Lasers used in the Confocal Microscope

Lasers are used in confocal microscopes because they provide: 1) Single wavelength (very pure color) light and 2) very bright light. These usually non-pulsed gas lasers. Two photon scanning fluorescence microscopy does used pulse lasers, but these are technically not confocal microscopes although they achieve the same effect using a different imaging principle. These microscopes will not be discussed here, but may be the subject of a future link.

Historically, the first type of laser to be used for confocal microscopy was the argon ion laser which has two very strong lines at 488 nm and 514 nm. These are blue and blue-green wavelengths, respectively. The blue line at 488nm is nearly an ideal wavelength for exciting fluorescein and its derivatives. It also works well on some red-shifted forms of Green Fluorescent Protein (see GFP). Originally the 514nm line was used to excite rhodamine. This wavelength did excite rhodamine, but it was not useful for studies of double labeled specimens because this wavelength also excited fluorescein; sometimes even better than rhodamine. In spite of efforts to use barrier filters specific for rhodamine, it was not possible to prevent the fluorescein signal from bleeding through into the rhodamine channel during rhodamine scanning.

An answer to this problem was developed by R. Todd Brelje working at the University of Minnesota. He looked for a laser which might produce a line at a wavelength in the true green part of the visible spectrum. A new mixed gas argon-krypton laser appeared to have the necessary line. This laser had strong lines at 488 nm (blue), 567 nm (yellow-green) and 647 nm (red) (an RYB laser). This laser had ideal wavelength characteristics for double-labeling experiments. The 567 nm line was far enough away from the excitation spectrum of fluorescein that the latter would not be excited, but excited rhodamine very well. Therefore fluorescein bleedthrough was no longer a problem. Furthermore, this laser had a red line far enough away from the rhodamine excitation spectrum so that a third fluorochrome such as allophycocyanin or Cy5 could be used and triple fluorescent probe experiments became possible.

In conjunction with Ion Laser Technology (ILT:Salt Lake City, UT), small tube argon-krypton lasers were produced and licensed for the Bio-Rad confocal microscopes. Another company Omnichrome (in Chino,CA) also began producing larger tube argon-krypton lasers to supply to other microscope companies for their confocal systems.

Almost immediately, problems began to arise with the argon-krypton lasers however. Several of the smaller tube ArKr lasers began to fail after 100-200 hrs of use. All of the ArKr lasers begin to lose the red line at 647 nm after a short time. The life of all of the Ar-Kr laser tubes was also a problem. They did not last nearly as long as the argon ion lasers (MTBF: 2000-4000 hrs).

Other confocal manufacturers looked at other lasers. One alternative was to use a small helium-neon laser. These could be manufactured to produce a line at either 543 nm or 633 nm. These were reasonable alternatives to the argon-krypton laser because while the lines produced were not exactly the same, the 543 nm green line and 633 red line were still in parts of the visible spectrum where there would not be overlapping of fluorochrome excitation and bleedthrough. Furthermore, helium-neon lasers were a proven technology. They last a long time ( up to 10,000 hrs or more) and have very low power consumption.

Argon-krypton lasers are still used extensively though. The 567 nm line is further away from the 488nm excitation line and therefore may be less likely to produce signal overlap from different fluorochromes.  The 547nm line would also provide a more efficient excitation of fluorochromes which are excited in the yellow to near red range such as Texas Red and Cy3.  One confocal system manufacturer is recommending a laser configuration employing an Ar-Kr laser which does not have the red line (which would fade out after a few hours of use anyhow) (a YB laser) and a 633 nm HeNe laser for a far red line.

A fourth area of the electromagnetic spectrum of interest to confocal microscopists is the near UV range for excitation. Several useful biological fluorescent probes are excited in the near UV such as the DNA probes Hoescht 33258 and 33325 (bis-benzimide) and DAPI, and the calcium probes Indo-1 and Fura-2 and the antibody conjugate AMCA. These all emit a silvery white or light blue wavelength upon excitation. The laser used for this type of excitation is a much more powerful (up to five times) argon ion laser. Besides the strong 488 and 514 nm lines, the argon laser also emits a weaker line at 367-368 nm. In the UV laser, the 488 and 514 nm lines are blocked and only the UV line is allowed to come through.

The fact that these lasers are so powerful requires that they be large and must have adequate cooling using water cooling systems. They are also rather expensive (~$50,000). To our knowledge, other laser types are rarely used in confocal microscope systems (however, double photon excitation systems do use other pulsed lasers). For specialized needs, though  (to cover other areas of the visible spectrum where excitation of certain fluorochromes might be useful to biologists in a particular field of interest), other laser configurations may be worthwhile investigating. 

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