Condenser-free contrast methods for transmitted-light microscopy.

K. F. WEBB

doi:10.1111/jmi.12181

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.

Highlights

Phase contrast microscopy is a ubiquitous imaging technique in the biological sciences, providing a cheap and effective solution for the visualization of transparent samples such as living cells
This paper presents, characterizes, and generalizes a condenser-free illumination schema which produces high-quality Zernike phase contrast and darkfield contrast using rings of light emitting diodes (LEDs); an image of which is projected into the objective back focal plane (BFP) using simple lens optics
The condenser-free phase contrast illumination schema presented here (Fig. 1B) exploits simple lens optics to ensure that a ring of LED sources placed at some distance from the sample opposite the objective lens produces an image of the emitters in the objective BFP

Summary

Introduction

Phase contrast microscopy is a ubiquitous imaging technique in the biological sciences, providing a cheap and effective solution for the visualization of transparent samples such as living cells. The basic principle is as follows: illuminating light from an extended source is collimated and an annulus of illumination is selected by inserting a ring-shaped mask into the aperture plane of the condenser assembly (Fig. 1 A). This illuminating annulus is focussed through the sample by the condenser lens as a hollow cone and, in interacting with the sample, is split into two partially coherent beams. In standard (‘positive’) phase contrast, the phase ring is made optically thinner than the rest such that direct light is phase advanced at the image plane by λ/2 relative to light diffracted by the sample (dashed lines), most of which misses the phase ring (Zernike, 1942b). Interference between the two beams produces constructive and destructive interference which creates intensity contrast at the image plane from minute differences in optical path within the sample (Zernike, 1942a)

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