Image improvement of temporal focusing multiphoton microscopy via superior spatial modulation excitation and Hilbert-Huang transform decomposition

Temporal focusing-based multiphoton excitation microscopy (TFMPEM) just provides the advantage of widefield optical sectioning ability with axial resolution of several micrometers. However, under the plane excitation, the photons emitted from the molecules in turbid tissues undergo scattering, resulting in complicated background noise and an impaired widefield image quality. Accordingly, this study constructs a theoretical model of TFMPEM utilizing Fourier optics and performs numerical simulations to determine the optimal spatial frequency and orientation of the structured pattern which maximize the axial excitation confinement. It is shown experimentally that the optimized pattern minimizes the intensity of the out-of-focus signal, and hence improves the quality of the image reconstructed using the Hilbert transform (HT). However, the square-like reflection components on digital micromirror device leads to pattern residuals in the demodulated image when applying high spatial frequency of structured pattern. Accordingly, the HT is replaced with Hilbert-Huang transform (HHT) in order to sift out the low-frequency background noise and pattern residuals in the demodulation process. The experimental results obtained using a kidney tissue sample show that the HHT yields a significant improvement in the TFMPEM image quality.