While in a normal microscope the image plane of the observation is isolated from the neighbouring axial planes by overlapping the focal plane of the objective lens with the detector, in a confocal microscope this isolation is achieved by illuminating the sample with a diffraction-limited point of light and filtering stray light by using a pinhole in the path of collected light at the conjugate focus of this point of light to achieve this isolation and thus improve resolution.
Only the light reflected back from the conjugate sample layer can pass through the pinhole in the collection light path, while the rest of the irrelevant sample layer reflections are blocked by the pinhole. This gives a significant increase in resolution. Shown below is a side-by-side comparison of multidimensional fluorescence microscopy and confocal microscopy of the same thick sample. When a series of images are taken at different focal planes, an image commonly referred to as a "z-stack" is produced, which shows the resolution and contrast gains provided by confocal microscopy and the underlying reasons for these gains. It can be seen that examining the image at the top of the stack where the imaging plane is above the tissue reveals a large amount of scattered light in the fluorescence image, whereas the confocal microscope image appears black. This reduction in PSF in the axial direction directly contributes to the difference in resolution observed at the optical interface in the middle of the z-stack.
Another feature of laser scanning confocal microscopy is that it is a scanning imaging technique. Traditional wide-field illumination techniques illuminate the entire sample so that the image can be captured directly by the naked eye or a detector, but LSCM uses one or more focused beams of light to scan through the sample, so that the resulting image is called an optical slice, as shown in the following diagram. The difference between traditional wide-field illumination and laser scanning confocal illumination is shown below.
Therefore, a practical way of modern confocal microscopy is shown in the figure below. The excitation light from the laser is passed through a dichroic mirror and scanned through a pair of galvanometer mirrors in the x-direction and y-direction of the sample, and the excitation (or reflection) of the sample passes through a pinhole into the PMT detector to be recorded, and the recorded scanned image is reconstructed by a computer to produce an actual image of the sample.
