Correlative light electron microscopy using gold nanoparticles as single probes
). AuNPs form small clusters and are no longer resolved as individual particles in FWM. Therefore, in this case, we determined the centroid position of the cluster in 3D from the FWM z-stack , and compared its 2D in-plane coordinates with the position of the geometrical centre of the cluster in TEM for the correlation analysis. The resulting correlation accuracy for the six clusters shown in Fig.
is 36 nm. Another example correlating 10 clusters is provided in Fig. S10, for which an accuracy of 44 nm is found.FWM-CLEM using 10 nm-radius AuNPs bound to EGF internalised in HeLa cells whereby the coordinates of the particles in FWM are measured in 3D via a z-stack. A large FWM overview in 2D with corresponding reflection image measured simultaneously is shown on the left . A TEM overview of the same region is shown in the centre, as indicated by the green dashed frame.
Generally, we found a correlation accuracy limited by systematics, in the range of 60 nm or less over areas larger than 10 µm. Systematic errors included a bending of the pioloform layer supporting the resin section, which changes from FWM in water to TEM in vacuum. This is difficult to correct for by coordinate transformations, and likely to require non-trivial methods beyond the linear transformation used by us.
The detection of individual AuNPs with FWM lends itself to applications in single particle tracking inside living cells
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