title: Condenser-free contrast methods for transmitted-light microscopy.
abstract: Phase contrast microscopy allows the study of highly transparent yet detail-rich specimens by producing intensity contrast from phase objects within the sample. Presented here is a generalized phase contrast illumination schema in which condenser optics are entirely abrogated, yielding a condenser-free yet highly effective method of obtaining phase contrast in transmitted-light microscopy. A ring of light emitting diodes (LEDs) is positioned within the light-path such that observation of the objective back focal plane places the illuminating ring in appropriate conjunction with the phase ring. It is demonstrated that true Zernike phase contrast is obtained, whose geometry can be flexibly manipulated to provide an arbitrary working distance between illuminator and sample. Condenser-free phase contrast is demonstrated across a range of magnifications (4-100×), numerical apertures (0.13-1.65NA) and conventional phase positions. Also demonstrated is condenser-free darkfield microscopy as well as combinatorial contrast including Rheinberg illumination and simultaneous, colour-contrasted, brightfield, darkfield and Zernike phase contrast. By providing enhanced and arbitrary working space above the preparation, a range of concurrent imaging and electrophysiological techniques will be technically facilitated. Condenser-free phase contrast is demonstrated in conjunction with scanning ion conductance microscopy (SICM), using a notched ring to admit the scanned probe. The compact, versatile LED illumination schema will further lend itself to novel next-generation transmitted-light microscopy designs. The condenser-free illumination method, using rings of independent or radially-scanned emitters, may be exploited in future in other electromagnetic wavebands, including X-rays or the infrared.
authors.affiliation: Royal Academy of Engineering Research Fellow, Institute of Biophysics, Imaging & Optical Science (IBIOS), School of Electrical and Electronic Engineering, The University of Nottingham, University Park, Nottingham, U.K.
authors.forename: K F
authors.initials: KF
authors.lastname: Webb
mesh_terms: D000818:Animals; D002460:Cell Line; D006801:Humans; D008027:Light; D051379:Mice; D008856:Microscopy, Fluorescence / Q000379:methods; D008858:Microscopy, Phase-Contrast / Q000379:methods*; D059828:Plant Cells
keywords: Condenser-free contrast enhancement; LED illumination; Rheinberg illumination; Zernike phase contrast microscopy; darkfield microscopy; scanning probe microscopy