Interactive Tutorials - Spinning Disk Fundamentals

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Superresolution Microscopy

The RESOLFT Concept

The theoretical foundation necessary for achieving resolution beneath the diffraction barrier, which is actually composed of a family of physical concepts, was first advanced by Stefan Hell and associates with their introduction of the idea of reversible saturable (or switchable) optical fluorescence transitions (RESOLFT). This scheme focuses on fluorescent probes that can be reversibly photoswitched between a fluorescent "on" state and a dark "off" state (or between any two states A and B). The exact nature of the these states is variable and can be the ground and excited singlet states (S0 and S1) of a fluorophore as will be discussed below for STED microscopy, the excited singlet and dark triplet states utilized in ground state depletion (GSD) and ground state depletion-individual molecule return (GSDIM) microscopy, or the bright and dark states of a reversibly photoswitchable fluorophore (such as Cy5, kindling fluorescent protein, or Dronpa). In contrast, many of the optical highlighter fluorescent proteins, such as Eos, Kaede, Dendra2, and PA-GFP, which are capable of being permanently photoconverted from one emission bandwidth to another through covalent molecular alterations to the polypeptide backbone (and served as the foundation for the first PALM experiments), are not suitable probes for RESOLFT because the changes in fluorescent state are not reversible. The RESOLFT concept also includes switching isomerization states (such as cis-trans) and other optically bistable transitions in fluorophores.

For any given fluorophore or class of fluorescent probes, the number of switchable states is strictly limited so it is not surprising that many of the RESOLFT and single-molecule superresolution techniques described below rely on similar fluorophore switching mechanisms. In order to better understand these common properties, a close examination of the basic principles behind incoherently driven optical transitions is useful. When a molecule photoswitches from one state to the other, the probability that the molecule will remain in the first state decreases in an exponential fashion with increasing excitation light intensity. The term saturation intensity is used to define the light intensity at which the switching transition occurs (for example, when 50 percent of the molecules have transitioned from dark to bright) and is inversely proportional to the lifetimes of the two states. In situations where the excitation light intensity exceeds the saturation intensity, it becomes highly probable that one of the incoming photons will initiate the photoswitch. Fluorophores with long lifetimes in the initial and final switching states will afford more latitude in selecting excitation intensities and often exhibit higher fatigue levels (a measure of the ability to repeatedly photoswitch before being destroyed). The various fluorescent probes used for superresolution microscopy have lifetimes that differ significantly, as does the saturation intensity necessary to invoke photoswitching.

Among the differences between the RESOLFT concept (encompassing the related techniques of STED and GSD) and the single-molecule methodology employed by PALM and STORM are the switching mechanisms and the excitation light intensities necessary to photoswitch fluorescent probes. Extremely high intensities are necessary for switching off the excited singlet state with STED (using what is termed a depletion laser), but switching to a metastable triplet or similar dark state (GSD) requires a light intensity between a thousand and a million times lower. Likewise, even lower excitation intensity is capable of switching between the metastable dark and bright states of photoswitchable fluorescent proteins. Thus, the single-molecule localization techniques can accommodate less powerful light sources while providing much larger fields of view. In order to acquire coordinates of the photoswitched molecules, specific areas of the specimen are either targeted by defining scanning regions (STED, GSD, RESOLFT) or the individual fluorophores are allowed to stochastically turn on and off throughout the field of view (PALM and STORM). The different factors involved with these techniques dictate experimental parameters, such as imaging speed, instrument complexity, and the sensitivity of detection.

The targeted photoswitching of RESOLFT (as generalized for STED and GSD) is performed by spatially controlling the depletion laser light intensity distribution so only that a limited number of molecules can be switched on while a majority of the others remain in the off state. Thus, when a specific wavelength having a defined intensity (that is much higher than the saturation intensity) is selected to switch the fluorophore off, applying this light in a spatially modulated manner is used to restrict the fluorophores remaining in the on state to a sharply defined region. The resolution (full width at half maximum; FWHM) for the point-spread function using these techniques is therefore defined by the following equation:

where λ is the wavelength of excitation light and the combined term η • sin(α) is the objective numerical aperture, as described above for the classical Abbe formula (Equation (1)). The variable a is a parameter that takes into consideration the shape of the spatially modulated depletion laser beam, which is often manifested in the form of a line shape or a "doughnut" having a central zero node. Under the square root, Imax is the peak intensity of the depletion laser and Is is the saturation intensity for the fluorophore being imaged. In cases where Imax equals zero, Equation (1) reduces to the Abbe diffraction limit. Conversely, when Imax is much greater than the fluorophore saturation intensity (in effect, the value of the square root increases), the point-spread function becomes very narrow and superresolution is achieved. For example, when Imax/Is equals 100, the improvement in resolution is about tenfold. Thus, in agreement with theory, sub-diffraction resolution beneath the Abbe limit scales with the square root of the light intensity depleting the ground state. The resolution of all methods that rely on targeted readout of fluorescent probes, including RESOLFT, STED, GSD, and SSIM, is governed by Equation (1) regardless of the fluorophore switching mechanism or the spatial modulation geometrical parameters (doughnut or line) dictated by the microscope configuration.

RESOLFT techniques require scanning the specimen with a zero node in the depletion laser field, but not necessarily using a single beam or a zero region that is geometrically confined to a point. Multiple dark lines or zeros can also be employed in conjunction with a conventional area-array (CCD) digital camera detector, provided the zeros or the dark lines are spaced further apart than the minimal distance required by the diffraction resolution limit. Scanning only with dark lines increases the resolution in a single lateral direction, but repeated scanning after rotating the pattern followed by mathematical deconvolution can provide sub-diffraction resolution across the lateral axis. The basic requirement for scanning the specimen is the reason why RESOLFT transitions (A/B or on/off) must be reversible. Molecules in one state must be able to return to their other state when they are scanned by the zero node. Note that saturated depletion of molecules in the excited state using a zero node focal spot produces a superresolution point-spread function that is not limited by the wavelength but only by the intensity of the depletion laser.

Another attractive feature of the RESOLFT concept is that the simplest mechanism for realizing a saturated optical transition is to excite the fluorophore intensely in what can be considered an "inverse" application of the approach. In this case, the ground state (S0) is depleted and the fluorophore is expected to reside largely in the fluorescent state (S1). The same RESOLFT principles discussed above still apply, except that the reverse state is now fluorescent such that highly defined dark regions are created that are surrounded by brightly fluorescent regions. The result is a "negative" image of the specimen details, which can subsequently be inverted to generate the final image through post-acquisition mathematical processing. The dark regions can either be lines generated by interference patterns or three-dimensional doughnuts. In the case of doughnuts, the result would be dark three-dimensional volumes that are confined by walls of intense fluorescence. Saturated structured illumination (SSIM) and saturated pattern excitation (SPEM) microscopy utilize this approach with line-shaped geometries to achieve superresolution images. The primary limitation of the inverse RESOLFT technique are the mandatory computations and high signal-to-noise ratio that are required in order to obtain the final image.

The intense excitation necessary for many of the RESOLFT techniques is compromised by the fact that high laser intensities often produce excessive rates of photobleaching so that fluorophores must be carefully chosen. Therefore, although the family of RESOLFT methods is not subject to the traditional diffraction barrier, the dependence of laser intensity on resolution gain installs another barrier that is governed by how much laser power the fluorophore can tolerate. The best remedy for this dilemma is to remove the necessity for strong intensities by implementing molecular transitions that occur with low depletion laser powers. Many bistable fluorescent probes fill this criterion and are able to be optically switched between fluorescent and non-fluorescent states through low-energy mechanisms such as photo-induced cis-trans isomerization. In cases where both of the states are stable, the optical transition back and forth between the states can be completed at arbitrary or otherwise very long time scales, enabling the illumination to be spread out in time and reducing the required intensity (as well as photobleaching artifacts) by many orders of magnitude. Such a strategy has enabled parallelization of RESOLFT methods to allow their use in large area widefield imaging. Note that as described above, one of the principal ideas behind RESOLFT is that superresolution imaging does not necessarily require extremely high light intensities.

Contributing Authors

Stephen P. Price and Michael W. Davidson - National High Magnetic Field Laboratory, 1800 East Paul Dirac Dr., The Florida State University, Tallahassee, Florida, 32310.