Noah Weber

Correlative light and electron microscopy (CLEM) combines two powerful visualization techniques; Fluorescent microscopy (FM) and cryo-electron microscopy (CryoEM). Combination of these techniques enables the visualization of specific cellular events in complex heterogeneous samples. Precise correlation between images acquired by these modalities requires fiducial markers that are visible in both methods. Current external fiducial markers exist (e.g. fluorescent beads), however internal cellular imaging techniques such as focused ion beam (FIB) milling raise the need for internal fiducials. Here, we propose the use of vaultosomes as a biological fiducial marker for CLEM in these internal images. The vaultosome is a large (13 MDa) multi-subunit protein complex widely distributed in normal tissues with a highly characteristic barrel-like structure. Its moderate cytoplasmic density and unambiguous morphology make it a prime candidate as a correlative fiducial marker. Our cloning strategy involves tagging major vault protein (MVP) with an HUH endonuclease. MVP’s high copy number in the vaultosome (78 copies per 1 vault) provides high fluorescence with low background, and the HUH endonuclease provides a mechanism for fluorescent staining. This combined MVP-HUH construct will be cloned into a lentiviral vector for transfection into a stable cell line. Following its expression, addition of quenched fluorescent oligonucleotides will interact with the HUH endonuclease to provide fluorescent vaultosomes. These modified vaults thus become fluorescent fiducial markers that have sufficient electron density for CryoEM to be used for internal cellular CLEM imaging. Experiments utilizing this novel fiducial marker will be able to correlate images from FM and CyroEM in cellular micro-environments not seen before.