Enhancing axial resolution and background rejection in line-scanning temporal focusing microscopy by focal modulation

Compared with two-photon point-scanning microscopy, line-scanning temporal focusing microscopy breaks the limitation on imaging rate and maintains the axial resolution, which makes it promising for various biomedical studies. However, for deep tissue imaging, it suffers from reduced axial resolution and increased background noise due to sample induced wavefront distortion. Here, we propose a spatio-spectral focal modulation technique to enhance axial resolution and background rejection by simply subtracting an aberrated image, which is induced by a spatial light modulator, from an unaberrated image. The proposed technique could improve the axial resolution by a factor of 1.3 in our implementation, verified by both simulations and experiments. Besides, we show that compared with spatial modulation alone, spatio-spectral modulation induces less peak intensity loss caused by image subtraction. We further demonstrate the performance of our technique on the enhanced axial resolution and background rejection by deep imaging of cleared mouse brains and in vivo imaging of living mouse brains.