4Pi microscope

Abstract: In a 4Pi confocal fluorescence microscope two opposing microscope objective lenses were used to illuminate a fluorescent object from both sides and to collect the fluorescence emissions on both sides. Constructive interference of either the illumination wave fronts in the common focus or the detection wave fronts in the common detector pinhole resulted in an axial resolution approximately four times higher than that in a confocal fluorescence microscope. A precise 4Pi confocal fluorescence microscope that uses simultaneous illumination was built. The full width at half-maximum of the depth discrimination was determined experimentally to be approximately 110 nm when lenses with a numerical aperture of 1.4, an excitation of 633 nm, and detection of approximately 725 nm were used.

Abstract: Utilizing single fluorescent molecules as probes, we prove the ability of a far-field microscope to attain spatial resolution down to 16 nm in the focal plane, corresponding to about 1/50 of the employed wavelength. The optical bandwidth expansion by nearly an order of magnitude is realized by a saturated depletion through stimulated emission of the molecular fluorescent state. We demonstrate that en route to the molecular scale, the resolving power increases with the square root of the saturation level, which constitutes a new law regarding the resolution of an emerging class of far-field light microscopes that are not limited by diffraction.

Abstract: Fourier imaging in the scanning microscope is considered. It is shown that there are two geometries of the microscope, which have been designated Type 1 and Type 2. Those of Type 1 exhibit identical imaging to the conventional microscope, whereas those of Type 2 (confocal microscopes) display various differences. Imaging of a single point object, two-point resolution and response to a straight edge are also considered. The effect of various arrangements using lenses with annular pupil functions is also discussed. It is found that Type 2 microscopes have improved imaging properties over conventional microscopes and that these may be further improved by use of one or two lenses with annular pupils.