscopy and deep imaging applications.