Axial gradient excitation accelerates volumetric imaging of two-photon microscopy

Two-photon excitation fluorescence microscopy(TPM),owing to its capacity for subcellular resolution,intrinsic optical sectioning,and superior penetration depth in turbid samples,has revolutionized biomedical research.However,its layer-by-layer scanning to form a three-dimensional image inherently limits the volumetric imaging speed and increases phototoxicity significantly.In this study,we develop a gradient excitation technique to accelerate TPM volumetric imaging.The axial positions of the fluorophores can be decoded from the intensity ratio of the paired images obtained by sequentially exciting the specimen with two axially elongated two-photon beams of complementary gradient intensities.We achieved a 0.63μm axial localization precision and demonstrate the flexibility of the gradient TPM on various sparsely labeled samples,including bead phantoms,mouse brain tissues,and live macrophages.Compared with traditional TPM,our technique improves volumetric imaging speed(by at least sixfold),decreases photobleaching(i.e.,less than 2.07±2.89%in 25 min),and minimizes photodamages.