Optical switch probes and optical lock-in detection (OLID) imaging microscopy: high-contrast fluorescence imaging within living systems

Abstract

Few to single molecule imaging of fluorescent probe molecules can provide information on the distribution, dynamics, interactions and activity of specific fluorescently tagged proteins during cellular processes. Unfortunately, these imaging studies are made challenging in living cells because of fluorescence signals from endogenous cofactors. Moreover, related background signals within multi-cell systems and intact tissue are even higher and reduce signal contrast even for ensemble populations of probe molecules. High-contrast optical imaging within high-background environments will therefore require new ideas on the design of fluorescence probes, and the way their fluorescence signals are generated and analysed to form an image. To this end, in the present review we describe recent studies on a new family of fluorescent probe called optical switches, with descriptions of the mechanisms that underlie their ability to undergo rapid and reversible transitions between two distinct states. Optical manipulation of the fluorescent and non-fluorescent states of an optical switch probe generates a modulated fluorescence signal that can be isolated from a larger unmodulated background by using OLID (optical lock-in detection) techniques. The present review concludes with a discussion on select applications of synthetic and genetically encoded optical switch probes and OLID microscopy for high-contrast imaging of specific proteins and membrane structures within living systems.

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

“…The intensity of the Cy3-fluorescence signal rstb.royalsocietypublishing.org Phil Trans R Soc B 368: 20120031 decreases by 13 + 0.1% immediately following the first and subsequent exposures of the field to 365 nm, and returns to approximately 3 per cent of the pre-365 nm level after 15 scans of the field at 543 nm (figure 5b). The normalized Cy3-intensity profile within each of the 5 cycles of optical switching of the preparation is identical (figure 5b), an indication that transitions between the SP and MC states of the NISO probe on the antibody are uniform and proceed via defined quantum yields [6]. The approximately 3 per cent decrease in the maximum intensity of red fluorescence for consecutive cycles of optical switching is likely due to photobleaching of Cy3, or the background, and not the NISO probe.…”

“…An ideal probe for OLID imaging of specifically labelled proteins within living cells is one that undergoes many cycles of orthogonal, rapid and reversible, optically driven transitions between the two states of the switch with high fidelity and over many cycles of optical switching [6,15]. For some types of reversible optical switch, typified by NitroBIPS, diarylethanes and Dronpa, the excited state of the coloured state of the switch can return to the same ground state with emission of fluorescence, or else isomerize to the non-coloured state of the switch [16,20,[24][25][26].…”

“…For some types of reversible optical switch, typified by NitroBIPS, diarylethanes and Dronpa, the excited state of the coloured state of the switch can return to the same ground state with emission of fluorescence, or else isomerize to the non-coloured state of the switch [16,20,[24][25][26]. It follows then that an optical switch probe having a quantum yield for fluorescence emission must be a poor switch, and vice versa [6]. We have previously shown that the fluorescence and switching functions of a probe can be uncoupled by imparting a switchable character on a conventional probe using a switchable acceptor probe via a FRET-based mechanism [18,19].…”

“…In OLID, the optical switch is optically manipulated between the fluorescent and non-fluorescent states to generate a defined fluorescence intensity waveform that is isolated from the unmodulated background [15]. An effective strategy to generate these unique time-dependent fluorescence signals or intensity waveforms from the fluorescent probe is schematized in figure 1c [6,15]. A fluorescence signal in a biological preparation that has a defined intensity waveform is clearly unique and differs significantly from background signals; we have shown that these modulated fluorescence signals can be isolated from other sources in the preparation by using OLID [15].…”

“…Consequently, NitroBIPS, Dronpa and related photochromes are either good fluorophores but poor switches, or good switches but poor fluorophores [6,15]. An ideal probe, however, should exhibit high efficiencies for both fluorescence and optical switching.…”