Macromolecular-scale resolution in biological fluorescence microscopy

Abstract

For more than a century, the resolution of a lens-based (far-field) optical microscope has been limited by diffraction (1). However, in the 1990s it became evident that the limiting role of diffraction can be broken in lens-based fluorescence microscopy if certain fluorophore properties are judiciously integrated into the image formation (2). The first viable concept of this kind is stimulated emission depletion (STED) microscopy (3), which, since its experimental validation (4, 5), has been key to solving a number of problems in biophysics (6) and cell biology (7, 8).

STED microscopy typically uses a scanning excitation spot that is overlapped with a doughnut-shaped counterpart for deexcitation of fluorophores by light, a phenomenon referred to as stimulated emission (9, 10). Oversaturating the deexcitation squeezes the fluorescence spot to subdiffraction dimensions (Fig. 1a) so that superresolved images emerge by scanning this spot through the object (5).

Fig. 1.

STED microscopy operation with interpulse time interval Δt. (a Left and Center) Measured focal spots (PSFs) for excitation (Exc; Left) (wavelength, 470 nm; blue) and STED (Center) (603 nm, orange). The blue spot of excitation light features a FWHM of 190 nm. (a Right) Applying a crest intensity Î_STED ^{yields the 22-nm effective (Eff) spot shown in green. (}b) Fluorophore energy levels and potential bleaching pathways. Fluorescence from S₁ is suppressed by stimulated emission. Stimulated emission may also excite the dye to a higher singlet state S_x, from which the molecule can cross to the triplet system T_x. In regions of weak STED pulse intensity, the molecule can directly cross to the T₁ featuring a lifetime τ_T = 0.5–3 μs. Excitation of T₁ molecules by subsequent pulses leads to reverse intersystem crossing or augmented photobleaching. (c) These adverse effects are counteracted by Δt > τ_T. Note the dramatic reduction in the focal spot area in a, resulting from STED.

The rate for deexcitation by stimulated emission is given by k_STED = σI_STED, with σ denoting the fluorophore cross-section and I_STED denoting the intensity of the stimulating beam. Oversaturating the deexcitation requires k_STED be much larger than the fluorescence decay given by the inverse of the lifetime, τ_Fl ≈ 1–5 ns, of the fluorescent state S₁. With σ ≈ 10 ^{cm, it follows that} I_STED ≫ 1/(στ_Fl) = 10 ^{photons per second per squared centimeter, which, at a wavelength of λ = 600 nm, amounts to Î}_STED ≫ 33 MW/cm^{. This intensity value is at least 10-fold lower than what is required for multiphoton excitation (}11), but still 10^{-fold larger than what is used for single-photon fluorescence excitation (}3).

Therefore, to operate with a moderate average power, the excitation and the STED beams are implemented as synchronized pulse trains (3). The duration of the STED pulse τ_STED is adjusted to a fraction of τ_Fl, typically ≈0.2 ns, in which case the depletion of the excited state is an exponential function of the stimulating intensity: exp[−σ·τ_STED·I_STED]. Hence, a doughnut-shaped focal distribution I_STED($\vec{r}$) featuring I_STED ^{≡ max[}I_STED($\vec{r}$)] at the doughnut crest and I_STED($\vec{r}$= 0) = 0 at the center suppresses the signal throughout the focal region, except at $\vec{r}$= 0. The remaining spot in the focal plane follows

with n sin α denoting the numerical aperture at which the doughnut is generated (12, 13). Thus, provided the doughnut zero is largely maintained, the resolution can be arbitrarily increased with increasing I_STED^{, in principle, down to the molecular scale (}3).

An obvious challenge toward maximizing I_STED ^{is elevated photobleaching of the fluorescent marker that usually scales nonlinearly with the applied intensity (}14, 15). For example, at the typical 80-MHz repetition rate of mode-locked lasers, the average focal power applicable to the green dye Atto532 for STED is ≈15 mW (7). The associated Î_STED ^{= 250 MW/cm yields a resolution of Δ}r = 50–70 nm. Larger Î_STED ^{and hence much narrower focal spots were reached only with a red dye under relatively favorable photochemical conditions (}13).

In STED microscopy, two bleaching pathways are imaginable (16): (i) the absorption of a fluorescent-state molecule, leading to a higher molecular singlet state, S₁ → S_x>1, as it has been proposed for multiphoton microscopy (17) and (ii) the excitation of excited molecules that have crossed to a triplet state, T₁ → T_x>1, or to another dark state with lifetime τ_T > 1 μs. The states S_x>1 and T_x>1 are well known starting points for bleaching reactions (Fig. 1b).

When considering the two routes, it becomes evident that the first one, S₁ → S_x>1, is counteracted by stimulated emission, S₁→ S₀. With a wavelength optimized for the latter, S₁ → S_x>1 excitation by the STED pulse is less probable. Nevertheless, because the cross-sections for S₁ → S_x>1 are finite both at the STED and at the excitation wavelength, higher singlet excitation is possible. The superexcited S_x>1 molecule may bleach, cross to the triplet system S_x>1 → T_x>1 → T₁, or return to the S₁ (18). Referred to as internal conversion, the last process is very effective because it occurs within a few picoseconds. Because of the STED pulse duration of τ_STED ≈ 0.2 ns, the putatively superexcited molecule is instantly quenched by the same pulse. Therefore, as long as the molecule remains in the singlet system, the STED pulse is able to protect the molecule from photobleaching.

Just the opposite is the case once the molecule has crossed to the triplet state T₁. Because of its prolonged typical lifetime, τ_T ≈ 1 μs at ambient conditions, the T₁ molecule is exposed to a train of intense excitation and STED pulses. Given the rather large molecular cross-sections (10 ^{to 10 cm) for triplet absorption over a broad wavelength range, the STED pulse can efficiently pump up the molecule to higher triplet states T}_x>1. The ≈80-MHz repetition rate of mode-locked lasers used so far for STED implies that an inherently fragile triplet molecule faces on average 80 intense STED pulses before relaxing to the S₀. Moreover, most fluorophores spontaneously undergo S₁ → T₁ crossings with a probability of 1–10% per excitation cycle (18). At the doughnut hole, where the STED pulse is weak and the molecules relax spontaneously, stimulated emission does not override the intersystem crossing. Therefore, a remedy against this bleaching pathway is to illuminate molecules as little as possible within the time span τ_T ≈ 1 μs after the excitation.

Based on this reasoning, we have explored the operational principles of STED microscopy at a pulse repetition rate of 0.25 MHz that is 320 times lower than the standard rate of 80 MHz. The Δt = 4 μs time gap between subsequent pulse pairs ensures that most triplet-state molecules of τ_T ≈ 1 μs have returned to the ground state before encountering a second or third pulse pair (Fig. 1c). We have termed this illumination scheme triplet relaxation (T-Rex) STED.

Acknowledgments

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