Development of far-red fluorescent proteins for temporal domain multiplexing and super-resolution imaging

Area of Science:

• Biophysics
• Cell Biology
• Microscopy

Background:

• Fluorescent proteins (FPs) are crucial tools for biological imaging, offering diverse spectral and photochemical properties.
• Existing FPs are used for applications including organelle labeling, super-resolution, and live-cell microscopy.

Purpose of the Study:

• To develop novel far-red fluorescent proteins with distinct photostability characteristics.
• To demonstrate the utility of these proteins for advanced imaging techniques, particularly temporal domain multiplexing (TDM).

Main Methods:

• Reported three far-red fluorescent proteins (mfRFP, mfRFP-A, mCardinal-A) with similar spectra but varying photobleaching rates.
• Utilized temporal domain multiplexing (TDM) for per-pixel unmixing of simultaneously imaged proteins.
• Established criteria for selecting FP pairs for efficient TDM and benchmarked TDM against other methods.
• Validated the photostable mfRFP variant in STED super-resolution microscopy and in vivo neuroimaging across multiple model organisms.

Main Results:

• Successfully performed simultaneous imaging and 3D reconstruction of cellular structures using TDM within a single imaging channel.
• Identified quantitative criteria for effective TDM FP pair selection.
• mfRFP demonstrated high photostability suitable for STED super-resolution and in vivo imaging.

Conclusions:

• The developed far-red FPs and TDM enable advanced multiplexed imaging without hardware modifications.
• Differential photostability is a key factor for efficient TDM-based imaging.
• These tools advance live-cell microscopy, super-resolution imaging, and in vivo studies across diverse biological systems.