Three-photon fluorescence microscopy with an axially elongated Bessel focus

Abstract

Volumetric imaging tools that are simple to adopt, flexible, and robust are in high demand in the field of neuroscience, where the ability to image neurons and their networks with high spatiotemporal resolution is essential. Using an axially elongated focus approximating a Bessel beam, in combination with two-photon fluorescence microscopy, has proven successful at such an endeavor. Here, we demonstrate three-photon fluorescence imaging with an axially extended Bessel focus. We use an axicon-based module that allowed for the generation of Bessel foci of varying numerical apertures and axial lengths, and apply this volumetric imaging tool to image mouse brain slices and for in vivo imaging of the mouse brain.

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.

Fig. 2

Three-photon fluorescence microscopy with Bessel focus scanning captures fine structures in volumes of mouse (Thy1-GFP line M) brain slices. (a,c) Maximum intensity projections of a 50-μm thick image stack obtained with a Gaussian focus, with a 2-μm axial step size, color-coded by depth, under 1300 nm excitation.Post-objective power: 2.8 mW. (b,d) Images of the same volumes as (a,c), respectively, obtained with a Bessel focus of NA 0.6 and axial FWHM of 36 μm. Post-objective powers: 25 and 38 mW, respectively.

Fig. 3

In vivo volumetric three-photon fluorescence microscopy of micebrains. Maximum intensity projection of a (a) 62-μm and (c) 52-μm thick volume at ∼80 μm below dura, color-coded by depth, imaged with a Gaussian focus, at a2-μm axial step size, under 1300 nm excitation. Post-objective powers: 3 and 4 mW, respectively. (b,d) Image of the same volume as (a,c), respectively, obtained with a Bessel focus of NA 0.6 and axial FWHM of 38 μm.White arrowheads label dendritic spines, and yellow arrowheads label axonal boutons.Post-objective powers: 30 and 40 mW, respectively.(e) Maximum intensity projection of a 30-μm thick volume at ∼290 μm below dura, color-coded by depth, imaged with a Gaussian focus, at a2-μm axial step size, under 1700 nm excitation. Post-objective power: 15 mW. (f) Image of the same volume as (e) obtained with a Bessel focus of NA 0.6 and axial FWHM of 27 μm. Post-objective power: 73 mW.A Thy1-GFP line M mouse was used for (a-d), and a Gad2-IRES-Cre × Ai14 for(e,f).