Improving two-photon excitation microscopy for sharper and faster biological imaging

Two-photon excitation laser scanning microscopy (TPLSM) has provided many insights into the life sciences, especially for thick biological specimens, because of its superior penetration depth and less invasiveness owing to the near-infrared wavelength of its excitation laser light. This paper introduces our four kinds of studies to improve TPLSM by utilizing several optical technologies as follows:(1) A high numerical aperture objective lens significantly deteriorates the focal spot size in deeper regions of specimens. Thus, approaches to adaptive optics were proposed to compensate for optical aberrations for deeper and sharper intravital brain imaging.(2) TPLSM spatial resolution has been improved by applying super-resolution microscopic techniques. We also developed a compact stimulated emission depletion (STED) TPLSM that utilizes electrically controllable components, transmissive liquid crystal devices, and laser diode-based light sources. The spatial resolution of the developed system was five times higher than conventional TPLSM.(3) Most TPLSM systems adopt moving mirrors for single-point laser beam scanning, resulting in the temporal resolution caused by the limited physical speed of these mirrors. For high-speed TPLSM imaging, a confocal spinning-disk scanner and newly-developed high-peak-power laser light sources enabled approximately 200 foci scanning.(4) Several researchers have proposed various volumetric imaging technologies. However, most technologies require large‐scale and complicated optical setups based on deep expertise for microscopic technologies, resulting in a high threshold for biologists. Recently, an easy‐to‐use light‐needle-creating device was proposed for conventional TPLSM systems to achieve one-touch volumetric imaging.