Label-free adaptive optics single-molecule localization microscopy for whole animals

Abstract

Specimen-induced aberration has been one of the major factors limiting the imaging depth in single-molecule localization microscopy (SMLM). In this study, we measured the wavefront of intrinsic reflectance signal at the fluorescence emission wavelength to construct a time-gated reflection matrix and find complex tissue aberration without resorting to fluorescence detection. Physically correcting the identified aberration via wavefront shaping with a liquid-crystal spatial light modulator (SLM)enables super-resolution imaging even when the aberration is too severe for initiating localization processes. We demonstrate the correction of complex tissue aberration, the root-mean-square (RMS)wavefront distortion of which is more than twice the 1 rad limit presented in previous studies; this leads to the recovery of single molecules by 77 times increased localization number. We visualised dendritic spines in mouse brain tissues and early myelination processes in a whole zebrafish at up to 102 μm depth with 28–39 nm localization precision. The proposed approach can expand the application range of SMLM to thick samples that cause the loss of localization points owing to severe aberration.

Aberration correction for deformable-mirror-based remote focusing enables high-accuracy whole-cell super-resolution imaging

“…In summary, the remote focusing enabled high-resolution whole-cell imaging, particularly in scenarios with high labeling density where large DOF PSF imaging could result in a strong background. With the precise in-situ PSF estimation, it can also be further extended to tissue imaging [37,38]. Although we showed superior performance of remote focusing compared to the whole-cell imaging with large DOF PSF, it is worth noting that there is a balance between the number of focus planes imaged and the number of molecules acquired due to photobleaching.…”

Adaptive optics in single objective inclined light sheet microscopy enables three-dimensional localization microscopy in adult Drosophila brains

“…Therefore, in 3D SMLM, in-situ PSF calibration should be combined with active aberration correction. To accomplish sample-induced aberration correction, two distinct approaches have been adopted: the first is based on a wave-front sensor to measure sample-induced aberrations and a DM to compensate for the measured aberration (Park et al, 2021 ); the second on a sensorless approach (Burke et al, 2015 ; Tehrani et al, 2015 ; Mlodzianoski et al, 2018 ; Siemons et al, 2021 ), where the aberrations are minimized by maximizing a metric for the image quality. Both algorithm and metric function should be tailored to an application to avoid non-convergent aberration corrections (Siemons et al, 2021 ) due to the non-convex dependency of the aberrations (Debarre et al, 2007 ; Soloviev, 2020 ).…”