Rapid Sequential in Situ Multiplexing with DNA Exchange Imaging in Neuronal Cells and Tissues

Abstract

To decipher the molecular mechanisms of biological function, it is critical to map the molecular composition of individual cells or even more importantly tissue samples in the context of their biological environment in situ. Immunofluorescence (IF) provides specific labeling for molecular profiling. However, conventional IF methods have finite multiplexing capabilities due to spectral overlap of the fluorophores. Various sequential imaging methods have been developed to circumvent this spectral limit, but are not widely adopted due to the common limitation of requiring multi-rounds of slow (typically over 2 hours at room temperature to overnight at 4 °C in practice) immunostaining. We present here a practical and robust method, which we call DNA-Exchange-Imaging (DEI), for rapid in situ spectrally-unlimited multiplexing. This technique overcomes speed restrictions by allowing for single-round immunostaining with DNA-barcoded antibodies, followed by rapid (less than 10 minutes) buffer exchange of fluorophore-bearing DNA imager strands. The programmability of DNA-Exchange-Imaging allows us to apply it to diverse microscopy platforms (with Exchange-Confocal, Exchange-SIM, Exchange-STED, and Exchange-PAINT demonstrated here) at multiple desired resolution scales (from ~300 nm down to sub-20-nm). We optimized and validated the use of DEI in complex biological samples, including primary neuron cultures and tissue sections. These results collectively suggest DNA-Exchange as a versatile, practical platform for rapid, highly multiplexed in situ imaging, potentially enabling new applications ranging from basic science, to drug discovery, and to clinical pathology.

Fluorescent in situ sequencing of DNA barcoded antibodies

“…In situ hybridization (ISH) of oligonucleotides conjugated to antibodies has emerged as a mild and reversible way to address a large number of targets with iterative staining. [6][7][8][9] Although effective, ISH only scales linearly with the number of iterative labelling reactions and does not take advantage of the data density of DNA (4 N where N = the number of bases in the oligos). For instance, one 20 nucleotidelong ISH probe encodes 1 bit of information whereas the sequencing of this length encodes 4 20 (over 1 trillion) bits.…”

Three dimensional nanoscopy of whole cells and tissues with in situ point spread function retrieval

“…We calibrated the deformable mirror (Supplementary Notes) to introduce individual Zernike-based aberration modes ranging from commonly experienced astigmatism and coma to high-order modes such as tertiary spherical aberration. We acquired single molecule blinking datasets in COS-7 cells by visualizing immuno-labeled mitochondrial marker TOM20 through DNA-PAINT (DNA point accumulation for imaging in nanoscale topography) [46][47][48][49]. The introduced aberrations distorted the single molecule emission patterns detected on the camera, which were then fed into INSPR to retrieve the in situ PSF and its corresponding pupil function.…”

About samples, giving examples: Optimized Single Molecule Localization Microscopy

“…Moreover, fluorophores are constantly renewed at the sample as new imagers interact with the docking strands, allowing to accumulate a large number of localizations. Finally, multi-color DNA-PAINT is straightforward by using orthogonal docking strands on distinct secondary antibodies and corresponding imagers, allowing to image 5-8 different targets Almada et al, 2019;Yu Wang et al, 2017;Werbin et al, 2017).…”