Choosing a Fluorescent Protein A Comprehensive Guide

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Understanding Fluorescent Proteins

Fluorescence Basics: FPs absorb light at a specific wavelength (excitation) and emit light at a longer wavelength (emission). These unique spectral properties allow for identification and quantification against biological backgrounds.
FP Evolution: Since the discovery of Green Fluorescent Protein (GFP), researchers have engineered numerous variants with improved brightness, photostability, and an expanded color spectrum.

Key Selection Criteria:

Spectral Properties: Choosing FPs with non-overlapping excitation and emission spectra is critical for multicolor imaging to prevent signal cross-talk.
Brightness and Photostability: Brightness influences detectability, while photostability, or resistance to photobleaching, is crucial for time-lapse imaging and high-intensity illumination.
Oligomeric State: Monomeric FPs are generally preferred for fusion constructs to minimize interference with the target protein's function.
Maturation Time: Rapidly maturing FPs are essential for observing dynamic processes, while slower maturation may be acceptable for static studies.
Cellular Localization: Engineered FPs with specific localization signals can target imaging to specific cellular compartments.

Popular FPs and Applications:

GFP and Variants: GFP and enhanced GFP (eGFP) are widely used for their brightness and stability.
Red Fluorescent Proteins (RFPs): RFPs like mCherry enable multicolor imaging with GFP due to their distinct spectral profiles.
Far-Red and Near-Infrared FPs: mPlum and iRFP are valuable for deep tissue imaging due to minimal overlap with autofluorescence and deeper tissue penetration.
Photoactivatable and Photoswitchable FPs: These FPs change fluorescence properties upon light exposure, facilitating super-resolution imaging and tracking protein dynamics. Examples include PA-GFP and Dronpa.