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.
A fluorescent emitter simultaneously transmits its identity, location, and cellular context through its emission pattern. We developed smNet, a deep neural network for multiplexed single-molecule analysis to enable retrieving such information with high accuracy. We demonstrate that smNet can extract three-dimensional molecule location, orientation, and wavefront distortion with precision approaching the theoretical limit and therefore will allow multiplexed measurements through the emission pattern of a single molecule.
Single-molecule localization microscopy is a powerful tool for
visualizing subcellular structures, interactions, and protein functions in
biological research. However, inhomogeneous refractive indices inside cells and
tissues distort the fluorescent signal emitted from single-molecule probes,
which rapidly deteriorates resolution with increasing depth. We propose a method
that enables the construction of an
in situ
3D response of
single emitters directly from single-molecule blinking datasets and therefore
allows their locations to be pin-pointed with precision that achieves the
Cramer-Rao lower bound and uncompromised fidelity. We demonstrate this method,
named
in situ
PSF retrieval (INSPR), across a range of cellular
and tissue architectures from mitochondrial networks and nuclear pores in
mammalian cells, to amyloid β plaques and dendrites in brain tissues, and
elastic fibers in developing cartilage of mice. This advancement expands the
routine applicability of super-resolution microscopy from selected cellular
targets near coverslips to intra- and extra-cellular targets deep inside
tissues.
Dynamic measurements of molecular machines can provide invaluable insights into their mechanism, but these measurements have been challenging in living cells. Here, we developed live-cell tracking of single fluorophores with nanometer spatial and millisecond temporal resolution in two and three dimensions using the recently introduced super-resolution technique MINFLUX. Using this approach, we resolved the precise stepping motion of the motor protein kinesin-1 as it walked on microtubules in living cells. Nanoscopic tracking of motors walking on the microtubules of fixed cells also enabled us to resolve the architecture of the microtubule cytoskeleton with protofilament resolution.
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