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Related Concept Videos

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,...
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.

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Related Experiment Video

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Multimodal Optical Imaging Platform for Studying Cellular Metabolism
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Multimodal Optical Imaging Platform for Studying Cellular Metabolism

Published on: June 6, 2025

Harmonic optical microscopy and fluorescence lifetime imaging platform for multimodal imaging.

Vitor B Pelegati1, Javier Adur, André A De Thomaz

  • 1Optics and Photonics Research Center, Gleb Wataghin Institute of Physics, State University of Campinas (UNICAMP), Departamento de Eletrônica Quôntica, Campinas, São Paulo. Brazil.

Microscopy Research and Technique
|June 1, 2012
PubMed
Summary

This study presents a novel multimodal optical setup integrating nonlinear microscopy (SHG, THG) and fluorescence lifetime imaging (FLIM) with a confocal microscope. The system enables advanced biological imaging with unprecedented versatility and ease of use.

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Area of Science:

  • Biomedical Optics
  • Microscopy
  • Advanced Imaging Techniques

Background:

  • Confocal microscopy is a standard tool, but integrating nonlinear optical (NLO) microscopy and fluorescence lifetime imaging microscopy (FLIM) presents challenges.
  • Simultaneous acquisition of two-photon excited fluorescence (TPEF) and second harmonic generation (SHG) is established, but integrating third harmonic generation (THG) and FLIM is less common due to technical hurdles.

Purpose of the Study:

  • To develop and demonstrate an easy-to-operate multimodal optical setup.
  • To integrate nonlinear microscopy (SHG, THG) and FLIM capabilities into a commercial confocal microscope.
  • To enable simultaneous imaging of TPEF, SHG, THG, and FLIM using a single laser source.

Main Methods:

  • Modification of a commercial confocal microscope (Olympus VF300) to incorporate NLO and FLIM modules.
  • Utilizing a single 80 MHz femtosecond Ti:sapphire laser source for all imaging modalities.
  • Verification of SHG and THG signals through physical evidence.

Main Results:

  • Successful integration of confocal, brightfield, NLO (SHG, THG), and FLIM imaging.
  • Demonstration of a versatile platform capable of acquiring TPEF, SHG, THG, and FLIM simultaneously.
  • Acquisition of images from vegetable and epithelial cancer biological samples using the integrated system.

Conclusions:

  • The developed multimodal optical setup offers a powerful and versatile platform for advanced biological imaging.
  • This integrated system overcomes previous limitations in combining THG and FLIM with other modalities.
  • The easy-to-operate nature of the platform facilitates broader application in biomedical research.