Abstract
SummarySuper-resolution microscopy requires that subcellular structures are labeled with bright and photostable fluorophores, especially for live-cell imaging. Organic fluorophores may help here as they can yield more photons—by orders of magnitude—than fluorescent proteins. To achieve molecular specificity with organic fluorophores in live cells, self-labeling proteins are often used, with HaloTags and SNAP-tags being the most common. However, how these two different tagging systems compare with each other is unclear, especially for stimulated emission depletion (STED) microscopy, which is limited to a small repertoire of fluorophores in living cells. Herein, we compare the two labeling approaches in confocal and STED imaging using various proteins and two model systems. Strikingly, we find that the fluorescent signal can be up to 9-fold higher with HaloTags than with SNAP-tags when using far-red rhodamine derivatives. This result demonstrates that the labeling strategy matters and can greatly influence the duration of super-resolution imaging.
Highlights
Super-resolution fluorescence microscopy, called ‘‘nanoscopy,’’ enables the visualization of cellular structures beyond the diffraction limit of light (Fornasiero and Opazo, 2015; Hell, 2007; Huang et al, 2009; Toomre and Bewersdorf, 2010; van de Linde et al, 2012)
Like Green fluorescent protein (GFP), these self-labeling SNAP-tags and HaloTags can be expressed as fusion proteins (Hinner and Johnsson, 2010) and selectively reacted with the substrates benzylguanine (BG) and chloralkane (CA), respectively, which are tagged with organic fluorophores
Cells were labeled with Halo or SNAP ligands conjugated to the near far-red fluorophore silicon rhodamine (SiR): SiR-CA and SiR-BG for HaloTags and SNAP-tags, respectively (Lukinavicius et al, 2013) (Figure 1A)
Summary
Super-resolution microscopy requires that subcellular structures are labeled with bright and photostable fluorophores, especially for live-cell imaging. To achieve molecular specificity with organic fluorophores in live cells, self-labeling proteins are often used, with HaloTags and SNAPtags being the most common. How these two different tagging systems compare with each other is unclear, especially for stimulated emission depletion (STED) microscopy, which is limited to a small repertoire of fluorophores in living cells. We find that the fluorescent signal can be up to 9-fold higher with HaloTags than with SNAP-tags when using far-red rhodamine derivatives. This result demonstrates that the labeling strategy matters and can greatly influence the duration of super-resolution imaging
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