![]() ![]() The most common confocal technology is that of confocal laser scanning microscopy (CLSM). Its use is popular because it has several benefits over conventional widefield microscopy, namely in its ability to remove out-of-focus signal, capture information from a reduced depth of focus, image discreet optical sections in thick samples and so create high contrast 3D image sets. A similar principal applies to a pinhole camera which uses a pinhole to obtain crisp images without a lens.Ĭonfocal microscopy is now a routinely used optical technique across a wide range of biological sciences, from plant science to mammalian models. Therefore only light from one plane is in focus to pass through the pinhole or as the name suggest 'confocal'. Conversely, light from the plane below the imaged plane (denoted in yellow) focuses before the pinhole and fails to pass through the pinhole. Light from the plane above the imaged plane (denoted in red) is focussed above the pinhole so most of the light fails to pass through the pinhole. It is these benefits that make confocal microscopy so popular and why you find one in most core imaging facilities.Īs can be seen in Figure 2 (inset), only light (denoted by green) from the right plane passes through the pinhole. The confocal principle rejects out-of-focus light through the use of a pinhole, with the added benefit of an improvement in lateral and axial resolution. As the sample increases in thickness, the ability to capture fine detail above out-of-focus signal becomes increasingly challenging. The fluorescence emitted by the specimen outside the focal plane of the objective interferes with the resolution of in focus features. In conventional widefield optical microscopy the specimen is bathed in the light used to excite fluorophores ( Figure 1a). ![]() An Overview of Spinning Disk Confocal Microscopy ![]()
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December 2022
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