In vivo two-photon microscopic observation and ablation in deeper brain regions realized by modifications of excitation beam diameter and immersion liquid.

Kohei Otomo,Ryoji Kitamura,Tomomi Nemoto,Kazushi Yamaguchi,Lawrence B Cohen,Ryosuke Kawakami

doi:10.1371/journal.pone.0237230

Abstract

In vivo two-photon microscopy utilizing a nonlinear optical process enables, in living mouse brains, not only the visualization of morphologies and functions of neural networks in deep regions but also their optical manipulation at targeted sites with high spatial precision. Because the two-photon excitation efficiency is proportional to the square of the photon density of the excitation laser light at the focal position, optical aberrations induced by specimens mainly limit the maximum depth of observations or that of manipulations in the microscopy. To increase the two-photon excitation efficiency, we developed a method for evaluating the focal volume in living mouse brains. With this method, we modified the beam diameter of the excitation laser light and the value of the refractive index in the immersion liquid to maximize the excitation photon density at the focal position. These two modifications allowed the successful visualization of the finer structures of hippocampal CA1 neurons, as well as the intracellular calcium dynamics in cortical layer V astrocytes, even with our conventional two-photon microscopy system. Furthermore, it enabled focal laser ablation dissection of both single apical and single basal dendrites of cortical layer V pyramidal neurons. These simple modifications would enable us to investigate the contributions of single cells or single dendrites to the functions of local cortical networks.

Highlights

Brain functions such as senses, behaviors, intelligence and emotions are believed to be achieved by intercellular communications in either local or global neural networks
To measure the focal volumes, we evaluated the full width at half maximums (FWHMs) of the fluorescence intensity profiles of the beads by fitting to the Gaussian distribution along the axial (z-axis) and lateral (x-axis) directions
We developed a method for evaluating the focal volume in living mouse brains and successfully revealed a condition that maximizes the two-photon excitation efficiency by achieving tighter focusing especially in deeper regions (Figs 1–4; S2 Table; the underfill and 1.36 conditions)

Summary

Introduction

Brain functions such as senses, behaviors, intelligence and emotions are believed to be achieved by intercellular communications in either local or global neural networks. Little is known about cellular mechanisms implementing such complex brain functions even in local neural networks [1]. Improvements of in vivo two-photon observation and ablation in living mice brains

Methods

Results

Conclusion