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Lab 3: Particle Tracking

Lab 3: Particle Tracking

Light microscopy is used to observe a vast array of moving particles, including endosomes, organelles, bacteria, viruses, lipid nanoparticles, micelles, proteins, ... The localization of these minute features with respect to cellular structure is very informative. More powerful is the ability to track the position and velocity of each over time. By doing so, one can begin to infer cellular transportation mechanisms, diffusion characteristics, and protein interactions, amongst others. Beyond tracking minute particles, the same technical mechanisms can be used to track macroscopic entities such as individual cells or even the movement of whole animals. Needless to say, the ability to track multiple fluorescent entities over time is an essential tool for today's researchers.

While it would be fun and interesting to perform particle tracking on the translocation of membrane proteins to the nucleus, or injection of virulence factors from infectious bacteria, in Lab 3 your are going to start with something more simple. You will perform two observations: 1) the natural Brownian movement of fluorescent beads in suspension and 2) track the movement of fluorescent beads within a simple flow chamber. While this may appear to not relate, many of the same techniques will be developed. You will need to identify the initial location of each particle within your field of view, track that particle over time, determine its trajectory and its velocity.

Useful Reading

Luckily for you (I hope), a lot of effort has already been put forth for particle tracking using Matlab. Here are a couple of interesting papers and two potentially useful links that may be of assistance (Caution: I have not actually used either for tracking particles):

  • Here is a nice overview and comparison of multiple particle tracking techniques (click here)
  • Here is a fun paper tracking the movement and interactions of mice (click here)
  • Click here for a Matlab plugin for particle tracking and velocimetry
  • Click here for an overview and description on how to track particles in Matlab

To Do:

The data acquisition for this lab should not take long.  You only need to acquire a couple of time lapses. That's it. And by a couple, I mean at least 2.

I will prepare for you two different imaging conditions

  1. An imaging dish with fluorescent microspheres (0.5 micron, red) suspended in PBS
  2. A flow cell through which fluorescent microspheres will pass through (10 microns, red)

You will need to acquire at least two videos, one for each imaging condition, with each video being between 10 and 90 seconds long. Be sure to note the frame rate! The videos will need to be acquired at a rapid frame rate. I will describe how to do this below. The 20x objective should be used for the Brownian motion acquisitions while the 4x objective should be used for the flow channel. You will use the TRITC acquisition settings for all of your videos. For each, you will set up the ND Acquisition tab to acquire a time lapse will the following conditions:

  • Interval: No Delay
  • Loops: 1000

A1R

Laser scanning confocal is inherently slow. Recent advances in the field have led to the development of resonant scanners (click here for a nice overview). Briefly, resonant galvonometers differ from traditional galvonometers in that, when a voltage is applied, it begins to vibrate in a manner akin to a tuning fork. The resonant galvonometer vibrates at a fixed frequency, allowing for the corresponding mirror to be quickly rotated, thereby enabling rapid acquisition rates. For this lab, the resonant scanner will be used. To enable the resonant scanner, select Resonant in the upper right hand corner of the Nikon Elements window. You will hear a high-pitch vibrating sound (it sounds like a mosquito - please do not hit the microscope).

NSTORM

The NSTORM has two camera options: An Andor 897 Ultra EMCCD and a Hamamatsu Flash4.0 sCMOS. The sCMOS has a much larger chip so the field of view is greater. Also, its pixels are much smaller (6.5 microns vs 16 microns). I will let you decide which camera to use. To chose, select the startup icon (Single Andor or Single Hamamatsu). If you go with the Hamamatsu, I highly recommend a 2x2 bin size, otherwise your file sizes will be HUGE. Once you have selected a camera, select an exposure time of 100 ms from the exposure time drop down menu. You will be approaching 10 frames per second. Remember, the Andor is on the Left camera port and the Hamamatsu is on the Right camera port be sure that your light path is in the correct orientation. You will most likely need to reduce your illumination power for the 10 micron beads, so insert the ND filters into the illumination path.

Spinning Disc

Once you have selected a camera, select an exposure time of 100 ms from the exposure time drop down menu. You will be approaching 10 frames per second. Adjust your laser power and gain accordingly.

All

  1. Trace the movement of your particles (click here for an example).
  2. Since you know the frame rate and the size of the image, calculate the average linear velocity of each particle (report as average and standard deviation). You can also report the average maximum and minimum instantaneous velocity (and the standard deviation as well)
  3. For the Brownian Motion acquisition, calculate the total distance each bead has moved (report as average and standard deviation).
  4. Test your code reducing the frame interval. For example, start with all 1000 frames, then use every other frame (500 total), every 3rd frame, then 4th, ... Continue until you cannot accurately track the particles any more. For reach, report the average linear velocity and distance traveled (Brownian Motion only for the latter). When does your code break?
  5. Have fun with your plots. There are a lot of interesting ways to plot out your data. The bead velocity at a given moment in time can be color coded, so can the distance traveled, and the current trajectory indicated by an arrow (the size and color of which can be color coded to indicate velocity).
  6. For those of you who are interested, you can calculate the diffusion coefficient of the PBS for the Brownian Motion (click here for a nice overview from Penn State University on how to do this). If you want a challenge, you can also do this using a different solution (like immersion oil).
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