Fig. 1.
Three-photon fluorescence microscopy with a Bessel focus. (a) Schematics of our homebuilt three-photon microscope with an axicon-based Bessel module. Flip mirrors M1 and M2 allow switching between Gaussian (yellow path) and Bessel (red path) imaging modalities. L, lenses; X and Y, galvanometers; PMT, photomultiplier tube.(b) Three-photon images of a 1-μm-diameter red fluorescent bead excited with different Bessel foci under 1300 nm excitation: (left) NA = 0.48, focal length L1 = 100 mm, L2 offset = -10 mm; (center) NA = 0.6, focal length L1 = 125 mm, L2 offset = -10 mm; (right) NA = 0.72, focal length L1 = 150 mm, L2 offset = 0 mm. Post-objective powers: 15.5, 4.9, and 3.2 mW, respectively. Note the different scales for the lateral and axial scale bars. (c) Images of a 0.2-μm-diameter red fluorescent bead taken under (left) two- and (right) three-photon excitation with a Bessel focus of 0.96 NA and axial FWHM of 10 μm (focal length of L1 = 200 mm, L2 offset = -10 mm), under 920 and 1300 nm excitation, respectively. Contrast was enhanced 7× to highlight the side rings in both images. Post-objective powers: 10 and 16.7 mW, respectively.(d) Signal profiles (obtained from a radial average and plotted in logarithmic scale) of images in (c). With three-photon excitation the side rings (typical of high-NA Bessel foci) are more effectively suppressed.