Optical Lock-In Detection of FRET Using Synthetic and Genetically Encoded Optical Switches

Abstract

The Förster resonance energy transfer (FRET) technique is widely used for studying protein interactions within live cells. The effectiveness and sensitivity of determining FRET, however, can be reduced by photobleaching, cross talk, autofluorescence, and unlabeled, endogenous proteins. We present a FRET imaging method using an optical switch probe, Nitrobenzospiropyran (NitroBIPS), which substantially improves the sensitivity of detection to <1% FRET efficiency. Through orthogonal optical control of the colorful merocyanine and colorless spiro states of the NitroBIPS acceptor, donor fluorescence can be measured both in the absence and presence of FRET in the same FRET pair in the same cell. A SNAP-tag approach is used to generate a green fluorescent protein-alkylguaninetransferase fusion protein (GFP-AGT) that is labeled with benzylguanine-NitroBIPS. In vivo imaging studies on this green fluorescent protein-alkylguaninetransferase (GFP-AGT) (NitroBIPS) complex, employing optical lock-in detection of FRET, allow unambiguous resolution of FRET efficiencies below 1%, equivalent to a few percent of donor-tagged proteins in complexes with acceptor-tagged proteins.

Hybridization kinetics is different inside cells

“…As a side note, the herein described optical lock-in (21) has to be distinguished from a more recent method with a similar name (44,45) (21). For temperature measurements, fluorescence amplitudes were translated to temperature changes via the Cy5 calibration.…”

Reversible optical control of cyanine fluorescence in fixed and living cells: optical lock-in detection immunofluorescence imaging microscopy

“…Optical switching of the high quantum yield fluorescence emission of Cy in the hybrid probe is achieved through a photochromic FRET mechanism [16,19,21]. In particular, excitation of the highly fluorescent SP state (no-FRET) with a short (100 ms), low-power pulse between 365 and 405 nm generates the non--fluorescent MC state (FRET) of Cy-NISO ( figure 1a,b).…”

“…The study also reveals that the power of the 546 nm light can be used to control the rate of the MC to SP transition, by increasing the number of MC molecules that enter the excited state during a fixed exposure to 546 nm, allowing the user to shape the waveform of the Cy signal. A Cy3-NISO-based suicide substrate is prepared for the Snap-tag (AGTase) [17,21], composed of a Cy3-NISO optical switch linked to BG, as shown in figure 4a. BG-Cy3-NISO is added to the culture medium of live NIH 3T3 cells expressing a SNAP-b2-adrenergic receptor (ADRB2) fusion protein [21].…”

Fluorescent Dyes Used in Polymer Carriers as Imaging Agents in Anticancer Therapy

“…The rational design of highly fluorescent fluorophore compounds with optical switches for high-contrast optical lock-in detection used in imaging microscopy in living cells was reported by Marriott et al [142,143]. They designed a highly fluorescent probe consisted of tetramethylrhodamine (TMR) and spironaphthoxazine (NISO) or (TMR-NISO) which was an optical switch to improve the image contrast.…”