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State-of-the-Art Optical Microscopy

The spatial resolution of optical microscopy is limited to a few hundred nanometers by the diffraction limit. Near-field optical microscopy overcomes the diffraction barrier. However, with diffraction limited optics, recent development of fluorescence imaging techniques enables us to obtain various information from a single molecule: the three-dimensional position and orientation of a single molecule with high resolution. Novel fluorescence microscopy systems are developed and applied to polymer physics.

Super-resolution observation of single molecules

    A single dye molecule is observed as a circular spot with a half of the wavelength in a fluorescence micrograph; therefore, the structure smaller than 200 nm cannot be directly resolved. However, when we focus on the 'position' of the fluorescent spot, the localization accuracy is very high. The intensity profile of a single molecule at a position r0 observed in a fluorescence image can be well approximated by a two-dimensional Gaussin distribution: 
        I(r, r0) = I0exp(-(r-r0)2/w2), 
where r indicates the position in a pixel of the fluorescence image and w is the observed size of the molecule. The right figure indicates the histogram of the localization result for independent 300 images for a single perylenediimide molecule immobilized in a PMMA film. The standard deviation of the localization was 3.1 nm, indicating the high localization accuracy. This characteristics of the fluorescence detection is sometimes called FIONA (Fluorescence Imaging with One-Nanometer Accuracy).

    The spatial resolution of the fluorescence imaging would be greatly enhanced by FIONA. As mentioned above, FIONA can be applied when there exists only a single molecule in a field of view.  In conventional fluorescence microscopy measurement, the fluorescence emission from lots of dye molecules labeled to a sample is imaged on a two-dimensional detector (CCD camera etc.); therefore, the molecules are overlapped in the observed image and the FIONA cannot be applied.
    The super-resolution imaging by FIONA is achieved by the sequential observation of individual dye molecules introduced to the sample. Such the observation is possible with a photochromic dye, which has fluorescent and non-fluorescent isomers. The conversion between the isomers should be triggered by the irradiation at a wavelength different from that for the excitation of the fluorescent state. The conversion to the non-fluorescent state is performed by the backward reaction or the irreversible photo-bleaching. The super-resolution by this mechanism is called STORM (STochastic Optical Reconstruction Microscopy) or PALM (Photo-Activated Localization Microscopy).
    The diagram at the right-hand side shows the schematic process of PALM. At first, all of the dye molecule is converted to a non-fluorescent state; therefore, nothing is observed (A, at the upper left corner). Then, only one dye molecule is converted to a fluorescent state, which is observed in the fluorescence image (C). The position of this molecule can be determined with nanometric accuracy by FIONA. The coordinate is recorded in the reconstruction image (D). The observed molecule is converted to the non-fluorescent state. Then another dye molecule is activated (E) and analyzed by FIONA (F). By repeating the activation-observation-erase process, the coordinate for all of the molecules is sequentially determined. Finally, a high resolution image is reconstructed (L, at the upper right). The spatial resolution of the reconstruction image is determined by the accuracy of FIONA; therefore, the fluorescence image with 10-nm resolution is available. The combination with interferometric or astigmatic detection schemes enables the three-dimensional observation.
The procedure to build a PALM system is described in the following page.

Orientational imaging of single molecules

    Fluorescence imaging techniques can provide the information on the molecular orientation as well as the position. In many cases, the orientation of a molecule is evaluated by the polarization detection of the fluorescence emission. However, the polarization provides only the in-plane orientation, the azimuthal angle φ shown in the lower left of the figure.
    The fluorescence intensity from a single molecule is dependent on the angle between the emission direction and the transition dipole moment; therefore, the orientation of the molecule can be determined from the angular distribution of the fluorescence intensity. In the fluorescence microscopy observation, the emission from a molecule is focused on a camera and observed as a circular spot. On the other hand, in a defocus condition, the fluorescence is not converged on one point, resulting in a blurred patten. As shown in the right figure, the observed pattern is strongly dependent on the molecular orientation: both of the polar and azimuthal angles, θ and φ, respectively. Therefore, the three-dimensional orientation of a single molecule can be evaluated by the analysis of a fluorescence image in a defocus condition. By using the defocus fluorescence imaging method, the dynamics of a molecule can be completely tracked because both of the position and the orientation, (x, y, z, θ, φ), of the molecule is recorded in real time. We investigate the dynamics of polymer chains at a single chain level with this method.