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.
Highlights
The advantages of multi-photon laser scanning microscopy (MPLSM) are well established [1,2,3,4] and developments in instrumentation and photochemistry are constantly on-going in order to obtain as much information as possible from the biological specimen
We present here a simple technique whereby spherical aberration can be greatly reduced for long wavelengths (λ>850 nm) used in MPLSM by changing the immersion medium
As is shown in (c) and (d), when exciting at a wavelength of λ = 1000 nm, methanol immersion (MI) provided a distribution close to Gaussian, unlike water immersion (WI), which produced a skewed Gaussian that is indicative of spherical aberration
Summary
The advantages of multi-photon laser scanning microscopy (MPLSM) are well established [1,2,3,4] and developments in instrumentation and photochemistry are constantly on-going in order to obtain as much information as possible from the biological specimen. An exciting development in MPLSM aberration correction technology has been presented This is known as the iterative multi-photon adaptive compensation technique (IMPACT) and is a system which takes advantage of the nonlinearity of the multi-photon process to determine, and compensate for, distortions and aberrations incurred throughout a specimen [21]. This technique has been shown to provide diffraction limited imaging at depths beyond the possibilities of conventional MPLSM. The approach was compatible with standard methods of specimen preparation for epi-fluorescence, including immunofluorescence and did not alter the morphology of fixed tissues
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