Photoactivation Yields _ Bleaching Yield Measurements for PA by wuyunyi


									                     1865      THE UNIVERSITY OF                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                        INSTITUTE
                                                                                                                             PHOTOACTIVATION YIELDS & BLEACHING YIELD MEASUREMENTS FOR PA FPs
                              MAINE                                                                                                                                                                                                   M.S. Gunewardene* and S.T. Hess*
                                                                                                                                                * Department of Physics & Astronomy and Institute for Molecular Biophysics, University of Maine,                                                                                                                                                                                                     Orono, ME 04469.
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                     M O L E C U LAR

                                                                 ABSTRACT                                                                                                                   THEORY & METHODOLOGY                                                                                                                                                                                     ANALYSIS                                                                                                                                           Total Number Vs. Frame #
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                       3600                                      3200
 Recent developments in single-molecule localization-based fluorescence methods are breaking the                                                                                                                                                                                                            Counting and Localizing Molecules6
                                                                                                                                                  The Photoactivation and Readout LASERs are used to control the number of active molecules in a
 diffraction barrier of light microscopy. Hence a better understanding of photophysical parameters for cell-                                      sample at a given time. Hence the following two major processes are of interest .                                                                                                                                                                                                                                                                                                                      A                                         B
 compatible photoactivatable/switchable fluorescent proteins (PA-FPs) is in demand. The Photoactivation                                                                                                                                                                                                                   A                                                             B                                                       C                                                                                                                3000
 yield (probability of activation of such a molecule upon receiving an activation photon), readout laser                                                                                                                                                                                                                                                                                                                                                                                                                                      τ =5.0 ± 0.4
                                                                                                                                                                        1. Activation/Bleaching by Photoactivation Photons.                                                                                                                                                                                                                                                                                            3200                                                                    τ =2.7 ± 0.3
 Photoactivation yield, and the photobleaching yield are important parameters of interest to optimize
 these microscopic techniques. A basic model for the dependence of the number of active molecules as                                                                                                                                                                                                                                                                                                                                                                                                                                                             2800
 a function of excitation time for two commonly used PA-FPs is derived using linear first-order kinetics.                                                               2. Activation/Bleaching by Readout Photons.                                                                                                                                                                                                                                                                                                    3000
 Single molecules were imaged by a high-sensitivity camera in a widefield fluorescence microscope
                                                                                                                                                  we propose a photoconversion scheme 3 using linear first order kinetics given by the following figure.
 under laser excitation. Analyzing measurements using the above model, the activation yields and the
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                              1   2   3   4   5     6    7   8   9   10 1     2    3   4   5   6   7   8      9   10
 bleaching yield for PAGFP and Wild type EosFP were determined under different conditions. Here we                                                 Ref: S.T.Hess et al, J. Phys. Chem. B 2004, 108, 10138-10148
 present the data and compare results for each of the species. Practical implications of the measured
 yields will be discussed.                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                               C       1600                              D
                                                                                                                                                    Photoconversion Scheme
                                                                                                                                                                                               Inactive State                     Active State                                                                                                                                                                                                                                                                                                τ =3.6 ± 0.4                                     τ =4.1 ± 0.4
                                                                INTRODUCTION                                                                                                                                                                                                                                                                                                                                                                                                                                           1600
   Photo-Activatable Green Fluorescent Protein (PAGFP)                                                                                                                                                                                                                                           Fig. 8. Molecule detection and localization procedure. (A) the raw image from the camera. (B) image
                                                                                                                                                                                                                                                                                                 after uniform background subtraction. (C) After setting upper and lower bound pixel thresholds to the
 The wild type of this protein is extracted from a Pacific Ocean jelly fish “Aequorea Victoria” The protein                                                                                                                                                                                      uniform background subtracted image, green boxes - Localized molecules, red boxes - spot with                                                                                                                         1400
 has 238 amino acids and the mutation at the amino acid site 203 Threonine with a Histidine (T203H) is                                                                                                              a                                                                           insufficient number of lower threshold pixels.
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                              1   2   3   4   5     6    7   8   9   10   1   2    3   4   5   6   7   8      9   10
 known as PAGFP.
                                                                                                                                                                                ka      λactivation                        λreadout               λfluorescence                                                                                                                                                                                                                                           Fig 13. Total Number vs. frame # curves. The data points were fitted to y = A exp(-x/τ) + c model to
                 A                                               B
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                          determine the decay time. (A)-PAGFP dry, (B)-PAGFP in pH7 buffer, (A)-EosFP dry, (B)-EosFP in pH7
                                                                                                                                                                                                                                                                                                                                      A                                                                                       B                                                                           buffer.

                                                                                                                                                                                                                          k dp                                                                                                                                                                                                                                                                                                                               RESULTS
                                                                Fig. 1. (A) β barrel structure of GFP. Has a diameter of ~30Å and                                   Fig. 5. Common Photoconversion Scheme for PAGFP and wEosFP kdp and kp
                                                                a length of ~40 Å. (B) chemical structure of the chromophore also                                   are the rate constants for ground state inter-conversions.                                                                                                                                                                                                                                                                                         PAGFP                         PAGFP in                          EosFP                       EosFP in
                                                                shown at the center in fig 1. A (yellow structure).
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                        Dry                         pH 7 buffer                         Dry                       pH 7 buffer
                                                                Figure from:                           Can show that the functional dependence of the active type molecules upon activation time is given by,
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                          Φpa   (1.77 ± 0.02) x 10-6          (1.10 ± 0.02) x 10-6            (1.19 ± 0.03) x 10-5 (1.64 ± 0.07) x 10-5
                                                                                                                                                                                                   k a a
    Eos Fluorescent Protein (EosFP)
                                                                                                                                                      n a ( t )  n0 a e         k bb t
                                                                                                                                                                                              k b b  k a  a
                                                                                                                                                                                                               n0 n e  k a a t
                                                                                                                                                                                                                                 e  k bb t
                                                                                                                                                                                                                                                                      ------- Eq. 1              Fig. 9. Distribution of the number of active molecules in each frame at different stages of the
                                                                                                                                                                                                                                                                                                  experiment done on PAGFP dry sample. (A) the initial decay shows photobleaching of molecules due
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                          Φra   (2.12 ± 0.02) x 10-6              (5.5 ± 0.2) x 10-7              (7.1 ± 0.2) x 10-8       (2.28 ± 0.05) x 10-8

 The wild type of this protein (wEosFP) is extracted from a scleractinian coral Lobophyllia hemprichii and                                                                                                                                                                                        to the readout laser. (B) a section of the activation stage with 2s activations till frame ~2850 and 5s                                                                                                                                     Bleaching Yield by Fitting Method
 been named after the goddess of dawn Eos in Greek mythology.                                                                                                                                                                                                                                     activations after frame ~2930. Each activation is followed by a readout .
                                                                                                                                                  Where, k a / kb – Activation / Bleaching excitation rates per molecule.                                                                                                                                                                                                                                                                                 Φb    (5.52 ± 0.03) x 10-6              (5.4 ± 0.1) x 10-6          (1.85 ± 0.02) x 10-5 (5.76 ± 0.08) x 10-6
                                                                Fig. 2. Molecular structure of EosFP.
                                                                (A) Ribbon diagram of the EosFP                                                          Φa / Φ b - Activation / Bleaching Yields.                                                                                                               Fitting Method
                                                                                                                                                         n a / nn - Number of Active / Inactive type molecules, the 0 subscript denotes the initial numbers.                                                                                                                                                                                                                                                                               Bleaching Yield by Histogram Method
                                                                tetramer. For one of the monomers,                                                                                                                                                                                               From the data extracted from the above routine, the number of active molecules detected vs. the
                                                                green and red colors indicate the                                                                                                                                                                                                activation time graphs were plotted, The average excitation rate per molecule was calculated using the                                                                                                   Φb      (2.7 ±0.3) x 10-5               (5.2 ±0.6) x 10-5               (3.1 ±0.4) x 10-5           (2.7 ±0.3) x 10-5
                                                                protein    fragments     created     by                                                                                                                                                                                          absorbance curves and an imaged laser profile for each fit. Hence a curve was fitted to the data to
                                                                photoconversion. The atoms in the                                                   Experimental Setup and Sample Preparation                                                                                                    extract the yields. The photoactivation laser power at the sample was assumed low enough for little or                                                                                                                                   Table 1. Summary of the measured Yields
                                                                chromophore are color-coded. (B and                                                                                                                                                                                              no photobleaching.
                                                                                                                                                   An Olympus IX71 inverted type microscope with                         Fig. 5. Experimental Geometry.                       Inverted type
                                                                C) The electron densities (2 Fo - Fc) of                                                                                                                                                                       Microscope
                                                                the green (B) and red (C)
                                                                chromophores are contoured at 1.2 σ.
                                                                                                                                                   NA 1.2 water immersed objective was used along
                                                                                                                                                   with    excitation   filters Z496/10X/Z514/10X,
                                                                                                                                                                                                                         Laser beams entering through
                                                                                                                                                                                                                         the back of the objective to the
                                                                                                                                                                                                                                                                                 System                                                                  Number of active molecules Na(t) Vs. Activation time t                                                                                                                                          CONCLUSIONS
                                                                (D and E) The hydrogen bonds                                                       emission filters ET525/50M/HQ590/75M, and                             sample. A lens is introduced to                                Sample                                                    Photoactivation                                                                Readout Activation and Bleaching
                                                                constraining the green (D) and red (E)                                             beam splitters. The lasers were aligned to have                       focus the beam to the focal plane                                                          Fitted                                                                                                                                                                                The wEosFP seems to be easily activated compared to PAGFP using 405nm photons.
                                                                chromophores are represented by                                                    the profiles centered. An Ar+ continuous-wave                         of the objective hence spreading                                                                                                     10000                                             500
                                                                                                                                                                                                                                                                                                                                                                                                                          A                                           B
                                                                                                                                                                                                                                                                                                                          A                                             B                                                                                   300
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                          For PAGFP the Readout activation Yield and Photoactivation Yield were the same order of magnitude.
                                                                dashed lines. The dashed arrow                                                     laser was used to generate the 496nm/514nm                            the beam at the sample which                                                                                                          8000                                             400
                                                                                                                                                                                                                                                                                                                                                                                                                          Φra = (2.12 ± 0.02) x 10-6                      Φra = (5.5 ± 0.2) x 10-7
                                                                depicts the interaction between the                                                Readout Laser lines with ~10mW at the back of                         allows greater imaging area.                                            3000

                                                                Glu-212 carboxyl and His-62 Cβ–H                                                   the objective and a 405nm semiconductor laser as                                                                               Objective                                                                    6000                                             300       Φb = (5.52 ± 0.03) x 10-6         200           Φb = (5.4 ± 0.1) x 10-6         Bleaching yields from the two methods were one order of magnitude different. The Fitting method
                                                                that is essential for cleavage of the                                              the Activation Laser with ~2.5mW at the back of                                   Excitation
                                                                                                                                                                                                                                                                                                                                                               4000                                             200
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                          seems less accurate since it uses small time scale fitting for evaluating the bleaching yield.
                                                                His-62 Nα–Cα bond.                                                                 the objective (less power to have insignificant                                                                                               1000                                                                                                                                                       100
                                                                                                                                                                                                                                                                                                                              Φpa = (1.77 ± 0.02) x 10-6 2000                   Φpa = (1.10 ± 0.02) x 10-6
                                                                                                                                                   amount of photobleaching) were used to image
                                                                                                                                                                                                                                                                                                                                                                                                                100                                                                                       The condition for same rate of Activation and Bleaching is, kaΦann(t)=kbΦbna(t). If an experiment is
Figure from: Karin Nienhaus et al. (Jun 28, 2005) PNAS. vol. 102 no. 26, 9156–9159                                                                                                                                       Readout
                                                                                                                                                   PAGFP/wEosFP samples both dry as well as in                            Laser                                                                     0                                                               0                                                                                                                                     performed at this condition for sufficient enough time to gather the data, a single laser is sufficient for
                                                                                                                                                                                                                                                                                                                 0             50     100         150   200             0        50     100         150   200         0        100             200    300         0         100             200     300
         Absorbance /Emission Spectral Characteristics                                                                                             Phosphate pH buffer. Protein samples were                                                                                                     10000                                                                                      t (s)               500                                         300                     t (s)
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                          both the readout and activation processes. (demonstrated in Hess et al., 2007 and Hess et al. 2006)
                                                                                                                                                                                                                                                                                                                                        t (s)                 5000
                                                                                                                                                   diluted and prepared at desired concentration to                                                                                                                       C                                                 D                                             C                                           D
                                                                                                         (x104)                                                                                                                                                               Emission Filter    8000                                                         4000                                              400
               0.14                                                                                                                                                                                                                                                                                                                                                                                                        Φra = (7.1 ± 0.2) x 10-8                       Φra = (2.28 ± 0.05) x 10-8
                                                                                                    16                                             have a S/N ratio >10. A Photometrics Cascade
                                                                                                                                                                                                                                              Photoactivation                                    6000                                                         3000                                                                                          200
                                                                                                                                                   512 B CCD Camera was used for imaging with                                                      Laser
                                                                                                                                                                                                                                                                                                                                                                                                                300        Φb = (1.85 ± 0.02) x 10-5                      Φb = (5.76 ± 0.02) x 10-6
           0.12           Active                                                                    14                                                                                                                                                                               CCD
                                                                                                                                                   frame time set to 150ms with minimal delay time.                                                                                 Camera
                                                                               Fluorescence (cps)

                                                                                                                                                                                                                                                                                                 4000                                                         2000
                          Inactive                                                                  12

                                                                                                                                                  The samples were prepared at room temperature and placed on 0.17mm thickness 22mm x 22mm                                                       2000                                                         1000
                                                                                                                                                                                                                                                                                                                                                                                                                100                                                                                       1. Patterson, G.H “A photoactivatable GFP for selective photolabeling of proteins and cells.” 2002
                                                                                                    10                                                                                                                                                                                                                         Φpa = (1.19 ± 0.03) x 10-5                       Φpa = (1.64 ± 0.07) x 10-5
               0.08                                                                                                                 475 nm ex     coverslips for imaging. The samples were diluted using UV treated HPLC water. For experiments with                                                    0
                                                                                                                                                                                                                                                                                                                                                                    0                                                                                                                                        Science 297(5588): 1873-1877.
                                                                                                    8                                             buffer, coverslips were coated with Poly-L-Lysine (PLL) and the proteins were allowed to attach before                                                                  0      50    100        150   200     250         0      50    100        150   200         0        100             200    300         0         100             200     300
               0.06                                                                                                                               the pH buffer solution was introduced.                                                                                                                                                  t (s)                                             t (s)                                      t (s)                                        t (s)
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                          2. Wiedenmann, J “EosFP, a fluorescent marker protein with UV-inducible green-to-red
                                                                                                    6                                                                                                                                                                                                                                                                                                           Fig. 11. Experimental data fitted to Eq 1. Readout
                                                                                                                                                                                                                                                                                                 Fig. 10. Experimental data fitted to Eq 1.                                                                                                                                                                  fluorescence conversion.” 2004 PNAS 101: 15905-15910.
           0.04                                                                                     4                                                                                                                                                                                                                                                                                                           activation Yield and Bleaching Yield denoted as Φra
                                                                                                                                                                                                                                                                                                 assuming no bleaching. Photoactivation Yield
           0.02                                                                                     2                                                Experimental Procedure                                                                                                                      denoted as Φpa (A)/(C) Experiment on                                                                           and Φb respectively. (A)/(C) Experiment on                                                3. Hess, S.T. et al. “Fluorescence Photoconversion Kinetics in Novel Green Fluorescent Protein pH
                                                                                                                                                                                                                                                                                                 PAGFP/wEosFP dried on a coverslip. (B)/(D)                                                                     PAGFP/wEosFP dried on a coverslip. (B)/(D)                                                   Sensors.” 2004 J. Phys. Chem. B 108: 10138-10148.
                 0                                                                                  0
                     250 300 350 400 450 500 550 600                                                      440 460 480 500 520 540 560 580 600                10 cycles                                                                                                                           Experiment on PLL attached PAGFP/wEosFP in                                                                     Experiment on PLL attached PAGFP/wEosFP in
                             Wavelength (nm)                                                                      Wavelength (nm)                                                                                                                                                                pH 7 buffer.                                                                                                   pH 7 buffer.                                                                              4. Lukyanov, K.A . et al. “Photoactivatable fluorescent proteins.” 2005(Nov.) Nat. Rev. Mol. cell Bio. 6:
                                                                                                                                                          Readout         Activation                    Start                     Readout
 Fig. 3. (A) Absorbance spectra. For PAGFP. Curve in Black/Green: Inactive/Active type (molecules                                                       LASER ON 5s      LASER OFF                    Imaging                    LASER ON
                                                                                                                                                                                                                                                              Readout                                                                                                                                                                                                                                        885-891.
                                                                                                                                                                                                                                                             LASER OFF                                                                                          5
                                                                                                                                                                                                                                   ~300s                                                                           Histogram Method
 before/after the illumination of UV radiation.) (B) Fluorescence spectrum at ~475nm excitation.
 Figure from: George H. Patterson, NIH                                                                                                                   Readout         Activation                                                                                                                                                                                                                                                                                                                       5. Hess, S.T. et al. “Dynamic clustered distribution of hemagglutinin resolved at 40nm in living cell
                                                                                                                                                                        LASER ON 5s                                                                                                              By plotting the number of molecules in a given radius from a detected molecule in subsequent frames a
                                                                                                                                                        LASER OFF                                                                                                                                                                                                                                                                                                                                            membranes discriminates between raft theories.” 2007 PNAS 104: 17370-17375.
                                                                                                                                                                                                                                                                                                 Probability distribution histogram for detecting a molecule after a time lapse is generated.

                                                                                                                                                                                                 Activation        Activation                       Readout            Readout                                                                                                                                                                                                                            6. Hess, S.T. et al. “Ultra-High Resolution Imaging by Fluorescence Photoactivation Localization
                                                                                                                                                                                                LASER ON 5s       LASER OFF                       LASER ON 5s         LASER OFF                                                                                                                                           Using this histogram the area was calculated                                       Microscopy.” 2006 Biophys. J. 91 4258.
                                                                                                                              Absorbance                                End                                                                                                                                                                                                                                               to get a relative magnitude of the total
                                                                                                                              Excitation                                                          Readout           Readout                         Activation         Activation                                                                                                                                         probability (Total number) of detecting a
                                                                                                                                                                                                 LASER OFF        LASER ON 5s                      LASER OFF          LASER ON 2s                                                                                                                                         molecule in a subsequent frame. By plotting
                                                                                                                                                                                                                                                                                                    Probability Density

                                                                                                                                                                                                                                                                                                                                                                                                                          this area vs. the frame # and fitting it to an
                                                                                                                                                                                                       30 cycles                                           30 cycles                                                                                                                                                      exponential decay model the decay time in
                                                                                                                                                  Fig. 7. Experimental Procedure. The first 10 cycles are used to bleach off any background fluorescent                                                                                                                                                                   terms of frames was found. Along with the                                       Dr. George Patterson at NIH for providing T203H mutant samples and spectral data.
                                                                                                                                                  molecules to a desirable level. Next, Imaging done for ~300s, the variation of active type molecules at                                                                                                                                                                 average excitation rate per molecule in the
                                                                                                                                                  this step will be used to find the Readout activation and Bleaching yields. The next activation cycles by                                                                                                                                                               region and the time per frame the bleaching                                     Dr.Uli Nienhaus and Dr.Jöerg Widenmann (University of Ulm) and Dr.Jöerg Bewersdorf (Jackson Lab,
                                                          300      400       500                          600     Wavelength (nm)                 the Photoactivation laser is used to determine the Photoactivation yield.                                                                                                                                                                                               yield was calculated.                                                           Bar Harbor) for the help in providing the wild-type EosFP samples..
 Fig. 4. Spectra of the green and red states of EosFP at pH 7. (A) Green species. (B) Red species                                                                                                                                                                                                                                                                                                                         Fig 12. Distribution of the Probability Density
 Excitation (emission) spectra were measured with emission (excitation) set to 520 (490) nm.                                                      Experiments were performed on the two fluorescent proteins, both dry and in pH 7 buffer. The captured                                                                                                                                                                   Vs. the radial displacement for the dried                                       Dr. Mike Mason at Chem. Eng. Dept. UMaine for the guidance and advice given on background signal
 ■: conversion yields scaled to the absorbance. (Inset) In vitro chromophore maturation at 27°C                                                   images were later analyzed to count the number of active type molecules in each frame. Hence the idea                                                                                                                                                                   EosFP sample. The colored lined shows the                                       reduction of solutions and coverslips for the experiment.
 determined from the absorbance at 506 nm (solid line, exponential fit).                                                                          is by getting the variation of such molecules with the activation time along with the theoretical model                                                                                                                                                                 distribution for each consecutive frame from
 Figure from: Jörg Wiedenmann et al. Nov 9, 2004 PNAS vol. 101 no. 45 15905–15910                                                                 derived to calculate the yields.                                                                                                                                                                                                                                        0-10.                                                                           Tom Tripp, Travis Gould, Ed Allgeyer of Dept. of Physics UMaine for technical support.
                                                                                                                                                                                                                                                                                                                                                  Radial Displacement (μm)

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