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Nat Commun. 2019 Sep 26;10(1):4377. doi: 10.1038/s41467-019-12372-6.

Multiplexed and high-throughput neuronal fluorescence imaging with diffusible probes.

Author information

1
Department of Biological Engineering, MIT, Cambridge, MA, USA.
2
Stanley Center for Psychiatric Research, Broad Institute of MIT and Harvard, Cambridge, MA, USA.
3
Broad Institute of MIT and Harvard, Cambridge, MA, USA.
4
Media Lab, MIT, Cambridge, MA, USA.
5
McGovern Institute for Brain Research, Department of Brain and Cognitive Sciences, MIT, Cambridge, MA, USA.
6
Stanley Center for Psychiatric Research, Broad Institute of MIT and Harvard, Cambridge, MA, USA. cottrell@broadinstitute.org.
7
Broad Institute of MIT and Harvard, Cambridge, MA, USA. cottrell@broadinstitute.org.
8
Department of Biological Engineering, MIT, Cambridge, MA, USA. mark.bathe@mit.edu.
9
Broad Institute of MIT and Harvard, Cambridge, MA, USA. mark.bathe@mit.edu.

Abstract

Synapses contain hundreds of distinct proteins whose heterogeneous expression levels are determinants of synaptic plasticity and signal transmission relevant to a range of diseases. Here, we use diffusible nucleic acid imaging probes to profile neuronal synapses using multiplexed confocal and super-resolution microscopy. Confocal imaging is performed using high-affinity locked nucleic acid imaging probes that stably yet reversibly bind to oligonucleotides conjugated to antibodies and peptides. Super-resolution PAINT imaging of the same targets is performed using low-affinity DNA imaging probes to resolve nanometer-scale synaptic protein organization across nine distinct protein targets. Our approach enables the quantitative analysis of thousands of synapses in neuronal culture to identify putative synaptic sub-types and co-localization patterns from one dozen proteins. Application to characterize synaptic reorganization following neuronal activity blockade reveals coordinated upregulation of the post-synaptic proteins PSD-95, SHANK3 and Homer-1b/c, as well as increased correlation between synaptic markers in the active and synaptic vesicle zones.

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