Photophysics of fluorescent probes for single-molecule biophysics and super-resolution imaging

Abstract

Single-molecule fluorescence spectroscopy and super-resolution microscopy are important elements of the ongoing technical revolution to reveal biochemical and cellular processes in unprecedented clarity and precision. Demands placed on the photophysical properties of the fluorophores are stringent and drive the choice of appropriate probes. Such fluorophores are not simple light bulbs of a certain color and brightness but instead have their own "personalities" regarding spectroscopic parameters, redox properties, size, water solubility, photostability, and several other factors. Here, we review the photophysics of fluorescent probes, both organic fluorophores and fluorescent proteins, used in applications such as particle tracking, single-molecule FRET, stoichiometry determination, and super-resolution imaging. Of particular interest is the thiol-induced blinking of Cy5, a curse for single-molecule biophysical studies that was later overcome using Trolox through a reducing/oxidizing system but a boon for super-resolution imaging owing to the controllable photoswitching. Understanding photophysics is critical in the design and interpretation of single-molecule experiments.

Figure 1

Three schemes of single-molecule based super-resolution imaging. (a) Probes are active initially and then are turned off. A small fraction is activated thermally or by light for localization. This process is repeated many times to result in an image. The smiley face is an actual image, adapted from ref. (118). (b) Probes are initially dark and then activated in small numbers. (c) Probes are active initially and turned off, and a small fraction becomes active by controlling additives in solution.

Figure 2

SmFRET time trajectories indicating the potential similarity of biomolecular dynamics and photophyscially induced FRET fluctuations. (a) FRET fluctuations representing conformational transitions between two conformers of a Holliday junction labeled with Cy3/Cy5. B: FRET fluctuations originating from transient off-states of the acceptor in double-stranded DNA labeled with ATTO647N and ATTO680.

Figure 3

Illustration of triplet-state blinking and its control. (a) Without oxygen removal, a single molecule spends only a small fraction of time in the off state (triplet state). (b) When oxygen is removed, the triplet state lifetime increases and the molecule spends long periods in the off state. (c) β-mercaptoethanol shortens the triplet state lifetime but not very well. (d) Trolox is highly efficient in shortening the triplet state lifetime. (e) Long triplet state lifetimes can cause early saturation of emission at high excitation rates under de-oxygenation condition, a problem that can be mitigated by using Trolox.

Figure 4

Simplified Jablonski diagram with accessible states under different conditions. The fluorophore is excited from its ground state (S₀) to the first excited state (S₁) with an excitation rate k_ex and fluoresces with the rate k_fl after a few nanoseconds. Competing processes are non-radiative decays to S₀ with rate constant k_nr and intersystem crossing to the triplet state k_isc with a lifetime in the millisecond range. If an oxidant or a reductant is added, the triplet state is depopulated quickly and radical cations (F^+∂) or anions (F^−∂) are formed with rate constants k_ox or k_red, respectively. Depending on their redox potential these dark states are comparatively stable but can be recovered to S₀ by the complementary process in a buffer containing a reducing and an oxidizing system. The resulting short lifetimes of the triplet and radical ion states additionally improve photostability, as photobleaching (P) usually occurs from excited states. Time trajectories were measured with enzymatic oxygen scavenging. Reductant: Ascorbic Acid (AA), Oxidant: Methylviologen (MV).

Figure 5

(a) Fluorescent transient of a TMR-ATTO647N pair attached to double stranded DNA at a distance of 6 base pairs. Intensity fluctuations are caused by direct donor-acceptor contact. (b) Only the FRET-pair Cy3/Cy5 shows comparably stable FRET even at short distances. Adapted with permission from ref. (95).

Figure 6

Illustration of how a single molecule fluorescence intensity trajectory can reveal the number of proteins (A) in a complex by showing three steps during photobleaching. Adapted with permission from ref. (7).