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

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.

PEO-CYTOP Fluoropolymer Nanosheets as a Novel Open-Skull Window for Imaging of the Living Mouse Brain

“…Second, a large cranial window using PEO-CYTOP nanosheet did not affect the natural curvature of the living brain surface but caused optical aberrations. To compensate them, adaptive optical techniques proposed previously should be adopted (Tanabe et al, 2016;Matsumoto et al, 2018;Yamaguchi et al, 2020).…”

Adaptive Optical Two-Photon Microscopy for Surface-Profiled Living Biological Specimens

“…Compensation of the Spherical Aberration. Using fluorescent beads embedded in agarose gel with a similar RI as the living mouse cortex (1.36), 8,27 we evaluated the performance of AO two-photon microscopy for compensating the spherical aberration (Figure 2a). The excitation laser intensity was maintained constant during all experiments.…”

Visualizing the Invisible: Advanced Optical Microscopy as a Tool to Measure Biomechanical Forces

“…However, it has also been implemented in LSFM configurations to combine the benefits of both techniques ( Truong et al, 2011 ; Mahou et al, 2014 ; Wolf et al, 2015 ). The predominant use of 2P microscopy in measuring biomechanical forces is through photoablation and measurement of subsequent tension relaxation ( Shen et al, 2005 ; Rauzi et al, 2008 , 2010 ; Ratheesh et al, 2012 ; Michael et al, 2016 ; Yamaguchi et al, 2020 ). While many studies leverage ultra-violet pulsed lasers to ablate specimen targets ( Kiehart et al, 2000 ; Hutson et al, 2003 ; Fernandez-Gonzalez et al, 2009 ; Colombelli and Solon, 2013 ; Smutny et al, 2015 ; Hara et al, 2016 ; Zhang et al, 2020 ), the use of 2P microscopy improves both ablation depth and precision.…”