RESOLFT Nanoscopy

Reversible Saturable Optical Fluorescence Transitions (RESOLFT) super resolution microscopy represents a powerful tool to decipher spatio-temporal information coded in the life sciences by (1) minimizing the illumination light intensities (2) employing genetically encoded markers (3) improving the spatial resolution of conventional fluorescence microscopy down to the nanoscale. In RESOLFT nanoscopy the spatial resolution is improved by taking advantages of “long-lived” (µs-ms) molecular states, which are populated/depopulated with light intensities in the range of W–kW/cm2 . The precious states are so far provided by reversible switchable fluorescent proteins (rsFPs). The available rsFPs for RESOLFT were generated by few mutations in the sequence of well-known fluorescent proteins such as Dronpa, EGFP, YFP or Cherry. rsFPs can be light driven in different molecular states which inhibit or permit their ability to fluorescence if exposed to visible light. These states are associated with cis-trans or hydrated-dehydrated molecular configurations. For RESOLFT to work these states need to be reversibly populated or depopulated multiple times.

The improvements in spatial resolution is in fact achieved by saturating the OFF switching transition using a light pattern featuring one or more intensity minima or “zeros” (ring or line shaped). The role of this light beam is to transiently silence the emission of rsFPs by switching them to the long lived off-state. Once switched off, the rsFPs cannot be excited anymore and remain dark. Only fluorophores residing in the direct vicinity of the zero-intensity minimum of the RESOLFT light pattern focus can effectively remain into the fluorescent (ON) state and hence contribute to the fluorescence signal. Importantly, the kinetics of the switching to the OFF state determines the final resolution.

Current point-scanning RESOLFT microscopy equipped with a ring shaped light pattern a lateral resolution down to 30-40nm. The axial resolution can be improved as well down to 70-90nm by adding an additional light pattern.

This system is comparably cheap and easy to use since it requires only few microwatts of continuous waves lasers for sample illumination (switching and excitation). Multi-channel variants employing spectral and lifetime information were also successfully realized enabling colocalization studies at the nanoscale. This microscope was also upgraded for brain tissue imaging using glycerin objective lens to match the refractive index of the tissue and additional lenses to correct for spherical aberrations.

A point-scanning implementation of the RESOLFT concept intrinsically compromises the temporal resolution of the technique, especially in large fields of view where many pixels are recorded. This caveat can be overcome by implementing parallelized illumination modes to switch and excite the rsFPs. Periodic line patterns rotated at different angles, as routinely done in structured illumination microscopy, or superimposed at 90 degrees can be successfully implemented to speed up the acquisition of large fields of view in RESOLFT microscopy. In fact, pioneering studies demonstrated super resolved recordings of a living cell in a field of view of about 100µm × 100µm happening in few seconds.

STED, Stimulated Emission Depletion

In STED microscopy the diffraction barrier is bypassed by controlling the switching of molecules in a deterministic way, point by point with the superimposition of a light pattern featuring local zero of intensities. STED microscopy uses a dedicated ‘STED’ beam to modulate the fluorescence capability of fluorophores residing closer than the diffraction barrier in order to make them distinguishable. In a typical implementation, a focused laser beam is used to raise the fluorophores to their excited state – as in confocal microscopy. However, the excitation spot is superimposed with the ‘STED beam’, which is typically ring-shaped. The role of this beam is to transiently silence the emission of the fluorophore by the phenomenon of stimulated emission. Stimulated emission means that the fluorophores are instantly sent to the ground state before they are capable of emitting a fluorescent photon spontaneously. The silencing takes place because the wavelength and intensity of the STED beam are chosen so as to take away the majority of the excitation energy in a copy photon of the STED beam, which is discarded.

Because at least a single de-exciting photon must be available within the lifetime (τ ≈ 1–5 ns) of the fluorescent molecular state, the intensity of the focal STED beam must exceed the threshold Is = Cτ−1 with C accounting for the probability of a STED beam photon to interact with the fluorophore. Only fluorophores residing in the direct vicinity of the zero-intensity minimum of the STED focus can effectively assume the fluorescent state and hence contribute to the fluorescence signal. If Δ is the conventional resolution (~200-300nm), the diameter of this area is given by

with Im (typically twice greater than Is) denoting the intensity at the ring crest. Hence, features that are (just slightly) more apart than d < λ cannot fluoresce at the same time even when simultaneously illuminated by excitation light. By scanning the superimposed excitation and ring-shaped beam focal spots jointly across the specimen, features that are closer than the diffraction barrier assume the fluorescent state sequentially and are thus readily separated. STED is a direct and immediate imaging method because it immediately achieves high spatial resolution (down to 15-20nm) without any requirements of image reconstruction. It is this feature that made STED the method of choice for many applications in cells and even in living mice.


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