Two-photon laser-scanning microscopy: tests of objective lenses and Ca2+ probes

The characteristics of objective lenses and Ca2+-sensitive probes were exam ined for imaging with a two-photon laser-scanning microscope (TP-LSM). The brightness of the images of beads taken by different objectives greatly var ied and depended predominantly on their numerical aperture (NA) and less on transmittance and chirping effects. Lateral and axial resolutions, dx and dz, defined as the half decay length of fluorescence intensity of the image of a spherical bead (0.3 m) were 0.12 and 0.42 mu m (objective; 40 x /0.75 ). They are far better than those of confocal microscopes (0.3 and 1.5 mu m , respectively) measured similarly (Kuba et al., 1994). dr linearly increas ed with an increase in 1/NA, while dz linearly increased with an increase i n n/(NA)(2) (n, refractive index) except for an objective of large NA (1.3) . The coverslip compensation of objective lenses greatly affected the shape of the X-Z scanned images of 5.0 mu m beads as well as resolutions, indica ting a large effect of spherical aberration. Two-photon excitation spectra of Ca2+-sensitive fluorescent probes, indo-1, fura-2 and Oregon Green BAPTA -1, lied in a wavelength range shorter than twice that activated by one-pho ton absorption, while emission spectra were unchanged. Three-dimensional im ages of a cultured hippocampal neurone loaded with Oregon Green BAPTA-1 sho wed fine structures of spines, dendrites and axons, while imaging with FM1- 43 localized presynaptic boutons and demonstrated synaptic vesicle turnover . Dyes bleached little during the recording of 100 sectioned images. These characteristics of TP-LSM as well as its ability to image deeper tissues pr ovide excellent means to study dynamic, spatial changes in intracellular su bstances and structures. To achieve the good performance of a TP-LSM, howev er, the relevant usage of appropriate objectives and fluorescent probes are required. (C) 1998 Elsevier Science Ireland Ltd. All rights reserved.