Abstract

Imaging single fluorescent proteins in a live cell is a challenging task because of the strong cellular autofluorescence. Autofluorescence can be minimized by reducing fluorescence excitation volume. Total internal reflection fluorescence (TIRF) microscopy has been routinely used to reduce excitation volume and detect single protein molecules in or close to cell membrane. However, the limited penetration depth of evanescent field excludes imaging of single fluorescent proteins that reside deep inside a eukaryotic cell. Here we report detection of single fluorescent proteins inside eukaryotic cells by two-photon fluorescence (TPF) microscopy. TPF has an excitation volume less than 0.1 femtoliter (fL). Cell autofluorescence under TPF is low and thus enables us to detect single enhanced green fluorescent proteins (EGFP) and single monomeric teal fluorescent proteins (mTFP1.0) that reside several microns deep inside the cell. Discrete stepwise photobleaching of TPF was observed for both proteins inside the cell. Quantitative analysis of single-molecule fluorescence trajectories show that mTFP1.0 is about twofold brighter than EGFP, while its fluorescence on-time before bleaching is about 10 fold shorter. These findings demonstrate the sensitivity of TPF for imaging of eukaryotic cells at single-molecule level and will be useful for measurement of protein stoichiometry inside the cell.

© 2012 OSA

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2011 (5)

G. W. Li and X. S. Xie, “Central dogma at the single-molecule level in living cells,” Nature475(7356), 308–315 (2011).
[CrossRef] [PubMed]

T. A. Planchon, L. Gao, D. E. Milkie, M. W. Davidson, J. A. Galbraith, C. G. Galbraith, and E. Betzig, “Rapid three-dimensional isotropic imaging of living cells using Bessel beam plane illumination,” Nat. Methods8(5), 417–423 (2011).
[CrossRef] [PubMed]

F. Cella Zanacchi, Z. Lavagnino, M. Perrone Donnorso, A. Del Bue, L. Furia, M. Faretta, and A. Diaspro, “Live-cell 3D super-resolution imaging in thick biological samples,” Nat. Methods8(12), 1047–1049 (2011).
[CrossRef] [PubMed]

M. Drobizhev, N. S. Makarov, S. E. Tillo, T. E. Hughes, and A. Rebane, “Two-photon absorption properties of fluorescent proteins,” Nat. Methods8(5), 393–399 (2011).
[CrossRef] [PubMed]

X. Hou and W. Cheng, “Single-molecule detection using continuous wave excitation of two-photon fluorescence,” Opt. Lett.36(16), 3185–3187 (2011).
[CrossRef] [PubMed]

2010 (3)

W. Cheng, X. Hou, and F. Ye, “Use of tapered amplifier diode laser for biological-friendly high-resolution optical trapping,” Opt. Lett.35(17), 2988–2990 (2010).
[CrossRef] [PubMed]

J. G. Ritter, R. Veith, A. Veenendaal, J. P. Siebrasse, and U. Kubitscheck, “Light sheet microscopy for single molecule tracking in living tissue,” PLoS ONE5(7), e11639 (2010).
[CrossRef] [PubMed]

P. D. Simonson, H. A. Deberg, P. Ge, J. K. Alexander, O. Jeyifous, W. N. Green, and P. R. Selvin, “Counting bungarotoxin binding sites of nicotinic acetylcholine receptors in mammalian cells with high signal/noise ratios,” Biophys. J.99(10), L81–L83 (2010).
[CrossRef] [PubMed]

2009 (2)

M. Drobizhev, S. Tillo, N. S. Makarov, T. E. Hughes, and A. Rebane, “Absolute two-photon absorption spectra and two-photon brightness of orange and red fluorescent proteins,” J. Phys. Chem. B113(4), 855–859 (2009).
[CrossRef] [PubMed]

J. R. Moffitt, Y. R. Chemla, K. Aathavan, S. Grimes, P. J. Jardine, D. L. Anderson, and C. Bustamante, “Intersubunit coordination in a homomeric ring ATPase,” Nature457(7228), 446–450 (2009).
[CrossRef] [PubMed]

2008 (4)

M. D. Cahalan and I. Parker, “Choreography of cell motility and interaction dynamics imaged by two-photon microscopy in lymphoid organs,” Annu. Rev. Immunol.26(1), 585–626 (2008).
[CrossRef] [PubMed]

W. Ji, P. Xu, Z. Li, J. Lu, L. Liu, Y. Zhan, Y. Chen, B. Hille, T. Xu, and L. Chen, “Functional stoichiometry of the unitary calcium-release-activated calcium channel,” Proc. Natl. Acad. Sci. U.S.A.105(36), 13668–13673 (2008).
[CrossRef] [PubMed]

J. T. Groves, R. Parthasarathy, and M. B. Forstner, “Fluorescence imaging of membrane dynamics,” Annu. Rev. Biomed. Eng.10(1), 311–338 (2008).
[CrossRef] [PubMed]

S. C. Kohout, M. H. Ulbrich, S. C. Bell, and E. Y. Isacoff, “Subunit organization and functional transitions in Ci-VSP,” Nat. Struct. Mol. Biol.15(1), 106–108 (2008).
[CrossRef] [PubMed]

2007 (2)

M. H. Ulbrich and E. Y. Isacoff, “Subunit counting in membrane-bound proteins,” Nat. Methods4(4), 319–321 (2007).
[PubMed]

E. Toprak and P. R. Selvin, “New fluorescent tools for watching nanometer-scale conformational changes of single molecules,” Annu. Rev. Biophys. Biomol. Struct.36(1), 349–369 (2007).
[CrossRef] [PubMed]

2006 (3)

M. C. Leake, J. H. Chandler, G. H. Wadhams, F. Bai, R. M. Berry, and J. P. Armitage, “Stoichiometry and turnover in single, functioning membrane protein complexes,” Nature443(7109), 355–358 (2006).
[CrossRef] [PubMed]

K. Svoboda and R. Yasuda, “Principles of two-photon excitation microscopy and its applications to neuroscience,” Neuron50(6), 823–839 (2006).
[CrossRef] [PubMed]

H. W. Ai, J. N. Henderson, S. J. Remington, and R. E. Campbell, “Directed evolution of a monomeric, bright and photostable version of Clavularia cyan fluorescent protein: structural characterization and applications in fluorescence imaging,” Biochem. J.400(3), 531–540 (2006).
[CrossRef] [PubMed]

2005 (4)

F. Helmchen and W. Denk, “Deep tissue two-photon microscopy,” Nat. Methods2(12), 932–940 (2005).
[CrossRef] [PubMed]

N. J. Carter and R. A. Cross, “Mechanics of the kinesin step,” Nature435(7040), 308–312 (2005).
[CrossRef] [PubMed]

P. J. Schuck, K. A. Willets, D. P. Fromm, R. J. Twieg, and W. E. Moerner, “A novel fluorophore for two-photon-excited single-molecule fluorescence,” Chem. Phys.318(1-2), 7–11 (2005).
[CrossRef]

N. C. Shaner, P. A. Steinbach, and R. Y. Tsien, “A guide to choosing fluorescent proteins,” Nat. Methods2(12), 905–909 (2005).
[CrossRef] [PubMed]

2003 (1)

W. R. Zipfel, R. M. Williams, and W. W. Webb, “Nonlinear magic: multiphoton microscopy in the biosciences,” Nat. Biotechnol.21(11), 1369–1377 (2003).
[CrossRef] [PubMed]

2002 (1)

T. Ha, I. Rasnik, W. Cheng, H. P. Babcock, G. H. Gauss, T. M. Lohman, and S. Chu, “Initiation and re-initiation of DNA unwinding by the Escherichia coli Rep helicase,” Nature419(6907), 638–641 (2002).
[CrossRef] [PubMed]

2001 (2)

N. Billinton and A. W. Knight, “Seeing the wood through the trees: a review of techniques for distinguishing green fluorescent protein from endogenous autofluorescence,” Anal. Biochem.291(2), 175–197 (2001).
[CrossRef] [PubMed]

E. Schaeffer, R. Geleziunas, and W. C. Greene, “Human immunodeficiency virus type 1 Nef functions at the level of virus entry by enhancing cytoplasmic delivery of virions,” J. Virol.75(6), 2993–3000 (2001).
[CrossRef] [PubMed]

2000 (1)

P. T. So, C. Y. Dong, B. R. Masters, and K. M. Berland, “Two-photon excitation fluorescence microscopy,” Annu. Rev. Biomed. Eng.2(1), 399–429 (2000).
[CrossRef] [PubMed]

1999 (4)

D. W. Piston, “Imaging living cells and tissues by two-photon excitation microscopy,” Trends Cell Biol.9(2), 66–69 (1999).
[CrossRef] [PubMed]

W. E. Moerner and M. Orrit, “Illuminating single molecules in condensed matter,” Science283(5408), 1670–1676 (1999).
[CrossRef] [PubMed]

E. J. Peterman, S. Brasselet, and W. E. Moerner, “The fluorescence dynamics of single molecules of green fluorescent protein,” J. Phys. Chem. A103(49), 10553–10560 (1999).
[CrossRef]

J. M. Squirrell, D. L. Wokosin, J. G. White, and B. D. Bavister, “Long-term two-photon fluorescence imaging of mammalian embryos without compromising viability,” Nat. Biotechnol.17(8), 763–767 (1999).
[CrossRef] [PubMed]

1998 (2)

1997 (1)

E. J. Sánchez, L. Novotny, G. R. Holtom, and X. S. Xie, “Room-temperature fluorescence imaging and spectroscopy of single molecules by two-photon excitation,” J. Phys. Chem. A101(38), 7019–7023 (1997).
[CrossRef]

1990 (1)

W. Denk, J. H. Strickler, and W. W. Webb, “Two-photon laser scanning fluorescence microscopy,” Science248(4951), 73–76 (1990).
[CrossRef] [PubMed]

1984 (2)

D. Axelrod, T. P. Burghardt, and N. L. Thompson, “Total internal reflection fluorescence,” Annu. Rev. Biophys. Bioeng.13(1), 247–268 (1984).
[CrossRef] [PubMed]

S. D. Smith, M. Shatsky, P. S. Cohen, R. Warnke, M. P. Link, and B. E. Glader, “Monoclonal antibody and enzymatic profiles of human malignant T-lymphoid cells and derived cell lines,” Cancer Res.44(12 Pt 1), 5657–5660 (1984).
[PubMed]

1961 (1)

W. Kaiser and C. G. B. Garrett, “2-Photon excitation in Caf2–Eu2+,” Phys. Rev. Lett.7(6), 229–231 (1961).
[CrossRef]

Aathavan, K.

J. R. Moffitt, Y. R. Chemla, K. Aathavan, S. Grimes, P. J. Jardine, D. L. Anderson, and C. Bustamante, “Intersubunit coordination in a homomeric ring ATPase,” Nature457(7228), 446–450 (2009).
[CrossRef] [PubMed]

Ai, H. W.

H. W. Ai, J. N. Henderson, S. J. Remington, and R. E. Campbell, “Directed evolution of a monomeric, bright and photostable version of Clavularia cyan fluorescent protein: structural characterization and applications in fluorescence imaging,” Biochem. J.400(3), 531–540 (2006).
[CrossRef] [PubMed]

Alexander, J. K.

P. D. Simonson, H. A. Deberg, P. Ge, J. K. Alexander, O. Jeyifous, W. N. Green, and P. R. Selvin, “Counting bungarotoxin binding sites of nicotinic acetylcholine receptors in mammalian cells with high signal/noise ratios,” Biophys. J.99(10), L81–L83 (2010).
[CrossRef] [PubMed]

Anderson, D. L.

J. R. Moffitt, Y. R. Chemla, K. Aathavan, S. Grimes, P. J. Jardine, D. L. Anderson, and C. Bustamante, “Intersubunit coordination in a homomeric ring ATPase,” Nature457(7228), 446–450 (2009).
[CrossRef] [PubMed]

Armitage, J. P.

M. C. Leake, J. H. Chandler, G. H. Wadhams, F. Bai, R. M. Berry, and J. P. Armitage, “Stoichiometry and turnover in single, functioning membrane protein complexes,” Nature443(7109), 355–358 (2006).
[CrossRef] [PubMed]

Arndt-Jovin, D. J.

Axelrod, D.

D. Axelrod, T. P. Burghardt, and N. L. Thompson, “Total internal reflection fluorescence,” Annu. Rev. Biophys. Bioeng.13(1), 247–268 (1984).
[CrossRef] [PubMed]

Babcock, H. P.

T. Ha, I. Rasnik, W. Cheng, H. P. Babcock, G. H. Gauss, T. M. Lohman, and S. Chu, “Initiation and re-initiation of DNA unwinding by the Escherichia coli Rep helicase,” Nature419(6907), 638–641 (2002).
[CrossRef] [PubMed]

Bai, F.

M. C. Leake, J. H. Chandler, G. H. Wadhams, F. Bai, R. M. Berry, and J. P. Armitage, “Stoichiometry and turnover in single, functioning membrane protein complexes,” Nature443(7109), 355–358 (2006).
[CrossRef] [PubMed]

Bavister, B. D.

J. M. Squirrell, D. L. Wokosin, J. G. White, and B. D. Bavister, “Long-term two-photon fluorescence imaging of mammalian embryos without compromising viability,” Nat. Biotechnol.17(8), 763–767 (1999).
[CrossRef] [PubMed]

Bell, S. C.

S. C. Kohout, M. H. Ulbrich, S. C. Bell, and E. Y. Isacoff, “Subunit organization and functional transitions in Ci-VSP,” Nat. Struct. Mol. Biol.15(1), 106–108 (2008).
[CrossRef] [PubMed]

Berland, K. M.

P. T. So, C. Y. Dong, B. R. Masters, and K. M. Berland, “Two-photon excitation fluorescence microscopy,” Annu. Rev. Biomed. Eng.2(1), 399–429 (2000).
[CrossRef] [PubMed]

Berry, R. M.

M. C. Leake, J. H. Chandler, G. H. Wadhams, F. Bai, R. M. Berry, and J. P. Armitage, “Stoichiometry and turnover in single, functioning membrane protein complexes,” Nature443(7109), 355–358 (2006).
[CrossRef] [PubMed]

Betzig, E.

T. A. Planchon, L. Gao, D. E. Milkie, M. W. Davidson, J. A. Galbraith, C. G. Galbraith, and E. Betzig, “Rapid three-dimensional isotropic imaging of living cells using Bessel beam plane illumination,” Nat. Methods8(5), 417–423 (2011).
[CrossRef] [PubMed]

Billinton, N.

N. Billinton and A. W. Knight, “Seeing the wood through the trees: a review of techniques for distinguishing green fluorescent protein from endogenous autofluorescence,” Anal. Biochem.291(2), 175–197 (2001).
[CrossRef] [PubMed]

Booth, M.

Brasselet, S.

E. J. Peterman, S. Brasselet, and W. E. Moerner, “The fluorescence dynamics of single molecules of green fluorescent protein,” J. Phys. Chem. A103(49), 10553–10560 (1999).
[CrossRef]

Burghardt, T. P.

D. Axelrod, T. P. Burghardt, and N. L. Thompson, “Total internal reflection fluorescence,” Annu. Rev. Biophys. Bioeng.13(1), 247–268 (1984).
[CrossRef] [PubMed]

Bustamante, C.

J. R. Moffitt, Y. R. Chemla, K. Aathavan, S. Grimes, P. J. Jardine, D. L. Anderson, and C. Bustamante, “Intersubunit coordination in a homomeric ring ATPase,” Nature457(7228), 446–450 (2009).
[CrossRef] [PubMed]

Cahalan, M. D.

M. D. Cahalan and I. Parker, “Choreography of cell motility and interaction dynamics imaged by two-photon microscopy in lymphoid organs,” Annu. Rev. Immunol.26(1), 585–626 (2008).
[CrossRef] [PubMed]

Campbell, R. E.

H. W. Ai, J. N. Henderson, S. J. Remington, and R. E. Campbell, “Directed evolution of a monomeric, bright and photostable version of Clavularia cyan fluorescent protein: structural characterization and applications in fluorescence imaging,” Biochem. J.400(3), 531–540 (2006).
[CrossRef] [PubMed]

Carter, N. J.

N. J. Carter and R. A. Cross, “Mechanics of the kinesin step,” Nature435(7040), 308–312 (2005).
[CrossRef] [PubMed]

Cella Zanacchi, F.

F. Cella Zanacchi, Z. Lavagnino, M. Perrone Donnorso, A. Del Bue, L. Furia, M. Faretta, and A. Diaspro, “Live-cell 3D super-resolution imaging in thick biological samples,” Nat. Methods8(12), 1047–1049 (2011).
[CrossRef] [PubMed]

Chandler, J. H.

M. C. Leake, J. H. Chandler, G. H. Wadhams, F. Bai, R. M. Berry, and J. P. Armitage, “Stoichiometry and turnover in single, functioning membrane protein complexes,” Nature443(7109), 355–358 (2006).
[CrossRef] [PubMed]

Chemla, Y. R.

J. R. Moffitt, Y. R. Chemla, K. Aathavan, S. Grimes, P. J. Jardine, D. L. Anderson, and C. Bustamante, “Intersubunit coordination in a homomeric ring ATPase,” Nature457(7228), 446–450 (2009).
[CrossRef] [PubMed]

Chen, L.

W. Ji, P. Xu, Z. Li, J. Lu, L. Liu, Y. Zhan, Y. Chen, B. Hille, T. Xu, and L. Chen, “Functional stoichiometry of the unitary calcium-release-activated calcium channel,” Proc. Natl. Acad. Sci. U.S.A.105(36), 13668–13673 (2008).
[CrossRef] [PubMed]

Chen, Y.

W. Ji, P. Xu, Z. Li, J. Lu, L. Liu, Y. Zhan, Y. Chen, B. Hille, T. Xu, and L. Chen, “Functional stoichiometry of the unitary calcium-release-activated calcium channel,” Proc. Natl. Acad. Sci. U.S.A.105(36), 13668–13673 (2008).
[CrossRef] [PubMed]

Cheng, W.

Chu, S.

T. Ha, I. Rasnik, W. Cheng, H. P. Babcock, G. H. Gauss, T. M. Lohman, and S. Chu, “Initiation and re-initiation of DNA unwinding by the Escherichia coli Rep helicase,” Nature419(6907), 638–641 (2002).
[CrossRef] [PubMed]

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S. D. Smith, M. Shatsky, P. S. Cohen, R. Warnke, M. P. Link, and B. E. Glader, “Monoclonal antibody and enzymatic profiles of human malignant T-lymphoid cells and derived cell lines,” Cancer Res.44(12 Pt 1), 5657–5660 (1984).
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Figures (9)

Fig. 1
Fig. 1

(a) Experimental setup for single-molecule TPF imaging. Cells were immobilized onto the inner surface of the microfluidic chamber. The laser focus was positioned inside the cell. Lateral scanning of the chamber driven by a 3D motion stage (ESP300, Newport) allows searching of EGFP molecules inside the cell. (b) Sup-T1 cells attached non-specifically onto the coverslip surface.

Fig. 2
Fig. 2

(a) Three independent repeats of cell autofluorescence recorded during one-dimensional scanning of the laser focus across a fixed sup-T1 cell. Cell boundaries were marked by arrows. The gray traces were raw data at 50 nm step size while the red traces were boxcar averaged and decimated with a window size of 10. Traces were shifted along y-axis for clarity of display. (b) Under constant laser illumination, the autofluorescence from a single spot within a fixed sup-T1 cells increases very slowly with time. The gray traces were raw data at 1 Hz while the red traces were boxcar averaged and decimated with a window size of 10. Traces were shifted along y-axis for clarity of display.

Fig. 3
Fig. 3

Representative photobleaching trajectories recorded from EGFP inside sup-T1 and 293T cells. Experimental traces are shown together with corresponding traces simulated with a custom-written Matlab program. (a) Single-step photobleaching in supT-1 cells. Traces are arbitrarily shifted along y axis for clarity of display. (b, c) Two-step photobleaching in supT-1 cells. (d) Single-step photobleaching in 293T cells. Traces are arbitrarily shifted along y axis for clarity of display. (e, f) Two-step photobleaching in 293T cells.

Fig. 4
Fig. 4

Statistics of single EGFP fluorescence inside sup-T1 cell. (a) Histogram of fluorescence intensity (bin size of 12 a.u.). The black curve is fitted by a Gaussian function. (b) Histogram of fluorescence on-time before photobleaching (bin size of 20 s). The black curve is fitted by a single exponential decay.

Fig. 5
Fig. 5

Simulation of single-molecule bleaching trajectories. For an ideal transition that occurs at 30 s, ~90% traces show clear transitions in the presence of Gaussian noise, as shown in (a), while ~10% traces show transitions with a slope as shown in (b), which is similar to the black trace in Fig. 3(a).

Fig. 6
Fig. 6

(a) The TPF excitation volume has a Gaussian profile. ‘r’ marks the radial distance of a fluorophore away from the center of the laser focus. (b) TPF intensity distribution in the absence of measurement noise for EGFP molecules that are randomly located within the excitation volume. (c) In the presence of uncertainties from repeated measurements (d = 10), the asymmetric distribution in (b) changes to become a Gaussian distribution, with peak centered around 50. (d) Increase in the experimental uncertainty d shifts the mean of the Gaussian profile to lower values (d value increases in the order: cyan 5, blue 5.5, green 6.2, red 7.1 and black10).

Fig. 7
Fig. 7

Measurement of molecule depth inside a stained sup-T1 cell. (a) Cartoon representation of the experimental method. (b-e) Four independent examples of single EGFP molecules identified from these measurements. For each example, the stepwise bleaching of the single molecule TPF trajectory is shown on the left. The bright field image of the cell is shown in the middle, which also corresponds to the z-plane at which the single-molecule TPF signal was collected. The blurred cell images on the right shows the cell images after the chamber was moved away from the objective by a distance z so that the laser focus fell on the edge of the cell. Red star marks the position of the laser focus. The scale bar is 5 micron.

Fig. 8
Fig. 8

EGFP fluorescence intensity as a function of laser power. Different symbols represent measurements from different spots within a 293T cell. Straight lines show linear fits in a double logarithmic scale, which give an average slope of 2.10 ± 0.01.

Fig. 9
Fig. 9

(a) Time courses of single-molecule fluorescence of mTFP1.0 inside supT-1 cells. Experimental traces are shown together with corresponding traces simulated with a custom-written Matlab program. Traces are arbitrarily shifted along y axis for clarity of display. (b) Histogram of fluorescence intensity (bin size of 15 a.u.). The black curve is fitted by a Gaussian function. (c) Histogram of fluorescence on-time before photobleaching (bin size of 3 s). The black curve is fitted by a single exponential decay.

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