Transport at Nanoscale Interfaces Laboratory
Ultrabright and stable luminescent labels for correlative cathodoluminescence electron microscopy (CCLEM) bioimaging
Keevend K, Puust L, Kurvits K, Gerken LRH, Starsich FHL, Li J-H, Matter MT, Spyrogianni A, Sotiriou GA, Stiefel M & Herrmann IK
Nano Letters, 19(9), 6013-6018.
https://doi.org/10.1021/acs.nanolett.9b01819
Abstract
The mechanistic understanding of structure-function relationships in biological systems heavily relies on imaging. While fluorescence microscopy allows the study of specific proteins following their labelling with fluorophores, electron microscopy enables holistic ultrastructural analysis based on differences in electron density. To identify specific proteins in electron microscopy, immunogold labelling has become the method of choice. However, the distinction of immunogold-based protein-labels from naturally occurring electron dense granules, and the identification of several different proteins in the same sample remains challenging. Correlative cathodoluminescence electron microscopy (CCLEM) bioimaging has recently been suggested to provide an attractive alternative based on labels emitting characteristic light. While luminescence excitation by an electron beam enables sub-diffraction imaging, structural damage to the sample by high energy electrons has been identified as potential obstacle. Here, we investigate the feasibility of various commonly used luminescent labels for CCLEM bioimaging. We demonstrate that organic fluorophores and semiconductor quantum dots suffer from a considerable loss of emission intensity, even when using moderate beam voltages (2 kV) and currents (0.4 nA). Rare-earth (RE) element doped nanocrystals, in particular Y2O3:Tb3+ and YVO4:Bi3+,Eu3+ nanoparticles with green and orange-red emission, respectively, feature remarkably high brightness and stability in the CCLEM bioimaging setting. We further illustrate how these nanocrystals can be readily differentiated from morphologically similar naturally occurring dense granules based on optical emission, making them attractive nanoparticle core materials for molecular labelling and (multi)color CCLEM.