Design and implementation of large field-of-view two-photon microscopy by dual objective system for biological study

Abstract

The study of brain connectivity, which describes how neurons and neural networks process information, is essential to elucidate the mechanisms of brain function and diseases such as stroke, Alzheimer’s, and neuropathic pain. Recently, two-photon laser scanning microscopy has attracted great attention to the brain network connectivity study. Unlike conventional whole brain functional imaging techniques such as positron emission tomography (PET) or functional magnetic resonance imaging (fMRI), two-photon laser scanning microscopy (TPLSM) enables cellular level imaging of the dynamics in individual neurons owing to high-resolution, deep penetration depth, and highly localized excitation. Also, two-photon microscopy can provide both functional imaging and structural imaging of the brain by introducing Ca2+ imaging. However, conventional TPLSM suffers from limited field-of-view (FOV) < 1 mm2. Various large FOV TPLSM methods have been introduced to overcome the small FOV of TPLSM, but the FOV of such TPLSM methods still remains at < 10 mm2, which can only cover half of the rodent brain. In this regard, this study proposes the state-of-the-art large FOV TPLSM by adopting dual objective lens system. This novel large FOV TPLSM has successfully achieved to provide 16mm × 8mm FOV two-photon imaging which enables scanning of the full extent of rodent animal brain at one take. The proposed system also can be extended to various biological studies and for controlled experiment using dual objective lens system.