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Biomedical Optics Express

Biomedical Optics Express

  • Editor: Joseph A. Izatt
  • Vol. 3, Iss. 12 — Dec. 1, 2012
  • pp: 3314–3324

Methanol immersion reduces spherical aberration of water dipping lenses at long wavelengths used in multi-photon laser scanning microscopy

Greg Norris, Ayman Gebril, Valerie A. Ferro, and Gail McConnell  »View Author Affiliations

Biomedical Optics Express, Vol. 3, Issue 12, pp. 3314-3324 (2012)

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Dipping objectives were tested for multi-photon laser scanning microscopy, since their large working distances are advantageous for thick specimens and the absence of a coverslip facilitates examination of living material. Images of fluorescent bead specimens, particularly at wavelengths greater than 850 nm showed defects consistent with spherical aberration. Substituting methanol for water as the immersion medium surrounding the beads corrected these defects and produced an increase in fluorescence signal intensity. The same immersion method was applied to two representative biological samples of fixed tissue: mouse brain labeled with FITC for tubulin and mouse gut in which the Peyer’s patches were labeled with Texas Red bilosomes. Tissue morphology was well preserved by methanol immersion of both tissues; the two-photon-excited fluorescence signal was six times higher than in water and the depth of penetration of useful imaging was doubled. No modification of the microscope was needed except the provision of a ring to retain a sufficient depth of methanol for imaging.

© 2012 OSA

OCIS Codes
(160.4670) Materials : Optical materials
(180.0180) Microscopy : Microscopy
(220.1000) Optical design and fabrication : Aberration compensation
(180.4315) Microscopy : Nonlinear microscopy

ToC Category:

Original Manuscript: August 29, 2012
Revised Manuscript: November 16, 2012
Manuscript Accepted: November 19, 2012
Published: November 21, 2012

Greg Norris, Ayman Gebril, Valerie A. Ferro, and Gail McConnell, "Methanol immersion reduces spherical aberration of water dipping lenses at long wavelengths used in multi-photon laser scanning microscopy," Biomed. Opt. Express 3, 3314-3324 (2012)

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