Spectroscopic DNA-PAINT for simultaneous multiplexed super-resolution microscopy.

Area of Science:

• Biophysics
• Optical Microscopy
• Nanotechnology

Background:

• Simultaneous multiplexed super-resolution imaging is crucial for understanding molecular interactions but faces challenges with spectral overlap and precision.
• Current single-molecule localization microscopy (SMLM) methods have limitations in distinguishing multiple fluorophores simultaneously.

Purpose of the Study:

• To introduce and validate spectroscopic DNA-PAINT (sDNA-PAINT) for high-fidelity, simultaneous multiplexed super-resolution imaging.
• To enhance spectral precision and photon budgets in SMLM for improved multiplexing capabilities.

Main Methods:

• Integration of DNA-PAINT with spectroscopic SMLM (sSMLM) to create the sDNA-PAINT framework.
• Utilized DNA Origami Nanorulers for *in vitro* and fixed-cell imaging experiments.
• Tested sDNA-PAINT with various fluorophore families (Rhodamine, Cyanine, Oxazine) and compared performance against conventional SMLM conditions.

Main Results:

• sDNA-PAINT significantly improved spectral precision and photon budgets compared to conventional methods.
• Demonstrated reliable single-molecule discrimination even for spectrally overlapping dyes.
• Achieved accurate spatial reconstruction and high classification accuracy in dual-target cellular imaging.

Conclusions:

• sDNA-PAINT enables high-fidelity simultaneous multiplexed super-resolution imaging within a single acquisition.
• This framework offers a pathway toward high-throughput simultaneous multiplexed super-resolution interaction imaging.
• sDNA-PAINT overcomes spectral limitations, advancing the field of super-resolution microscopy.