( fluorophore (fluorochrome) is a fluores...)A new wide field-of-view confocal imaging system and its applications in drug discovery and pathology

1.1.2. Confocal laser scanning microscopy

There are two technical aspects in confocal laser scanning microscopy (CLSM),^2-6 i.e., confocality and scanning. In an ideal confocal imaging system, both the illumination and the detection are confined to a diffraction-limited spot. The image is recorded by precisely controlled raster-scanning of the focused laser spot across the specimen plane or vice versa. The scanning can be realized in various ways, such as x-y stage-stage scanning, x-y beam- beam scanning, x-y beam-stage scanning, or objective lens scanning.²⁵

In a basic confocal imaging configuration, the point illumination source, the specimen, and the detector pinhole are located at conjugate focal planes, hence the name "confocal". Although the practical confocal imaging systems today are somewhat different from the basic form, the operating principle is still the same, i.e., both the detection pinhole and objective lens take part in the image formation. The confocal effect comes from the confocal pinhole aperture, which prevents out-of-focus light from being detected. In reflectance and fluorescence imaging, the rejection of such optical "noise" is realized in two ways. The point illumination at the specimen reduces and the confocal pinhole at the detector further rejects the \noise" from the areas adjacent to the detection volume being imaged and the sections away from the focal plane. Therefore, confocal microscopy offers greater spatial resolution (a theoretical improvement of a factor of 1.4) than conventional widefield microscopes. With confocal microscopy, it is possible to look deep into cells and tis
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