There are many aspects to consider regarding the functionality and operation of a confocal microscope.
How Does a Confocal Microscope Work?
The following section explains the confocal principle in four steps.
1. Light transmission
Confocal microscopes transmit light via the objective lens onto the target. The light is focused, via an objective lens, in the focal point of the desired measurement area of the target.
2. Reflected light
The target positioned at the focal point reflects light back through the optical system, to a half mirror that directs the light to the light receiving element.
3. Reflected light passes through the pinhole
A pinhole placed in front of the light receiving element blocks out all out of focus light reflected from the sample.
Only light passing through the pinhole is focused; the rest of the light from the confocal microscope is absorbed by the mask area. If the target is not at the focal point, the light is focused on a point offset from the pinhole, which widens the light beam from the confocal microscope so that the light is no longer focused on the pinhole itself. This means that the amount of light that hits the light receiving element is reduced. The confocal microscope detects whether the target is at the focal point based on the intensity of the reflected light that returns to the light receiving element. As a result, light only reaches the light receiving element from the area of the target which is in focus, and is blocked from all other areas.