Image Scanning Microscopy

A new microscopy technique is introduced, image scanning microscopy (ISM), which combines conventional confocal-laser scanning microscopy with fast wide-field CCD detection. The technique allows for doubling the lateral optical resolution in fluorescence imaging. The physical principle behind ISM is similar to structured illumination microscopy, by combining the resolving power of confocal-laser scanning microscopy with that of a wide-field imaging microscopy. This Letter describes the theoretical foundation and experimental realization of ISM.

Figure 1

ISM Setup, (1) Excitation with supercontinuum white light source and acousto-optic tunable filter, (2) $90/10$ nonpolarizing beam splitter cube, (3) major dichroic mirror, (4) piezo scan mirror, (5) $4f$ telescope, (6) UPL APO 60x W microscope objective, (7) beam diagnostic camera, (8) confocal aperture, and (9) EM CCD detection camera system.

Figure 2

Image of a single fluorescent bead of 100 nm diameter. Left panel: CLSM image; middle panel: ISM image; right panel: Fourier-weighted ISM image. The horizontal bar in the left panel has a length of $1\mu \mathrm{m}$.

Figure 3

Cross section through one of the bead images in Fig. 2.