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. 2010 Mar 15;82(6):2192-203.
doi: 10.1021/ac9024889.

Single-molecule Spectroscopy and Imaging of Biomolecules in Living Cells

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Single-molecule Spectroscopy and Imaging of Biomolecules in Living Cells

Samuel J Lord et al. Anal Chem. .
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Abstract

The number of reports per year on single-molecule imaging experiments has grown roughly exponentially since the first successful efforts to optically detect a single molecule were completed over two decades ago. Single-molecule spectroscopy has developed into a field that includes a wealth of experiments at room temperature and inside living cells. The fast growth of single-molecule biophysics has resulted from its benefits in probing heterogeneous populations, one molecule at a time, as well as from advances in microscopes and detectors. This Perspective summarizes the field of live-cell imaging of single biomolecules.

Figures

Figure 1
Figure 1
Generalized microscope configurations for single-molecule imaging in living cells.
Figure 2
Figure 2
Some general approaches to SMS in live cells.
Figure 3
Figure 3
Single molecules in cell membranes. (A) Cell-penetrating peptides labeled with a DCDHF organic fluorophore in the plasma membrane of mammalian cells. At low enough concentrations of labeled peptides, single molecules can be are visible as they interact with the top surface of the cell; at higher concentrations, it is obvious that the fluorophores are bright while in the membrane and in particular regions of the cytosol. These images were taken in epifluorescence mode, required in order to probe the top surface of the cell in widefield. From reference . (B) (left) A snapshot of single transmembrane proteins on the surface of a living mammalian cell. Antigen ligand peptides were labeled with Cy5 and allowed to bind strongly to the transmembrane proteins. This epifluorescence image represents 12×12 μm at the sample plane, with an integration time of 100 ms. (right) Examples of single-molecule trajectories. From references , . (C) Several successive trajectories of single copies of a fluorescently labeled substrate interacting with a nuclear pore complex in living cells. These measurements used a widefield configuration but added a 400-μm pinhole to restrict the imaging area and reduce background fluorescence. From reference (© 2004 The National Academy of Sciences of the USA).
Figure 4
Figure 4
Single molecules in bacteria. (A) FP-labeled MreB, an actin homolog, shows treadmilling through short MreB filaments in a living C. crescentus cell. Directional motion of MreB–FP was measured by imaging single copies of MreB–FP. Single molecules trace out the filaments and the cytoskeletal structure, exhibiting direction and zig-zag motions (bottom left). The diagrams in the center depict the mechanism of treadmilling and motion of MreB monomers in filaments. The cells in the upper right represent several trajectories of the movements of single MreB–FP, tracing out filaments. The + (toward the so-called “stalked” pole of the cell) and – (toward the “swarmer” pole) signs indicate the direction of the movement. See reference . (B) Gene expression visualized on the individual-cell and single-molecule scale. (top) Time-lapse movie of fluorescence images (yellow) overlaid with simultaneous white-light images (gray) show E. coli cells expressing single FP-labeled proteins (sporadic bursts of yellow). (bottom) Time traces of the expression of proteins along three particular cell lineages extracted from time-lapse fluorescence movies. The vertical axis is the number of protein molecules newly synthesized during the last three minutes. The dotted lines mark the cell division times. The time traces show that protein production occurs in random bursts, within which variable numbers of protein molecules are generated. From reference . (C) Single PopZ–FP molecules in a living C. crescentus cell. (left) Time-lapse visualization of two molecules in a cell, with colored lines tracking the distance moved between frames. One molecule (red) remains localized to the pole, and the other (green) has increased mobility. The tracks are overlaid on a transmitted light image of the cell, outlined in black. (right) A representation of the data from the experiment, showing the distance of the molecules from one pole as a function of time. The black horizontal dotted line marks the opposite pole; the red and green lines follow the stationary and mobilized molecules, respectively. From reference .
Figure 5
Figure 5
First live-cell super-resolution SMS experiments. (A) Clusters of hemagglutinin in the membrane of a living fibroblast cell. The right frame is a zoomed-in portion of the left image. The jagged border of the cluster helped eliminate some models for membrane rafts. From reference (© 2007 The National Academy of Sciences of the USA). (B) Super-resolution fluorescence image of C. crescentus stalks labeled with a Cy3–Cy5 covalent pair (yellow) superimposed on a white-light image of the cells (gray). From reference . (C) Time-lapse super-resolution images of FP-labeled MreB in living C. crescentus cell. (left) Quasi-helical structure of MreB in a stalked cell. (right) Midplane ring of MreB in a predivisional cell. Scale bars, 300 nm. From reference . (D) Imaging dynamics of an adhesion complex. Time-lapse super-resolution images in a small region of a live NIH 3T3 cell expressing a photoswitchable FP fused to paxillin. Multiple super-resolution images were obtained over an extended period to observe the morphologies and dynamics of the signal-transduction protein paxillin as the adhesion complex formed and elongated at the edge of the cells. These snapshots are high magnification of a single adhesion complex, revealing molecular organization during the elongation process. Scale bar, 500 nm. From reference .

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