Active PSF shaping and adaptive optics enable volumetric localization microscopy through brain sections

Abstract

Application of single-molecule switching nanoscopy (SMSN) beyond the coverslip surface poses substantial challenges due to sample-induced aberrations that distort and blur single-molecule emission patterns. We combined active shaping of point spread functions and efficient adaptive optics to enable robust 3D-SMSN imaging within tissues. This development allowed us to image through 30-μm-thick brain sections to visualize and reconstruct the morphology and the nanoscale details of amyloid-β filaments in a mouse model of Alzheimer's disease.

When can we Believe what we see?

“…A recent noteworthy example comes from researchers at Purdue University (IN, USA), who have developed a super-resolution nanoscope that provides a 3D view of brain molecules with much greater detail than previously possible [5]. …”

Three dimensional nanoscopy of whole cells and tissues within situpoint spread function retrieval

“…The resulting PSFs generated by the in situ pupil show high degree of similarity with those generated by the known pupil ( Figure 2D). The performance is further tested through retrieving a previously estimated wavefront distortion at various imaging depths above the coverslip, up to ~45 μm [30] ( Figures S2A and S2B). We find that INSPR enables retrieval of these in situ PSFs that vary significantly throughout the depths (0, 6.7, 14.35, 27.55, and 45.4 μm).…”

“…Such information loss cannot be recovered by algorithm but rather requires a physical element that modifies the distorted wavefront prior to the detection of the fluorescent signal. The combination of adaptive optics (AO) [29,30,[58][59][60][61][62][63][64] with INSPR will allow restoring emission pattern information and pin-pointing 3D location of single molecules with high accuracy simultaneously. In addition, INSPR can be combined with light-sheet illumination approaches [65][66][67] and tissue clearing and expansion methods [68][69][70] to further reduce the fluorescence background and increase the achievable resolution, therefore opening doors to observe the static and dynamic conformation of complex intra-and extra-cellular constituents over large tissue volumes at the nanoscale level.…”

“…It is, therefore, imperative to obtain an accurate model of the 3D PSF, which can reflect the complex biological and physical context constituting the path that the emitted photons travel through before being detected. Inaccurate models will give rise to imaging artifacts and significant deteriorations of the super-resolved 3D volume [29,30].…”

Targeted volumetric single-molecule localization microscopy of defined presynaptic structures in brain sections

“…In addition, 3D-SMLM imaging was achieved in brain sections using oil-immersion objectives to visualize the distribution of synaptic proteins a few micrometers above the coverslip 10,13 . More recently, self-interference 3D super-resolution microscopy and active point-spread function (PSF) shaping in combination with adaptive optics were introduced to enable 3D localization of emitters in tissue with a thickness of up to 50 µm 15,16 . The latter approach allowed reconstructing super-resolution volumes with an axial depth of several micrometers.…”