Multiphoton versus confocal high resolution z-sectioning of enhanced green fluorescent microtubules: Increased multiphoton photobleaching within the focal plane can be compensated using a Pockels cell and dual widefield detectors

D. R. Drummond, N. Carter, R. A. Cross

Research output: Contribution to journalArticle

23 Citations (Scopus)

Abstract

Multiphoton excitation was originally projected to improve live cell fluorescence imaging by minimizing photobleaching effects outside the focal plane, yet reports suggest that photobleaching within the focal plane is actually worse than with one photon excitation. We confirm that when imaging enhanced green fluorescent protein, photobleaching is indeed more acute within the multiphoton excitation volume, so that whilst fluorescence increases as predicted with the square of the excitation power, photobleaching rates increase with a higher order relationship. Crucially however, multiphoton excitation also affords unique opportunities for substantial improvements to fluorescence detection. By using a Pockels cell to minimize exposure of the specimen together with multiple nondescanned detectors we show quantitatively that for any particular bleach rate multiphoton excitation produces significantly more signal than one photon excitation confocal microscopy in high resolution Z-axis sectioning of thin samples. Both modifications are readily implemented on a commercial multiphoton microscope system.

Original languageEnglish
Pages (from-to)161-169
Number of pages9
JournalJournal of Microscopy
Volume206
Issue number2
DOIs
Publication statusPublished - May 30 2002
Externally publishedYes

All Science Journal Classification (ASJC) codes

  • Pathology and Forensic Medicine
  • Histology

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