Single-molecule resolution

PS-P2VP copolymers with a miktoarm starlike architecture deposited onto mica or Si wafers have been investigated by Kiryi et al. [116]. These authors studied, using AFM with molecular resolution, single-molecule conforma-... 

Greenleaf, W. J., Woodside, M. T., Block, S. M., High Resolution, Single molecule Measurements of Biomolecular Motion, Annu. Rev. Biophys. Biomol. Struct. 2007, 36, 171 190. 

Koopman, M., Cambi, A., de Bakker, B. I., Joosten, B., Figdor, C. G, van Hulst, N. R, and Garcia-Parajo, M. R, Near-field scanning optical microscopy in liquid for high resolution single molecule detection on dendritic cells, FEBS Letters, 573, 6-10, 2004. 

Campos, L.A., Liu, J., Wang, X., Ramanathan, R., English, D.S. A photoprotectirai strategy for microsecond-resolution single-molecule flumescence spectroscopy. Nat. Methods 8, 143-146 (2011)... 

An important point for all these studies is the possible variability of the single molecule or single particle studies. It is not possible, a priori, to exclude bad particles from the averaging procedure. It is clear, however, that high structural resolution can only be obtained from a very homogeneous ensemble. Various classification and analysis schemes are used to extract such homogeneous data, even from sets of mixed states [69]. In general, a typical resolution of the order of 1-3 mn is obtained today. 

One interesting new field in the area of optical spectroscopy is near-field scaiming optical microscopy, a teclmique that allows for the imaging of surfaces down to sub-micron resolution and for the detection and characterization of single molecules [, M]- Wlien applied to the study of surfaces, this approach is capable of identifying individual adsorbates, as in the case of oxazine molecules dispersed on a polymer film, illustrated in figure Bl.22,11 [82], Absorption and emission spectra of individual molecules can be obtamed with this teclmique as well, and time-dependent measurements can be used to follow the dynamics of surface processes. 

Figure Bl.22.11. Near-field scanning optical microscopy fluorescence image of oxazine molecules dispersed on a PMMA film surface. Each protuberance in this three-dimensional plot corresponds to the detection of a single molecule, the different intensities of those features being due to different orientations of the molecules. Sub-diffraction resolution, in this case on the order of a fraction of a micron, can be achieved by the near-field scaiming arrangement. Spectroscopic characterization of each molecule is also possible. (Reprinted with pennission from [82]. Copyright 1996 American Chemical Society.)...

Moerner W E 1996 Fligh-resolution optical spectroscopy of single molecules in solids Acc. Chem. Res. 29 563-71... 

Figure Cl.5.4. Comparison of near-field and far-field fluorescence images, spectra and lifetimes for the same set of isolated single molecules of a carbocyanine dye at a PMMA-air interface. Note the much higher resolution of the near-field image. The spectmm and lifetime of the molecule indicated with the arrow were recorded with near-field excitation and with far-field excitation at two different excitation powers. Reproduced with pennission from Trautman and Macklin [125].

A significant development in the FRET imaging field has been the systematic implementation of spectral resolution [15-20], including D-A population analysis [8, 19] (see also Chapter 8), often in the context of single-molecule determinations [21-26] see focus issue on this subject, Nature Methods, June 2008. Invariably, photobleaching phenomena [21, 27, 28] intervene either as a hindrance (that can be minimized, CLEM [29]) or a facilitation of the FRET determination [1, 30], The equally important issue of background suppression or compensation can be achieved by novel means photon-free (bio)chemical instead of photonic excitation... 

FIGURE 2-10 Tracking a gold particle attached to a single molecule of phosphatidyl ethanolamine. What appears to be simple Brownian diffusion at a time resolution of 33 ms per video frame (A) is revealed to actually consist of fast hop diffusion by recording 300 times faster (B) at 110 ps per video frame. In (A) each color represents 60 frames = 2 seconds. In (B) each color indicates an apparent period of confinement within a compartment and black indicates intercompartmental hops. The residency time for each compartment is indicated. The hypothetical explanations are illustrated in part (C) and discussed in the text. Adapted from [29]. 

LuskinsPB, OatesT. Single molecule high-resolution structure and electron conduction of photosystem II from scanning tunneling microscopy and spectroscopy. Bio-chem Biophys Acta 1998 1409 1-11. 

Ultimate temporal and spatial resolution femtoseconds, femtoliters, femtomoles and single-molecule detection... 

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