Triple‐Color Super‐Resolution Imaging of Live Cells: Resolving Submicroscopic Receptor Organization in the Plasma Membrane

Abstract

In living color: Efficient intracellular covalent labeling of proteins with a photoswitchable dye using the HaloTag for dSTORM super‐resolution imaging in live cells is described. The dynamics of cellular nanostructures at the plasma membrane were monitored with a time resolution of a few seconds. In combination with dual‐color FPALM imaging, submicroscopic receptor organization within the context of the membrane skeleton was resolved (see figure).

Restricted diffusion of OXPHOS complexes in dynamic mitochondria delays their exchange between cristae and engenders a transitory mosaic distribution

“…In dual color super-resolution experiments a combination of fluorescent paGFP and Halo/TMR was used as described recently (Appelhans et al, 2012;Wilmes et al, 2012). paGFP and TMR were excited in parallel allowing a recording rate of 30 Hz.…”

Identification of the factors affecting co-localization precision for quantitative multicolor localization microscopy

“…In 2011 another application of the mEos2-PSCFP2 pair (Yao et al 2011), as well as two works using mEos2-Dronpa combination were published (Hsu and Baumgart 2011) (Lehmann et al 2011). A successful multi-color strategy was also demonstrated using dSTORM suitable dyes (Baddeley et al 2011) or a combination of PALM/STORM approaches, as in the recent work by Hess and coworkers (Wilmes et al 2012), leading to three color live cell super-resolution imaging. Since the original development of PALM/STORM in 2006, a large number of photoswitchable fluorescent proteins or labeling strategies with organic dyes are now available .…”

Localization microscopy coming of age: from concepts to biological impact

“…However, most of these methods still use large protein tags comparable to the size of fluorescent proteins that can sterically interfere with protein function, such as the HaloTag with a molecular mass of 35 kDa (Los et al, 2008), SNAP/CLIP-tags with a molecular mass of 20 kDa (Gautier et al, 2008) and the DHFR/TMP tag with a molecular mass of 18 kDa (Gallagher et al, 2009) (Box 1). So far, different combinations of SNAP-, Halo-, TMP-, CLIP-tags and organic fluorophores have also been successfully used in combination with photoactivatable fluorescent proteins for multicolor live-cell localization microscopy (Appelhans et al, 2012;Benke et al, 2012;Klein et al, 2012;Wilmes et al, 2012). The FlAsH tag, consisting of only 12 amino acids, is much smaller than fluorescent proteins and has been used successfully for intracellular labeling.…”

“…As a logical consequence, photoactivated localization microscopy (PALM) (Betzig et al, 2006;Shroff et al, 2007;Shroff et al, 2008), fluorescence photoactivation localization microscopy (FPALM) Hess et al, 2007) and stochastic optical reconstruction microscopy (STORM) using special pairs of fluorophores (Rust et al, 2006;Bates et al, 2007) were introduced shortly afterwards. A short time later, the use of commercially available organic fluorescent probes, such as fluorophore-labeled antibodies or phalloidin probes, was demonstrated for super-resolution imaging by direct STORM (dSTORM), facilitating the acceptance and broad applicability of localization microscopy Heilemann et al, 2009;van de Linde et al, 2011a;Jones et al, 2011;Dempsey et al, 2011;Sillibourne et al, 2011;Williamson et al, 2011;Kaminski-Schierle et al, 2011;van de Linde et al, 2012;Zessin et al, 2012;Lampe et al, 2012;Wilmes et al, 2012;Rossy et al, 2013;Mattila et al, 2013). Furthermore, other localization microscopy methods using photoswitching of standard organic fluorophores under slightly different experimental conditions emerged under the names of GSDIM (ground-state depletion microscopy followed by individual molecule return) (Fölling et al, 2008) and blink microscopy .…”