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.

Multi‐Photon Microscopy

“…Unlike confocal microscopy, multi-photon imaging is effective with low-magnification objectives of moderate NA (multi-photon has a much smaller focal volume than confocal, which is more effective when using low NA objectives). Therefore, water-dipping objectives are commonly used for in vivo imaging with multi-photon microscopy, although methanol immersion (Norris, Gebril, Ferro, & McConnell, 2012) has also been used successfully. Dipping objectives have long working distances exceeding 1 mm, with a high NA, to achieve efficient focusing and excitation.…”