Abstract
Research in cell biology demands advanced microscopy techniques such as confocal fluorescence microscopy (FM), super-resolution microscopy (SRM) and transmission electron microscopy (TEM). Correlative light and electron microscopy (CLEM) is an approach to combine data on the dynamics of proteins or protein complexes in living cells with the ultrastructural details in the low nanometre scale. To correlate both data sets, markers functional in FM, SRM and TEM are required. Genetically encoded markers such as fluorescent proteins or self-labelling enzyme tags allow observations in living cells. Various genetically encoded tags are available for FM and SRM, but only few tags are suitable for CLEM. Here, we describe the red fluorescent dye tetramethylrhodamine (TMR) as a multimodal marker for CLEM. TMR is used as fluorochrome coupled to ligands of genetically encoded self-labelling enzyme tags HaloTag, SNAP-tag and CLIP-tag in FM and SRM. We demonstrate that TMR can additionally photooxidize diaminobenzidine (DAB) to an osmiophilic polymer visible on TEM sections, thus being a marker suitable for FM, SRM and TEM. We evaluated various organelle markers with enzymatic tags in mammalian cells labelled with TMR-coupled ligands and demonstrate the use as efficient and versatile DAB photooxidizer for CLEM approaches.
Highlights
The red fluorescent dye TMR can be used as a fluorochrome bound to ligands of the widely applicable HaloTag, SNAP-tag and CLIP-tag and these systems are already well established as reliable and versatile tags for fluorescence microscopy (FM) and SRM20–23
In this study we introduced the TMR-conjugated ligands as DAB-based EM markers, extending the use of HaloTag, SNAP-tag and CLIP-tag as general-purpose markers suitable for correlative microscopy of all three microscopic techniques
CLEM markers based on DAB oxidation exist and, to our knowledge, only three of them were reported to work as general-purpose-marker
Summary
Most of the known genetically encoded live cell-CLEM markers based on DAB oxidation have limitations, and to our knowledge only three general-purpose-markers for correlative approaches of all microscopic techniques were described (Fig. 1), i) ReAsH15–17, ii) resorufin ligase[18] and iii) FLIPPER19 For those three markers some constraints were reported, such as nonspecific labelling and cellular toxicity for ReAsH15–17, efficiency problems for the resorufin ligase[18] and restricted functionality for FLIPPER19. We show that the highly specific TMR ligands of HaloTag, SNAP-tag or CLIP-tag can, in addition to the fluorescent properties, photooxidize DAB to an osmiophilic polymer visible on TEM sections, displaying all-in-one markers suitable for all three microscopic modalities (Fig. 1). We evaluated the TMR ligands for HaloTag, SNAP-tag and CLIP-tag fused to several organelle markers in mammalian cells and provide for the first time results for its use as DAB photooxidizer for EM
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
