A unique series of reversibly switchable fluorescent proteins with beneficial properties for various applications

We studied the single-molecule photo-switching properties of Dronpa, a green photo-switchable fluorescent protein and a popular marker for photoactivated localization microscopy. We found the excitation light photoactivates as well as deactivates Dronpa single molecules, hindering temporal separation and limiting super resolution. To resolve this limitation, we have developed a slowswitching Dronpa variant, rsKame, featuring a V157L amino acid substitution proximal to the chromophore. The increased steric hindrance generated by the substitution reduced the excitation light-induced photoactivation from the dark to fluorescent state. To demonstrate applicability, we paired rsKame with PAmCherry1 in a two-color photoactivated localization microscopy imaging method to observe the inner and outer mitochondrial membrane structures and selectively labeled dynamin related protein 1 (Drp1), responsible for membrane scission during mitochondrial fission. We determined the diameter and length of Drp1 helical rings encircling mitochondria during fission and showed that, whereas their lengths along mitochondria were not significantly changed, their diameters decreased significantly. These results suggest support for the twistase model of Drp1 constriction, with potential loss of subunits at the helical ends.photo-physics | PALM | suborganelle structures P hotoactivated localization microscopy (PALM) allows for subdiffraction optical resolution via the stochastic temporal separation of individual photoactivatable or reversibly photoswitchable fluorescent proteins (PA-FPs or PS-FPs) and their subsequent localization in space. Successful PALM imaging requires two conditions: (i) temporally separated stochastic activation of a population of PA-FPs or PS-FPs, and (ii) accurate localization of each molecule in space (1, 2).For PALM imaging to be more biologically applicable, robust two-color PALM is necessary. Several published protocols for two-color PALM exist, one featuring EosFP and Dronpa, a GFPlike photo-switchable protein (3-5). Initially dark, upon photoactivation by 405-nm light, Dronpa becomes fluorescent with an excitation maximum at 503 nm and an emission maximum at 515 nm. When excited by 488 nm, activated Dronpa emits green fluorescence ("ON" state) until it is photo-induced back into the dark state ("OFF" state) and can be reactivated multiple times before photobleaching (Fig. 1A). In addition, Dronpa was observed to spontaneously recover from the OFF to the ON state in tens of seconds even under 488-nm illumination alone, which hypothetically has been ascribed to thermal activation (4, 6, 7). As a result, large overlapping populations of Dronpa molecules can be excited simultaneously, especially in densely labeled samples, hindering single-molecule identification and localization. rsFastLime, a Dronpa variant, has the amino acid mutation V157G (8). The valine, not directly part of the chromophore, is involved in the photoactivated isomerization of the chromophore. The V157G mutation is thought to reduce the steric ...

Section: Resultsmentioning

confidence: 99%

Optimized two-color super resolution imaging of Drp1 during mitochondrial fission with a slow-switching Dronpa variant

Rosenbloom

Lee

To³

et al. 2014

“…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.…”

mentioning

confidence: 86%

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

Wang¹,

Moffitt²,

Dempsey³

et al. 2014

332

382

Photoactivatable fluorescent proteins (PAFPs) have been widely used for superresolution imaging based on the switching and localization of single molecules. Several properties of PAFPs strongly influence the quality of the superresolution images. These properties include (i) the number of photons emitted per switching cycle, which affects the localization precision of individual molecules; (ii) the ratio of the on-and off-switching rate constants, which limits the achievable localization density; (iii) the dimerization tendency, which could cause undesired aggregation of target proteins; and (iv) the signaling efficiency, which determines the fraction of target-PAFP fusion proteins that is detectable in a cell. Here, we evaluated these properties for 12 commonly used PAFPs fused to both bacterial target proteins, H-NS, HU, and Tar, and mammalian target proteins, Zyxin and Vimentin. Notably, none of the existing PAFPs provided optimal performance in all four criteria, particularly in the signaling efficiency and dimerization tendency. The PAFPs with low dimerization tendencies exhibited low signaling efficiencies, whereas mMaple showed the highest signaling efficiency but also a high dimerization tendency. To address this limitation, we engineered two new PAFPs based on mMaple, which we termed mMaple2 and mMaple3. These proteins exhibited substantially reduced or undetectable dimerization tendencies compared with mMaple but maintained the high signaling efficiency of mMaple. In the meantime, these proteins provided photon numbers and on-off switching rate ratios that are comparable to the best achieved values among PAFPs.P hotoactivated localization microscopy, stochastic optical reconstruction microscopy, and related imaging methods take advantage of photoswitching and imaging of single molecules to circumvent the diffraction limit of spatial resolution in light microscopy (1-3). In these methods, only a subset of the fluorescent labels in the sample is switched on at any given time such that the positions of individual fluorophores can be localized from their images with high precision. Iteration of this process allows numerous fluorescent labels to be localized and an image with sub-diffraction-limit resolution to be reconstructed from the fluorophore localizations. Fluorescent proteins that can be activated from dark to fluorescent or converted from one color to another are widely used for such imaging approaches (4, 5). Although photoactivatable fluorescent proteins (PAFPs) are generally dimmer than photoswitchable dyes (6, 7) and hence give lower image resolution, the ease and high specificity of labeling protein targets in living cells with fluorescent proteins makes PAFPs highly appealing probes for imaging the dynamics of cellular structures (4,8).For single-molecule-based superresolution imaging methods, several properties of PAFPs are particularly important for the image quality. Here, we focus on four such key properties. (i) The first property is the photon budget, defined as the average number of ph...

“…The protocol used was described previously (16). Purified protein of Skylan-NS was first photoswitched to its off states using a 60-mW, 488-nm LED.…”

Section: Methodsmentioning

confidence: 99%

Highly photostable, reversibly photoswitchable fluorescent protein with high contrast ratio for live-cell superresolution microscopy

Zhang

Dong

et al. 2016

Self Cite

Two long-standing problems for superresolution (SR) fluorescence microscopy are high illumination intensity and long acquisition time, which significantly hamper its application for live-cell imaging. Reversibly photoswitchable fluorescent proteins (RSFPs) have made it possible to dramatically lower the illumination intensities in saturated depletion-based SR techniques, such as saturated depletion nonlinear structured illumination microscopy (NL-SIM) and reversible saturable optical fluorescence transition microscopy. The characteristics of RSFPs most critical for SR live-cell imaging include, first, the integrated fluorescence signal across each switching cycle, which depends upon the absorption cross-section, effective quantum yield, and characteristic switching time from the fluorescent "on" to "off" state; second, the fluorescence contrast ratio of on/off states; and third, the photostability under excitation and depletion. Up to now, the RSFPs of the Dronpa and rsEGFP (reversibly switchable EGFP) families have been exploited for SR imaging. However, their limited number of switching cycles, relatively low fluorescence signal, and poor contrast ratio under physiological conditions ultimately restrict their utility in time-lapse live-cell imaging and their ability to reach the desired resolution at a reasonable signal-to-noise ratio. Here, we present a truly monomeric RSFP, Skylan-NS, whose properties are optimized for the recently developed patterned activation NL-SIM, which enables low-intensity (∼100 W/cm 2 ) live-cell SR imaging at ∼60-nm resolution at subsecond acquisition times for tens of time points over broad field of view. Skylan-NSI n the last two decades, the power of fluorescence microscopy has been enhanced by the addition of superresolution (SR) imaging techniques (1-7). Although every SR technique has been successfully demonstrated to resolve ultrastructures beyond the diffraction limit, many of them encounter practical limitations when imaging nano-scale dynamics in living biological samples, especially over long times and large fields of view. For example, the thousands of raw frames typically acquired for a single-molecule localizationbased SR image greatly restrict the temporal resolution of the technique (1, 2, 8) and make it susceptible to blurring induced by cellular motion. On the other hand, structured illumination microscopy (SIM) is capable of live-cell time-lapse imaging for tens to hundreds of time points, at speeds as fast as 11 frames per second (9) and illumination intensities of only 1-100 W/cm 2 . However, its major shortcoming is that it improves resolution only twofold, to ∼100 nm. Stimulated emission depletion (STED) microscopy (4) and saturated SIM (SSIM) (7, 10, 11) are not subject to this constraint but rather provide diffraction unlimited resolution by exploiting a nonlinear dependence of the fluorescence emission rate upon an illumination intensity. Saturation of the excited electronic state S 1 to ground state S 0 (S 1 →S 0 ) via stimulated emission is used in STED,...