ID-1-O-2747 Correlative Light and Electron Microscopy Without Fluorescent Probes

Correlative Light and Electron Microscopy (CLEM) combines the optical capability of imaging dynamic physiological specimens with the nanoscale three dimensional resolution of electron microscopy. Most methods for CLEM use a metal nanoparticle (mNP) bound to a fluorescent marker (Fig. 1), but the fluorescence is often quenched in the EM prep meaning that preparation artefacts can not be checked. Presented here is a novel Correlative Microscopy approach that integrates Four Wave Mixing (FWM) microscopy into a CLEM experiment only using the gold nanoparticle tag.

FWM is a multiphoton technique which exploits the nonlinear optical response of mNPs at their surface plasmon resonance (SPR) to image these mNPs as absorbers, rather than fluorescence emitters. This technique has high sensitivity, photo-stability and depth accuracy, and in addition is also background free (Fig. 2). Additional advantages are that the absorption is linear to the number of particles in the voxel, and shifts in the peak can be used to detect inter-particle distances at a macro-molecular precision. Here we demonstrate this concept by combining FWM with standard fluorescent-mNP CLEM and comment on any artefact from high pressure freezing and Lowicryl embedding.

HeLa cells were grown on 1.48mm diameter sapphire disks (with a carbon finder grids) glued into a homemade carrier suitable for high pressure freezing (Leica EMPACT2 + rapid transfer system). The cells were serum starved and then incubated (20min) in Epidermal Growth Factor-biotin bound to Alexa Fluor 488 streptavidin – 10nm colloidal gold. One cell of interest on each disk was then imaged using a confocal microscope system, high pressure frozen and Lowicryl embedded at low temperature (without additional metal staining). The embedded blocks had the sapphire disk carefully removed and were trimmed as if for EM. The 1st and 2nd 5µm sections containing the cells, placed in glycerol between a glass slide and coverslip, were imaged with FWM. After FWM imaging the sections were washed, remounted, thin sectioned and imaged with EM.

The preliminary results demonstrate the viability of FWM as a future standard CLEM technique and for testing aretfact in fluorescently labelled CLEM techniques in general.