PT  - JOURNAL ARTICLE
AU  - Niclas Gimber
AU  - Sebastian Strauss
AU  - Ralf Jungmann
AU  - Jan Schmoranzer
TI  - Simultaneous multicolor DNA-PAINT without sequential fluid exchange using spectral demixing
AID  - 10.1101/2021.11.19.469218
DP  - 2021 Jan 01
TA  - bioRxiv
PG  - 2021.11.19.469218
4099  - http://biorxiv.org/content/early/2021/11/19/2021.11.19.469218.short
4100  - http://biorxiv.org/content/early/2021/11/19/2021.11.19.469218.full
AB  - Several variants of multicolor single-molecule localization microscopy (SMLM) have been developed to resolve the spatial relationship of nanoscale structures in biological samples. The oligonucleotide-based SMLM approach ‘DNA-PAINT’ robustly achieves nanometer localization precision and can be used to count binding sites within nanostructures. However, multicolor DNA-PAINT has primarily been realized by ‘Exchange-PAINT’ that requires sequential exchange of the imaging solution and thus leads to extended acquisition times. To alleviate the need for fluid exchange and to speed up the acquisition of current multichannel DNA-PAINT, we here present a novel approach that combines DNA-PAINT with simultaneous multicolor acquisition using spectral demixing (SD). By using newly designed probes and a novel multichannel registration procedure we achieve simultaneous multicolor SD-DNA-PAINT with minimal crosstalk. We demonstrate high localization precision (3 – 6 nm) and multicolor registration of dual and triple-color SD-DNA-PAINT by resolving patterns on DNA origami nanostructures and cellular structures.Competing Interest StatementThe authors have declared no competing interest.SMLMSingle-molecule Localization MicroscopySTORMStochastic Optical Reconstruction MicroscopySDSpectral DemixingPAINTPoints Accumulation in Nanoscale Topographyfm-DNA-PAINTFrequency Multiplexing DNA-PAINTNeNANearest Neighbor Based AnalysisNNNearest NeighborABAntibodyNBNanobodyF(ab’)1Antigen Binding Fragment