Localization microscopy using noncovalent fluorogen activation by genetically encoded fluorogen-activating proteins.

Area of Science:

• Cell biology
• Biochemistry
• Microscopy

Background:

• Genetically encoded proteins are densely tagged, posing challenges for single-molecule detection and super-resolution imaging.
• Existing labeling methods often require photoswitching or specific buffer conditions.

Purpose of the Study:

• To develop a novel sparse labeling strategy for genetically encoded proteins.
• To enable single-molecule detection and super-resolution imaging in fixed and living cells.
• To simplify multicolor labeling applications.

Main Methods:

• Utilized noncovalent equilibrium activation of a fluorogenic malachite green dye with a cognate fluorogen-activating protein (FAP).
• Achieved sparse labeling by controlling dye concentration without photoswitching or photoactivation.
• Employed physiological buffers and cellular media for labeling.

Main Results:

• Demonstrated sparse labeling of actin using a FAP fusion.
• Achieved super-resolution imaging with 10-30 nm localization precision per object.
• Reported photon counts per object between those of fluorescent proteins and switching-dye pairs.

Conclusions:

• The developed labeling strategy effectively enables sparse labeling and super-resolution imaging.
• This method simplifies cellular labeling by eliminating the need for additives or switching buffers.
• The approach complements existing techniques and may facilitate multicolor labeling.