Total internal reflection fluorescence microscopy (TIR-FM) is one of the techniques widely used for single molecule detection both in vitro and in vivo (2, 5). When a light beam illuminates the meniscus of two media obliquely from a high to a low diffractive index with an incident angle greater than the critical angle of total internal reflection, an evanescent electromagnetic field rises from the interface into the medium with the lower diffractive index (12). The decay length of the evanescent field along the depth of the field, which is dependent on the incident angle, is typically one hundred to several hundred nanometers. Using the evanescent field for excitation, the excitation depth of a fluorescence microscope can be limited to a vary narrow range. This is one of the most effective ways to reduce background of fluorescence microscopy to achieve single-molecule imaging (13). This type of microscopy is called TIRFM. Various configurations are possible for TIR-FM (12). The setup of an “objective-type” TIR-FM (14), in which the excitation laser beam illuminates the specimen through the objective lens, is shown in Fig. 1A. As well as TIR-FM, conventional epifluorescence microscopy using a laser for excitation can also be used to visualize single fluorescent molecules in living cells (7).
Single-molecule imaging of EGF on the surface of living cells
A TIR-FM image of single molecules of EGF conjugated with a fluorophore Cy3 (Cy3-EGF) was visualized on the surface of living A431 cells (Fig. 1B). EGF is a small peptide hormone that induces cell proliferation. Since mouse EGF has only one reactive amino residue (at the amino terminus), it can be labeled with aminoreactive dyes with a dye / protein ratio of exactly 1 to 1. Binding of Cy3-EGF to the cell surface was completely inhibited by adding an excess amount of non-labeled EGF simultaneously with Cy3-EGF, indicating specific binding of Cy3-EGF to cell surface EGFR.
Changes of the fluorescence intensity from each spot were measured over time. Constant fluorescent intensity followed by single-step photobleaching was strong evidence of single-molecule detection (Fig. 1C). Singlemolecule detection was also confirmed by the quantum nature of the intensity distribution of fluorescent spots (Fig. 1D). A monoclonal anti-EGFR antibody (EGFR1) was conjugated with Cy3 (Cy3-EGFR1) and bound to cells in the same manner as Cy3-EGF, and the intensity distributions of the fluorescent spots were examined. Since EGFR1 can be labeled with multiple Cy3 molecules, the intensity distribution of Cy3-EGFR1 spots on the cell surface could be fitted to the sum of 2 or 3 Gaussian distribution functions. The results after fitting are consistent with Poisson distributions of the number of Cy3 to EGFR1, assuming the first, the second and the third components represent EGFR1 conjugated with one, two, and three Cy3 molecules, respectively. Using the same method, the fluorescence intensity distribution of Cy3-EGF on the cell surface was fitted to the sum of two Gaussian functions. The mean of the intensity distribution of the first component of Cy3-EGF was the same as that for Cy3-EGFR1, suggesting that this component represents single molecules of Cy3-EGF (6). Using similar techniques, we have observed singlemolecules of nerve growth factor and cAMP, which were both conjugated with chemical fluorophores, Ras and Rho family small G-proteins and a serine/threonine kinase Raf1, which were tagged with green fluorescent proteins (GFP) in living cells (8, 15).0%;
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