Practical Advice for Acquiring Multiphoton Lasers

par | Mai 22, 2024

In our previous posts in this series, we discussed the broad range of excitation wavelengths for 2P fluorescence and described how many fluorophores can be simultaneously excited by a single excitation wavelength. We have covered the basics of two-photon microscopy previously, and over the next few posts, we will share some tips when choosing a laser for optimal 2P imaging. 

Often microscopes are sold as integrated systems, but when buying 2P microscopes, there are more decisions to make because the laser is often purchased separately. Although some multiphoton microscopes are designed for specific laser models, most have a choice of lasers. While an integrated system can offer a more seamless user experience, having options offers more flexibility. A wider selection adds overhead when decision-making, but can be critical for specialized imaging needs. 

When choosing multiphoton lasers, the decision-making process starts with the biological questions and model. What kind(s) of samples are you planning to investigate? This will guide the type of multiphoton microscope you need (upright, inverted, in-vivo?). From there, consider what fluorophores are applicable to your sample type and investigate the compatible lasers. Does the microscope you are considering support lasers that can match the wavelengths needed to excite your intended fluorophores?

Most multiphoton systems are equipped with a laser that can be tuned over a broad wavelength range, from red (~700 nm) to the near-infrared (NIR). The operator then can adjust the wavelength (also known as “tuning the laser”) as needed. Extending the tuning range increases cost, but this allows the excitation of orange, red, and far-red emitting dyes while still being able to excite blue and green emitters.

Fibre lasers also can be used for 2P imaging. Fibre lasers are more economical, but they emit a single wavelength. Yet as many 2P excitation spectra are broad, one or two fibre lasers may be sufficient if they can excite the fluorophores of interest.

If your multiphoton system is intended for an imaging core facility, it is important to pick a system that is flexible and meets the needs of a wide variety of users in the community. Here a laser with very broad tuning capabilities or dual outputs is useful.  In contrast, individual labs may only intend to work with one or two dyes that can be excited by a single wavelength. In this situation, a single-wavelength fiber laser or a laser with a more limited tuning range may be a better choice. 

When purchasing a separate laser, it is important to select a company that provides responsive service coverage for your area to minimise downtime headaches. Also, consider the fine details of your service contract with the microscope vendor with respect to the laser and the third-party vendor to ensure there are no surprises with the laser’s warranty and coverage duration.

Our next blog post will cover the care and feeding of your multiphoton laser, including best maintenance practices and operator safety.

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