Tissue refractive-index heterogeneity limits the imaging quality of two-photon fluorescence microscopy in vivo by distorting its excitation wave-front and resulting in an enlarged focal vol­ume and reduced focal intensity. For imaging fine structures at depth, it is essential to measure and correct these sample-induced aberrations through adaptive optics (AO). An ideal aberration-measurement method should work with signal from structures intrinsic to the sample, under any labeling density, with any labeling strategy (from bright synthetic dye to dim fluorescent proteins), in any sample (from transparent to strongly scatter­ing), and, if the sample permits, generate a corrective pattern that improves image quality over a large volume. Researcher at State Key Laboratory of High Field Laser Physics, Shanghai Institute of Optics and Fine Mechanics, Chinese Academy of Sciences,describe a multiplexed aberration-measurement method that satisfies all of the above criteria.[Nature Methods.11:1037–1040,(2014) http://www.nature.com/nmeth/journal/v11/n10/full/nmeth.3068.html]

They describe an adaptive optics method that modulates the intensity or phase of light rays at multiple pupil segments in parallel to determine the sample-induced aberration. Applicable to fluorescent protein–labeled structures of arbitrary complexity, it allowed us to obtain diffraction-limited resolution in various samples in vivo. For the strongly scattering mouse brain, a single aberration correction improved structural and functional imaging of fine neuronal processes over a large imaging volume.