Deep Tissue Imaging with Multiphoton Fluorescence Microscopy

Abstract

We present a review of imaging deep-tissue structures with multiphoton microscopy. We examine the effects of light scattering and absorption due to the optical properties of biological sample and identify 1,300 nm and 1,700 nm as ideal excitation wavelengths. We summarize the availability of fluorophores for multiphoton microscopy as well as ultrafast laser sources to excite available fluorophores. Lastly, we discuss the applications of multiphoton microscopy for neuroscience.

Keywords: Fluorophores for multiphoton microscopy; Laser sources for multiphoton microscopy; Multiphoton Microscopy Fluorescence Imaging; Scattering and absorption in brain tissue.

Figure 1. Effects of scattering and absorption

Photon fraction at a depth of 1 mm for average brain tissue optical properties (g=0.9, a=1.1 mm⁻¹, b=1.37, water content=75%) [9] is demarcated by the blue line. Regions shaded in red indicate areas in which 50% or more of photons are absorbed, as calculated by the red line (dashed red indicates below 50%; solid red indicates over 50%). Available laser options are outlined for their respective wavelength range (Yb = ytterbium, 2C2P = two-photon two-color of Yb fiber laser and diamond laser, Er=Erbium, OPO=optical parametric oscillator, OPA=optical parametric amplifier).

Figure 2. Cross Sections of various fluorophores

Peak two-photon absorption cross sections of common fluorophores compiled from published literature. Dark blue circles are from Dana et al. (2016) [26], black squares are from Drobizhev et al. (2011) [27], pink diamonds are from Janelia Harris [28], teal triangles are from Kobat et al. (2009) [29], and gray inverted triangles are from Xu et al. (1996) [30]. The calculated normalized photon fraction of excitation light that reaches 1 mm into brain tissue (normalized to 1300 nm, analogous to Figure 1 solid blue line) is overlaid as background fill, where the color scale ranges from 0.01 (red) to 0.5 (white) to 1.6 (blue).

Figure 3. Examples of in vivo multiphoton microscopy

(a) Image stacks of dimensions 185 × 185 × 900 μm³ collected by two-photon excitation with an ytterbium-doped fiber laser labeling for layer V pyramidal neurons with YFP (left), and vasculature with Texas Red (right). (b) Merged z-projections of the stacks shown in a with neurons displayed in yellow and vasculature in red; the image at each plane is a maximum intensity projection of the nearest 50 μm. (c) Image stack of dimensions 365 × 365 × 1535 μm³ collected by an optical parametric amplifier labeling for vasculature with Texas Red. (d) Two-dimensional z-projections of the volume shown in c. (e) Vectorization of deep microvasculature in the volume shown in c. Scale bar is 50 μm.

Figure 4. Recording of hippocampal neurons

(a) Image of CA1 hippocampal neuron population from an 18–20 week old transgenic mouse (CAMKII-tTA/tetO-GCaMP6s) with a chronic cranial window preparation taken with three-photon microscopy at 984 μm beneath the dura. Individual neurons are indexed for references to traces. Scale bar, 20 μm. (b) Recording of spontaneous activity from labeled neurons in a. Reproduced with permission from Ouzounov et al. (2017) [40].