Visualizing Synaptic Multi-Protein Patterns of Neuronal Tissue With DNA-Assisted Single-Molecule Localization Microscopy

Abstract

The development of super-resolution microscopy (SRM) has widened our understanding of biomolecular structure and function in biological materials. Imaging multiple targets within a single area would elucidate their spatial localization relative to the cell matrix and neighboring biomolecules, revealing multi-protein macromolecular structures and their functional co-dependencies. SRM methods are, however, limited to the number of suitable fluorophores that can be imaged during a single acquisition as well as the loss of antigens during antibody washing and restaining for organic dye multiplexing. We report the visualization of multiple protein targets within the pre- and postsynapse in 350–400 nm thick neuronal tissue sections using DNA-assisted single-molecule localization microscopy (SMLM). In a single labeling step, antibodies conjugated with short DNA oligonucleotides visualized multiple targets by sequential exchange of fluorophore-labeled complementary oligonucleotides present in the imaging buffer. This approach avoids potential effects on structural integrity when using multiple rounds of immunolabeling and eliminates chromatic aberration, because all targets are imaged using a single excitation laser wavelength. This method proved robust for multi-target imaging in semi-thin tissue sections with a lateral resolution better than 25 nm, paving the way toward structural cell biology with single-molecule SRM.

Fast DNA-PAINT imaging using a deep neural network

“…1d ). DNA-PAINT data was recorded in semi-thin structurally conserved tissue labelled for α-tubulin and the mitochondrial protein TOM20 23 . The predicted images were compared to their respective GT images and the prediction quality was assessed using several quantitative metrics.…”

“…We applied the trained model to predict multi-colour SMLM super-resolution images. Structurally preserved semi-thin (~350 nm) cryosectioned rat neuronal tissue sections in the medial nucleus of the trapezoid body (MNTB) region 25 were stained for α-tubulin and TOM20 using DNA-labelled antibodies (see Methods; Table 1 ) and imaged sequentially following the Exchange-PAINT protocol 22 , 23 . A super-resolution image reconstructed from low emitter density DNA-PAINT data (0.5 nM imager strands P1/P5; 10,000 frames) served as the ground truth (Fig.…”

“…Third, low-density and high-density emitter data can be generated on the same sample to obtain a ground truth image for each prediction, hence bypassing using simulated datasets for NN assessments. Fourth, Exchange-PAINT permits multi-colour imaging by exchanging fluorophore-labelled oligonucleotide strands from the imaging buffer, which facilitates multi-target prediction with only a single NN model 22 , 23 . Finally, the bleaching-independent nature of DNA-PAINT permits the acquisition of large-sample areas in a short time.…”

Quantitative Imaging With DNA-PAINT for Applications in Synaptic Neuroscience

“…In one recent study, the concept of Exchange-PAINT has also been applied to neuronal tissue sections. Using four-target Exchange-PAINT, Narayanasamy et al (2021) were able to show the distribution of the pre-and postsynaptic scaffold proteins Bassoon and Homer1, as well as the glutamate vesicle marker VGlut1 in the calyx of Held synapse active zones with up to 25 nm resolution (Figure 2D).…”

“…The middle panel shows the entire field of view with a zoom-in in the bottom panel and the overlayed image on the right side. Adapted with permission(Narayanasamy et al, 2021).…”