Cloneable tag for FWM-CLEM

Combining information acquired by different (imaging) modalities from a single experiment can provide more insight into biological processes than from each single technique applied separately. Such Correlative Multimodal Imaging (CMI) workflows are one of the most powerful imaging approaches capitalising on the strengths of each technique. Correlative Light Electron Microscopy (CLEM) is the best-established CMI method where, generally, the (live) light microscopy (typically fluorescence) capabilities are coupled to the high-resolution ultrastructural reference space of Electron Microscopy (EM). One of the major issues in a CLEM experiment is the marker(s) used. In most cases, a marker is only detected in one modality and either the localisation of that marker in the other mode is inferred or a second type of marker is used. Both approaches come with its drawbacks.

We have recently developed a new CLEM workflow where we only use a single marker (gold nanoparticles) for both light and electron microscopy. To visualise the gold nanoparticles in light microscopy we apply a multiphoton method called Four Wave Mixing (FWM) imaging. This technique provides high spatial (<10nm) localisation precision in 3D, a correlation accuracy of less than 40nm with EM, and importantly detects each individual gold nanoparticle, owing to their strong FWM response.

With this approach, currently we have to add gold particles to cells from the outside, either via endocytosis or cell permeabilisation, significantly limiting the application spectrum of FWM-CLEM. In this project, we will overcome this issue by genetically tagging gold nanoparticles directly inside cells. We will express protein constructs, based on Metallothionein, that are able to scavenge gold ions and turn them into a particulate gold cluster. This will be achieved following robust and reliable approaches that have been developed recently, including in our lab.

We will then explore whether gold clusters formed this way, can be visualised by FWM microscopy and utilised in FWM-CLEM experiments, including inside living cells. To this end we will express the gold-binding constructs onto Sorting Nexin-1 (SNX-1) which has a well-established and discrete localisation on recycling endosomes.

Merging the advantages of FWM-CLEM, whereby the same individual gold particle probe is detected in both modalities, with the possibility to genetically tag the gold probe to endogenous proteins directly inside a cell, has the potential to revolutionise CLEM, tackling biological questions virtually impossible to address with current fluorescence-based technologies.