Application of an advanced maximum likelihood estimation restoration method for enhanced-resolution and contrast in second-harmonic generation microscopy.

Second-harmonic generation (SHG) microscopy has gained popularity because of its ability to perform submicron, label-free imaging of noncentrosymmetric biological structures, such as fibrillar collagen in the extracellular matrix environment of various organs with high contrast and specificity. Because SHG is a two-photon coherent scattering process, it is difficult to define a point spread function (PSF) for this modality. Hence, compared to incoherent two-photon processes like two-photon fluorescence, it is challenging to apply the various PSF-engineering methods to improve the spatial resolution to be close to the diffraction limit.

Cardiac muscle organization revealed in 3-D by imaging whole-mount mouse hearts using two-photon fluorescence and confocal microscopy.

The ability to image the entire adult mouse heart at high resolution in 3-D would provide enormous advantages in the study of heart disease. However, a technique for imaging nuclear/cellular detail as well as the overall structure of the entire heart in 3-D with minimal effort is lacking. To solve this problem, we modified the benzyl alcohol:benzyl benzoate (BABB) clearing technique by labeling mouse hearts with periodic acid Schiff (PAS) stain.