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Related Experiment Videos

Instrumentation for multiwavelengths excitation imaging

P Messler1, H Harz, R Uhl

  • 1Abteilung Physikalische Biologie der Ludwig-Maximilians-Universität München, Germany.

Journal of Neuroscience Methods
|November 1, 1996
PubMed
Summary

A new fluorescence ratio-imaging system uses a fast-switching polychromatic illumination system and a slow-scan CCD camera for advanced microscopy. This system enables rapid, computer-controlled wavelength selection for precise multi-dye measurements and optimal experimental conditions.

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Area of Science:

  • Microscopy and Imaging Technologies
  • Biophotonics
  • Fluorescence Spectroscopy

Background:

  • Accurate and efficient fluorescence ratio-imaging is crucial for quantitative biological studies.
  • Existing systems often face limitations in speed, wavelength flexibility, and illumination control.
  • Optimizing experimental conditions requires adaptable and high-performance imaging tools.

Purpose of the Study:

  • To develop and characterize a novel fluorescence ratio-imaging system with enhanced speed and wavelength control.
  • To enable precise multi-dye measurements and facilitate the determination of optimal experimental parameters.
  • To improve image acquisition speed and data processing capabilities in fluorescence microscopy.

Main Methods:

  • Development of a patented polychromatic illumination system with rapid wavelength switching (260-680 nm in <3.5 ms).

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  • Integration of a 12-bit, 2 MHz slow-scan CCD camera for high-resolution image acquisition.
  • Utilized critical illumination with a UV condenser and reflective optics for homogeneous specimen illumination.
  • Implemented a computer-controlled DOS program for simultaneous monochromator and camera operation.

    • Main Results:

    • The system achieves high-intensity monochromatic light (>3 mW between 300-500 nm) with a 12 nm bandwidth.
    • Fluence rates up to 10^23 photons m^-2 s^-1 at 340 nm were achieved with a 40x objective.
    • Image acquisition rates of up to 13 full frames/s and 100 subframes/s (binning/skipping mode) were demonstrated.
    • Simultaneous image acquisition at different wavelengths and data transfer was enabled by the camera's frame-transfer structure.

    Conclusions:

    • The developed fluorescence ratio-imaging system offers significant advantages in speed, flexibility, and illumination control.
    • It facilitates advanced multi-dye measurements and optimization of experimental protocols.
    • The system provides a powerful tool for quantitative fluorescence microscopy in various scientific applications.