Resolution beyond the diffraction limit: 4PI-confocal-, STED-, and GSD- fluorescence microscopy

Abstract

In far-field light microscopy resolution is determined by diffraction. In a far-field light microscope such as the confocal scanning light microscope, the resolution is governed by the extent of the squared intensity distribution in the focal region. Precise measurements of the confocal PSF have shown that the axial and lateral resolution of a confocal microscope (NA=1.4 oil, 1= 633 nm) is 520nm and 200nm (FWHM), respectively. At a wavelength of 375nm, this amounts to a resolution of 300 nm (axial) and 120 nm (lateral), obtainable with a standard confocal microscope of high aperture.A 3-7 fold increase in axial resolution is achieved with a 4Pi-confocal microscope. The 4Piconfocal microscope uses two high numerical aperture objective lenses that are used coherently for illuminating or detecting the same point in the object space. The present paper deals with the latest developments in the field of 4Pi-confocal microscopy. The Optical Transfer Functions (OTF) of 4Piconfocal microscopies with 4Pi-illumination (type A), 4Pi-detection (type B), and 4Pi illumination and detection (type C) are measured and compared with their standard confocal counterpart.