Single-molecule observation of a higher order structure of a proton channel in influenza virus

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A laser-scanning confocal microscope was developed for three-dimensional (3D) imaging and co-localization of single biomolecules immobilized at a temperature of 1.5 K. The performance was demonstrated by taking 3-color 3D emission image of single quantum dots and 3D co-localization of single dots. The 3D image size of single dot with excitation wavelength of 635, 532, and 405 nm were all within 1.2 times of the diffraction-limited size. The 3D co-localization precision of single dot in images taken with 635 and 532 nm excitation was 3 nm in the lateral (x, y) and 18 nm in the axial (z) directions.