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Simultaneous two-photon spectral and lifetime fluorescence microscopy.

Damian K Bird1, Kevin W Eliceiri, Ching-Hua Fan

  • 1Laboratory for Optical and Computational Instrumentation, Department of Molecular Biology, 271 Animal Sciences, 1675 Observatory Drive, University of Wisconsin-Madison, Madison, Wisconsin 53706, USA. dbird@wisc.edu

Applied Optics
|October 12, 2004
PubMed
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This study introduces a novel microscope combining spectral and lifetime imaging to analyze fluorescence photons from living cells. This advanced technique offers detailed molecular and microenvironmental insights for cellular physiology research.

Area of Science:

  • Cellular Biology
  • Biophysics
  • Microscopy

Background:

  • Fluorescence photons from molecules in living cells contain unique signatures.
  • These signatures can identify molecules and their microenvironments, offering insights into cell physiology.
  • Unambiguous identification and monitoring require exploiting comprehensive fluorescent signature information.

Purpose of the Study:

  • To develop and implement a novel microscopy technique for detailed analysis of cellular fluorescence.
  • To enable unambiguous identification of fluorescent probes and monitoring of their physiological environment.
  • To visualize cellular structures and processes using spectrally and temporally resolved fluorescence data.

Main Methods:

  • Development and implementation of a combined two-photon spectral and lifetime microscope.

Related Experiment Videos

  • Acquisition of fluorescence lifetime images across 16 individual wavelength components.
  • Achieving 10-nm spectral resolution on a pixel-by-pixel basis.

    • Main Results:

    • The developed microscope provides spectrally and temporally resolved fluorescence information.
    • Detailed visualization of cellular structures and processes is achieved.
    • The instrument enables pixel-by-pixel analysis of fluorescence signatures.

    Conclusions:

    • The combined spectral and lifetime microscope offers a unique approach to analyzing cellular fluorescence.
    • This technology enhances the understanding of cellular physiology by providing detailed molecular and microenvironmental data.
    • Potential applications include advanced live cell and tissue imaging.