Confocal (fluorescence) microscopy is a microscopic technique that provides true three dimensional imaging and resolution. The 3D resolution is attained by designing a microscope in such a way that all the light which is not coming from an in-focus plane is being blocked or removed afterwards. The way to achieve this is by putting a pinhole in front of the detector. In this way all the light originating from an in-focus plane will pass freely through the pinhole, whereas light coming from a out-of focus plane will largely be blocked by the pinhole. The light coming from the laser passes an excitation pinhole and is reflected by a dichroic mirror and focused by a microscope objective to a small spot on the sample. A dichroic mirror has the property that it reflects one wavelenght while transmitting others. Specific dichroic mirrors can be made for the relevant wavelength regions of excitation and emission. When the sample is excited, it will start to emit light in a random direction. A fraction of the emitted photons is collected by the microscope objective and imaged onto the detector. By putting a pinhole in front of the detector blurring form out-of focus planes is greatly reduced. The position of this pinhole is such that it is in a conjugate plane with both the plane of focus of the microscope objective and the point of the excitation of the laser, which is defined by the excitation pinhole. The effect of blocking out the out of focus contributions is also known as optical sectioning. It permits the imaging of separate (axial) slices within the specimen. The size of the pinhole, of course, determines how much background reduction can be realized. For a specific setting of the microscope only a single point in the sample is imaged at one time. In other words, confocal microscopy is a serial technique rather than a parallel one. To obtain a single optical section some kind of scanning is required.
There are two different ways in which this can be done: sample scanning and laser scanning. With sample scanning the beam is kept stationary and the sample is moved, while in laser scanning the sample is kept fixed and the laser beam is moved. Nowadays laser scanning is the preferred way of scanning, because a lot of biological samples are used. Cells are very flexible objects so when they move a lot of blurring will occur. Laser scanning is usually denoted as Confocal Scanning Laser Microscopy (CSLM).