New Ways to Super-Resolution: Reversible Chemical Reactions Control Fluorescent States
- Fig. 1: Diffraction barrier limits the resolution in light microscopy.(a) Two objects in a distance above resolution limit (dmin) are easily resolvable.(b) Superposition of two fluorescence signals at a distance of around the resolution limit already shows an overlap but the origin of the signal is still distinguishable.(c) Two fluorescence signals overlap completely below the resolution limit and blur the origin of the signals (c).
- Fig. 2: (a) To resolve objects below the resolution limit by localization microscopy based approaches it is necessary to switch off most fluorescent labels to localize their position with higher precision.(b) By saving their positions and repeating the procedure for other fluorophores it is possible to reconstruct an image with much higher resolution.
- Fig. 3: (a) STED microscopy is based on the activation of fluorophores in a confocal volume followed by a depopulation of excited fluorophores outside the central region by a donut shaped depletion beam. (b) In localization microscopy approaches (e.g. STORM and PALM) fluorophores are deactivated or bleached by a laser beam with high power and (re)activated by a laser pulse at different wavelength. (c) This principle can be generalized to random cycling of on/off states by arbitrary reversible reactions that might or might not be light driven.
- Fig. 4: (a) For CHIRON blinking of the probe is induced by binding of copper(II) ions to a bipyridine ligand in spatial proximity to the dye (tetramethylrhodamine) TMR.(b) The full probe consists of a double-stranded DNA which is biotinylated on the one end for labeling while the other end carries chelating ligand and dye.
- Fig. 5: (a) A micrograph can be reconstructed from movies recording the random blinking of the probes. (b) The improvement in resolution (~30 nm) in comparison to (c) the raw data reveals more details of the cellular structures.
Recently, light microscopy has been revolutionized by novel approaches that circumvent the diffraction barrier the resolution limit of optical microscopes. Most of these novel methods are based on light-controlled switching of the labels fluorescent states and therefore require use of additional or more intense laser excitation lines. To overcome these demands a new probe has been developed which is controlled by a reversible chemical reaction thereby reducing the demands on the microscopes.
Limitations of Light Microscopy
In 1873 Ernst Abbe postulated one of the fundamental limitations of microscopy . He suggested that it is impossible to resolve two objects within a distance of about half of the wavelength used. The so called diffraction barrier results in a resolution limit of roughly 200 nm for visible light (fig. 1). In the past decade different methods have been developed to bypass this limitation.
Most prominent among these methods are the Stimulated Emission Depletion (STED) microscopy  and the single-molecule based localization microscopy approaches (Stochastic Optical Reconstruction Microscopy - STORM, Photo-Activation and Localization Microscopy - PALM) and their variations [3,4,6]. Localization microscopy methods are based on the precise localization single fluorophores by use of mathematical fitting algorithms to an accuracy of about 1 nm. By driving most fluorophores of the observed sample into a non-fluorescent state it is possible to localize the central point of a few remaining fluorescent spots - and the position of the fluorophore respectively - with high accuracy . By repeating this procedure several times a high resolution image can be obtained (fig. 2).
Microscopy to Nanoscopy - Switching is Key
While all localization techniques share similar mathematical algorithms to localize single fluorophores their main difference is the method used to switch the fluorophores to a dark state.
In PALM a few photo-activatable proteins are statistically activated by use of more energetic light and afterwards those activated fluorescent proteins are imaged till they are photo-bleached with an imaging laser . In STORM all fluorophores are in a bright state before they are deactivated by a laser pulse with high power. Reactivation of the fluorophores is driven either by a blue shifted laser pulse with low power to activate only a small subpopulation or by use of reagents like mercaptoethylamine (MEA) [3,7].
Obviously, the use of high power lasers or of laser lines with different energy leads to the necessity of special microscopes. To generalize localization microscopy methods it is possible to say that the fundamental underlying principle is the cycling of probes between a fluorescent (on) and a non-fluorescent (off) state (fig. 3). Additionally the ratio of activated fluorophores to those in an off state strongly depends on the density of fluorophores in the observed sample and a value of 1:200 has been suggested as a good parameter for most biological samples . The ultimate probe would therefore be based on a random switching process which is light-independent but can be controlled by some other mechanism (fig. 3c).
Chemistry Provides Alternative Switching Mechanisms
Uncoupling of the switching process from light has many promising advantages. Due to the high laser power needed for photo-switching the introduction of alternative mechanisms lowers the radiation and therefore the photo toxicity for biological samples. Another advantage is the minimization of requirements for the experimental setup. To realize the idea of light-independent switching processes an ion sensor has been modified. The requirements of a probe suitable for localization microscopy are: (i) the on/off ratio has to be adjustable (ii) the probe has to show bright on and dark off states (iii) the on times of the probe have to be around 100 ms and (iv) the probe has to be labeled to some biological target. A probe which has been developed a few years ago to observe chemical reactions in thermodynamic equilibrium at single-molecule level fulfilled all these requirements .
The essential parts of the probe are on the one hand a fluorophore and on the other hand a ligand which is able to bind free ions. The chelating ligand used is a bipyridine which efficiently binds copper(II) ions. Due to the spatial proximity of the copper-bipyridine-complex fluorescence of the dye is quenched (fig.4a). By increasing the concentration of copper(II) ions in the measurement buffer a strong influence on the on-duration could be observed while the off-duration of the fluorophores remained unaltered. This can be explained by the fact that with increasing ion concentration the probability to bind an ion to the ligand increases while the energy of the bond is unaffected. Due to the nature of the probe, this novel technique was named chemically improved resolution for optical nanoscopy (CHIRON) . The probe design is based on a double stranded DNA containing a biotinylated end allowing attachment of the probe to streptavidin tagged targets (fig. 4b) to fulfill the requirement for biological applicability.
Application to Biological Targets
As the localization microscopy approaches are evaluated by their ability to resolve structures in cellular environments an interesting target that can easily be addressed is the cytoskeleton. To test CHIRON, tubulin was chosen as target. To do so a biotinylated antibody against tubulin was labeled with the probe and applied on fixed mouse fibroblasts. By addition of a low concentration of copper ions to the measuring buffer (12 µM) it was possible to induce blinking of the probe (fig. 5a) allowing reconstruction of a high-resolution image (fig. 5b). In contrast to a regular micrograph the nanoscopic image reveals much more information, achieving a resolution of ~ 30nm.
Versatility and Development
As the probe design implies, it holds great versatility. Either dye, ligand or ion can be exchanged providing a high degree of multiplexing and adaptability in various experimental conditions. While the color of excitation and detection can be changed by using different dyes it is important to determine the quenching efficiency to be sure that the probe still has an easily distinguishable dark state. Substitution of the chelating ligand will change the binding energy of copper(II)ions and therefore influence the duration of dark states. This additional parameter can be used to balance the on/off ratio of the probe with the samples labeling density.
The initial design can be further improved and optimized by reducing its complexity, while maintaining essential parts like the spatial proximity of ligand and dye. A more compact probe which can be attached to targets via biochemical standard methods instead of biotin/streptavidin will make the probe suitable for fluorescence microscopy in a more general manner and will also help to further improve the resolution. And of course, the presented probe marks only the beginning of a development where other reversible reactions might be combined with different quenching mechanisms to design fluorescent probes for future microscopy.
We acknowledge financial support from the DFG (EXC 81, SFB 623) and the BMBF (VIROQUANT).
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Dr. Konstantinos Lymperopoulos
PD Dr. Dirk-Peter Herten
Cellnetworks Cluster of Excellence and Physical Chemistry Department