Spiropyrans as molecular optical switches

Abstract

Optical microscopes use visible light and an arrangement of lenses to provide us with magnified images of small samples. Combined with efficient fluorescent probes and highly sensitive fluorescence detection techniques they allow the non-invasive 3D study of subcellular structures even in living cells or tissue. However, optical microscopes are subject to diffraction of light which limits optical resolution to approximately 200 nm in the imaging plane. In the recent past, powerful methods emerged that enable fluorescence microscopy with subdiffraction optical resolution. Since most of these methods are based on the temporal control of fluorescence emission of fluorophores, photochromic molecules that can be switched reversibly between a fluorescent on- and a non-fluorescent off-state are the key for super-resolution imaging methods. Here, we present our approach to use spiropyran-fluorophore conjugates as efficient molecular optical switches (photoswitches). In these photochromic conjugates fluorescence emission of the fluorophore is controlled by the state of the spiropyran, which can be switched reversibly between a colorless spiropyran and a colored merocyanine form upon irradiation with light. Thus, the efficiency of energy transfer from the fluorophore to the spiropyran can be modulated by the irradiation conditions. We present ensemble data of the switching process of various spiropyrans and spiropyran-fluorophore conjugates and demonstrate photoswitching at the single-molecule level. Our data suggest that spiropyrans have to be immobilized in polymers to stabilize the merocyanine form in order to be useful for super-resolution fluorescence imaging based on precise localization of individual emitters. Special emphasis is put on photobleaching of donor fluorophores due to UV irradiation, i.e. photoswitching of the photochromic acceptor. Furthermore, we present a water soluble switchable spiropyran derivative and demonstrate the first intermolecular single-molecule photoswitching experiments in polymers.

Probes for Nanoscopy: Photoswitchable Fluorophores

“…In addition, the linker should be not too short in order to avoid quenching effects by a nonresonant mechanism [31]. Examples of fluorescent switchable dyads operating under this principle can be found in the literature combining a large variety of conventional fluorophores and different kinds of photochromic dyes, such as fulgides [26], diheteroarylethenes [29,30], azobenzenes [38,39], and spyropirans [40,41].…”

“…The merocyanine isomer is rarely fluorescent unless it is embedded in apolar environments. Thus, many reported examples of fluorescent switches are based in a 6-NO 2 -BIPS used as a FRET-modulator of the fluorescent signal of a bridged fluorophore (e.g., Lucifer Yellow [40], rhodamines [41], BODIPY [63], or QDs [64]). A slow thermal reaction constant (slow switching-on) and poor fatigue resistance are probably the reasons why these dyads have not found yet applications in fluorescence nanoscopy [41].…”

“…Thus, many reported examples of fluorescent switches are based in a 6-NO 2 -BIPS used as a FRET-modulator of the fluorescent signal of a bridged fluorophore (e.g., Lucifer Yellow [40], rhodamines [41], BODIPY [63], or QDs [64]). A slow thermal reaction constant (slow switching-on) and poor fatigue resistance are probably the reasons why these dyads have not found yet applications in fluorescence nanoscopy [41]. [42,43] The direct fluorescence of the merocyanine has recently been exploited for imaging beyond the diffraction limit using a stochastic approach [65], by embedding the probe in hydrophobic cavities of polymeric nanoparticles (Fig.…”

Single-molecule Photoswitching and Localization

“…In the past years, a huge amount of photoswitchable fluorophores has been developed including photochromic compounds, [54][55][56] conventional organic fluorophores, [31,35,36,40,47,57] caged fluorescent dyes, [58] push-pull fluorophores, [59] and a variety of photoswitchable FP. [4,[60][61][62] In the following, we restrict our considerations to irreversible photoswitching of photoactivatable or photoconvertible FP and reversible photoswitching of FP and synthetic organic fluorophores since they are currently the most promising candidates for single-molecule based localization microscopy.…”

Photochromic and thermochromic spiranes. 34.* synthesis of photochromic 5-(4,5-diphenyl-1,3-oxazol-2-yl)-substituted spirobenzochromeneindolines

“…The insertion of various functional fragments into the spiropyran molecule opens up the possibility of obtaining a wide range of polyfunctional photochromic molecular systems exhibiting magnetic [5], fluorescent [6][7][8][9], and complexing [9][10][11][12] properties switched on by optical radiation. Earlier we reported on the synthesis of photochromic 5-(4,5-diphenyl-1,3-oxazol-2-yl)-substituted spiropyrans, the acyclic isomer of which forms reversibly complexes with the divalent cations of heavy metals [13].…”