Condenser-free contrast methods for transmitted-light microscopy

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 il- luminating 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

Problematic pigmented lesions: approach to diagnosis

A number of pigmented lesions are difficult to classify and raise the possibility of a melanoma diagnosis. Care should be exercised to exclude non-melanocytic lesions, and benign melanocytic entities, both of which can mimic melanoma histologically. In addition, the possibility of the lesion being a melanoma variant or epidermotropic metastasis should be considered. There will still be some cases that are difficult to resolve. These usually fall into one of three categories: atypical junctional melanocytic lesion versus early melanoma; naevus versus naevoid melanoma; and atypical Spitz, cellular blue, and deep penetrating naevi versus thick melanoma. These will pose problems even for experts. The atypical Spitz lesions are perhaps the most important category because they tend to be from younger individuals, the differential diagnosis is thick melanoma, and there is no single discriminating histological feature. Key Words: difficult diagnosis • pigmented lesions • melanom