Fig. 1. Definition of optical invariants. (a) Optical invariants of the imaging objectives; (b) Optical invariants of the scanning relay; (c) Optical invariants of the fluorescence collection system

Fig. 2. Design of a large-FOV two-photon microscope system using optical invariant analysis. (a) Optical layout; (b) Cerebral vasculature imaged over the mouse cortex with the large-FOV two-photon microscopy

Fig. 3. Large field-of-view two-photon microscopy by optimizing the off-axis aberrations using lens series. (a) Optical layout; (b) Experimental measurements of the PSF (point-spread-function) as a function of FOV, measured by 0.5 µm fluorescence beads; (c) Large-FOV two-photon image (λ= 800 nm, max intensity project) of vasomotion in cortical arterioles across both hemispheres of an awake, head-fixed mouse through dual transcranial windows

Fig. 4. Large-FOV two-photon random access microscopy. (a) Optical layout; (b) PSF at middle position and edge position of the FOV, measured by 0.5 µm microbeads; (c) Large-FOV two-photon image of fluorescent proteins in anesthetized thy-1 mice (max intensity project) and higher magnification image (dashed box in the large-FOV image)

Fig. 5. Large field-of-view and multi-region two-photon microscopy. (a) Optical layout; (b) PSF at middle position and edge position of the FOV, measured by 0.2 µm microbeads; (c) Large-FOV imaging was used to examine neuronal activity of a transgenic mouse expressing the genetically encoded fluorescent calcium indicator GCaMP6 s; (d) Segmenting the image sequence can yield 5,361 active neurons; (e) Simultaneous two region imaging to monitor neuronal activity between two cortical visual areas

Fig. 6. Extending the field of view of two-photon microscopy using adaptive optics. (a) Schematic diagram; (b) Imaging optical path diagram; (c) Large-FOV two-photon image of the brain slice of thy-1 mice (max intensity project); (d) Comparison before and after adaptive optics correction in the yellow solid area of Fig. (c)