Abstract. Scanning confocal microscopes offer improved rejection of out-of-focus noise and greater resolution than conventional imaging. In such a microscope, the imaging and condenser lenses are identical and confocal. These two lenses are replaced by a single lens when epi-illumination is used, making confocal imaging particularly applicable to incident light microscopy. We describe the results we have obtained with a confocal system in which scanning is performed by moving the light beam, rather than the stage. This system is considerably faster than the scanned stage microscope and is easy to use. We have found that confocal imaging gives greatly enhanced images of biological structures viewed withepifluorescence. The improvements are such that it is possible to optically section thick specimens with little degradation in the image quality of interior sections.

A history of the early development of the confocal laser scanning microscope in the MRC Laboratory of Molecular Biology in Cambridge is presented. The rapid uptake of this technology is explained by the wide use of fluorescence in the 80s. The key innovations were the scanning of the light beam over the specimen rather than vice-versa and a high magnification at the level of the detector, allowing the use of a macroscopic iris. These were followed by an achromatic all-reflective relay system, a non-confocal transmission detector and novel software for control and basic image processing. This design was commercialized successfully and has been produced and developed over 17 years, surviving challenges from alternative technologies, including solid-state scanning systems. Lessons are pointed out from the unusual nature of the original funding and research environment. Attention is drawn to the slow adoption of the instrument in diagnostic medicine, despite promising applications.

Current optical microscope objectives of low magnification have low numerical aperture and therefore have too little depth resolution and discrimination to perform well in confocal and nonlinear microscopy. This is a serious limitation in important areas, including the phenotypic screening of human genes in transgenic mice by study of embryos undergoing advanced organogenesis. We have built an optical lens system for 3D imaging of objects up to 6 mm wide and 3 mm thick with depth resolution of only a few microns instead of the tens of microns currently attained, allowing sub-cellular detail to be resolved throughout the volume. We present this lens, called the Mesolens, with performance data and images from biological specimens including confocal images of whole fixed and intact fluorescently-stained 12.5-day old mouse embryos.DOI: http://dx.doi.org/10.7554/eLife.18659.001

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