Live-cell monochromatic dual-label sub-diffraction microscopy by mt-pcSOFI

Area of Science:

• Microscopy
• Biophysics
• Cell Biology

Background:

• Super-resolution microscopy techniques are crucial for visualizing cellular structures.
• Photochromic super-resolution optical fluctuation imaging (pcSOFI) offers high spatial resolution.
• Distinguishing spectrally similar fluorescent probes remains a challenge in live-cell imaging.

Purpose of the Study:

• To expand pcSOFI for monochromatic dual-channel sub-diffraction microscopy.
• To develop a method for unmixing spectrally identical reversibly switchable fluorescent proteins (RSFPs).
• To enable simultaneous imaging of two distinct structures in living cells using RSFPs.

Main Methods:

• Development and application of multi-tau pcSOFI (mt-pcSOFI).
• Utilizing blinking kinetics of RSFPs for spectral unmixing.
• Imaging experiments in living cells using existing and novel RSFPs (ffDronpa-F).

Main Results:

• Successfully demonstrated mt-pcSOFI for monochromatic dual-channel imaging.
• Achieved unmixing of spectrally identical RSFPs based on their distinct blinking kinetics.
• Showcased simultaneous imaging of two cellular structures using mt-pcSOFI and ffDronpa-F.

Conclusions:

• mt-pcSOFI is a powerful extension of pcSOFI for dual-channel super-resolution imaging.
• The method allows for simultaneous visualization of multiple targets labeled with spectrally similar RSFPs.
• This technique advances live-cell imaging capabilities by overcoming spectral overlap limitations.