Photoactivatable mCherry for high-resolution two-color fluorescence microscopy

Abstract

The reliance of modern microscopy techniques on photoactivatable fluorescent proteins prompted development of mCherry variants that are initially dark but become red fluorescent after violetlight irradiation. Using ensemble and single-molecule characteristics as selection criteria, we developed PAmCherry1 with excitation/emission maxima at 564/595 nm. Compared to other monomeric red photoactivatable proteins, it has faster maturation, better pH stability, faster photoactivation, higher photoactivation contrast and better photostability. Lack of green fluorescence and single-molecule behavior make monomeric PAmCherry1 a preferred tag for twocolor diffraction-limited photoactivation imaging and for super-resolution techniques such as oneand two-color photoactivated localization microscopy (PALM). We performed PALM imaging using PAmCherry1-tagged transferrin receptor expressed alone or with photoactivatable GFPtagged clathrin light chain. Pair correlation and cluster analyses of the resulting PALM images identified ≤200 nm clusters of transferrin receptor and clathrin light chain at ≤25 nm resolution and confirmed the utility of PAmCherry1 as an intracellular probe.Genetically encoded `photoactivatable' fluorescent proteins (PAFPs) make up a small category of fluorescent proteins 1 , but are beginning to find uses far and above those of normal' fluorescent proteins 2 . With initially little or no fluorescence within their associated spectral detection window, photoactivatable proteins can be switched on by irradiation with violet light. Thus they are useful for spatially pulse-labeling subpopulations of molecules in cells in complement to photobleaching applications and can provide other useful features such as a high contrast over background in the photoactivated region and circumvention of fluorescence contributions from newly synthesized, nonactivated PAFPs. PAFPs and photoswitchable dyes also provide probes necessary for high-resolution optical techniques, such as photoactivated localization microscopy (PALM) 3 , fluorescence photoactivated localization microscopy (FPALM) 4 , stochastic reconstruction microscopy (STORM) 5 , PALM with independent running acquisition (PALMIRA) 6 RESULTS Development of photoactivatable mCherry variantsWe analyzed data on color transitions of red fluorescent proteins to the respective nonfluorescent chromoproteins that had been generated by mutagenesis 1 and identified the corresponding crucial amino acid positions on the basis of the mCherry structure 18 . Positions spatially close to the chromophore, such as 148, 165, 167 and 203 (numbering is in accordance with GFP alignment; Supplementary Fig. 1 online), appear to be major molecular determinants of color 10,19 . We hypothesized that mutagenesis of mCherry at these positions might convert it to a photoactivatable red probe and performed saturating mutagenesis at these positions using the overlap extension approach.We screened the resulting bacterial library of the site-specific mCherry mutants by fluorescence-activated ...

Characterization and development of photoactivatable fluorescent proteins for single-molecule–based superresolution imaging

“…Here, we compare four properties of PAFPs that are critical for superresolution imaging and report two new PAFPs that exhibit excellent performance in all four properties. (14), mEos3.2 (15), tdEos (16), mKikGR (17), PAmCherry (18), PAtagRFP (19), mMaple (20), PSCFP2 (13,21), Dronpa (22), and mGeosM (23). From this screen, we found that none of these PAFPs was simultaneously optimal in all four criteria described above.…”

A reversibly photoswitchable GFP-like protein with fluorescence excitation decoupled from switching

“…Still, photoswitchable proteins have not displayed their full potential, because proteins that are just photoactivatable [11][12][13] can be switched only once, which implies that repeated measurements with the same molecule are impossible. On the other hand, photochromic or reversibly switchable fluorescent proteins (RSFPs) can be repeatedly photoswitched between a fluorescent and a nonfluorescent state by irradiation with light of two different wavelengths.…”

Single-molecule evaluation of fluorescent protein photoactivation efficiency using an in vivo nanotemplate

“…Alexa 647, Dronpa, Dreinklang and rsEGFP) 3,[18][19][20] and (ii) irreversibly photoactivatable and photoconvertible (e.g. PA-GFP, PA-mCherry, mEos2, mEos3.1, mEos3.2, Dendra2, mClavGR2 and mMaple) [21][22][23][24][25][26][27] . These different categories provide complementary advantages.…”