Condenser-free contrast methods for transmitted-light microscopy

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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.

Lay Description

Biological cells are difficult to image using standard transmission microscopy as they are often very transparent to visible light. One solution to this problem, which won its inventor Frits Zernike the Nobel prize for Physics in 1953, is the phase contrast microscope. In this method a ring-shaped pattern of illumination is focussed through the sample in a hollow coneby a condenser lens. Light passing straight through the sample is slowed down slightly by a ring-shaped “phase plate” to bring it exactly out of step with light diffracted by the cells. When mixed together by the optics, the two waves create constructive and destructive interference, which creates contrast and therefore makes the cells visible. Even very modern microscopes still use the same method to perform this operation as has been used since the 1950s. Live cells are usually imaged using an “inverted” microscope, where the objective lens approaches the cells from underneath. The condenser lens can cause a problem as it is both physically bulky and must usually be positioned quite close to the sample, restricting access to the cells from above. This paper reports the development of a new way of creating phase contrast, by removing the condenser assembly entirely and instead creating the required circle of illumination using a ring of separate light emitting diodes (LEDs). By this method the system is made much simpler to align, and can be made in any physical size desired. This gives the flexibility to miniaturise the microscope significantly, or to build much larger rings which leave the space above the cells free for introducing electrodes to study their behaviour. The new design also allows the system to be used with cutting-edge scanning-probe microscopy methods, all the while maintaining excellent phase contrast imaging. In addition we show that removing the condenser lens allows several related methods such as darkfield microscopy and Rheinberg illumination, which create contrast allowing the sample to scatter light into the objective lens and therefore to appear as illuminated objects lit in front of a dark or contrasting-coloured background. As a whole, condenser-free illumination seems to provide a general solution to simplify several methods of contrast enhancement in biological imaging, and will be very useful when applied to the design of next generation microscopes in the future, using both visible light as well as other wavelengths such as X-rays and the infrared.

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