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

Research output: Contribution to journalArticle

Abstract

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.
LanguageEnglish
Pages3314-3324
Number of pages11
JournalBiomedical Optics Express
Volume3
Issue number12
DOIs
Publication statusPublished - 21 Nov 2012

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Immersion
dipping
Photons
Confocal Microscopy
submerging
Lenses
Methanol
aberration
methyl alcohol
lenses
microscopy
beads
scanning
mice
Water
photons
Fluorescence
wavelengths
water
lasers

Keywords

  • multi-photon laser scanning microscopy
  • wavelengths
  • fluorescent beads
  • tissue morphology
  • methanol

Cite this

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title = "Methanol immersion reduces spherical aberration of water dipping lenses at long wavelengths used in multi-photon laser scanning microscopy",
abstract = "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.",
keywords = "multi-photon laser scanning microscopy, wavelengths , fluorescent beads, tissue morphology, methanol",
author = "Greg Norris and Gebril, {Ayman M} and Valerie Ferro and Gail McConnell",
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AU - Norris, Greg

AU - Gebril, Ayman M

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AU - McConnell, Gail

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