Confocal Raman microscopy makes it possible to sample a precisely defined area at the focus of an instrument while excluding signals from the surrounding area. This is achieved by placing an aperture at a secondary focus in the optical path between the sample and the detector. One can think of this as projecting the confocal aperture onto the sample plane. Only Raman signals originating within this projected aperture can pass through the confocal aperture; all out-of-focus Raman signals are rejected.
Ideally, the Raman excitation laser is focused on the same confocal area in the sample plane. Under these conditions, the laser creates the maximum Raman scattering exactly in the desired sampling location.
Not all lasers, however, produce the best results for confocal scattering. At high magnification, such as with a 50× or 100× microscope objective, the diameter of a confocal aperture projected on the sample is on the order of 1 μm. To focus a laser into such a small spot size, the laser must be what is termed high-brightness. The brightness in this context refers to the laser beam quality — its ability to achieve a high power density by focusing all of its energy into the smallest possible spot size.Diode lasers can also be "low brightness" because of their design parameters. Such lasers cannot be focused to a small spot size regardless of the optics used. The result is that areas of the sample outside of the confocal aperture are illuminated by the laser. Spatial resolution may be reduced, but the immediate effect is much lower Raman signal strength than might otherwise be expected. High-brightness lasers are readily available with output power levels of 30–80 mW. Low-brightness lasers have much higher power levels, from 250 mW to several watts. However, at high magnifications and with small confocal apertures, high-brightness lasers can produce equal or higher signal-to-noise ratios (S/N). This improved S/N can make the difference between success or failure in energy-starved applications such as Raman microscopy where every photon of Raman signal counts.
The focusing quality of a laser beam is characterized by its diameter and divergence. These parameters are often combined in a laser beam figure of merit known as the M 2 factor.
Beam diameter, also known as beam waist, is the minimum diameter of a laser beam along its axis of propagation. The beam waist is sometimes reported as the beam waist radius, which is half the beam diameter.
Beam divergence is a measure of how much the laser beam diameter spreads along its propagation axis. It is usually reported as a half-angle in mrad (where mrad is milliradians).
M2 factor is the product of beam diameter times divergence relative to that of an ideal Gaussian (TEM00) beam. For this reason, this factor can be thought of as "times diffraction limit" of the laser beam. A laser beam that is diffraction-limited has ideal beam quality and can be focused into the smallest possible spot size. This ideal beam quality is designated by an M2 value of 1.0. For example, a laser with an M2 of 2 can only focus to a spot diameter that is twice the theoretical minimum.