Principles of Two-photon Excitation
At a high photon density, a fluorophore absorbs two long-wavelength photon simultaneously, then after a short relaxation process, emits a shorter-wavelength photon. The similar effect can also be elicited by using one photon with a half of the long-wavelength.
Remarkable Advantages of Two-photon Excitation in Biological Imaging
Photons of long wavelength are used for excitation , in which less laser scatrering permits deeper pentration and enables thicker tissue imaging
Better optical section capbility allows a higher axial resolution
It elicits less photobleaching and less photodamage, which is suitable for long-term image
It exhibits high fluorescence collection efficiency, thus high resolution imaing data can be obtained
It is suitable for in vivo imaging
How to Achieve Fast, High- resolution Imaging in Freely Behaving Animals?
The signals of the most commonly used fluorescent indicators, GFP and GCaMP are acquired with a specially customized optical fiber, which can effectively transmit 920 nm femtosecond laser pulses without any distortion
Optical interference caused by the freely behaving mice was minimized by using a supple fiber bundle (SFB) while collecting the emitted fluorescence signal. Thus, the imaging quality will be promoted without affecting the freely moving animal
High-speed imaging at a frame rate of 9Hz with a resolution of 512×512 pixels was achieved by high-speed scanning with a micro-electromechanical system (MEMS)scanning galvanometer. ; Supple scanning galvanometer cables were used to supply scanning power for the MEMS to minimize the gross weight of the module without sacrificing good anti-interference effect
Sub-cellular resolution imaging data was captured with a miniature objective of a high numerical aperture
The whole system can maintain the stability and robustness of itself during the experiment, ensuring that the imaging quality will not be severely influenced by vigorous movements of the animal
The small, light and detachable optical probe enables complex behavioral experiments as well as monitoring functional and morphological changes in the brain of a freely moving animal in a long period of time