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

Area of Science:

• Biophysics
• Super-resolution microscopy
• Cellular imaging

Background:

• DNA point accumulation for imaging in nanoscale topography (DNA-PAINT) is a super-resolution technique.
• Multiplexed bio-imaging in dense cellular environments remains challenging.
• Existing methods like Exchange-PAINT require fluid exchange, potentially disturbing samples.

Purpose of the Study:

• To develop a novel multiplexing strategy for DNA-PAINT using fluorescence lifetime.
• To enable simultaneous multi-target imaging in cells without fluid exchange.
• To enhance the capabilities of super-resolution microscopy for complex biological samples.

Main Methods:

• Integration of fluorescence lifetime imaging microscopy (FLIM) with DNA-PAINT.
• Utilizing fluorescence lifetime as a parameter for target identification.
• Demonstration using wide-field FLIM and 3D time-resolved confocal laser scanning microscopy (CLSM).

Main Results:

• Simultaneous imaging of up to three targets within a single cell was achieved.
• Fluorescence lifetime PAINT (FL-PAINT) demonstrated effective multiplexing without fluid exchange.
• The method proved compatible with both wide-field and confocal microscopy setups.

Conclusions:

• FL-PAINT offers a robust and non-invasive method for multiplexed super-resolution bio-imaging.
• The technique overcomes limitations of existing methods in cellular imaging.
• FL-PAINT is a promising tool for high-throughput multi-target bio-imaging and can be combined with other techniques.