The effects of spherical aberration on multiphoton fluorescence excitation microscopy

P. A. Young, S. G. Clendenon, J. M. Byars, R. S. Decca, Kenneth Dunn

Research output: Contribution to journalArticle

13 Citations (Scopus)

Abstract

Multiphoton fluorescence excitation microscopy is almost invariably conducted with samples whose refractive index differ from that of the objective immersion medium, conditions that cause spherical aberration. Due to the quadratic nature of multiphoton fluorescence excitation, spherical aberration is expected to profoundly affect the depth dependence of fluorescence excitation. In order to determine the effect of refractive index mismatch in multiphoton fluorescence excitation microscopy, we measured signal attenuation, photobleaching rates and resolution degradation with depth in homogeneous samples with minimal light scattering and absorption over a range of refractive indices. These studies demonstrate that signal levels and resolution both rapidly decline with depth into refractive index mismatched samples. Analyses of photobleaching rates indicate that the preponderance of signal attenuation with depth results from decreased rates of fluorescence excitation, even in a system with a descanned emission collection pathway. Similar results were obtained in analyses of fluorescence microspheres embedded in rat kidney tissue, demonstrating that spherical aberration is an important limiting factor in multiphoton fluorescence excitation microscopy of biological samples.

Original languageEnglish
Pages (from-to)157-165
Number of pages9
JournalJournal of Microscopy
Volume242
Issue number2
DOIs
StatePublished - May 2011

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Multiphoton Fluorescence Microscopy
Refractometry
Fluorescence
Photobleaching
Immersion
Microspheres
Kidney
Light

Keywords

  • Multiphoton fluorescence excitation microscopy
  • Refractive index mismatch
  • Spherical aberration
  • Two-photon microscopy

ASJC Scopus subject areas

  • Histology
  • Pathology and Forensic Medicine

Cite this

The effects of spherical aberration on multiphoton fluorescence excitation microscopy. / Young, P. A.; Clendenon, S. G.; Byars, J. M.; Decca, R. S.; Dunn, Kenneth.

In: Journal of Microscopy, Vol. 242, No. 2, 05.2011, p. 157-165.

Research output: Contribution to journalArticle

Young, P. A. ; Clendenon, S. G. ; Byars, J. M. ; Decca, R. S. ; Dunn, Kenneth. / The effects of spherical aberration on multiphoton fluorescence excitation microscopy. In: Journal of Microscopy. 2011 ; Vol. 242, No. 2. pp. 157-165.
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