Lasers were once called “a solution looking for a problem.” The word—which is an acronym for Light Amplification by Stimulated Emission of Radiation—used to conjure up images of deadly weapons from Sci-Fi movies and TV series. However, their increasing use in everyday life, first in CD players and then in barcode scanners and pointers, have made them seem more down-to-earth.
They are also perfect for confocal microscopy.
The Perfect Light Source for Confocal Microscopy
The first laser emission was generated by Theodore Maiman on May 16th 1960, and the work was published in Nature —after having been rejected by Physical Review Letters.
But what is it that makes lasers so special? The answer is easy: their ability to generate an intense, very narrow beam of light of a single wavelength. This beam stays narrow over very long distances, which makes it especially useful for long–distance applications, like bouncing it off a small reflector on the surface of the moon. The small diameter of the laser beam makes them also especially appropriate as light sources for confocal scanning microscopy.
A light source that is suitable for confocal illumination needs to be bright, stable, easily focused and, of course, at the appropriate wavelength. Other considerations include the amount of heat and noise generation and, obviously, purchase and running costs, including energy consumption.
The power per unit area, or brightness, is characteristic of lasers. As the laser beam diameter is usually in the range of less than a millimeter, even a laser of a few mW produces a spot of very bright light, exactly because all its energy is focused on that spot. This, by the way, is what makes lasers dangerous for the eyes.
Uniformity and Stability of Illumination
Intensity, beam profile, and direction, are all important factors during image acquisition that ensure homogenous illumination. The illumination should be stable not just during acquisition but also from day to day to enable comparisons of measurements performed on different days.
The ability to focus the light to a diffraction-limited spot is of the utmost importance in confocal microscopy. A laser that emits light with a non-divergent beam is very easy to focus.
Wavelength, which is connected to the type of laser, merits the most discussion. In fact, the type of laser warrants its own section, and choice of laser type is arguably one of the most important decisions.
There are 3 major types of lasers used in confocal microscopy:
- Gas: glass or quartz tubes filled with gas, pumped by high-voltage electrical discharges
- Semiconductor: diodes pumped by the application of current across the junction of layers of semiconductors
- Crystal (solid state): rods of fluorescent crystal pumped by light at the appropriate wavelength
Both diode and crystal devices are solid state, of course, but their mode of operation is different. Therefore, they are categorized separately—usually “solid state” refers to crystal lasers. This is rather confusing for those of us that are into hi-fi, where “solid state” always refers to diode (transistor) equipment, as opposed to vacuum-tube devices!
When confocal microscopy started to be commercially available, gas, helium-neon (HeNe), lasers were used as light sources. These lasers were rather weak, around 0.5 mW, with lines at 633 nm and 543 nm, the latter being more useful for the limited fluorochromes that were then available. Another gas laser, the Argon (Ar) laser with two prominent emissions at 488 nm and 514 nm was better, especially as its 488 nm line is perfect FITC and its derivatives, as well as GFP. Ar lasers are still used today, giving five different excitation lines in the blue/blue-green range.
Crystal and Diode
Although gas lasers are still in use, crystal and diode units are taking over. They are more stable and produce less heat. Thus, crystal and diode lasers have no need for active fan cooling. This is very good for your ears, if you spend long hours using a confocal. They also emit a large variety of wavelengths. Most of them are pulsed lasers, which makes them also appropriate for fluorescence lifetime imaging (FLIM). They can only emit in one wavelength, so a number of laser sources is needed for multiple wavelengths, with five different lines usually enough for a broad range of dyes excited in the visible range.
405 nm diode lasers are used for UV today, as they can excite DAPI so strongly (albeit at the edge of its excitation curve) that shorter wavelengths are not necessary. These lasers can also be used for deep-blue excited fluorescent proteins.
Lasers can be used in confocal microscopy in more advanced and versatile ways than simply exciting fluorophores using a small, set number of wavelengths. This is the result of the development of new classes of lasers, which we will discuss in the another article.