What are the principles of confocal microscopy?
The principle of confocal microscopy is the elimination of out-of-focus light, thus producing a high z-resolution image. Confocal fluorescence microscopes achieve this via two principal mechanisms. First, incident light is focused to a particular point within the specimen by passing it through a very small pinhole. The focusing helps to limit the excitation of fluorophores above and below the plane of focus. Second, any emission that is above or below the plane of focus is blocked from reaching the detector by passing it through the same pinhole. The specimen is placed in the light path at a conjugate focal plane such that movement in the vertical (z) direction keeps the focus at a fixed distance from the objective, which effectively scans in layers through the specimen. Passing light through a pinhole limits the illumination of the sample to one particular spot, a spot much smaller than the typical field of view. To fully develop an image, light must be delivered to every point of the
