Biol. It can be excited using a 532 nm laser paired with a 575/25 nm bandpass filter, a configuration that can be . rhodamine b extinction coefficient in water 05 Jun. In addition, PA-SiR-Actin was tested for SMLM in fixed COS-7 cells revealing stress fibers and connecting thinner fibers (Supplementary Fig. Rev. Potassium ferrioxalate as a standard chemical actinometer. Take this solution in the cuvette for FCS . Nat. Scale bar, 100nm. provided the U-2 OS NUP96-Halo cell line. We demonstrate the utility of these fluorophores for live-cell SMLM of intracellular targets and single-particle tracking experiments. Rev. PubMed Central Single-molecule assay data were further processed by a costume written MatLab script provided by Dr. Christian Sieben (EPFL) based on the Crocker, Weeks, and Grier Algorithm70. All proteins were purified using affinity-tag Ni-NTA (Qiagen) leading to higher than 95% pure proteins (verified by sodium dodecyl sulfate polyacrylamide gel electrophoresis (PAGE) coomassie staining). Where given X2 the reduced chi-squared corresponds to the residual sum of square (RSS) and R2 is the squared correlation coefficient. Colorless needle shaped crystals were grown from slow evaporation of a CH2Cl2/MeOH solution at 4C. Wysocki, L. M. & Lavis, L. D. Advances in the chemistry of small molecule fluorescent probes. PA-SiR(1mg, 2.0mol) was dissolved in PBS/D2O (1mL, 90:10) and NaOH (1L, 5m) was added to achieve better solubility as PA-SiR was isolated as its TFA salt (pH=78, pH paper). Soc. . Crystal data and details of the structure determinations are compiled in Supplementary Table11. Sydor, A. M., Czymmek, K. J., Puchner, E. M. & Mennella, V. Super-resolution microscopy: from single molecules to supramolecular assemblies. 1e). 16). Richard Lincoln, Mariano L. Bossi, Stefan W. Hell, Jonas Buceviius, Rta Gerasimait, Gravydas Lukinaviius, Joyce Woodhouse, Gabriela Nass Kovacs, Martin Weik, Elias A. Halabi, Dorothea Pinotsi & Pablo Rivera-Fuentes, Francesca Pennacchietti, Ekaterina O. Serebrovskaya, Ilaria Testa, Nature Communications Measure the absorbance in the spectrophotometer. Moreover, both photoactivation of PA-SiR as well as the equilibrium between 2 and 3 are pH sensitive (Fig. Nat. Google Scholar. Efficient homogeneous illumination and optical sectioning for quantitative single-molecule localization microscopy. 45, 849854 (2012). Homologous recombination between the FRT sites in pcDNA5-FRT-TO-GOI and on the host cell chromosome, catalyzed by the Flp recombinase expressed from pOG44, produced the U-2 OS FlpIn TREx cells expressing stable and inducible the GOI. Nuclear pores as versatile reference standards for quantitative superresolution microscopy. Chem. ISSN 2041-1723 (online). This buffer was supplemented with 50mM KCl, 2mM MgCl2, 5mM guanidine carbonate and 1mM ATP to obtain F-actin. Am. Ed. It should be noted that other PA-SiR probes can be generated (Supplementary Figs. Cite this article. U-2 OS cells were seeded on 24mm glass coverslips and transiently transfected (TOMM20-Halo or -2-adrenergic-receptor-Halo). Article Article PA-SiR-Halo was measured with addition of BSA or HaloTag (20M). 12df)20. Chem. The rhodamine is incorporated into the animal's whiskers and teeth. The absorption values were collected using a spectral bandwidth of 1.0 nm, a signal averaging time of 0.133 sec, a data interval of 0.25 nm, and a scan rate of 112.5 nm/min. Briefly, they were fixed and extracted for 1min using a solution of 0.3% [w/v] glutaraldehyde and 0.25% [v/v] Triton X-100 in CB buffer (CB: 10mM MES, pH 6.1, 150mM NaCl, 5mM EGTA, 5mM glucose and 5mM MgCl2), and then postfixed for 10min in 2% [w/v] glutaraldehyde in CB. Chem. B. et al. 109, 1397813983 (2012). Phys. [4], Rhodamine B (BV10) is mixed with quinacridone magenta (PR122) to make the bright pink watercolor known as Opera Rose. 2fh). Rep. 5, 15348 (2015). The extent, to which the undesired side-product is formed, depends on the structure and environment of the fluorophore complicating applications of the diazoketone approach. Lavis, L. D. & Raines, R. T. Bright building blocks for chemical biology. Methods 6, 343345 (2009). 1a, c and Supplementary Figs. CrysAlisPro. Chem. 91, 42584272 (2006). PBS solutions of different pH were adjusted by addition of HCl or NaOH solution using a pH meter. Karstens, T. and K. Kobs (1980) Rhodamine B and Rhodamine 101 as reference substances for fluorescence quantum yield measurements. Sauer, M. & Heilemann, M. Single-molecule localization microscopy in eukaryotes. Rhodamine B - an overview | ScienceDirect Topics The microscope was equipped with a CO2 and temperature controllable incubator (Life Imaging Services, 37C). (For assignment of peaks see Supplementary Fig. Briefly, 1818mm high-precision coverslips (Carl Roth) were sonicated for 10min in MQ water, 10min in acetone, 10min in MeOH, 10min in KOH (1m, prepared from 99.98% purity Carl Roth) and rinsed with MQ water after each step. In addition, photoactivation of fluorophores caged with the diazoketone group proceeds through a carbene, which can react with intracellular nucleophiles (Supplementary Fig. Raymo, F. M. Photoactivatable synthetic dyes for fluorescence imaging at the nanoscale. Fixed-cell samples were mounted in PBS on cavity slides (VWR) sealed with twinsil 22 (Picodent) and imaged therein. Lateral drift was minimized by the suppressed motion (SuMo) stage of the Leica SR GSD and by keeping the temperature of the environment stable via an incubation box (T=210.1C, instrument parameter) covering the entire microscope. The Rhodamine-B dye purchased from the Ases chemicals (Jodhpur, India) has been used as dye photo-sensitizer material. Nat. 1a, d, f and Supplementary Fig. In order to determine these numbers, we immobilized HaloTag labeled with PA-SiR-Halo on coated glass coverslips and imaged the fluorophore using total-internal reflection (TIRF) microscopy (Supplementary Fig. Molar Extinction Coefficient:3 70,000 (495 nm) Storage Temperature -20 C Physical Properties Of Phalloidin, Tetramethyl-rhodamine B isothiocyanate (TRITC) (Catalog Number P1951): Molecular Formula: C 60 H 70 N 12 O 13 S 2 Molecular Weight: 1231.41 Excitation:3,5 540-545 nm Emission:3,5 570-573 nm Molar Extinction Coefficient:3 80,000 (545 nm) Biophysical J. Article Ed. Rhodamine dyes fluoresce and can thus be detected easily and inexpensively with fluorometers. Further information on research design is available in theNature Research Reporting Summary linked to this article. J. Luminescence 27, 455-462. The extinction coefficient for different concentration of Rhodamine B Rhodamine B is spectrally similar to MMP Red, Cy3 . Buffer A: 0.05% HCOOH in H2O Buffer B: 0.05% HCOOH in ACN. Using this photoactivatable fluorophore, we create probes for HaloTag and actin for live-cell single-molecule localization microscopy and single-particle tracking experiments. 14a, g)8,31. March 2004; Russian Journal of Applied Chemistry 77(3):414-422; . Optimization of the PA-SiR structure as well as HOMO/LUMO calculations were performed at the B3LYP/6-31G(d) level of theory by using the software package Gaussian 0942. Schnell, U., Dijk, F., Sjollema, K. A. Scale bar, 1m. The toxicity associated with the . The absorption values were collected using a spectral bandwidth of 1.0 nm, a signal averaging time of 0.133 sec, a data interval of 0.25 nm, and a scan rate of 112.5 nm/min. Du, H., R.-C. A. Fuh, J. Li, L. A. Corkan and J. S. Lindsey (1998) PhotochemCAD: A computer-aided design and research tool in photochemistry. Photoactivatable fluorophores are important for single-particle tracking and super-resolution microscopy. Rhodamine B is a xanthene dye, which functions as a water tracer fluorescent. Provided by the Springer Nature SharedIt content-sharing initiative. Persistence of Vision Pty. In these experiments, we used a 405nm laser for photoactivation, generally used to create a sparse subset of fluorescent molecules in SMLM. Proteins were used from glycerol stocks and were further diluted. A pET51b(+) vector (Novagen) was used for protein production in Escherichia coli. 67, 17531755 (1994). Annu. [nt 1] Chlorinated tap water decomposes rhodamine B. Rhodamine B solutions adsorb to plastics and should be kept in glass. (e) Molar extinction coefficient and (l) wavelength. Tinevez, J.-Y. This optical absorption measurement of Rhodamine B were made by R.-C. A. Fuh on 06-20-1995 using a Cary 3. Its extinction coefficient of 646=90,00018,000m1cm1 and fluorescence quantum yield =19.02.4% in aqueous buffer (mean95% confidence interval, N=3 and 4 samples, respectively) were only marginally smaller than those of the previously described SiR-carboyxl22 (Fig. Sample preparation was adapted from two literature procedures61,62. Representative measurements are displayed. 11e, f) were performed using a Solis405C High-Power LED (Thorlabs) coupled to a 3mm liquid light guide. All requests for the NUP96-Halo cell line should be directed to Jan Ellenberg. Database of Absorption and Fluorescence Spectra of >300 Common Methods 15, 755756 (2018). However, in the absence of structural information on HaloTag labeled with PA-SiR-Halo we cannot provide more detailed insights on the nature of these interactions. [15], In California, rhodamine B is suspected to be carcinogenic and thus products containing it must contain a warning on its label. 9, 10c, 12df, 13b, 14g, and 15ac and Supplementary Tables1, 2, 5, 7, and 9 are provided as a Source Data file. Scale bar, 10m. Further information can be found in Supplementary Table11. c Normalized absorption spectra of PA-SiR in PBS (10M) before and after UV irradiation as well as emission spectra after activation. Detailed procedures for the synthesis of all compounds and their characterization are given in theSupplementary Methods. PA-SiR-Halo might prove to be beneficial over PA-JF646-Halo in intracellular single-particle tracking experiments, where high signal-to-background ratios are required. Super-resolution microscopy was performed on a Leica SR GSD (Leica Microsystems) microscope equipped with an Andor iXon3 897 EMCCD camera (Andor) using a central 180 180 pixel or 400 400 pixel subregion of the camera chip. Data were corrected for air and detector absorption, Lorentz and polarization effects43 and scaled essentially by application of appropriate spherical harmonic functions43,45,46. Further studies are needed to clarify the mechanism of this remarkable reaction and to the best of our knowledge this type of light-induced protonation has not previously been reported for rhodamine derivatives or other xanthenes. The extinction coefficients used for TRITC/ConA conjugates under native and denaturing conditions were 6.52 x 104 M-~ cm-~ and 6.96 x 104 M-1 cm-1, respectively. Gaussian 16 Rev. PDF OH CH H CH N O O O CH C CH OH - Sigma-Aldrich PA-SiR was dissolved in MQ water (~20m). Sheldrick, G. Crystal structure refinement with SHELXL. 117, 74787509 (2017). The recently published ImageJ plugin HAWK71 was used to achieve imaging at high emitter densities to capture fast structural changes. Wldchen, S., Lehmann, J., Klein, T., van de Linde, S. & Sauer, M. Light-induced cell damage in live-cell super-resolution microscopy. Chem. However, with the many strategies available to tune the HOMOs and LUMOs of xanthene derivatives, we believe it should be feasible to generate photoactivatable rhodamine and carbopyronine derivatives, thereby expanding the scope of this reaction. Int. Google Scholar. Genome-edited U-2 OS cells with Halo-tagged NUP9632 were seeded on 24mm round coverslips (No. Saturation experiments under 405nm irradiation (Supplementary Fig. The summed stacks were analyzed as follows: the mean of a rectangular ROI within the nucleus was divided by the mean of a rectangular ROI adjacent to the nucleus. Fluorescence was collected through a high-numerical aperture (NA) oil-immersion objective (160/1.43 NA; Leica), filtered by a 700/100 bandpass filter (AHF) and focused onto an Evolve512D EMCCD camera (Photometrics). Peer review information Nature Communications thanks Zhiqian Guo and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. 179, 298310 (1996). Nat. Wysocki, L. M. et al. Nat. CAS Chem. [13] The fluorescence yield is temperature dependent;[14] the compound is fluxional in that its excitability is in thermal equilibrium at room temperature. Rhodamine 6G (R6G) is an organic laser dye and can be used to study the probes as it has a high quantum yield for fluorescence. Liquid chromatographymass spectrometry (LC-MS) was performed on a Shimadzu MS2020 connected to a Nexera UHPLC system equipped with a Supelco Titan C18 80 (1.9m, 2.150mm) column. Snare, M. J., F. E. Treloar, K. P. Ghiggino and P. J. Thistlethwaite (1982) The photophysics of rhodamine B. J. Photochem. Another pollutant that is extensively used for dyeing purpose is Rhodamine B (RhB). Chem. and J.R.), and the National Institutes of Health Common Fund 4D Nucleome Program (Grant U01 EB021223/U01 DA047728 to J.E. 6 and 10, Table5 and 7), assuming that during the activation the decay (k2 and k-2) is negligible, and the absorbance reached in equilibrium in the saturation experiment (Supplementary Fig. Use the absorbance (A) of the solution at = 555 nm. Prepare 2 mL solution of 1 M BSA in PBS buffer with no rhodamine B in it. Lukinaviius, G. et al. The data supporting the findings of this study are available within the paper and its Supplementary Information and are available from the corresponding author upon reasonable request. However, small-molecule fluorophores are generally brighter and more photostable than fluorescent proteins9, and therefore of advantage for SMLM experiments10,11. Normalized absorbance values Amax directly after activation and Aeq in equilibrium at different pH values are given, reflecting changes in activation (Amax) and equilibrium constant (Aeq). Rev. Chem. Exploratory insight into the stability of Rhodamine B and crude aqueous Nature Communications (Nat Commun) 1)21. In contrast to other photoactivatable fluorophores, no caging groups are required, nor are there any undesired side-products released. However, PA-SiR underwent protonation upon ultraviolet (UV) irradiation in aqueous solution, re-establishing the fluorescent xanthene core of SiR 2 (Fig. the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in 10 = Recommended molar ratio of NHS-Rhodamine to protein 528 = Molecular weight of NHS-Rhodamine 100 = Microliters of solvent in which the 1mg of NHS-Rhodamine is dissolved EXAMPLE: For 1mL of a 1mg/mL solution of IgG (150,000Da), 3.5L NHS-Rhodamine will be used. The next day the cells were stained with PA-SiR-Halo as described above and the coverslips were mounted into attofluor cell chambers (Life technologies) and the imaging medium was supplemented with HEPES (20mM). To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. Nuclear pores possess a regular circular shape with an internal diameter of about 100nm33,34. This apparent fluorogenicity of the probe should prove beneficial for live-cell imaging as unconjugated PA-SiR-Halo is not fluorescent, which increases the signal-to-background ratio. (extinction coefficient) 104500-115800 at 542-554 nm in methanol. Bioinformatics 30, 23892390 (2014). PA-SiR-Halo, PA-SiR-SNAP and PA-SiR-Actin probes (10M) were directly added to the target protein (20M SNAP-tag, 20M HaloTag or 0.4mgmL1 G-actin), or to a bovine serum albumin (Sigma) solution in PBS. Sect. Slider with three articles shown per slide. 14c). They were treated with freshly prepared 0.1% sodium borohydride for 7min. EMBO J. All other photochemical terms are used in accord with standard IUPAC recommendations (34,35). The photophysical properties such as the number of detected photons per frame and fluorophore are decisive for SMLM as the attainable localization precision scales with the inverse square root of the number of detected photons30. (OriginLab Corporation, Northampton, MA, USA). SCALE3 ABSPACK, C. CrysAlisPro, Agilent Technologies UK Ltd., Oxford, UK (20112014) and Rigaku Oxford Diffraction, Rigaku Polska Sp.z o.o., Wrocaw, Poland (20152019). 1H NMR spectra were measured on a Bruker AV 600 spectrometer at 600MHz and 298K. Chemical shifts are reported in ppm downfield from tetramethylsilane using the DMSO signal (H=2.50ppm) instead of the residual deuterated solvent signal as an internal reference. Article application(s) diagnostic assay manufacturing hematology . These cell lines were regularly tested for mycoplasma contamination. Rhodamine B is used in biology as a staining fluorescent dye, sometimes in combination with auramine O, as the auramine-rhodamine stain to demonstrate acid-fast organisms, notably Mycobacterium. [17], Rhodamine 610, C.I. PubMed 45170, InChI=1S/C28H30N2O3.ClH/c1-5-29(6-2)19-13-15-23-25(17-19)33-26-18-20(30(7-3)8-4)14-16-24(26)27(23)21-11-9-10-12-22(21)28(31)32;/h9-18H,5-8H2,1-4H3;1H, CCN(CC)C1=CC2=C(C=C1)C(=C3C=CC(=[N+](CC)CC)C=C3O2)C4=CC=CC=C4C(=O)O. (extinction coefficient) 104500-115800 at 542-554 nm in methanol. J. Nat. Soc. The Flp-In T-REx System (ThermoFisher Scientific) was used to generate stable cell lines exhibiting tetracycline-inducible expression of the gene of interest (GOI). It is sometimes also known as Basic Red 1. Sci. Structure of Rhodamine-B. Trends Cell Biol. 10b and 12c)27,28. The highly dynamic mitochondria form thin tubules between neighboring mitochondria (blue arrowheads) and disconnect (fission) in other areas (yellow arrowheads). Molar extinction coefficient of R6G in water at room temperature. A near-infrared fluorophore for live-cell super-resolution microscopy of cellular proteins. Specifically, we attached PA-SiR to the F-actin-binding natural product jasplakinolide, yielding PA-SiR-actin, and used it successfully for live-cell imaging of actin filaments (Supplementary Figs. Both buffers are components of the actin polymerization fluorescence assay kit (Cytoskeleton). Photobiol. 2004, 30463056 (2004). The output of a commercial LightHub laser box (Omicron-Laserage Laserprodukte) with 405, 488, 561, and 640nm laser lines and an additional 640nm booster laser (Toptica) were focused on a speckle reducer (LSR-3005-17S-VIS; Optotune) and coupled into a multi-mode fiber (M105L02S-A; Thorlabs). Pigment Violet 1, Basic Violet 10, C.I. 9) can be very efficiently activated and its fluorescent form 2 is stable over hours at physiological pH, whereas PA-SiR-Halo not conjugated to HaloTag is inefficiently activated and the activated probe decays quickly (Fig. In addition, PA-SiR-Halo conjugated to HaloTag and photoactivated showed much greater stability toward other nucleophiles such as cysteamine than free PA-SiR (Fig. Moreover, the data is consistent with previously reported data (Fig. Images were reconstructed by plotting all localized emitters at the fitted positions as Gaussians with a width proportional to their localization precision. Express 24, 2808028090 (2016). Dim localizations (localization precision>30nm) and out-of-focus localizations (fitted size of the Gaussian>160nm) were filtered out. Measurements were performed in triplicates expect for the saturation experiments with 405nm irradiation these were performed in duplicates. 22 (IUCr and Oxford University Press, Oxford, UK, 1988). The cells were incubated for 2448h before imaging. For stability measurement images were taken in the Cy5 (500ms, ex: 10%), transmission (100ms) and the GFP channel (100ms, ex: 5%) every 30s. Activation was performed for 1s once. 1.5H; 117640; Marienfeld). Furthermore, we imaged a HaloTag fusion of Nup9632, a protein of the nuclear pore complex. Acta Crystallogr. solved the crystal structure. Methods 9, 582584 (2012). This optical absorption measurement of Rhodamine B were made by Use the extinction coefficient () of rhodamine B . Bull. Super-resolution microscopy and single-particle tracking experiments. Selective chemical imaging of static actin in live cells. The coverslips were cleaned with piranha solution (1:3, H2O2/H2SO4) twice for 30min. For visibility, only tracks that have an overall displacement larger than 0.28m are shown (30ms exposure time, 0.3kWcm2 at 642nm excitation). Biol. Biol. A Math. using a Spex FluoroMax. Experimental database of optical properties of organic compounds - Nature Phosphate-buffered saline (PBS) (6.7mM, Lonza) was used in all experiments. Chem. 30 10 !4 , and (3) 70 10 !4. Lukinaviius, G. et al. Structural modifications on PA-SiR can influence this equilibrium as demonstrated by several synthetized analogs (Supplementary Fig. Science 313, 16421645 (2006). ACS Chem. Gibson, D. G. et al. [9] Its luminescence quantum yield is 0.65 in basic ethanol,[10] 0.49 in ethanol,[11] 1.0,[12] and 0.68 in 94% ethanol. 10). If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate. Photoactivatable (or caged) small-molecule fluorophores are known throughout many of the different fluorophore families and are mainly synthesized using photolabile protecting groups11,12,13,14. international journal with low publication fee > cardiff university resit policy > rhodamine b extinction coefficient in water. Grimm, J. Briefly, pcDNA5-FRT-TO-GOI and pOG44 were co-transfected into the host cell line U-2 OS FlpIn TREx59. HaloTag protein (4M) was labeled using PA-SiR-Halo (0, 1, 2, 3, 4, or 6M) in activity buffer (50mM HEPES, 50mM NaCl, pH 7.3) for 2h at room temperature. ized water and the . Background: Herein we report the multigram-scale synthesis, characterization and application of a rhodamine B-based fluorophore (ROSA) suitable for fluorescent studies in biological applications. 0.00007 mmol NHS-Rhodamine mmol IgG 10 mmol NHS-Rhodamine 150,000 mg IgG Data were acquired until no more activated fluorophores were observed. Curr. Prepare a stock solution of rhodamine B in PBS buffer. The microscope was equipped with a CO2 and temperature controllable incubator (PeCon, 37C). mEos3.2-Halo was purified analogously but using an additional Strep-Tactin (IBA)column purification step to reach higher purity and following the suppliers instructions. Chem. It is often used as a tracer dye within water to determine the rate and direction of flow and transport. PLOS ONE 3, e3944 (2008). By submitting a comment you agree to abide by our Terms and Community Guidelines. J. Appl. 7). A fiber coupled LED (Omicron, 340nm, 3mm liquid light guide) was used to perform UV irradiation unless otherwise stated. Acta Crystallogr. PubMed Central Chem. Cysteamine concentrations were adjusted by the addition of concentrated cysteamine solution (1M). Methods 115, 8090 (2017). A general design of caging-group-free photoactivatable fluorophores for live-cell nanoscopy, Switchable stimulated Raman scattering microscopy with photochromic vibrational probes, A general highly efficient synthesis of biocompatible rhodamine dyes and probes for live-cell multicolor nanoscopy, A synergistic strategy to develop photostable and bright dyes with long Stokes shift for nanoscopy, Photoswitching mechanism of a fluorescent protein revealed by time-resolved crystallography and transient absorption spectroscopy, Photoregulated fluxional fluorophores for live-cell super-resolution microscopy with no apparent photobleaching, Super-resolution imaging of non-fluorescent molecules by photothermal relaxation localization microscopy, Surface-dependent quenching of Qdot emission can be a new tool for high resolution measurements, Fast reversibly photoswitching red fluorescent proteins for live-cell RESOLFT nanoscopy, https://doi.org/10.1038/s41592-019-0574-9, Description of Additional Supplementary Files, http://creativecommons.org/licenses/by/4.0/, Engineered HaloTag variants for fluorescence lifetime multiplexing. Hess, S. T., Girirajan, T. P. K. & Mason, M. D. Ultra-high resolution imaging by fluorescence photoactivation localization microscopy. 61, 345367 (2010). Source data are provided as a Source Data file. Article 43, 825836 (2010). PA-SiR-Halo and the influence of HaloTag on its equilibrium system. These measurements were scaled to make the molar . Spectrum [Rhodamine 6G] | AAT Bioquest Get the most important science stories of the day, free in your inbox. Google Scholar. J. Org. J. Luminesc. The fluorescence emission spectrum of Rhodamine B The equilibrium between 2 and 3, as measured by recording the absorbance at equilibrium and correcting for Amax at 646nm (Aeq), was shifted toward 3 at higher pH values (Fig. These measurements were scaled to make the molar extinction coefficient match Enzymatic assembly of DNA molecules up to several hundred kilobases. The generated fluorescent product had an extinction coefficient of 646=180,00030,000m1cm1 and a fluorescence quantum yield of =29.21.2% in aqueous buffer (mean95% confidence interval, N=3 samples) making it an outstanding fluorophore. Advances in super-resolution microscopy (SRM) have led to insights into cellular structures and processes over the past decade1,2. Matsui, M., Tsuge, M., Shibata, K. & Muramatsu, H. Photochromism of 1,1-diaryl-1-alkanols. and K.J. This work was supported by the Max Planck Society, the cole Polytechnique Fdrale de Lausanne, a grant from the Swiss Commission for Technology and Innovation (CTI), the NCCR Chemical Biology, and the European Molecular Biology Laboratory (to P.H., M.L., B.N., M.K., J.E. PDF Determining the extent of labeling for tetramethylrhodamine protein