2-photon standard operating procedure (SOP)

Van Hooser Lab Two-photon Microscope from Bruker (formerly Prairie Technologies)

Basic User's Guide

Winter 2020

Table of contents:

A. Institutional requirements for all users

B. Typical usage instructions (Standard operating procedure)

C. Personnel and equipment safety checklists

D. What to do in the case of an injury

E. Basic principles behind two-photon microscopy

A) Institutional requirements for all users

We have two institutional requirements for using the two-photon system.

    1. First, the two-photon uses a Class IV Ti-Sapphire laser (product Mai Tai from Spectra-Physics/Newport). All users must take the laser safety class from Robin Bell (bell@brandeis.edu). Note that the laser beam associated with the two-photon is fully enclosed during normal operation but it is important to understand general laser safety principles.

    2. Second, all users must be trained by the Van Hooser lab or be under direct supervision of a user who has been trained by the Van Hooser lab. This is very important for the safety of the user as well as for the equipment. See Steve Van Hooser (vanhooser@brandeis.edu) to schedule training.

B) Typical usage instructions

This section is not intended to replace a hands-on training session but rather to remind each user about important steps for using the two-photon.

Before starting, please follow the "Before" safety checklist to ensure the equipment is on and has not been left in a dangerous configuration.

1) Position the tissue to be sampled on the table under the microscope. If you like, you can manually hunt around for the tissue you'd like to sample using the fluorescent lamp or a manual light. To do this with a manual light, turn the microscope turret to position "2" and push in the rod on the right side of the turret (that toggles between "BP" and "LSM"). If you'd like to use the fluorescent lamp, open the shutter on the right side of the turret and turn on the lamp. The lamp must remain on for 30 minutes after it is turned on and remain off for 15 minutes after it is turned off to avoid rapid temperature changes that shorten the life of the bulb. Use the X, Y, and Z controls to find the tissue you'd like to image.

2) Float the table by turning on the nitrogen gas. Wait for a minute or so for the table legs to come up to pressure. It is normal for the valves to hiss a bit as each leg initially regulates its height but sustained hissing may indicate a leak that needs to be repaired.

3) Close the light cage, pushing in to the table slightly at the end so the cage will depress the interlock safety switches. If you must image with the light box open, then cover the eye pieces with the curtain on the ceiling and use the interlock override magnets to depress the interlock safety switches. For your safety it is important that your sample be installed before using the two-photon with the interlock override. The sample must be capable of absorbing infrared light.

4) Start the PrairieView application on the computer. Initially, the photomultipliers gain and Pockel's cell will be at their lowest settings.

5) Using the PrairieView software, you may now open the shutter on the MaiTai laser (Under the "two-photon laser" tab in the middle of the command window) and increase the Pockel's cell intensity and increase photomultiplier gain to image your sample.

6) When you are finished imaging, follow the "After" checklist below.

C) Personnel and equipment safety checklists

Before use:

1) After your sample is set up, be sure the computer, all devices on the two-photon equipment rack, and the laser power meter from Miles-Griot are on before opening the MaiTai shutter. It's important to check that the PrairieView application has not crashed. It occasionally crashes if devices are turned off and this can result in the safety shutter being left open. [If the Mai Tai shutter is closed then the program or computer can be safely restarted.]

2) Make sure the photomultiplier filter cube is installed.

3) NEVER touch the screw drivers that are deep in the light shield box on the far right or remove any panels from the microscope.

After use:

1) Be sure the Mai Tai laser is disabled and the Mai Tai shutter is closed (via the “2-photon” panel on the PrairieView software).

2) Turn off any gases used.

3) Clean and store any objectives that were used according to their instructions. The 40XIR or 16X lens should be cleaned by gently touching the business end to lens paper and moving the paper slowly until it is dry.

4) Make sure the fluorescent lamp is off

5) Clean any surfaces that are dirty.

6) Shut down the computer.

7) Power down the devices on the two-photon equipment rack (big, Hollywood-looking power switch).

D) What to do in the case of an injury

In Case of Suspected Eye, Skin or Other Injury:

    1. Keep the injured person as calm as possible

    2. If laser burn or other injury has no active bleeding, go to the Brandeis University Health Center (6-3677) or, after hours, go to the Urgent Care Center at 9 Hope Ave (at Children’s Hospital, corner of South St. and Highland) or Newton-Wellesley Hospital (exit 21A off of 95/128). If bleeding is excessive, call campus safety’s emergency number at 6-3333.

    3. Shut down the laser system until evaluation and corrective actions are implemented.

    4. Notify the Laser Safety Manager at 617-504-3109. Notify Robin Bell at 6-4261.

    5. Notify the injured person's supervisor.