Optical imaging is a powerful tool in the biomedical sciences, allowing direct inspection and biological processes analysis. Such imaging becomes even more powerful when implemented in three dimensions (3D), and can be further augmented if combined with super-resolution techniques that uncover fine details otherwise obscured by diffraction.  

Unfortunately, neither 3D nor super-resolution- techniques could reach their full potential in thick samples, as optical aberrations (distortions due to sample induced inhomogeneity in refractive index) severely degrade resolution and signal.  

ZHENG Wei, the Principle Investigator of Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences, together with Hari Shroff, the Professor in National Institute of Biomedical Imaging and Bioengineering, developed a new two-photon excitation Instant Structured Illumination Microscope (2P-ISIM) that incorporates rapid adaptive optics (AO) correction enabling 3D and super-resolution imaging achieve unprecedented depth (> 250μm from the coverslip). 

This instrument enables ‘real time’ super-resolution imaging. The higher resolution information derived from the excitation and emission point spread functions is directly ‘written’ into the image without the post-processing required in traditional SIM.  

Adaptive correction is also convenient and fast, since the two photon induced fluorescence functions as a guide star that allows direct wavefront sensing and subsequent aberration correction with a deformable mirror.  

This instrument was tested by imaging more than ten different samples, including bead phantoms, cells in gels, drosophila brain lobe, excised mouse tissue, and live zebrafish and nematode larvae. In all these cases, AO is crucial for maintaining super-resolution at depth (up to 176±10 nm laterally and 729±39 nm axially, more than doubling resolution relative to conventional 2P imaging), and in boosting signal (up to 40-fold) that would otherwise be lost due to aberrations.