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Lasers for Confocal Microscopy

Since its inception, confocal microscopy has required light sources with high brightness and a beam profile which is easily focused into a diffraction limited spot. These criteria are best met by lasers (acronym for Light Amplification by Stimulated Emission of Light), which is why all commercial confocal microscopes use lasers as light sources.

One can distinguish different types of lasers in terms of technical realization. Important classes are semi-conductor lasers, solid state lasers, gas lasers and fiber lasers. For simplicity, semi-conductor lasers shall be viewed as solid state lasers as well. Leica employs all kinds of these laser types depending on the applicative requirement. The following provides an overview with links for further reference.

General principles

The common underlying principle of all lasers is stimulated emission. This term refers to a kind of “forced” emission, e.g. by light, as opposed to spontaneous emission which is the basis for fluorescence. Stimulated emission requires
(over-)population of an excited quantum state from which stimulated emission can occur
. Under normal conditions of a Boltzmann distribution between ground state and excited state, stimulated emission is a very rare process. Thus, lasers require an inversion of the equilibrium population, i.e. the excited state has to be populated more than the ground state. In order to reach this non-equilibrium distribution, energy is transferred from a coupled higher energy level, e.g. in another material, a process known as pumping. Depending on the physical make of the laser, pumping can be achieved by light (optical pumping) or an electrical field. The emitted photons induce further emission that leads to self-amplification. Therefore, lasers generally produce coherent light (light being in-phase, since emitted at the same time). As a side note, stimulated emission is also the underlying principle of stimulated emission depletion (STED) applied to super-resolution microscopy. A general overview of lasers used for confocal microscopy is found on the Leica Science Lab.

Solid state lasers

By definition, the lasing medium (also known as active laser medium) in a solid state laser is a solid (as opposed to a gas, for example). Solid-state lasers are an established laser type. In fact, the first laser, built by Theodore Maiman in 1960, used an optically pumped ruby crystal as lasing material. If the pumping is done electrically in a semi-conductor, the laser is called a diode laser. In case the pumping is done through light (optical pumping), one refers to it as an optically pumped or diode pumped solid state laser.

There is a large variety of diode lasers available at a reasonable price which makes them ideal for entry-level confocal imaging. In the Leica TCS SP8 several kinds of solid state lasers are used. They are very stable, produce only little heat and therefore need no cooling. The Compact Supply Unit of the Leica TCS SP8 contains a set of different diode lasers and optically pumped solid state lasers and offers a small footprint, while the Flexible Supply Unit  allows to combine gas lasers with  optically pumped solid state lasers, but requires more space and better cooling.

Solid state laser for wavelengths higher than 680 nm

At the opposite end of the scale, both technically and in terms of wavelength, are femtosecond pulsed infrared lasers such as Ti:Sapphire lasers using titanium doted sapphire crystals as lasing material (see photograph on Wikipedia). With their tunable emission wavelengths from around 680 – 1050 nm they are used for multiphoton imaging deeper within tissues as scattering of longer wavelengths is reduced. Also, Ti:Sapphire lasers are used for photoactivation and laser microdissection.

A special variant are optical parametric oscillators (OPO), which extend the wavelength range to 1300 nm and higher. They are not lasers per se, but require a laser, usually Ti:Sapphire, as pump laser. In multiphoton microscopy they are used for the excitation of red fluorescent proteins or for  specific applications such as CARS that yields chemical contrast.

Both Ti:Sapphire lasers and OPOs are pulsed lasers with pulse lengths of around 70-200 fs. This ensures that the peak powers are high enough to produce multiphoton effects, but the average power is still sufficiently low to maintain cell viability.

Further information on infrared lasers and their application is found in the following Science Lab articles:

Gas lasers

Here, the lasing material is a gas-filled cavity. Due to the nature of the atomic energy transitions, laser lines produced by a gas laser are particularly monochromatic thus providing the best image contrast of continuous wave lasers. Some gases such as the mixture of argon and krypton possess several energy levels which can lase simultaneously, thus providing a multi-line source from 458 to 514 nm. In the case of the argon laser the gas is transformed into a plasma, i.e. superheated, ionized gas with electrical conductivity, of which the Leica TCS SP8 can utilize three to five wavelengths depending on the beam splitter.

Another important type of gas lasers used with the Leica TCS SP8 is the Helium-neon (HeNe) laser. In this type of laser, helium serves as the pump medium, while neon is the lasing material. Helium is excited electrically and transfers the pump energy to neon atoms by collisions. HeNe lasers are mostly employed in the orange and red range while green HeNes have been replaced by more  powerful solid state lasers. For further reading, see the article on helium-neon laser on Wikipedia.

Fiber lasers

The term fiber laser refers to the construction of the lasing medium or a fiber for the modification of laser light. For Leica TCS SP8 X and Leica TCS SP8 STED with gated STED so-called supercontinuum lasers (white light lasers) are employed. The term supercontinuum refers to the presence of non-linear effects in the photonic fiber that convert the single wavelength of a pump source into a broad spectrum. The implemented supercontinuum laser produces a white spectrum which is freely tunable from 470 to 670 nm.

Conventional laser sources provide only one or a small number of single wavelength lines. As a consequence, large gaps in the excitation spectrum limit the flexibility to excite non-standard dyes and new fluorescent proteins. On the other hand, light sources such as halogen or metal-halogenide lamps produce a wide spectrum but are not suitable for confocal microscopy due to constraints on brightness and the lack of coherence.

The white light laser effectively offers the flexibility and broadband characteristics of a lamp source that is combined with the brightness and coherence of a laser. Leica’s implementation combines this tunable source with the likewise tunable beam splitter AOBS and multi-spectral detection. This puts all aspects of the fluorescence process, namely, excitation wavelength, line selection, color combination and detection band under the full control by the user. Real-time tuning with up to eight lines simultaneously literally offers trillions of combinations for laser excitation settings. Since the white light laser is a pulsed source, it can be used as a flexible source for fluorescence lifetime imaging, too, especially to cover the difficult green and yellow to orange part of the spectrum.

Leica goes a step beyond these more obvious applications and makes use of the WLL’s properties for plenty of new applications. Due to the capability of tuning within the µs-range, the system can record two-dimensional excitation-emission spectra, which involves an additional dimension for separation of fluorophores and characterization of a sample’s fluorescent components. Moreover, in combination with HyDs as photon counting detectors, fluorescence light can be separated into different components by means of a time-gating mechanism (LightGate). Hence, each channel can provide more information on components derived from reflection, fluorescent dyes, and autofluorescence – depending on the respective fluorescence lifetime. Filter-free suppression of reflection and disentangling complex mixtures of fluorescence species are possible. In combination with STED systems this gating technology increases super-resolution to lower than 50 nm. Further reading on the WLL:

  • For details on supercontinuum generation, wavelength tuning, excitation-emission scans and Lightgate please refer to article on Leica Science Lab by Rolf Borlinghaus and Lioba Kuschel.