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. Take this solution in the cuvette for FCS . PubMed ADS Use the Previous and Next buttons to navigate the slides or the slide controller buttons at the end to navigate through each slide. U-2 OS-CEP41-Halo cells were seeded on 24mm glass coverslips and stained with PA-SiR as described above. 10). Nat. C 71, 38 (2015). 134, 84808486 (2012). 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. In addition, PA-SiR-Actin was tested for SMLM in fixed COS-7 cells revealing stress fibers and connecting thinner fibers (Supplementary Fig. Bull. We demonstrate the utility of these fluorophores for live-cell SMLM of intracellular targets and single-particle tracking experiments. Visit ChemicalBook To find more Rhodamine B(81-88-9) information like chemical properties,Structure,melting point,boiling point,density,molecular formula,molecular weight, physical properties,toxicity information,customs codes. CAS The microtubule diameter was determined to be FWHMPA-SiR-Halo=38.77.7nm (mean95% confidence interval, N=20 tubules) which corresponds well to the microtubule diameter of 25nm if one takes the size of Cep41-Halo (74kDa, ca. Sauer, M. & Heilemann, M. Single-molecule localization microscopy in eukaryotes. Rhodamine B is spectrally similar to MMP Red, Cy3 . The constant \(\epsilon\) is called molar absorptivity or molar extinction coefficient and is a measure of the probability of the electronic transition. 1c and Supplementary Table1). Biophys. the value of 106,000cm-1/M at 542.8nm (Eastman, 1993). Chem. Selective chemical imaging of static actin in live cells. After extensive rinsing with MQ water they were dried under a N2 stream. Ed. We, therefore, incubated U-2 OS cells expressing a histone H2B-HaloTag fusion protein with 0.5 M PA-SiR-Halo for 2h and imaged the cells prior and after UV activation at 365nm (Fig. Article Bonds C6C7 and C7C8 are considerably longer. Photobiol., 81, 212-213. COS-7 cells were seeded on 24mm glass coverslips and stained with PA-SiR-Actin as described above. 1e and 2c, Supplementary Figs. 22 (IUCr and Oxford University Press, Oxford, UK, 1988). Other properties listed for this fluorescent dye include the physical form, solubility, absorption maxima, emission maxima, molar extinction coefficient and quantum yield. 3b)36. Posted at 16:45h in chris bell powerlifter by colonoscopy prep magnesium citrate vomiting. 5). ADS volume10, Articlenumber:4580 (2019) March 2004; Russian Journal of Applied Chemistry 77(3):414-422; . J. Chem. Proteins were expressed in Escherichia coli strain BL21(DE3)-pLysS. Photobiol. Cysteamine concentrations were adjusted by the addition of concentrated cysteamine solution (1M). Marsh, R. J. et al. Chem. In this study different concentrations (10-2,10-3,10-4) mol./l were prepared for Rhodamine B dye in solvent water at room temperature, then the optical linear properties for example transmission . Opin. Scale bar, 2m. Lond. Scale bar, 10m. Table 1 . Scale bar, 10m. Scale bar, 5m. 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. Article The coverslips were cleaned with piranha solution (1:3, H2O2/H2SO4) twice for 30min. 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. [2], It is also being tested for use as a biomarker in oral rabies vaccines for wildlife, such as raccoons, to identify animals that have eaten a vaccine bait. Opt. LuriaBertani broth cultures were grown at 37C to optical density at 600nm (OD600nm) of 0.8, induced by the addition of 0.5mM isopropyl--d-thiogalactopyranoside and grown at 17C overnight in the presence of 1mM MgCl2. Proc. CAS Proteins were used from glycerol stocks and were further diluted. Remaining authors declare no competing interests. PA-SiR was dissolved in MQ water (~20m). Flow chambers were assembled at need from one glass slide (Carl Roth) and one coated coverslip separated by double sided tape and fixed with epoxy glue. The molar extinction coefficient () for Rhodamine B is 106000 cm-1M-1 in Methanol. Before sample preparation, the respective dye was added to the medium to a final concentration of 1M and incubated for 2h. All following incubations were carried out at room temperature and all incubations longer than 1min were performed on an orbital shaker in the dark to prevent preactivation of the dye. Wysocki, L. M. et al. TRITC is widely used by other companies to prepare most of their so-called "rhodamine" immunoconjugates; however . We found that PA-SiR-Halo showed an excellent signal-to-background ratio after activation under no wash conditions (325, mean95% confidence interval, N=119 cells) and that the fluorescence signal after activation was stable over time. High-speed computation of the absorption correction for single-crystal diffraction measurements. In summary, PA-SiR is a photoactivatable, cell-permeable, far-red fluorophore that is activated by an unusual light-induced protonation. Costume written MatLab code was used to produce the rolling frame video. First, localizations were detected using a difference of Gaussians algorithm and a dynamic threshold to exclude random signal fluctuations. At physiological pH only about 10% of the activated PA-SiR was present as SiR 2 in comparison to 80% at pH=6.1. Angew. Chem. f 1H nuclear magnetic resonance (NMR) spectra of PA-SiR (2.0mm in PBS) before UV irradiation, after complete conversion to 3 and after further 48h in the dark. Ed. Halabi, E. A., Thiel, Z., Trapp, N., Pinotsi, D. & Rivera-Fuentes, P. A photoactivatable probe for super-resolution imaging of enzymatic activity in live cells. & Giepmans, B. N. G. Immunolabeling artifacts and the need for live-cell imaging. Soc. J. SCALE3 ABSPACK, C. CrysAlisPro, Agilent Technologies UK Ltd., Oxford, UK (20112014) and Rigaku Oxford Diffraction, Rigaku Polska Sp.z o.o., Wrocaw, Poland (20152019). Sample preparation was adapted from two literature procedures61,62. 2fh). It is used as a colorant . Betzig, E. et al. and JavaScript. Fluorogenic probes for live-cell imaging of the cytoskeleton. 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. Scale bar, 20m. Scale bar, 5m. BMC Bioinforma. and J.R.), and the National Institutes of Health Common Fund 4D Nucleome Program (Grant U01 EB021223/U01 DA047728 to J.E. The excitation and emission monochromators were set at 1 mm, giving a spectral bandwidth of 4.25 nm. These measurements were scaled to make the molar . UV light shows higher phototoxicity than red light38,39 and should be used only at low intensities and/or low pulse frequencies. 61, 345367 (2010). Rhodamine dyes fluoresce and can thus be detected easily and inexpensively with fluorometers.. Rhodamine B is used in biology as a staining fluorescent dye, sometimes in combination with auramine O, as the auramine . Samples were prepared in 1cm pathlength quartz cells with absorbance less than 0.1 at the excitation and all emission wavelengths to uniformly illuminate across the sample, and to avoid the inner-filter effect. 25, 730748 (2015). PubMed Central et al. PA-SiR-Halo was measured with addition of BSA or HaloTag (20M). The cells were harvested by centrifugation (4500g, 10min, 4C) and lysed by sonication. Data were acquired until no more activated fluorophores were observed. A near-infrared fluorophore for live-cell super-resolution microscopy of cellular proteins. The dimerization constant, K D , molar extinction coefficient of monomer, M , and dimer, D (in terms of P for H-dimer and N for J-dimer), twist angle, , the oscillator strength, f , dipole moments of monomer and dimer, and interaction energy, U of R6G . CAS In these experiments, we used a 405nm laser for photoactivation, generally used to create a sparse subset of fluorescent molecules in SMLM. On most of the diagrams you will come across, the absorbance ranges from 0 to 1, but it can go higher than that. 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). The dark counts were subtracted and the spectra were corrected for wavelength-dependent instrument sensitivity. ACS Chem. PA-SiR possesses an exocyclic double bond and the two aromatic ring systems are not conjugated, reflected by its abs,max value of 290nm and demonstrated by the X-ray crystal structure of PA-SiR analog 4 (Fig. 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 . Eur. Proteins were finally concentrated using an Ultra-0.5mL centrifugal filter device (Amicon) with a molecular weight cut-off according to the protein size and then stored in a glycerol 45% (v/v)solution at 20C. Schnell, U., Dijk, F., Sjollema, K. A. and P.H. 2c). 8 and 10). The samples were incubated for 23h at 37C. PubMed It can be used as a fluorescence tracker which helps in defining the spectroscopic characteristics for achieving a high conversion efficiency and precision of measurements. Nature Communications (Nat Commun) Rhodamine B | C28H31ClN2O3 | CID 6694 - structure, chemical names, physical and chemical properties, classification, patents, literature, biological activities . The image is reconstructed from 14,083 frames (100ms exposure time, 2.9kWcm2 at 642nm excitation). any of the graphs by clicking and dragging a rectangle. The C7C14 bond is the shortest bond followed by the aromatic bonds exemplified by C4C5 or C5C6. In total, 100L of a 0.2mgmL1 solution of streptavidin (Life Technologies) in PBS was applied to the flow chamber and incubated for 10min. These experiments also revealed that the photoactivation is reversible on a time scale of days (Fig. It is often used as a tracer dye within water to determine the rate and direction of flow and transport. 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. Endogenously tagged Nup96-Halo in U-2 OS cells was stained with PA-SiR-Halo (1M for 2h). A pcDNA5/FRT/TO vector (ThermoFisher Scientific) was used for transient expression in mammalian cells and generation of stable cell lines. Article The rhodamine is incorporated into the animal's whiskers and teeth. Internet Explorer). 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. Wysocki, L. M. & Lavis, L. D. Advances in the chemistry of small molecule fluorescent probes. Its quantum yield of activation was found to be act=0.860.07% at 340nm and act=0.090.04% at 405nm (meanstandard error of the mean, N=3 samples), similar to that of PA-JF646 (Supplementary Tables1 and 2)29. We attribute the remarkable stability of PA-SiR-Halo attached to HaloTag to specific interactions between HaloTag and the activated probe. Persistence of Vision Pty. Nat. They are defined as follows: Fluorescence spectra were measured on a JASCO FP-8600 fluorimeter in 1.4mL fluorescence cuvettes (Hellma Analytics). The fluorescence emission spectrum of Rhodamine B 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). C 28 H 31 Cl N 2 O 3, M.W. Chem. rhodamine b extinction coefficient in water. Crystallogr. After each UV irradiation step the NMR sample was transferred to the NMR spectrometer. 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. Angew. In the case of the actin probe, buffer containing 5mM Tris-HCl (pH 8.0), 0.2mM CaCl2 and 0.2mM ATP was used. 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. Genome-edited U-2 OS cells with Halo-tagged NUP9632 were seeded on 24mm round coverslips (No. Google Scholar. After washing three times for 5min each in PBS, the coverslips were mounted and imaged in PBS. Use the absorbance (A) of the solution at = 555 nm. Jain, A., Liu, R., Xiang, Y. K. & Ha, T. Single-molecule pull-down for studying protein interactions. Efficient homogeneous illumination and optical sectioning for quantitative single-molecule localization microscopy. Fixed-cell samples were mounted in PBS on cavity slides (VWR) sealed with twinsil 22 (Picodent) and imaged therein. 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. Methods 13, 985988 (2016). The specific parameters can be found in Supplementary Table10. A general method for the covalent labeling of fusion proteins with small molecules in vivo. Methods 9, 185188 (2012). 67, 17531755 (1994). The movie and the snapshots taken thereof revealed intermediate formation of thin tubules between mitochondria (blue arrowheads), as was previously seen with SMLM imaging of MitoTracker Red40. Angew. Charge-transfer interactions in 4-donor 4-acceptor substituted 1,1-diphenylethenes. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate. 92, 6590-6594. Int. NUP96-Halo samples were imaged on a custom-built epi-fluorescence microscope with homogenous high-power illumination63. . Chem. Localizations that were found within 75nm of each other in consecutive frames with maximum one frame dark time were grouped into one localization. PubMed Both buffers are components of the actin polymerization fluorescence assay kit (Cytoskeleton). Google Scholar. performed the SMLM on NUP96-Halo. Acta Crystallogr. Patterson, G., Davidson, M., Manley, S. & Lippincott-Schwartz, J. Superresolution imaging using single-molecule localization. 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. Manley, S. et al. PubMed A new sensitive chemical actinometer - II. A fiber coupled LED (Omicron, 340nm, 3mm liquid light guide) was used to perform UV irradiation unless otherwise stated. Unless otherwise specified, molar extinction coefficient () values are typically measured in ddH 2 O or PBS. Department of Chemical Biology, Max Planck Institute for Medical Research, Jahnstrasse 29, 69120, Heidelberg, Germany, Institute of Chemical Sciences and Engineering (ISIC), cole Polytechnique Fdrale de Lausanne (EPFL), 1015, Lausanne, Switzerland, Cell Biology and Biophysics Unit, European Molecular Biology Laboratory (EMBL), Meyerhofstrasse 1, 69117, Heidelberg, Germany, Philipp Hoess,Bianca Nijmeijer,Moritz Kueblbeck,Jan Ellenberg&Jonas Ries, Collaboration for joint PhD degree between EMBL and Heidelberg University, Faculty of Biosciences, Heidelberg, Germany, Advanced Light Microscopy Facility (ALMF), European Molecular Biology Laboratory (EMBL), Meyerhofstrasse 1, 69117, Heidelberg, Germany, Anorganisch-Chemisches Institut, University of Heidelberg, Im Neuenheimer Feld 270, 69120, Heidelberg, Germany, Spirochrome AG, Chalberweidstrasse 4, CH-8260, Stein am Rhein, Switzerland, Biomolecular Screening Facility, cole Polytechnique Fdrale de Lausanne (EPFL), 1015, Lausanne, Switzerland, National Centre of Competence in Research (NCCR) in Chemical Biology, 1015, Lausanne, Switzerland, You can also search for this author in PubMed M.S.F. 11, Table8). 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. Annu. 7). Article 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. 21, 8689 (2002). Leukemia-associated mutations within the NOTCH1 heterodimerization domain fall into at least two distinct mechanistic classes. Typically, we acquire 15,00030,000 frames with 50ms exposure time and laser power densities of about 13kWcm2. They are color coded in order to distinguish the individual tracks. and J.R.), the EMBL International PhD Program (to P.H. The cells were incubated for 2448h before imaging. 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 PA-SiR-Halo and the influence of HaloTag on its equilibrium system. PubMedGoogle Scholar. c Fluorescence signal after addition of cysteamine (0.001100mm) to fully activated PA-SiR or PA-SiR-Halo on HaloTag solutions in equilibrium (1M dye on 2M HaloTag). Natural photo-sensitizer (aqueous spinach extract) Aqueous spinach extract . Natl Acad. Rhodamine B is a xanthene dye, which functions as a water tracer fluorescent. Synthesis of a farred photoactivatable siliconcontaining rhodamine for superresolution microscopy. Acta Crystallogr. 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. 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. Biochem. 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. Gibson, D. G. et al. 45, 49004921 (2006). Article Chem. 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 . This optical absorption measurement of Rhodamine 6G were made by R.-C. A. Fuh in the summer of 1995 using a Cary 3. TrackMate: an open and extensible platform for single-particle tracking. Methanol fixation was performed as follows: growth medium was removed, cells were incubated for 7min in 20C cold methanol and washed twice with PBS. Article J. Phys. with rhodamine derivatives that contain a succin- . Nat. PA-SiR-Halo labeled proteins were imaged using red epi illumination (695/55nm). Zhang, Z., Park, S. R., Pertsinidis, A. The Rhodamine-B dye purchased from the Ases chemicals (Jodhpur, India) has been used as dye photo-sensitizer material. b Sum projection over the first 10s mimicking the diffraction limited image. 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). Cells were directly imaged after mounting. Ovesn, M., Kek, P., Borkovec, J., vindrych, Z. [13] The fluorescence yield is temperature dependent;[14] the compound is fluxional in that its excitability is in thermal equilibrium at room temperature. Nat. Emission spectra were collected from 610 to 1000nm exciting at 580nm; excitation spectra were recorded at 664nm exciting from 400 to 655nm unless otherwise stated. Another pollutant that is extensively used for dyeing purpose is Rhodamine B (RhB). the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Int. Crystal data and details of the structure determinations are compiled in Supplementary Table11. Aggregation of Rhodamine B in Water. Szymborska, A. et al. The human nuclear pore complex as revealed by cryo-electron tomography. export the graph in other formats. Kilian, N. et al. It should be noted that other PA-SiR probes can be generated (Supplementary Figs. Sci. . Super-resolution images and TIRF data from the single-molecule assay were processed with the ImageJ plugin ThunderSTORM68. The microscope was equipped with a CO2 and temperature controllable incubator (PeCon, 37C). 7, 289293 (2012). 10b and 12c)27,28. Sci. The recently published ImageJ plugin HAWK71 was used to achieve imaging at high emitter densities to capture fast structural changes. 30 10 !4 , and (3) 70 10 !4. 139, 1320013207 (2017). The channel was washed with 400L PBS and filled with PBS. Rhodamines have also been rendered photoactivatable through a diazoketone group19, leading to the introduction of the photoactivatable Janelia Fluor dyes PA-JF549 and PA-JF64620, which have been successfully used for fixed-cell and live-cell SMLM. [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. Measure the absorbance in the spectrophotometer. 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. 2d, e). Quite conveniently, RWT is a fluorescent xanthene dye, facilitating highly sensitive . Cloning was performed by Gibson assembly56. ADS Rhodamine B; Rhodamine B has been conjugated to silver nanoparticles to detect their skin penetration in mice. Photoactivation is prevented by protonation of the aniline groups and is therefore highest at pH values above pH=6 as revealed by measuring the maximal absorbance at 646nm reached directly after activation (Amax). Bio-Protocol 6, e1784 (2016). 8 and 10)25,26,27,28, we discovered that PA-SiR-Halo attached to HaloTag (Supplementary Fig. Mol. using a Spex FluoroMax. All images except the NUP96-Halo images were processed with ImageJ/Fiji66,67. J. Phys. a Super-resolved image of microtubules in fixed U-2 OS cells stably expressing Cep41-Halo stained with PA-SiR-Halo (1M for 2h). Google Scholar. Express 24, 2808028090 (2016). Lukinaviius, G. et al. We next tested the performance of PA-SiR-Halo in live-cell single-particle tracking photoactivated localization microscopy (sptPALM) (Fig. (e) Molar extinction coefficient and (l) wavelength. 3a, Supplementary Fig. 5, 132139 (2013). 15). Dim localizations (localization precision>30nm) and out-of-focus localizations (fitted size of the Gaussian>160nm) were filtered out. planned the experiments and co-wrote the paper. Taken together, these experiments validate that PA-SiR-Halo is suitable for live-cell imaging. Rev. ACS Chem. Nuclear pore scaffold structure analyzed by super-resolution microscopy and particle averaging. Rev. CAS Prepare a stock solution of rhodamine B in PBS buffer. Acta Crystallogr. Rhodamine B; Rhodamine B has been conjugated to silver nanoparticles to detect their skin penetration in mice. e Single nuclear pores from boxed regions in (d) following the same order. There is more than one type of Rhodamine dye, and the specific dye of relevance here is Rhodamine WT, where the "WT" stands for "water tracer.". Nat. 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. B. et al. Instead of the desired fluorescent SiR 2 we isolated the non-fluorescent analog PA-SiR (1) (Supplementary Fig. and K.J. performed the chemical synthesis and characterization as well as the widefield and confocal measurements. west ham player dies on pitch,