The movie presented was convoluted with a Gaussian function (sigma=12nm). Burla, M. C. et al. PA-SiR-Halo was measured with addition of BSA or HaloTag (20M). CAS Rhodamine B is tunable around 610nm when used as a laser dye. B. et al. Lukinaviius, G. et al. 1. Continuous lines are drawn representing the movement of individual receptors. Nat. R.-C. A. Fuh on 06-20-1995 using a Cary 3. [3] Rhodamine B is an important hydrophilic xanthene dye well known for its stability and is widely used in the textile industry, leather, paper printing, paint, coloured glass and plastic industries. Tinevez, J.-Y. You can resize 2004, 30463056 (2004). 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). and K.J. Fitted parameters such as decay constants etc. An absorbance of 0 at some wavelength means that no light of that particular . Cells were seeded on glass bottom 35mm dishes (Mattek or Greiner bio-one), 10-well glass bottom dishes (Greiner bio-one) or 24mm high precision round coverslips #1.5 (Carl Roth GmbH) one day before imaging. A new sensitive chemical actinometer - II. Furthermore, we imaged a HaloTag fusion of Nup9632, a protein of the nuclear pore complex. The chapter also includes references on the use, properties, and safety/toxicity of Rhodamine 6G (RH 6G). The cells were fixed as previously described60. Take this solution in the cuvette for FCS . Crystallogr. & Giepmans, B. N. G. Immunolabeling artifacts and the need for live-cell imaging. 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. Remaining authors declare no competing interests. Use the Previous and Next buttons to navigate the slides or the slide controller buttons at the end to navigate through each slide. Busing, W. R. & Levy, H. A. Int. Both the mixed-isomer and single-isomer TRITC preparations typically have extinction coefficients above 80,000 cm-1 M-1, whereas some competitive sources of TRITC have extinction coefficients reported to be below 50,000 cm-1 M-1. Rhodamine B | C28H31ClN2O3 | CID 6694 - structure, chemical names, physical and chemical properties, classification, patents, literature, biological activities . Google Scholar. Typically, we acquire 15,00030,000 frames with 50ms exposure time and laser power densities of about 13kWcm2. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The C7C14 bond is the shortest bond followed by the aromatic bonds exemplified by C4C5 or C5C6. Its outstanding spectroscopic properties make it well suited for SMLM in both fixed and live-cells and enabled us to create powerful probes for HaloTag and actin. Quantum yields of activation were determined using standard ferroxialate actinometry41 along with the activation rates determined in the saturation experiments (Supplementary Fig. Article The microscope was operated by the Leica LAS X software (version 1.9.0.13747). It is used as a colorant . Rhodamine WT is distinct from Rhodamine B, for example, because Rhodamine WT (RWT) is water soluble. Nuclear pores as versatile reference standards for quantitative superresolution microscopy. Correspondence to performed the chemical synthesis and characterization as well as the widefield and confocal measurements. Raymo, F. M. Photoactivatable synthetic dyes for fluorescence imaging at the nanoscale. An illumination correction was performed as part of the numerical absorption correction45. [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. 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. In these experiments, we used a 405nm laser for photoactivation, generally used to create a sparse subset of fluorescent molecules in SMLM. The samples were incubated for 23h at 37C. Structural modifications on PA-SiR can influence this equilibrium as demonstrated by several synthetized analogs (Supplementary Fig. Potassium ferrioxalate as a standard chemical actinometer. Source data are provided as a Source Data file. Angew. 45, 849854 (2012). For visibility, only tracks that have an overall displacement larger than 0.28m are shown (30ms exposure time, 0.3kWcm2 at 642nm excitation). Fixed-cell samples were mounted in PBS on cavity slides (VWR) sealed with twinsil 22 (Picodent) and imaged therein. Genome-edited U-2 OS cells with Halo-tagged NUP9632 were seeded on 24mm round coverslips (No. PubMed Chem. [15], In California, rhodamine B is suspected to be carcinogenic and thus products containing it must contain a warning on its label. 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 . 6). Malecki, M. J. et al. Sign up for the Nature Briefing newsletter what matters in science, free to your inbox daily. 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. 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. 3). Chem. Rhodamine B is spectrally similar to MMP Red, Cy3 . March 2004; Russian Journal of Applied Chemistry 77(3):414-422; . Use the absorbance (A) of the solution at = 555 nm. It has been previously observed that HaloTag possesses a high affinity toward zwitterionic rhodamine derivatives, including SiR-carboxyl22. The image is reconstructed from 14,083 frames (100ms exposure time, 2.9kWcm2 at 642nm excitation). High-speed computation of the absorption correction for single-crystal diffraction measurements. Biol. Use the absorbance (A) of the solution at = 555 nm. Assessing photodamage in live-cell STED microscopy. Matsui, M., Tsuge, M., Shibata, K. & Muramatsu, H. Photochromism of 1,1-diaryl-1-alkanols. PA-SiR-Halo labeled proteins were imaged using red epi illumination (695/55nm). It is a water-soluble fluorescent xanthene dye used to dye various substances. 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. 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. A 51, 3338 (1995). 1a). Furthermore, 2AR labeled with either PA-SiR-Halo or PA-JF646-Halo moved with comparable mean speeds (Supplementary Fig. Grimm, J. Science 341, 655658 (2013). 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. 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. 22 (IUCr and Oxford University Press, Oxford, UK, 1988). 92, 14.20.1114.20.17 (2010). 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. UV irradiation was performed directly inside the spectrophotometer during the ongoing experiment for 12s unless otherwise stated. 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. planned the experiments and co-wrote the paper. For the quantification of microtubule width, we constructed a perpendicular line profile from a 250nm long section of the microtubule. Google Scholar. Int. B.N., M.K. 2fh). 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). application(s) diagnostic assay manufacturing hematology . Casey, K. G. and E. L. Quitevis (1988) Effect of solvent polarity on nonradiative processes in xanthene dyes: Rhodamine B in normal alcohols. The microscope was equipped with a CO2 and temperature controllable incubator (Life Imaging Services, 37C). b Image of cumulative single-particle tracks of -2-adrenergic-receptor-Halo stained with PA-SiR-Halo (0.5M, 1h) measured during 2min. HeLa, U-2 OS (both ATCC), COS-7 (Gift from Dr. R. Sprengel, MPI for Medical Research) or U-2 OS NUP96-Halo (generously provided by the Ellenberg lab, EMBL) cells were cultured in high-glucose phenol-red free Dulbecco's Modified Eagle Medium (DMEM) (Life Technologies) medium supplemented with GlutaMAX (Life Technologies), sodium pyruvate (Life Technologies) and 10% fetal bovine serum (FBS) (Life Technologies) in a humidified 5% CO2 incubator at 37C. The switching can be achieved by using photoactivatable or switchable fluorophores6,7,8. We then fitted a Gaussian distribution (bin width 2nm) to the profile and plotted its FWHM in a boxplot (Supplementary Fig. 5, 132139 (2013). Rev. Angew. J. Photoactivatable fluorophores are important for single-particle tracking and super-resolution microscopy. Carbopyronine and fluorescein derivatives with such an exocyclic double bond have been described (see Supplementary Fig. Dixon, J. M., M. Taniguchi and J. S. Lindsey (2005), "PhotochemCAD 2. Both buffers are components of the actin polymerization fluorescence assay kit (Cytoskeleton). 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. Mol. Bioorg. Molar extinction coefficient of R6G in water at room temperature. 3a, Supplementary Fig. Edelstein, A., Amodaj, N., Hoover, K., Vale, R. & Stuurman, N. Computer control of microscopes using Manager. Artifact-free high-density localization microscopy analysis. All requests for the NUP96-Halo cell line should be directed to Jan Ellenberg. 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. 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. Nat. B. et al. Photobiol. Both PA-SiR-Halo and PA-SiR-Actin are cell-permeable and make it possible to label live-cells, circumventing permeabilization steps during fixation and therefore reducing potential sources of artifacts35. Another pollutant that is extensively used for dyeing purpose is Rhodamine B (RhB). B., Miyawaki, A. It is often used as a tracer dye within water to determine the rate and direction of flow and transport. A Refined Program with Accompanying Spectral Databases for Photochemical Calculations, Photochem. with rhodamine derivatives that contain a succin- . Nat. performed the SMLM on CEP41-Halo, F-actin, and mitochondria with assistance from M.L. Pigment Violet 1, Basic Violet 10, C.I. Acta Crystallogr. Hess, S. T., Girirajan, T. P. K. & Mason, M. D. Ultra-high resolution imaging by fluorescence photoactivation localization microscopy. Rhodamine B; Rhodamine B has been conjugated to silver nanoparticles to detect their skin penetration in mice. Med. Nat. Sheldrick, G. Crystal structure refinement with SHELXL. Chem. Methods 9, 676682 (2012). 3 for an overview of related structures and reactions), but have not been reported to undergo light-induced protonation. Ovesn, M., Kek, P., Borkovec, J., vindrych, Z. Nuclear pores possess a regular circular shape with an internal diameter of about 100nm33,34. Bioinformatics 30, 23892390 (2014). 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. 3b)36. A 64, 112122 (2008). 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. It was possible to follow fission events of mitochondria highlighting the dynamic network of connecting and disconnecting mitochondrial units (yellow arrowheads). In addition, PA-SiR-Actin was tested for SMLM in fixed COS-7 cells revealing stress fibers and connecting thinner fibers (Supplementary Fig. Sci. ACS Chem. M.S.F. They are color coded in order to distinguish the individual tracks. Phys. The generated fluorescent product had an extinction coefficient of 646 = 180,000 30,000 m 1 cm 1 and a fluorescence quantum yield of = 29.2 1.2% in aqueous buffer (mean 95% . Photoactivation of silicon rhodamines via a light-induced protonation, $${\it{{X}}}^2 = {\rm{RSS}} = \mathop {\sum}\limits_{i = 1}^n {(y_i - \widehat {y_i})^2},$$, $$R^2 = 1 - \frac{\rm{RSS}}{\rm{TSS}} = 1 - \frac{{\mathop {\sum }\nolimits_{i = 1}^n (y_i - \widehat {y_i})^2}}{{\mathop {\sum }\nolimits_{i = 1}^n (y_i - \overline {y_i} )^2}}.$$, $$y\left( x \right) = y_0 + A \cdot e^{ - (x - x_{\mathrm{c}})^2/2 \cdot s^2},$$, $${\mathrm{FWHM}} = 2 \cdot \sqrt {2 \cdot \ln 2} \cdot s.$$, https://doi.org/10.1038/s41467-019-12480-3. Protoc. Rhodamine Green and Rhodamine Green-X Dyes Carboxyrhodamine 110, which we have named Rhodamine Green dye, is the nonsulfonated analog of Alexa Fluor 488 dye. 109, 1397813983 (2012). Photoactivation of silicon rhodamines via a light-induced protonation. Aggregation of Rhodamine B in Water. Schindelin, J. et al. C 28 H 31 Cl N 2 O 3, M.W. Where given X2 the reduced chi-squared corresponds to the residual sum of square (RSS) and R2 is the squared correlation coefficient. ACS Chem. and J.R.). The solutions were incubated for 5min at room temperature, then mixed and incubated for additional 20min at room temperature. Sheldrick, G. M. SHELXL-20xx,University of Gttingen and Bruker AXS GmbH, Karlsruhe, Germany, (20122018). 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 cell lysate was cleared by centrifugation (20,000g, 20min, 4C). ), the European Research Council (ERC CoG-724489, to P.H. J. Phys. 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. Rhodamine dyes are also used extensively in biotechnology applications such as fluorescence microscopy, flow cytometry, fluorescence correlation spectroscopy and ELISA. It is used as a colorant . Keppler, A. et al. Rational design of true monomeric and bright photoactivatable fluorescent proteins. (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). PubMed Acta Crystallogr. volume10, Articlenumber:4580 (2019) Scale bar, 20m. 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. 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. Structure of Rhodamine-B. Sydor, A. M., Czymmek, K. J., Puchner, E. M. & Mennella, V. Super-resolution microscopy: from single molecules to supramolecular assemblies. 19, 10231029 (2011). However, small-molecule fluorophores are generally brighter and more photostable than fluorescent proteins9, and therefore of advantage for SMLM experiments10,11. 15). 82, 27752783 (2002). Selection was performed using 100gmL1 hygromycin B (ThermoFisher Scientific) and 15gmL1 blasticidine (ThermoFisher Scientific). (For assignment of peaks see Supplementary Fig. 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. 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. 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. Cloning was performed by Gibson assembly56. Absorption spectra. 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 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. 54, 80548066 (2015). h the outer mitochondrial membrane (TOMM20-Halo). 8 and 10)25,26,27,28, we discovered that PA-SiR-Halo attached to HaloTag (Supplementary Fig. 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. 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. J. Chem. CAS J. Org. PubMed Short additional poststaining was performed with 0.5M PA-SiR-Actin (1h, 25C). 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. Provided by the Springer Nature SharedIt content-sharing initiative. Each frame is reconstructed from 200 frames (10s). PubMed Enzymatic assembly of DNA molecules up to several hundred kilobases. b Sum projection over the first 10s mimicking the diffraction limited image. Cysteamine concentrations were adjusted by the addition of concentrated cysteamine solution (1M). Natl Acad. Up to date, fluorescent proteins and small-molecule fluorophores are the two most commonly used fluorophore classes in SMLM. export the graph in other formats. Ed. Habuchi, S., Tsutsui, H., Kochaniak, A. 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. 55 (1993-94), Fisher Scientific. A near-infrared fluorophore for live-cell super-resolution microscopy of cellular proteins. 2c). 25, 730748 (2015). Chem. 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. 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. Bonds C6C7 and C7C8 are considerably longer. Jpn. This optical absorption measurement of Rhodamine B were made by Rhodamine B; Rhodamine B has been conjugated to silver nanoparticles to detect their skin penetration in mice. 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. 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. Mayer, G. & Heckel, A. Biologically active molecules with a Light Switch. Puliti, D., Warther, D., Orange, C., Specht, A. If you 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. 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. 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. PBS solutions of different pH were adjusted by addition of HCl or NaOH solution using a pH meter. Van Walree Cornelis, A. et al. The molar extinction coefficient () for Rhodamine B is 106000 cm-1M-1 in Methanol. You are using a browser version with limited support for CSS. Article Nat. CCDC 1942173 contains the supplementary crystallographic data for this paper. Moreover, PA-SiR-Halo was used to image various other intracellular HaloTag fusion proteins (Fig. A Math. Rev. A pET51b(+) vector (Novagen) was used for protein production in Escherichia coli. d Super-resolved image from the boxed region in (c). The dark counts were subtracted and the spectra were corrected for wavelength-dependent instrument sensitivity. [citation needed], Rhodamine B is often mixed with herbicides to show where they have been used. Rhodamine B 4- (3-carboxypropionyl . Lavis, L. D. Teaching old dyes new tricks: biological probes built from fluoresceins and rhodamines. Angew. Robinson, W. & Sheldrick, G. M. in Crystallographic Computing 4 (eds Isaaks, N. W. & Taylor M. R.) Ch. Cells were stained with 0.21M PA-SiR (12h, 37C) in phenol-red free DMEM medium supplemented with GlutaMAX, sodium pyruvate and 10% FBS (all Life Technologies), washed with the same medium or PBS (once for 3min, 37C) and imaged in the same medium. 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). Absorption by the crystal was treated numerically (Gaussian grid)45,47. Spectrum [Rhodamine 6G] Rhodamine 6G is a maroon powder with a molecular weight of 479.02. 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. 9, 10c, 12df, 13b, 14g, and 15ac and Supplementary Tables1, 2, 5, 7, and 9 are provided as a Source Data file. 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. Eastman Laboratory Chemicals Catalog No. This will eventually help to study interactions between the inner and outer membrane of mitochondria by two color SMLM. Methods 3, 793795 (2006). Jain, A., Liu, R., Xiang, Y. K. & Ha, T. Single-molecule pull-down for studying protein interactions.
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