2. What to do during the lab?

2.1 Equipment and materials

• Light microscope with digital camera and video capture software. Bertrand lens. Calibration slide.
• Polystyrene beads of different diameters (typically: 0.5 μm, 0.75 μm, 1 μm, 2 μm, 3 μm, 15 μm) in a stock solution.
• Micropipettes, tips, 1.5 ml Ependorf tubes, kimwipes.
• Pre-assembled microscope slide and coverslip.
• Clear nail polish.
• Water.
• Sharpies.
• A container to dispose of used tips.

2.2 Sample preparation

• Select beads with a size that might be appropriate to study Brownian motion.
• Place a small amount of solution (~ 5 μl) in a pre-assembled sample slide and check the concentration of particles under the microscope (see below for optimal microscope settings).
• Seal the sample with nail polish. Make sure the sample is completely sealed. Make sure the nail polish is completely dry before placing the sample under the objective again.

2.3 Video recording

• In order to capture the motion of the beads, the setting of the microscope and camera will need to be just right. Start by using regular illumination (not phase, i.e. place the condenser dial on “A”) and the 40× objective. Make sure you align the microscope according to the procedure we used in a previous lab. For alignment, you might want to first focus into the parafilm (the material used as a spacer between the slide and coverslip) which is easy to find and image, then translate the sample so that the beads are in your field of view.
• You will need to play with the camera settings in order to obtain a good image quality (which you will need to automatically track the beads) at a high frame rate (so you can catch fast bead motions). You can check how many frames per second (FPS) the camera is collecting by looking at the top left of the live image preview in the Motic Images software. Finding the right balance between a high image quality and high enough FPS (at least 50 to 100 frames per second) is crucial to later achieving good tracking with the particle tracking software. In order to do so, increasing the intensity of the illumination as much as you can will be helpful.
  • Start by lowering the exposure time (the length of time for which each image in the video is recorded) in order to increase the FPS (to get an FPS around 50 to 100, the exposure time should be in the range ~10 to 20 ms). Note that lowering the exposure time also results in poorer image quality.
  • Play with the gain (which determines the sensitivity of the camera – a high gain will give you more signal but also more noise) to improve image quality.
  • On the right hand side panel, there is a drop down menu called Resolution. The resolution here is determined by how many pixels are used on the camera sensor. A lower resolution (less pixels) results in a lower quality picture but also increases the frame rate. If you find the your frame rate is too slow, try lowering the resolution and assessing the quality of the image.
  • Decreasing the field of view can also help increase the frame rate. To do so, crop the image by drawing a region of interest (ROI) on the live image preview and then hitting “ROI Preview” in the top panel. The software then only shows and capture the smaller selected ROI. Use the smallest possible ROI (it will still need to be large enough to capture the motion of a bead for the entire duration of the movie) when recording your movie.
• Record one or several movies, each for a duration of ~10 to 15 s.
• If there is time, repeat with beads of different sizes.
• In order to meaningfully analyze the motion of the beads, you will need to be able to measure times and distances. So don’t forget to take a picture of the calibration slide before you leave the lab, at the same resolution as the movies you captured. And don’t forget to write down the exposure time and the total duration of your movie(s).