Lasers for Confocal Microscopy: Part II

Continuing from our first article on lasers for confocal microscopy, we will now discuss two specialized types of lasers: lasers for two-photon excitation and tunable, white light lasers. We will also discuss the applications of the two lasers.

Lasers for Two-Photon Excitation

The two-photon absorption phenomenon was first described for microscopy in 1931. Here, the simultaneous absorption of two photons of identical or different frequencies results in the excitation of a molecule from one state to a higher energy electronic state, just like ‘normal’ (one-photon) fluorescence excitation.

The Physics Behind the Two-Photon Phenomenon

What makes this phenomenon exceptional is that the difference between the lower and upper states of the molecule is equal to the sum of the energies of the two photons and not just of a single photon.

For this synergistic excitation to happen, the absorption of the two photons must be simultaneous. Unlike one-photon absorption, this is a nonlinear process and the strength of absorption depends on the square of the light intensity—which intuitively makes sense. If the excitation is strong enough, three-photon and multi-photon excitation is also possible.

Two-Photon Excitation in Practice

The first practical demonstration of the multi-photon phenomenon in microscopy dates to 1990. The technique is intrinsically confocal, because all excitation happens at the focal plane and all emission comes from the focal plane. Fluorescence occurs only at the microscope focal volume, where the photon density is high enough for the probability of two-photon excitation occurrence to be considerable. This probability drops exponentially with decreasing intensity outside the focal volume. Consequently, there is no need for pinhole apertures to reject the out-of-focus signal, because such a signal is practically non-existent. Typically, a two-photon excitation point spread function has a radial full width at half-maximum of 0.3 mm and an axial full width of 0.9 mm, using a 960 nm excitation light and an objective with a numerical aperture of 1.25.

The creation of the two-photon phenomenon requires a very high photon density. Consequently, the method became practical only when short, pulsed lasers of sufficient power became available. The laser of choice is a titanium-sapphire (Ti:Sa) laser with pulse lengths of about a hundred femtoseconds, emitting in the range between 650–1100 nm. Most standard two-photon microscopes are equipped with Ti:Sa lasers that generate near-infrared radiation between 680–1040 nm. However, this limits the use of such systems to blue, green, and yellow fluorophores. Longer wavelength (red) fluorophores require pulsed laser radiation in the far infrared (IR) range (>1100 nm), and this requires special instrumentation (e.g., Optical Parameter Oscillators or OPOs). These are passive laser-like devices that convert the short Ti:Sa laser radiation into tunable radiation of longer wavelengths. In OPO-equipped two-photon microscopy setups, the Ti:Sa laser pumps the OPO at a fixed wavelength between 740–880 nm, and the OPO delivers tunable femtosecond laser radiation from 1100 to 1600 nm.

You can read a lot more about two-photon microscopy here, in one of my previous articles.

Tunable and White Light Lasers

Laser Source Types

Ti:Sa lasers offer the advantage of tunability for pulsed and continuous light delivery, as well as solid-state dependability, delivering 80 to 100 femtosecond light pluses at high repetition rates (100 MHz). The range of tunable wavelengths extends from the far red to the near-infrared spectral regions (700–1000 nm), making them useful for two-photon microscopy. Most of these lasers operate via optical pumping using high-power argon lasers, which require water cooling and are complex and expensive. However, a new type of tunable laser has been developed in recent years. It uses a variation of the solid-state laser, namely the fiber laser, with the medium being a fiber. An important feature of fiber lasers is that the fiber has a large surface-to-volume ratio so that heat can dissipate more effectively. Fiber lasers are optically pumped, usually with laser diodes.

White-light lasers employ a fiber IR laser, which emit light pulses up to 80 MHz. This compound constitutes a “seed” (i.e., it provides a precise clock but has quite low energy). The pulses are subsequently amplified in a diode-pumped laser amplifier. The amplification laser is also fiber based, and both systems are coupled seamlessly by fibers. The output of the amplifier can be as high as 10 W of IR light average power, divided in 200 ps pulses at 80 MHz. These pulses are finally focused on the entry surface of a structure called photonic crystal fiber (PCF). These fibers feature a pattern of hollow tubes in the center of the fiber in which nonlinear processes cause monochromatic light to be spread into a broad spectrum, up to several hundreds of nanometers. It is not a flat spectrum but all colors are present, and the desired ones can then be selected, ideally by means of an Acousto-Optical Tunable Filter (AOTF).

Pros and Cons of White Light Lasers

These laser sources yield complete spectral freedom to the user, especially when combined with an AOTF for step-less detection. Their main disadvantage is that their intensity on any given wavelength is rather low, because power is spread over the entire spectrum. For this reason, users who need higher laser power, for example, those employing FRAP, should also have an alternative laser source for use on these occasions. So, a complete set up could consist of a UV diode laser, an Argon laser with 5 lines, and a white light laser to cover all possible needs.

All in all, there is much more to laser sources than just Argon. Before you shop for a laser source, think about whether a single source could cover more than one application. If the answer is yes, then that source is probably a good place to start spending your lab budget!

Further Reading

Svoboda K, Yasuda R. (2006) Principles of two-photon excitation microscopy and its applications to neuroscience. Neuron. 50(6):823–39.

Frank JH, Elder AD, Swartling J, Venkitaraman AR, Jeyasekharan AD, Kaminski CF. (2007) A white light confocal microscope for spectrally resolved multidimensional imaging.J Microsc. 227(Pt 3):203–15.

Image credit: Douglas Muth

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