Investigation of the synaptic ultrastructure with multicolor STORM

We are interested in the spatial distribution of proteins at the presynaptic terminal and how the molecular composition of the terminal regulates the synaptic vesicle cycle. Single-molecule localization microscopy allows us to observe these structures on the nanoscale. Due to the high frame numbers required to achieve good localization precision, multi-colour experiments can however be challenging. Here, we present two methods that exploit the spectral information of single emission event to record up to four standard organic fluorescent dyes simultaneously with a single excitation wavelength over a large field of view. Our approach tackles challenges such as image registration, chromatic aberrations and sample drift common to other multi-color approaches.

Optical setup

We use a 638 nm diode laser and a microlens-based Köhler integrator¹ for homogenous epi-illumination of the entire field of view. This ensures field-independent image resolution². Stochastic emission events are detected through an image splitter on a sCMOS camera. For 3D localization, a cylindrical lens introduces a depth-dependent astigmatism in one detection path. The optical setup can be implemented on any inverted epi-fluorescence microscope. Microscope control and localization fitting are performed in open-source software µManger³ and Fiji/ ThunderSTORM^4,5.

sdSTORM

CF647 and CF680 have been shown to form an optimal dye pair for spectral demixing (sdSTORM), where emission photons are split by a dichroic emission splitter of approximately 700 nm into a short and long wavelength channel. Colour is assigned to individual localizations based on non-linear filtering of the intensity histogram from both image channels.

Spectral STORM

Instead of a dichroic emission splitter, a grating or prism can split photon from single emission events into spectra. Custom spectral analysis assigns a false colour to each localization based on its spectroscopic signature. We intend to publish the spectral analysis as an open source package in the future. In contrast to previous spectroscopic super-resolution microscopy efforts^6,7 we can achieve nanometer resolution in 3D with a single objective and camera over a field of view of 130 x 60 µm².

References

1. Völkel, R. & Weible, K. J. Laser Beam Homogenizing: Limitations and Constraints. Proc. SPIE 7102 (2008).
2. Douglass, K. M. et al. Super-resolution imaging of multiple cells by optimized flat-field epi-illumination. Nat. Photonics 10 (2016).
3. Edelstein, A. D. et al. Advanced methods of microscope control using μManager software. J. Biol. Methods (2014).
4. Ovesný, M. et al. ThunderSTORM: a comprehensive ImageJ plug-in for PALM and STORM data analysis and super-resolution imaging. Bioinformatics 30 (2014).
5. Schindelin, J. et al. Fiji: an open-source platform for biological-image analysis. Nat. Methods (2012).
6. Dong, B. et al. Super-resolution spectroscopic microscopy via photon localization. Nat. Commun. 7 (2016).
7. Zhang, Z. et al.  Ultrahigh-throughput single-molecule spectroscopy and spectrally resolved super-resolution microscopy. Nat. Methods 12 (2015).