Abstract
The limits of conventional light microscopy (“Abbe-Limit“) depend critically on the numerical aperture (NA) of the objective lens. Imaging at large working distances or a large field-of-view typically requires low NA objectives, thereby reducing the optical resolution to the multi micrometer range. Based on numerical simulations of the intensity field distribution, we present an illumination concept for a super-resolution microscope which allows a three dimensional (3D) optical resolution around 150 nm for working distances up to the centimeter regime. In principle, the system allows great flexibility, because the illumination concept can be used to approximate the point-spread-function of conventional microscope optics, with the additional benefit of a customizable pupil function. Compared with the Abbe-limit using an objective lens with such a large working distance, a volume resolution enhancement potential in the order of 104 is estimated.
Highlights
Due to novel developments in optical technology and photophysics[1] it has become possible to radically overcome the classical diffraction limit for high numerical aperture (NA) objective lenses of conventional far-field microscopy[2]
Due to the high NA objective lenses used in these studies, the thickness of an object which can be analyzed in 3D with such a high resolution in many approaches is presently restricted to a maximum of several tens of μm
N) emitting collimated beams are arranged at defined positions rnio=t p(rxoiv, iydi,eztir)ualryocuonlldimthaeteodpbtiecaaml asx;iisnosftetahde focusing distributed aperture microscopy (DAM) system. (Note that real illumination systems will often Gaussian beam optics may be used to attribute for this deficiency; see Supplementary Notes 3 and 4.1)
Summary
Due to novel developments in optical technology and photophysics[1] it has become possible to radically overcome the classical diffraction limit for high NA objective lenses (ca. 200 nm laterally, 600 nm along the optical axis; called the Abbe-limit) of conventional far-field microscopy[2]. For developing single fluorophore detection as the basis for single molecule localization microscopy using photoactivated proteins; and to Stefan Hell for the development of Stimulated Emission Depletion (STED) Microscopy, a “focused nanoscopy” method[3] Using these approaches, both optical resolution (smallest distance detectable between two adjacent point sources) and structural resolution (smallest structural detail determined based on the density of point sources resolved) has been enhanced very substantially. Due to the high NA objective lenses used in these studies, the thickness of an object which can be analyzed in 3D with such a high resolution in many approaches is presently restricted to a maximum of several tens of μm This means that in most cases, only individual cells arranged in monolayers on glass substrates, or thin tissue sections can be studied at highest resolution. In many applications a field of view many times larger than 100 μm and a specimen thickness in the millimeter to centimeter range should be highly desirable[7]
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