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). 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. Biol. Soc. 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. 1e and Supplementary Fig. The standard Leica filter sets for SR GSD systems were usedin brief: Leica set 488 for 405 and 488nm excitation: DBP 405/10 488/10 excitation filter, LP 505 dichroic mirror and 555/100 suppression/emission filter; Leica set 532 for 405 and 532nm excitation: DBP 405/10 532/10 excitation filter, LP 550 dichroic mirror and 600/100 suppression/emission filter; Leica set 642 for 405 and 642nm excitation: DBP 405/10 642/10 excitation filter, LP 650 dichroic mirror and 710/100 suppression/emission filter. 61, 345367 (2010). The different components of the microscope are managed by a field-programmable gate array (Mojo; Embedded Micro) which is controlled using a custom-written plugin for Manager64. Chem. PA-SiRs were diluted in PBS (10M unless otherwise stated). the value of 106,000cm-1/M at 542.8nm (Eastman, 1993). Lukinaviius, G. et al. Methods 11, 731733 (2014). 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. The specific parameters can be found in Supplementary Table10. Continuous lines are drawn representing the movement of individual receptors. They are color coded in order to distinguish the individual tracks. Hell, S. W. Nanoscopy with focused light (nobel lecture). 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. Imaging intracellular fluorescent proteins at nanometer resolution. Rhodamine B | C28H31ClN2O3 | CID 6694 - structure, chemical names, physical and chemical properties, classification, patents, literature, biological activities . CAS Methods 6, 343345 (2009). Publishers note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Biol. a Reaction scheme for photoactivation of PA-SiR (1), and equilibrium between 2 and 3. b Chemical structure of compound 4 together with its Oak Ridge Thermal Ellipsoid Plot (ORTEP), arbitrary numbering. EMBO J. Annu. Lavis, L. D. & Raines, R. T. Bright building blocks for chemical biology. We found that the photon numbers per particle per frame for PA-SiR-Halo at a power density of 1.2kWcm2 suitable for live-cell single-particle tracking were roughly 30% higher than for PA-JF646-Halo and considerably higher than those measured for mEos3.2 (Supplementary Fig. 16). CrysAlisPro. In total, 100L of a 0.2mgmL1 solution of streptavidin (Life Technologies) in PBS was applied to the flow chamber and incubated for 10min. Banala, S., Maurel, D., Manley, S. & Johnsson, K. A caged, localizable rhodamine derivative for superresolution microscopy. 41, 3042-3045. All nonhydrogen atoms were given anisotropic displacement parameters. Lond. The Rhodamine-B dye purchased from the Ases chemicals (Jodhpur, India) has been used as dye photo-sensitizer material. d Absorption spectra of PA-SiR in PBS (10M) before activation and directly after UV irradiation measured every 1min, revealing the reaction from 2 to 3. e pH dependence of the equilibrium system of PA-SiR in PBS (10M) at different pH after brief photoactivation through UV irradiation. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate. & Medalia, O. The output of this fiber is magnified by an achromatic lens, cleaned up by a quadband filter (390/482/563/640 HC Quad; AHF) and focused into the sample. Using 2AR fused to HaloTag and labeled with PA-SiR-Halo, we were able to track 2AR for several hundreds of milliseconds before photobleaching (Fig. 50, 1120611209 (2011). PA-SiR-Halo possesses a number of properties that make it an attractive candidate for live-cell imaging such as the absence of side-products during photoconversion, the absence of caging groups that affect solubility and permeability, the efficiency of photoactivation and stability of the HaloTag-bound probe compared to unconjugated probe, and its outstanding spectroscopic properties. Mol. Chem. 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. Scale bar, 100nm. Nat Commun 10, 4580 (2019). Int. 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. Atomic displacement parameters are drawn at 50% probability level. Science 313, 16421645 (2006). Lpez Arbeloa, F., P. Ruiz Ojeda and I. Lpez Arbeloa (1989) Fluorescence self-quenching of the molecular forms of rhodamine B in aqueous and ethanolic solutions. The mixture was incubated for 1h (HaloTag) or 2h (SNAP-tag) at room temperature. Scale bar, 1m. Briefly, pcDNA5-FRT-TO-GOI and pOG44 were co-transfected into the host cell line U-2 OS FlpIn TREx59. Peer reviewer reports are available. Rev. Rhodamine dyes are also used extensively in biotechnology applications such as fluorescence microscopy, flow cytometry, fluorescence correlation spectroscopy and ELISA. ISSN 2041-1723 (online). Methods https://doi.org/10.1038/s41592-019-0574-9 (2019) in press. Shim, S.-H. et al. 117, 74787509 (2017). Bonds C6C7 and C7C8 are considerably longer. Rhodamine Green dye offers a combination of desirable properties, including good photostability, a high extinction coefficient (>75,000 cm -1 M -1 ) and a high fluorescence quantum yield . A solution of SNAP-tag:EGFP:HaloTag (5M), fluorophore (2.5M), biotin-ligand (5M; SNAP-Biotin (NEB), HaloTag Biotin (Promega)), in PBS was prepared and incubated for 1h. In total, 100L of a 1:10001:500 dilution thereof was applied to the flow chamber and incubated for 10min. Blessing, R. An empirical correction for absorption anisotropy. d Super-resolved image from the boxed region in (c). Moreover, the data is consistent with previously reported data (Fig. Phys. Rhodamine B is a xanthene dye, which functions as a water tracer fluorescent. 6). Thank you for visiting nature.com. Costume written MatLab code was used to produce the rolling frame video. After washing three times for 5min each in PBS, the coverslips were mounted and imaged in PBS. These were prepared as stock solutions in dry DMSO and diluted in the respective buffer such that the final concentration of DMSO did not exceed 5% v/v. The authors thank Dr. H. Farrants, Dr. J. Hiblot for sharing reagents, Dr. B. Koch for help with the establishment of the stable CEP41-Halo cell line, Dr. C. Sieben (EPFL) for valuable discussions and sharing of the Matlab analysis script, Dr. Rolf Sprengel (MPI for Medical Research) for the donation of the COS-7 cells, the electronic workshop of the Max Planck Institute for Medical Research for technical assistance, the NMR service of EPFL for assistance with the NMR experiments, Heidrun Haungs for technical assistance with the X-ray crystallography, and the Advanced Light Microscopy Facility (ALMF) at the European Molecular Biology Laboratory (EMBL) and Leica Microsystems for support. The unusual mechanism of photoactivation and the fluorophores outstanding spectroscopic properties make it a powerful tool for live-cell super-resolution microscopy. The average microtubule diameter was found to be 38.77.7nm (mean95% confidence interval, N=20 tubules). Karstens, T. and K. Kobs (1980) Rhodamine B and Rhodamine 101 as reference substances for fluorescence quantum yield measurements. In such experiments, we could follow the fast dynamics of mitochondria (TOMM20-Halo) labeled with PA-SiR-Halo over one minute in 10s snapshots without artificial narrowing and collapsing of structures (Fig. Transient transfection of cells was performed using Lipofectamine 2000 reagent (Life Technologies) according to the manufacturers recommendations: DNA (2.5g) was mixed with OptiMEM I (100L, Life Technologies) and Lipofectamine 2000 (6L) was mixed with OptiMEM I (100L). ized water and the . Pigment Violet 1, Basic Violet 10, C.I. PubMed Central To determine the extinction coefficient ( max), the absorbance (A) and the concentration (c) of chromophores should be known based on the Beer's law (A = bc where b is the pathlength . A solution of 2% [v/v] N-[3-(trimethoxysilyl)propyl]ethylendiamine (Sigma-Aldrich) in dry acetone was prepared and the clean coverslips were immersed in the dark for 1h. The coverslips were rinsed with acetone, MQ water and then dried with N2. Furthermore, they result in the stoichiometric formation of very electrophilic nitroso-aldehydes or ketones as reactive byproducts, which are toxic and of concern in live-cell imaging18. Endogenously tagged Nup96-Halo in U-2 OS cells was stained with PA-SiR-Halo (1M for 2h). Bio-Protocol 6, e1784 (2016). For the quantification of microtubule width, we constructed a perpendicular line profile from a 250nm long section of the microtubule. We expect that the exceptional properties of PA-SiR will be exploited in the future to create various other photoactivatable probes for live-cell imaging. Cloning was performed by Gibson assembly56. Biol. 1a, d, f and Supplementary Fig. Live-cell SMLM data were additionally processes using the HAWK plugin using three levels and time grouping, followed by multi-emitter fit in ThunderSTORM allowing for five emitters per fitting region71. A blank was measured before starting the measurement. 3ce). U-2 OS-CEP41-Halo cells were seeded on 24mm glass coverslips and stained with PA-SiR as described above. Up to date, fluorescent proteins and small-molecule fluorophores are the two most commonly used fluorophore classes in SMLM. Puliti, D., Warther, D., Orange, C., Specht, A. The cells were incubated for 2448h before imaging. J. Phys. 2b, Supplementary Figs. After labeling, the proteins were separated by PAGE (420% 10-well Mini-Protean TGX, BioRad) as recommended by the manufacturer and revealed by in gel fluorescence using a ChemiDoc MD Imaging System (BioRad). Rhodamine B - an overview | ScienceDirect Topics Chem. C 28 H 31 Cl N 2 O 3, M.W. g F-actin (LifeAct-Halo). Photoactivatable fluorophores are important for single-particle tracking and super-resolution microscopy. Hatchard, C. G. & Parker, C. A. & van Oijen, A. M. mKikGR, a monomeric photoswitchable fluorescent protein. Acta Crystallogr. Article (For assignment of peaks see Supplementary Fig. For clarity, snapshots are shown only every 5s. Several mitochondria are perceived to be hollow as Tomm20 is localized to the outer membrane of mitochondria (red arrowheads). Subsequently, the FA was quenched by incubating the coverslip for 5min in 100mM NH4Cl in PBS. It is used as a colorant . Rhodamine 6G (RH 6G) - Handbook of Fluorescent Dyes and Probes - Wiley hover the mouse over the graph, you will see a pop-up showing the ADS A full shell of intensity data were collected at low temperature with an Agilent Technologies Supernova-E CCD diffractometer (Mo-K radiation, microfocus X-ray tube, multilayer mirror optics). Stable cell lines were seeded on glass bottom dishes as described in the previous section, and induced using 100gmL1 doxycycline (Sigma-Aldrich) for 2448h previous to imaging. In light of the limitations of the existing caging strategies, alternative chemical strategies are needed to generate photoactivatable fluorophores. Article export the graph in other formats. A solution of 1mg biotin-PEG-SVA (MW 5000, Laysan Bio) and 54mg mPEG-SVA (MW 5000, Laysan Bio) was prepared in 230L sodium bicarbonate buffer (10mm freshly prepared) and applied to three coverslip pairs. Biophys. Int. 84, 1871-1872. 9, 10c, 12df, 13b, 14g, and 15ac and Supplementary Tables1, 2, 5, 7, and 9 are provided as a Source Data file. Anyone you share the following link with will be able to read this content: Sorry, a shareable link is not currently available for this article. PubMed 86, 825843 (2017). Ed. B. et al. COS-7 cells were seeded on 24mm glass coverslips and stained with PA-SiR-Actin as described above. Scale bar, 10m. UV irradiation was performed for 1min in a quartz cuvette (Hellma Analytics) and aliquots were taken to measure LCMS at defined time points using an analytical gradient from 10 to 90% B within 6min with 0.5mLmin1 flow. 109, 1397813983 (2012). Structure of Rhodamine-B. Colorless needle shaped crystals were grown from slow evaporation of a CH2Cl2/MeOH solution at 4C. Fixed-cell samples were mounted in PBS on cavity slides (VWR) sealed with twinsil 22 (Picodent) and imaged therein. Synthesis and Characterization of ROSA Dye - A Rhodamine B-type This fluorophore is devoid of rhodamine spirolactone formation and furthermore characterized by a high molar extinction coefficient (=87250 1630 M-1cm-1) and quantum yield () of 0.589 0.070 . Where given X2 the reduced chi-squared corresponds to the residual sum of square (RSS) and R2 is the squared correlation coefficient. d, e Maximum projection of a z-stack of U-2 OS cells stably expressing H2B-Halo (nucleus) stained with PA-SiR-Halo (0.5m for 2h) before d and after UV irradiation e Scale bar, 40m. Thompson, R. E., Larson, D. R. & Webb, W. W. Precise nanometer localization analysis for individual fluorescent probes. You can resize To obtain Snare, M. J., F. E. Treloar, K. P. Ghiggino and P. J. Thistlethwaite (1982) The photophysics of rhodamine B. J. Photochem. 14a, g)8,31. Malecki, M. J. et al. J. application(s) diagnostic assay manufacturing hematology . Further information on research design is available in theNature Research Reporting Summary linked to this article. 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) Lavis, L. D. Teaching old dyes new tricks: biological probes built from fluoresceins and rhodamines. In contrast to other photoactivatable fluorophores, no caging groups are required, nor are there any undesired side-products released. Lett. All absorbance measurements (spectra and time traces) were performed in 1.5mL stirrable quartz cuvettes (Hellma Analytics) on a JASCO V770 spectrophotometer with a Peltier element (PAC743R) under continuous stirring and at 21C. Methods 3, 793795 (2006). The channel was washed with 400L PBS. Moreover, both photoactivation of PA-SiR as well as the equilibrium between 2 and 3 are pH sensitive (Fig. B., Miyawaki, A. Conjugate fluorescence is determined by measuring the fluorescence quantum yield of the conjugated dye relative to that of the free dye and multiplying by the number of fluorophores per . Sheldrick, G. Crystal structure refinement with SHELXL. PDF Determining the extent of labeling for tetramethylrhodamine protein Correspondence to (e) Molar extinction coefficient and (l) wavelength. Use the Previous and Next buttons to navigate the slides or the slide controller buttons at the end to navigate through each slide. Ovesn, M., Kek, P., Borkovec, J., vindrych, Z. Schindelin, J. et al. 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. They are defined as follows: Fluorescence spectra were measured on a JASCO FP-8600 fluorimeter in 1.4mL fluorescence cuvettes (Hellma Analytics). The reconstruction of super-resolved images of NUP96-Halo was done using the custom-written software SMAP (Super-resolution Microscopy Analysis Platform, https://github.com/jries/SMAP). Eastman Laboratory Chemicals Catalog No. Sect. [5], Rhodamine B can exist in equilibrium between two forms: an "open"/fluorescent form and a "closed"/nonfluorescent spirolactone form. 11.3 (Kluwer Academic Publishers, Dordrecht, The Netherlands, 2001). Phototoxicity caused by activation of small-molecule synthetic probes with UV light can be due to the UV light itself as well as toxic side products of the photoactivation. et al. These experiments also revealed that the photoactivation is reversible on a time scale of days (Fig. Rhodamine B - an overview | ScienceDirect Topics 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. The cell lysate was cleared by centrifugation (20,000g, 20min, 4C). Ries, J., Kaplan, C., Platonova, E., Eghlidi, H. & Ewers, H. A simple, versatile method for GFP-based super-resolution microscopy via nanobodies. Synthesis of a farred photoactivatable siliconcontaining rhodamine for superresolution microscopy. The images or other third party material in this article are included in the articles Creative Commons license, unless indicated otherwise in a credit line to the material. application(s) diagnostic assay manufacturing hematology . We next tested the performance of PA-SiR-Halo in live-cell single-particle tracking photoactivated localization microscopy (sptPALM) (Fig. Sign up for the Nature Briefing newsletter what matters in science, free to your inbox daily. Chem. The effective concentrations at which half maximal fluorescence intensity was reached (EC50 values) were determined to be 0.1920.019mM for PA-SiR and 3.10.5mM for PA-SiR-Halo (mean95% confidence interval, both N=24 samples), error bars correspond to 95% confidence intervals. Use the absorbance (A) of the solution at = 555 nm. Crystallogr. The human nuclear pore complex as revealed by cryo-electron tomography. Sauer, M. & Heilemann, M. Single-molecule localization microscopy in eukaryotes. Gibson, D. G. et al. 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. 68, 141-142. Short additional poststaining was performed with 0.5M PA-SiR-Actin (1h, 25C). The excitation and emission monochromators were set at 1 mm, giving a spectral bandwidth of 4.25 nm. are inventors on a patent filed by EPFL and Spirochrome AG. 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. Express 24, 2808028090 (2016). 2c). PA-SiR-Halo and the influence of HaloTag on its equilibrium system. H.W. 1a, c and Supplementary Figs. Staehelin, M. & Simons, P. Rapid and reversible disappearance of beta-adrenergic cell surface receptors. Med. BMC Bioinforma. SNAP-tag and HaloTag7 were fused to the N or C terminus of the genes of interest (GOI) and a T2A-EGFP sequence was introduced. 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). All images except the NUP96-Halo images were processed with ImageJ/Fiji66,67. 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. M.S.F. PA-SiR-Halo was measured with addition of BSA or HaloTag (20M). Rhodamine 6G is a fluorescent compound with an excitation peak at 525 nm and an emission peak at 548 nm. After 12h incubation in a humidified 5% CO2 incubator at 37C the medium was changed to fresh medium. A 51, 3338 (1995). 1)21. Use the absorbance (A) of the solution at = 555 nm. The movie presented was convoluted with a Gaussian function (sigma=12nm). March 2004; Russian Journal of Applied Chemistry 77(3):414-422; . Proc. Chem. and J.R.), the EMBL International PhD Program (to P.H. [16] Cases of economically motivated adulteration, where it has been illegally used to impart a red color to chili powder, have come to the attention of food safety regulators. 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. Acta Crystallogr. Fluorescein, Oregon Green and Rhodamine Green DyesSection 1.5 11, Table8). J. Colloid Interface Sci. [4], Rhodamine B (BV10) is mixed with quinacridone magenta (PR122) to make the bright pink watercolor known as Opera Rose. Article PDF 46406 2083.1 Number Description - Thermo Fisher Scientific Database of Absorption and Fluorescence Spectra of >300 Common Spectrum [Rhodamine 6G] Rhodamine 6G is a maroon powder with a molecular weight of 479.02. PubMed 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. Crystallogr. 13a). Molar extinction coefficient of R6G in water at room temperature. Detailed procedures for the synthesis of all compounds and their characterization are given in theSupplementary Methods. ADS Thevathasan, J. V. et al. J. Phys. Lavis, L. D. Chemistry is dead. 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. The microscope was equipped with a CO2 and temperature controllable incubator (PeCon, 37C). 3 for an overview of related structures and reactions), but have not been reported to undergo light-induced protonation. A general method for the covalent labeling of fusion proteins with small molecules in vivo. Spectra were measured with NS=128 using a water suppression presaturation sequence. Use the extinction coefficient () of rhodamine B ( 555 = M. The cells were fixed as previously described60. It has been previously observed that HaloTag possesses a high affinity toward zwitterionic rhodamine derivatives, including SiR-carboxyl22. The microscope was equipped with a CO2 and temperature controllable incubator (Life Imaging Services, 37C). Rhodamine B is a xanthene dye, which functions as a water tracer fluorescent. Extinction coefficients at 646nm after activation were calculated from the equilibrium constants (K2) obtained in the 12s activation experiments (Fig. 22 (IUCr and Oxford University Press, Oxford, UK, 1988). One of these SRM approaches is single-molecule localization microscopy (SMLM), which relies on the switching of fluorophores between an off and an on state3,4,5. coordinates. Internet Explorer). Sydor, A. M., Czymmek, K. J., Puchner, E. M. & Mennella, V. Super-resolution microscopy: from single molecules to supramolecular assemblies. CCDC 1942173 contains the supplementary crystallographic data for this paper. ACS Chem. Rhodamine 6G - omlc.org 55 (1993-94), Fisher Scientific. volume10, Articlenumber:4580 (2019) c Time series of boxed region in (a). The Beer-Lambert Law - Chemistry LibreTexts Each frame is reconstructed from 200 frames (10s). Phys. A pcDNA5/FRT/TO vector (ThermoFisher Scientific) was used for transient expression in mammalian cells and generation of stable cell lines. Manley, S. et al. 118, 94129454 (2018). Determination of phosphorescence efficiencies. The pulse-length of the 405nm laser was adjusted during the acquisition to maintain a similar number of localizations per frame. Burla, M. C., Giacovazzo, C. & Polidori, G. From a random to the correct structure: the VLD algorithm. Xu, K., Babcock, H. P. & Zhuang, X. Dual-objective STORM reveals three-dimensional filament organization in the actin cytoskeleton. 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. 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. CAS ), the European Research Council (ERC CoG-724489, to P.H. Use the extinction coefficient ( ) of rhodamine B ( 555 = 106,000) to determine the concentration of the rhodamine B solution. Rhodamine B = 95 HPLC 81-88-9 - Sigma-Aldrich Habuchi, S., Tsutsui, H., Kochaniak, A. Photoactivation of silicon rhodamines via a light-induced protonation. Remaining authors declare no competing interests. B.01 (Wallingford, CT, 2016). Calculations of the frontier molecular orbitals of model compound PA-SiR 4 and data published on cross-conjugated 1,1-diphenyl alkenes23,24 indicate that the photoactivation could proceed through a twisted intramolecular charge transfer followed by protonation of the intermediate (Supplementary Fig. Chem. The fluorescence emission spectrum of Rhodamine B Here we present a photoactivatable fluorophore that forms a bright silicon rhodamine derivative through a light-dependent protonation. Selected bond lengths () and torsion angles (): C4C5 1.380(2), C5C6 1.3999(19), C6C7 1.4896(19), C7C8 1.4929(19), C7C14 1.344(2), C1C6C7C14 145.94(15), C5C6C7C14 31.9(2), C7C8C13Si1 2.91(17), C14C7C8C9 33.4(2).
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