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Single-Molecule Spectroscopy and Imaging of Biomolecules in Living Cells

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Single-Molecule Spectroscopy and Imaging of Biomolecules in Living Cells Anal. Chem. 2010, 82, 2192–2203 Single-Molecule Spectroscopy and Imaging of Biomolecules in Living Cells Samuel J. Lord, Hsiao-lu D. Lee, and W. E. Moerner* Department of Chemistry, Stanford University, Stanford, California 94305-5080 The number of reports per year on single-molecule imaging instance, the shape of the distribution may be skewed or reveal experiments has grown roughly exponentially since the first multiple subpopulations, which may offer insight into underlying successful efforts to optically detect a single molecule were mechanisms. Each single molecule is a local reporter on the completed over two decades ago. Single-molecule spectros- makeup and conditions of its immediate surroundings, its “na- copy has developed into a field that includes a wealth of noenvironment”, and thus acts as a readout of spatial heterogene- experiments at room temperature and inside living cells. The ity of a sample. SMS also measures time-dependent processes that fast growth of single-molecule biophysics has resulted from are not necessarily synchronized throughout the sample or its benefits in probing heterogeneous populations, one population. For example, multiple catalytic states of an enzyme molecule at a time, as well as from advances in microscopes will be convolved with all the states of other copies in an ensemble, and detectors. This Perspective summarizes the field of live- whereas a SMS experiment can measure uncorrelated stochastic cell imaging of single biomolecules. transitions of a single enzyme. SMS also has the ability to observe intermediate states or rare events, given that the instruments have Single-molecule biophysics spans a range of experiments, from sufficient time resolution. force studies of single macromolecules using tweezers1-3 or Because living systems are highly complex samples, with cantilevers4 to in vitro assays of fluorogenic enzymatic turnovers.5 spatial and temporal heterogeneities that have biological relevance For instance, by decorating a biomolecule with many copies of a and with a wealth of processes that operate at the single- probe, researchers have studied single DNA strands,6,7 membrane biomolecule level, SMS is a powerful tool to better understand molecules,8 motors,9 and viruses.10 In this Perspective, we focus the processes involved in life. Without needing to synchronize instead on single-molecule spectroscopy and imaging (SMS) populations of biomolecules or cells, SMS is able to record the experiments that measure the signal from each individual fluo- time evolution of these samples, for instance showing the rescent label in living cells. Moreover, in the interest of space, sequence of events in a pathway. In many situations, fluctuations we will not discuss the related area of fluorescence-correlation and rare events may be essential to biological function, making 11 spectroscopy, although the method can probe the ensemble studying each single molecule that much more powerful. Finally, 12 dynamics of single emitters and has been applied to living cells. sparsely labeling a population of biomolecules (as is sufficient for The main reason for performing SMS is the ability to measure many SMS experiments) reduces the chances that the probe will the full distribution of behavior instead of a single population interfere with the higher-level biology one is studying. For these average, thus exposing normally hidden heterogeneities in com- reasons, SMS is quickly becoming a popular technique in plex systems. A full distribution of an experimental parameter biophysics and cell biology. provides more information than the ensemble average; for History of SMS and Biophysics. The optical absorption of single molecules was originally detected in solids at cryogenic * To whom correspondence should be addressed. E-mail: wmoerner@ 13 stanford.edu. temperatures by direct sensing of the absorbed light; subse- (1) Smith, S. B.; Finzi, L.; Bustamante, C. Science 1992, 258, 1122–1126. quently, researchers detected optical absorption by measuring the (2) Svoboda, K.; Schmidt, C. F.; Schnapp, B. J.; Block, S. M. Nature 1993, fluorescence from single emitters under similar conditions.14 In 365, 721–727. (3) Strick, T. R.; Allemand, J.; Bensimon, D.; Bensimon, A.; Croquette, V. the early experiments, optical saturation, spectral diffusion, photon Science 1996, 271, 1835–1837. antibunching, resonant Raman, electric field effects, and magnetic (4) Florin, E.; Moy, V.; Gaub, H. Science 1994, 264, 415–417. 15 (5) Rotman, B. Proc. Natl. Acad. Sci. U.S.A. 1961, 47, 1981–1991. resonances of single molecules were observed. Optical detection (6) Morikawa, K.; Yanagida, M. J. Biochem. 1981, 89, 693–696. of single molecules was eventually performed at room temperature (7) Perkins, T. T.; Quake, S. R.; Smith, D. E.; Chu, S. Science 1994, 264, from burst analysis in solution,16-18 in microdroplets,19 using near- 822–826. (8) Barak, L. S.; Webb, W. W. J. Cell Biol. 1982, 95, 846–852. (9) Mehta, A. D.; Reif, M.; Spudich, J. A.; Smith, D. A.; Simmons, R. M. Science (13) Moerner, W. E.; Kador, L. Phys. Rev. Lett. 1989, 62, 2535–2538. 1999, 283, 1689–1695. (14) Orrit, M.; Bernard, J. Phys. Rev. Lett. 1990, 65, 2716–2719. (10) Lakadamyali, M.; Rust, M. J.; Babcock, H. P.; Zhuang, X. Proc. Natl. Acad. (15) Moerner, W. E. J. Phys. Chem. B 2002, 106, 910–927. Sci. U.S.A. 2003, 100, 9280–9285. (16) Shera, E. B.; Seitzinger, N. K.; Davis, L. M.; Keller, R. A.; Soper, S. A. (11) Rigler, R.; Elson, E.; Elson, E. Fluorescence Correlation Spectroscopy, Chem. Phys. Lett. 1990, 174, 553–557. Springer Series in Chemical Physics; Schaefer, F. P., Toennies, J. P., Zinth, (17) Eigen, M.; Rigler, R. Proc. Natl. Acad. Sci. U.S.A. 1994, 91, 5740–5747. W., Eds.; Springer: Berlin, 2001; Vol. 65. (18) Nie, S.; Chiu, D. T.; Zare, R. N. Science 1994, 266, 1018–1021. (12) Hess, S. T.; Huang, S.; Heikal, A. A.; Webb, W. W. Biochemistry 2002, (19) Barnes, M. D.; Ng, K. C.; Whitten, W. B.; Ramsey, J. M. Anal. Chem. 41, 697–705. 1993, 65, 2360–2365. 2192 Analytical Chemistry, Vol. 82, No. 6, March 15, 2010 10.1021/ac9024889 2010 American Chemical Society Published on Web 02/17/2010 field tips,20 and by 3D nanoscale tracking of single emitters in environment inside cells, many bioconjugation reactions used in porous gels.21 vitro (e.g., maleimide with cysteine or N-hydroxysuccinimide ester As single-molecule techniques addressed biologically relevant with lysine) are not sufficiently selective. Therefore, bioorthogonal systems and samples at room temperature, biophysics quickly labeling reactions are necessary for targeting organic fluorophores 15,22,23 became an active target of SMS research. Single copies of to biomolecules of interest in the cell.31,32 More commonly, fluorescent proteins (FPs) were imaged, and the ability to control researchers have relied on the genetic expression of FPs for 24 FP photoswitching was demonstrated. Fo¨rster-resonance-energy single-molecule biophysics in living cells. (4) After an exogenous 25 transfer (FRET) was observed on the single-pair level, and the probe is targeted inside the cell, the difficulty of washing out diffusion of single emitters was recorded in a phospholipid unbound copies introduces further complications. Therefore, there 26 27-29 membrane. Single motor proteins were imaged, and the must be a method to reject spurious signal from nontargeted nucleotide-dependent orientations of single kinesin motors were fluorophores. For instance, this can be accomplished by employing measured.30 fluorogenic labeling reactions33,34 or by adjusting the detector Studying living cells can be significantly more difficult than in integration times to average out the signal (spread over many vitro samples or fixed cells, because a living cell is a complex pixels) from quickly diffusing unbound fluorophores.35,36 (5) environment with elaborate interactions among components and Finally, it is imperative that the experiment does not significantly cells exhibit continually changing states. Nevertheless, the reasons interfere with the relevant biology of the cell. High labeling ratios, that make living cells tricky to study are fundamental character- istics of biology, and better understanding these attributes is large probe size, photoradical production, and genetic manipula- critical to a deeper understanding of actual biological processes. tion can change phenotypes or even kill cells. Thus, careful See Table 1 for a selected timeline of SMS experiments with controls are necessary to ensure that the labeling technique, relevance to living cells. sample preparation, and imaging conditions do not alter the Basic Requirements for SMS in Living Cells. SMS tradi- physiology of interest. tionally requires the following: a transparent, nonfluorescent host matrix; molecules that are resolved by separating them in space PROBES AND LABELING (by more than the diffraction limit of ∼200 nm), time, or Probes. As FPs are powerful tools used extensively in wavelength; and probes that are efficient absorbers, highly biological imaging,37,38 it is not surprising that they are also fluorescent, and exceptionally photostable. 39,40 Imaging single emitters in living cells introduces further important for live-cell SMS. To minimize background, longer- challenges. (1) In order to maintain low background counts, one wavelength FPs (e.g., EYFP and other orange- or red-emitting must avoid cellular autofluorescence, the result of exciting FPs) are most desirable; therefore, there has been a sustained endogenous cellular fluorophores (e.g., flavins, NADH, tryp- effort to tune FPs to longer wavelengths and to impart other tophan). Autofluorescence can be avoided by selecting an imaging beneficial qualities (e.g., photostability, brightness, monomeric wavelength longer than about 500 nm, where biological fluoro- or tandem dimers).41 phores typically do not absorb, and using cell growth media and The major drawback to FPs is that they are generally an order imaging buffers that are free of fluorophores. (2) The cell of magnitude less photostable than many small organic fluoro- membrane is a significant barrier to cell entry, because the probe phores, which can emit millions of photons before photoblea- must be able to pass through the hydrophobic lipid bilayer.
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