Fluorescence lifetime DNA-PAINT for multiplexed super-resolution imaging of cells

Oleksiievets et al. combine single-molecule localization super-resolution microscopy technique DNA-PAINT with fluorescence lifetime imaging to allow fast multi-target super-resolution imaging in one spectral region by separating two or three different targets by fluorescence lifetime of the associated dyes. The method is demonstrated on two complementary experimental techniques, a standard confocal laser scanning setup, and a widefield microscope employing a novel TCSPC camera as the detector. DNA point accumulation for imaging in nanoscale topography (DNA-PAINT) is a powerful super-resolution technique highly suitable for multi-target (multiplexing) bio-imaging. However, multiplexed imaging of cells is still challenging due to the dense and sticky environment inside a cell. Here, we combine fluorescence lifetime imaging microscopy (FLIM) with DNA-PAINT and use the lifetime information as a multiplexing parameter for targets identification. In contrast to Exchange-PAINT, fluorescence lifetime PAINT (FL-PAINT) can image multiple targets simultaneously and does not require any fluid exchange, thus leaving the sample undisturbed and making the use of flow chambers/microfluidic systems unnecessary. We demonstrate the potential of FL-PAINT by simultaneous imaging of up to three targets in a cell using both wide-field FLIM and 3D time-resolved confocal laser scanning microscopy (CLSM). FL-PAINT can be readily combined with other existing techniques of multiplexed imaging and is therefore a perfect candidate for high-throughput multi-target bio-imaging.

Automated summary

The authors combined DNA-PAINT with fluorescence lifetime imaging to achieve fast multi-target super-resolution imaging in one spectral region. By utilizing fluorescence lifetime as a multiplexing parameter, this method enables simultaneous imaging of multiple targets without fluid exchange.