Serial Block Face Sectioning & Light Sheet Fluorescence Expansion Microscopy: Complete Mapping of Extended Neuronal Circuits at Super Resolution

Juan Eduardo Rodriguez-Gatica1, Jens Schweihoff2, Irina Pavlova2, Daniel Poétes1, Jan Peter Siebrasse1, Martin Karl Schwarz2, Ulrich Kubitscheck1

1 Institute of Physical and Theoretical Chemistry, University of Bonn, Bonn
2 Institute Experimental Epileptology and Cognition Research (EECR), University of Bonn Medical School, Bonn

The combination of tissue expansion and Light Sheet Fluorescence Microscopy (LFSM) allows extended volumetric super resolution imaging of mouse brain samples at high speed (Gao et al., 2019). Recently, we demonstrated the capabilities of this method by performing three color imaging of mouse CA1 and dentate gyrus molecular-, granule cell- and polymorphic layers (Bürgers et al., 2019). This approach features high imaging rates, high contrast, low photobleaching, lateral sample extensions in the centimeter range and effective optical super resolution. A careful sample preparation allows preserving the fluorescence of autofluorescent proteins and thus avoids the use of antibodies, which do not penetrate thick samples well and produce considerable background noise. When imaging expanded brain slices, their thickness easily exceeds the working distance of high-resolution objective lenses. Sectioning the sample before imaging results in artifacts and distortions that prohibit reconstructing intact neuronal circuits.

Here we approach this problem by combining serial block face sectioning and LFSM of the expanded samples in the same instrument using a custom-developed microtome featuring a 51 µm thin stainless steel wire. Using the microtome we cut and eliminated physically the specimen slab already imaged and could therefore examine samples with a practically unlimited axial extension. Distortions introduced by the cutting process reached approximately 200 micrometers deep into the sample. We present super resolved images of expanded samples with a thickness greater than 4 mm expressing autofluorescent proteins expressing endogenous fluorescent proteins generated by Tetbow, a stochastic multicolor labeling that uses a tetracycline-operator system (Sakagushi et al., 2018).