Care and Feeding of Your Multiphoton Laser Part 1: Laser Safety

by | Jun 25, 2024

In our previous post, we discussed some practical details of choosing a multiphoton laser for your microscope. Although the technology continues to evolve and improve, multiphoton lasers still require special care and handling. Over the next two posts, we will provide some pointers for operation and maintenance, starting next with the basics of laser safety.


Lasers are classified according to their wavelength(s) and power, ranging from Class 1 to 4. The classification specifies the necessary safety measures and precautions for each class and the safety measures increase with each class. Most multiphoton lasers are Class 4, the most dangerous class of laser. As Class 4, multiphoton lasers are critical eye, skin, and fire hazards. You should refer to your specific institution’s laser safety procedures and regulations as these can vary by jurisdiction

When purchasing a multiphoton microscope, check with the vendor to see what safety measures are in place. Laser safety typically involves multiple layers of controls to prevent injury. In the ideal setup, exposure risks are eliminated when used for regular imaging. Most systems enclose the laser beam path in metal tubes or pipes that cover the route between the laser and the microscope. Further, the microscope itself including the stage is typically contained within an enclosure.

Only properly trained and qualified staff (usually determined by an institutional laser safety officer) should be permitted to align and otherwise adjust the laser beam. Most multiphoton lasers emit pulsed invisible infrared light, so often the danger is not apparent until injury has already occurred. Safety glasses or goggles blocking the correct wavelength range (multiphoton lasers are often tuneable over a wide range) with sufficient optical density designed for high energy pulses should be worn. Often sites require prominent signage as well (Figure 1 is an example from our lab). Consult your institution’s laser safety officer and documentation for more details.

Always err on the side of caution when working with lasers. Here’s a personal note from one of the authors (Pina): “Early in my career, I was aligning a laser beam and inadvertently directed the beam in my left eye. As the beam hit, I felt that my eye was made of wax, and it felt like my vision was melting away. I first saw a small black spot that opened up rapidly, leaving me with a black crater in my vision. The crater was a burn hole in my retina. Fortunately, my eye healed within a few weeks, but I would not recommend this experience to anyone.” 

Of course, looking directly into a high-powered laser beam is a bad idea but sometimes people overlook the risk of reflections or scattered high-intensity light at the sample. When imaging, it is critical to shield and avoid examining the sample when the laser is scanning as even reflected or scattered light can be powerful enough to cause eye or tissue injury. 

Commercial multiphoton microscopes should completely enclose the beam and include interlock systems that disable the laser if there is any risk of exposure. For example, interlocks are triggered automatically when eyepieces are enabled, or when a door or access hatch is open. Automated systems typically control the interlocks through the image acquisition software, while other systems require the operator to engage the interlocks for the laser to fire. Be aware of the interlock mechanisms in your system as they must be satisfied before you can use the laser for imaging. Even after years of imaging, both of us sometimes forget this step. So if you are wondering why your image acquisition looks black, checking the interlock is the first step when troubleshooting.

Unless you are an engineer, physicist, or other professional with specific laser safety training, do not attempt to align lasers or perform other operations that could expose you to the beam. This includes power measurements at any point in the optical path, including at the sample plane. This last point may be controversial, as power measurements are important for rigor and reproducibility in optical imaging. Yet without specific training, we argue that the risks outweigh the benefits. We recommend that people who want to carry out power measurements on multiphoton microscopes seek out guidance from their laser safety officers, and also implement workplace controls. 

By consulting with your workplace laser safety officer, it is feasible to develop a standard operating procedure that can be carried out with minimal risk by a trained operator. Typical measures include carrying out power measurements at the sample plane, and not in other parts of the light path. Further, the power sensor should be an absorptive rather than reflective material. Other controls include controlling access to the room housing the system, avoiding bending at the waist (this brings your eye down to the level of the laser on the table!), wearing appropriate laser safety goggles, and removing jewelry and other reflective accessories. Also don’t operate heavy machinery or Class 4 lasers when fatigued! These are only a few suggestions, and it is critical you develop guidelines in accordance with your organization’s safety regulations.

In our next post, we cover some important yet often overlooked maintenance procedures for multiphoton lasers.


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