Laser advances allow visualization of living cells
Confocal fluorescence imaging has long been applied to the study of living cells or tissues. Unfortunately, the dyes used to stain the cells release toxins when they are excited by laser light. This situation limits the amount of excitation and time that living tissues can be observed in fluorescence. Also, although the whole sample in the field of view is irradiated, only one focal section is observed at a time.
These problems can now be minimized with a new technique known as two-photon excitation. By illuminating the sample with a wavelength twice the wavelength of the absorption peak of the dye being used, no excitation occurs. But, when a high-power pulsed laser with short picosecond, subpicosecond, or femtosecond pulses is used, mean power levels are moderate and do not damage the specimen.
By using such lasers, the photon density is sufficiently high so that two photons can be absorbed by the dye simultaneously. Then, excitation only occurs at the point of focus of the microscope, thereby eliminating any unnecessary phototoxicity. Although such multiple-photon excitation can extend fluorescence imaging to living tissue, high-power, subpicosecond lasers have proved expensive, bulky, and power-consuming.
Thanks to researchers at the Institute of Photonics at the University of Strathclyde (Glasgow, Scotland), small, inexpensive, all-solid-state lasers have been developed that help reduce the cost of two-photon microscopy. Collaborating with Biorad Microscience (Hemel Hempstead, England), Allister Ferguson, technical director of the institute, has developed a 20-mW, 70-fs solid-state laser. "At typical repetition rates of 100 MHz, the laser`s average power and pulse duration produce enough peak power for excitation states that are similar to those produced by one-photon systems," says Ferguson.
Based on Cr:LiSAF, the laser is still in a prototype stage, although Ferguson hopes that volume applications will soon drive the price down to between $20,000 and $30,000. "Two-photon microscopy already has had a massive impact on biological sciences and is beginning to become widely adopted," Ferguson says. "Better still, the peak power available from femtosecond sources is capable of producing three-photon and higher-order processes.
In the three-photon excitation process, three photons are absorbed simultaneously, effectively tripling the excitation energy. Using this technique, ultraviolet excited dyes may be imaged with infrared (IR) excitation. Because excitation levels are dependent on the cube of the excitation power, resolution is improved compared to two-photon excitation, where there is a quadratic power dependence.
"Dyes can be selected so that samples can be imaged by combination of two- and three-photon excitation using a single IR excitation source," remarks Ferguson. For more information, contact Ferguson at email@example.com.