Boosting Super-Resolution by Mirror-Enhanced dSTORM
Maximizing Precision and Signal-to-Noise Ratio
- Fig. 1: meSTORM of the nuclear pore complex. (a) Scheme of a nuclear pore complex labeled with red fluorescent dyes, engulfed by the nuclear membrane and placed on a mirror coating designed to place emission (solid line) and excitation (dashed line) enhancement maximum at the height of the central ring. (b) Conventional (left) and meSTORM (right) images of NPC rings in the nuclear membrane (upper panel) and of single NPC rings (lower panel). (c) Resolution estimation by Fourier ring correlation (FRC) of the different image configurations: sunny-side-down (gray solid line), TIRF- (gray dashed line) and meSTORM (red line).
- Fig. 2: meSTORM provides sharp images and a 3D view. 3D information of cellular microtubule network labeled with red fluorescent dyes in vicinity of a tailored mirror coating based on the distance dependent localization uncertainty, overview image (left) and magnified image of one region of interest (right).
Mirror-enhanced stochastic optical reconstruction microscopy (meSTORM) maximizes precision and signal-to-noise ratio in single molecule localization microscopy (SMLM): A coating on the microscopy glass cover slip creates a “mirror effect” . The key for enhancement is a metal-dielectric nanocoating that acts as a tuned mirror for emitters in the vicinity.
Mirror, Mirror on the Wall: Which Image Is the Sharpest of them All?
SMLM methods open a unique window to visualize the cellular architecture at a molecular level pushing the lateral resolution to ~20 nm [2-5]. As SMLM approaches rely on the determination of the single fluorophores’ position, the final resolution gain simply depends on the localization precision which in turn is dependent on the number of fluorescence photons detected per localization event . Here, mirror-enhanced fluorescence — a concept that has been around for centuries and implemented for various approaches [7-10]— comes in to play as it pushes photon efficiency, and makes meSTORM a straight-forward SMLM approach to further boost resolution by easy-to-fabricate nanocoatings on standard glass coverslips. The method is not only spectrally and spatially tunable by the layer design and wavelength but also live cell compatible. It neither needs any tailored microscope nor software, and, in this respect, outperforms most other attempts improving localization precision [11-14].
Every Photon Counts
First, we show meSTORM for the nuclear pore complex (NPC), an aqueous channel which provides access to the nucleus and regulates the transport of proteins and RNA across the nuclear envelope . The NPC is well studied and several super-resolution techniques have demonstrated their capability to resolve the eightfold symmetry of the NPC [16,17]. The layer design (fig. 1a) ensures that the enhancement region matches the effective fluorophores’ region above the coverslip, which is expected at a distance of ~ 50 nm above the coverslip for classical immunolabeling of the pore anchoring protein gp210. To selectively enhance this region, a 2 nm germanium (Ge) layer, followed by a 50 nm silver (Ag) layer covered by 10 nm of silicon nitride (Si3N4) (fig.
1a) was nanofabricated on standard glass coverslips using a table-top thin film deposition system under standard laboratory conditions. With that the maximum axial extension of the enhancement window (~ 120 nm) is wavelength-dependent  and comparable to those reached by other evanescent techniques such as total internal reflection fluorescence (TIRF) microscopy, so that similar advantages and limitations occur for meSTORM.
Next, the nuclear envelopes are spread on the metal-dielectric substrate and on a bare glass coverslip for comparison. Note that for meSTORM the nanocoating with the specimen is directed towards the front lens of the water objective (NA 1.15) in a “sunny-side-down” (SSD) configuration, while the direct STORM (dSTORM) experiments on bare glass were performed in both SSD (NA 1.15) and TIRF (NA 1.46) configurations. The SSD dSTORM image on bare glass appears blurrier than the mirror-enhanced dSTORM image where the eight gp210 elements can be distinguished (fig. 1b). Fourier ring correlation (FRC) analysis  of the overview images (fig. 1b) reveals an overall resolution enhancement of 150% (fig. 1c). Importantly, the meSTORM resolution exceeds TIRF-based dSTORM by 25% without the requirement of a TIRFM setup.
2D and 3D meSTORM
The NPC sample exhibits a planar architecture placing the features of interest directly in the enhancement region. In contrast, a three-dimensional sample will partly exceed the enhancement region so that features located in and outside the enhancement maximum become distinguishable as the height-dependent profile can be translated into axial distances. For a three-dimensional microtubule network of Cos7 cells (fig. 2) the average localization uncertainty can be used to pinpoint the height of single filaments (fig. 2, filaments <130 nm distance in yellow), which is not possible in conventional dSTORM where the localization uncertainty is consistent within a wide axial range. Thus, meSTORM allows axial distinction of crossing microtubules (fig. 2, crossing points marked by white arrows).
In summary, meSTORM on easy-to-fabricate metal-dielectric nanocoatings suppresses background noise and improves the photon yield of the fluorophores in vicinity of the biocompatible coatings, meaning below 200 nm distance. Thus, the precision of dSTORM can be improved by a factor of two using a standard epifluorescence setup and without the need for specialized software other than typically used in dSTORM approaches (e.g. ThunderSTORM  or RapidSTORM ). As the emitted and the reflected light waves are superimposed the resulting interference effects further supports super-resolution imaging. Depending on the distance to the mirror, emitted light is amplified or attenuated so that structures in a certain image plane can be highlighted.
Keep it Simple: No Added Complexity to the Microscope, Software or Analysis Routine
The method is surprisingly easy to use. Except for the cheap metal-dielectric coated coverslip there is no need of any additional microscope hardware or software to boost the localization precision, which makes meSTORM a powerful add-on for advanced microscopy.
Katrin G. Heinze1 and Hannah S. Heil1
1 Molecular Microscopy Group, Rudolf Virchow Center, University of Würzburg, Germany
Prof. Dr. Katrin G. Heinze
Molecular Microscopy Group
Rudolf Virchow Center
University of Würzburg