(282d) Novel Multicolour Immuno-Labels for Correlative Cathodoluminescence Electron Microscopy

Modern biological imaging relies strongly on fluorescence and electron microscopy. Fluorescence microscopy reveals cellular functions, while electron microscopy enables holistic understanding of the ultrastructure. However, correlation between these methods is complicated because of the resolution mismatch and different sample preparation requirements.¹ Identification of specific entity in electron microscope strongly relies on the electron dense labels. Immunogold, i.e. the use of small gold nanoparticles conjugated to antibodies, is an established method for protein localization in electron micorscopy², but it poses challenges. For example, immunogold labelling does not facilitate co-localization studies (i.e., simultaneous observation of two epitopes)².

Interestingly, a broad range of materials emit light when exposed to accelerated electrons, creating an effect called cathodoluminescence (CL). Cathodoluminescence enables multicolour sub-diffraction imaging, since labels are discriminated based on their characteristic emission spectra. Here, we showcase rare earth element doped nanocrystals as novel CL labels. We demonstrate the synthesis of monodisperse nanoparticles and further surface functionalization prior to the immunolabeling. We report the use of these nanoparticles as immuno-labels and showcase their ability to identify several epitopes in biosamples with nanometre resolution. Additionally, we present their usability also in resin-embedded samples, as their emission is not negatively affected by heavy metal staining or resin embedding³ unlike fluorescent labels⁴.

1. Hell, S. W.; Sahl, S. J.; Bates, M.; Zhuang, X.; Heintzmann, R.; Booth, M. J.; Bewersdorf, J.; Shtengel, G.; Hess, H.; Tinnefeld, P.; Honigmann, A.; Jakobs, S.; Testa, I.; Cognet, L.; Lounis, B.; Ewers, H.; Davis, S. J.; Eggeling, C.; Klenerman, D.; Willig, K. I.; Vicidomini, G.; Castello, M.; Diaspro, A.; Cordes, T. Journal of Physics D: Applied Physics 2015, 48, (44), 443001.
2. Melo, R. C. N.; Morgan, E.; Monahan-Earley, R.; Dvorak, A. M.; Weller, P. F. Nat. Protocols 2014, 9, (10), 2382-2394.
3. Keevend, K.; Stiefel, M.; Neuer, A. L.; Matter, M. T.; Neels, A.; Bertazzo, S.; Herrmann, I. K. Nanoscale 2017, 9, (13), 4383-4387.
4. Paez-Segala, M. G.; Sun, M. G.; Shtengel, G.; Viswanathan, S.; Baird, M. A.; Macklin, J. J.; Patel, R.; Allen, J. R.; Howe, E. S.; Piszczek, G.; Hess, H. F.; Davidson, M. W.; Wang, Y.; Looger, L. L. Nature Methods 2015, 12, 215.