Detection sensitivity enhancements for fluorescence imaging with multi-photon excitation microscopy

David L. Wokosin*, W. Brad Amos, John G. White

*Corresponding author for this work

Research output: Contribution to journalConference article

10 Scopus citations

Abstract

Multiphoton excitation microscopy (MPEM) offers several distinct advantages over laser scanning confocal microscopy (LSCM). One is that the resolution and the thickness of the optical section are determined by the excitation beam; a confocal detection aperture is therefore unnecessary. This feature allows the photodetector to be relocated from its usual location in the descanned emission beam within a confocal scan head so as to directly intercept the emitted fluorescent signal emerging from the microscope. The enhancement in signal gained by changing from internal to whole area (external) detection in this way has been evaluated quantitatively. In particular, longitudinal chromatic aberration has been shown to reduce the efficiency of internal detection relative to external. Also, a novel optical design is described, which allows increased collection efficiency with MPEM by capturing light emitted away from the objective lens. Together, these two enhancements typically provide an improvement in efficiency of at least 4.5 times, and even greater inside thick and scattering specimens. Such improvements are crucial for extending the longevity of living preparations by minimizing phototoxic exposure during observation. They also enable deeper imaging inside specimens where signal loss by scattering has previously limited the obtainable sectioning depth.

Original languageEnglish (US)
Pages (from-to)1707-1714
Number of pages8
JournalAnnual International Conference of the IEEE Engineering in Medicine and Biology - Proceedings
Volume4
StatePublished - Dec 1 1998
EventProceedings of the 1998 20th Annual International Conference of the IEEE Engineering in Medicine and Biology Society. Part 4 (of 6) - Hong Kong, China
Duration: Oct 29 1998Nov 1 1998

ASJC Scopus subject areas

  • Signal Processing
  • Biomedical Engineering
  • Computer Vision and Pattern Recognition
  • Health Informatics

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