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Single-Molecule Imaging in Live Cells

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Handbook of Single-Molecule Biophysics

Abstract

This chapter provides a comprehensive overview of how single-molecule imaging is achieved in live cells. The main focus is on fluorescent proteins, which are the most widely used fluorescent labels for live-cell imaging. The chromophore structures and the associated photochemical and photophysical properties of fluorescent proteins are discussed in detail, with a particular focus on how they influence single-molecule imaging in live cells. A few fluorescent proteins in the yellow-to-red spectral range, including newly discovered photoinducible ones, are selected for more detailed discussions due to their superior properties in single-molecule imaging. Special considerations for live-cell imaging and general instrumentations for single-molecule detection are also described. Finally, a few representative applications using single-molecule imaging in live cells are provided to illustrate how important biological knowledge can be obtained using this powerful technique.

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Notes

  1. 1.

    Here and throughout, single-molecule imaging refers to single-fluorophore imaging. Alternatively, a single molecule can be imaged by labeling a single molecule using multiple fluorophores or detected by amplifying the fluorescent signal using a fluorogenic substrate [3–10]. These approaches are discussed briefly at the end of the chapter.

  2. 2.

    In the field of fluorescent proteins, the wavelength of each color is defined slightly differently from what is defined in physics. Following the convention of the field, in this chapter the wavelength of each color is as follows: violet: ∼400 nm; blue: ∼480 nm; green: ∼510 nm; yellow: ∼550 nm; orange: ∼580 nm; red: ∼620 nm.

  3. 3.

    It is unclear why Tyr66 is universally conserved in all GFP-like proteins in nature. In engineered FPs it was found that other two aromatic groups, Trp and Phe, or even His, can substitute Tyr66 and produce blue-shifted fluorescence as in CFPs and BFPs [47,62,63].

  4. 4.

    There is one recent study reporting the generation of the first red fluorescence-emitting derivative (excitation and emission maxima at 555 and 585 nm, respectively) of avGFP [106]. Purified proteins or cells expressing this mutant, however, showed both green (strong) and red (dim) emission. It is likely that only a small population of the mutant matures to the red chromophore. Nonetheless, this study indicates that the full mutagenesis potential of GFP has yet to be reached.

  5. 5.

    Here the term “photoinducible FPs” is introduced to characterize the common feature of this class of fluorescent proteins: They all undergo light-induced processes to change their fluorescence emission properties. In the literature, the terms photoactivation, photoswitching, and photoconversion are used, sometimes interchangeably. In the context of this chapter photoactivation refers specifically to the process of fluorescence turn-on from a dark state irreversibly (off → on); photoswitching to the process of reversible fluorescence turn-on and turn-off cycles (off ↔ on); and photoconversion to the process of light-induced change of excitation/emission spectra between two color regions (on1 on2).

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  1. Johns Hopkins University School of Medicine, 708 WBSB, Baltimore, MD 21205, USA

    Jie Xiao

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  1. Institut für Biophysik, Universität Linz, Altenberger Straße 69, Linz, 4040, Austria

    Peter Hinterdorfer

  2. Harvard Medical School, Harvard University, Longwood Ave. 250, Boston, 02115, U.S.A.

    Antoine Oijen

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Xiao, J. (2009). Single-Molecule Imaging in Live Cells. In: Hinterdorfer, P., Oijen, A. (eds) Handbook of Single-Molecule Biophysics. Springer, New York, NY. https://doi.org/10.1007/978-0-387-76497-9_3

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