What is an OPO?

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 (titaniumsapphire), start with red wavelengths, e.g., 680 nm, and range into the infrared, e.g., 1080 nm. Second, you need two photons to reach the fluorescent dye at approximately the same time. Then the two photons of, for example, 1000 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 1080 nm, the longest reachable excitation in this twophoton 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 1080 nm is used. With an optical parametric oscillator, or OPO, you can now use excitation wavelengths up to 1300 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 a non-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.

What does Leica Microsystems offer?
We have fully integrated the control of the Coherent Compact OPO with our Leica LAS AF software, which greatly facilitates its operation. The Leica TCS SP8 MP, designed for infrared imaging with OPO, extends the choice of excitation wavelengths from what was formerly up to 1080 nm, to now up to 1300 nm. The user has the choice of three operation modes: single or sequential excitation with 1040 to 1300 nm with the OPO alone; 680 to 1040 nm with the Ti:Sa IR laser alone; or simultaneous excitation with both excitation sources, at 740 to 880 nm with the Ti:Sa laser, and 1030 to 1300 nm with the OPO.

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