What is an OPO?

November 02, 2010

Imaging thick tissue sections as well as whole animals plays a growing role in life science research. Obtaining spatial information in deep tissue areas is crucial to fully understanding biological processes. However, image quality decreases the deeper you image in the tissue, as light is scattered by biological specimens. Current methods allow light to reach about one hundred microns deep with standard widefield or confocal fluorescence microscopy by using excitation sources in the visible range. Unfortunately, it becomes impossible to penetrate hundreds of microns into the tissue while using visible light. Because light scattering is dependent on the wavelength, better tissue penetration can be achieved by using longer excitation wavelengths. This is where excitation with infrared light, two-photon processes, and the OPO (optical parameter oscillator) can dramatically improve image quality.

How do we get longer excitation wavelengths?

First, you need laser sources in the red and infrared. Normally, these sources, called Ti:Sa lasers (titanium-sapphire), start with red wavelengths, e.g., 680 nm, and range into the infrared, e.g., 1,080 nm. Second, you need two photons to reach the fluorescent dye at approximately the same time. Then the two photons of, for example, 1,000 nm, together equal the energy of an excitation wavelength of approximately 500 nm. This process is called multiphoton or two-photon imaging.

When the maximum wavelength of the IR laser is at 1,080 nm, the longest reachable excitation in this two-photon process equals approximately 540 nm. However, many labels and dyes used in biological research need to be excited at longer wavelengths and cannot be used for two-photon imaging, unless an excitation wavelength longer than 1,080 nm is used. With an optical parametric oscillator, or OPO, you can now use excitation wavelengths up to 1,300 nm in the two-photon imaging process. This allows exciting dyes with an excitation maximum in standard one-photon microscopy of up to approximately 650 nm, which is a great improvement to confocal imaging. The more dyes that are possible and reachable with the two-photon process, the more information obtained from specimens with large imaging depths.

What are the applications for OPO?

If you look at the neurosciences, there is a field called connectomics, which is related to the connections between neurons, or between cells in general. To obtain a roadmap of connections between cells you need both a large overview and detailed resolution. The aim is to understand the function of the tissue – to look at how the circuits work. Many other research areas can benefit from the OPO. For example, in developmental biology it is crucial to protect tissue from photodamage during intravital embryo imaging as well as deep penetration of highly scattering tissues. Here, the longer excitation wavelengths generated by the OPO are optimal. Additionally, the OPO is useful for using red and far red dyes for multiphoton imaging. Even simultaneous excitation of two dyes at two different wavelengths is possible with the OPO.

How does an OPO work?

It is important to note that an OPO utilizes non-linear optics, which underlying physics are not easy to explain. However, think of single photons from a pump laser, which leave the IR source. In an optical resonator and anon-linear crystal, the pump photons overlap and produce a signal and an idler. Those three waves, the pump, the signal and the idler, interact in the non-linear crystal. The signal – which is what you want – gains power with every round trip in the resonator. This is called parametric amplification of the signal, and the pump loses power accordingly. The signal is then coupled out and used for IR imaging.

Tubulin stained with Atto647N. Two-photon excitation with OPO at 1,200 nm.
Tubulin in epithelial cells stained with DsRed. Excitation 1200 nm generated by an optical parametric oscillator (OPO). Image taken with a Leica TCS SP5 MP with OPO.
Mouse mammary gland. Blood vessels labelled with 70 kD-Texas Red excited with OPO at 1,150 nm (red). Simultaneous excitation at 800 nm results in second harmonic generation (SHG) signal of type I collagen (purple) and autofluorescence of single cells (green).

Courtesy of Evelyne Beerling, Jacco van Rheenen, Hubrecht Institute, Utrecht, The Netherlands