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.
Highlights
Super-resolution microscopy (SRM) has revolutionized our understanding of cell biology
Α-tubulin, mitochondria (TOM20), microtubuleassociated protein 2 (MAP2), and vesicular glutamate transporter (VGLUT1) were labeled with primary antibodies (Ab) and secondary Ab conjugated to DNA docking strands (P1, P5, R1, or R4; see section “Materials and Methods”) (Figure 1)
This set of steps was repeated with R1 and R4 strands until all labeled proteins were imaged within the same region of interest (ROI)
Summary
Super-resolution microscopy (SRM) has revolutionized our understanding of cell biology. Singlemolecule localization microscopy (SMLM) is one branch of SRM, which employs photoswitchable or transiently binding fluorophore labels and has demonstrated a near-molecular spatial resolution (Sauer and Heilemann, 2017) allowing molecular quantification (Dietz and Heilemann, 2019). The short oligonucleotides act as transiently hybridizing pairs, with one coupled to a target protein (the “docking strand”, attached to e.g., an antibody) and a second carrying a fluorophore (the “imager strand”) suspended in the imaging buffer. The transient hybridization of both oligonucleotides generates a temporally short and spatially localized signal, which at low concentration of imager strands is recorded as a single-molecule emission event. Multiplexing and the excellent spatial resolution achieved with DNA-PAINT is beginning to evolve as a tool in cell biology (Harwardt et al, 2020; Schröder et al, 2020; Strauss and Jungmann, 2020)
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