Difference between revisions of "Optical Microscopy Part 4: Particle Tracking"

Introduction and Background

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Experiment Details

Stability and Setup

The major challenge of particle tracking microrheometery is the small scale of the thermal forces and the associated nanometer scale displacements. A few things you can do to ensure the experiment works: Be sure all the microscope components are rigidly assembled and firmly tightened. Poorly built scopes shake. It is also vital that when you perform this experiment that the optical tables are floating so that building noise is isolated. Of course, avoid touching the optical table and the microscope during the measurement. There are also cardboard boxes available that you can put over your microscope to isolate it from air currents. Finally, make sure that you and the people around you are not talking too loudly during the experiment, because acoustic noise is significant.

Proper camera gain settings for particle tracking: Choose "Properties..." under the "Device" menu and set the gain and brightness level to zero. Change the exposure time until the intensity of the fluorescent beads is just high enough to achieve good contrast, but do not saturate the intensity value to 255.

System Verification

To verify that your system is suficiently rigid/stable, first measure a specimen containing red fluorescent beads (Molecular Probes) dried in a cell dish. Chose a field of view in which you can see at least 3-4 beads. Using a 40x objective record an .avi movie for about 3 min. at a frame rate of 30 frames/sec. From your experience with image processing, you already know how to import .avi movie data into matlab. To improve signal to noise ratio, sum every 30 frames together, which will make your sequence have a temporal interval of 1 sec. Use the bead tracking processing algorithm on two beads to calculate two trajectories. To further reduce common-mode motion from room vibrations, calculate the differential trajectory from the individual trajectories of these two beads. Calculate the MSD <$ \Delta r^2 $> from this differential trajectory. Your MSD should start out less than 10 nm$ ^2 $ at $ \tau $ = 1 sec and still be less than 100 nm$ ^2 $ for $ \tau $ = 180 sec. If you don't get this, do not proceed further and ask for help.

Live Cell Measurements

Now that your system is su±ciently stable, you can run the experiment on cell samples. A key technique to keep in mind when working with live cells - to avoid shocking them with "cold" at 20°C, be sure that any solutions you add are pre-warmed to 37°C. We will keep a warm-water bath running on the hotplate for this purpose, in which we will keep the various media. You are provided with NIH 3T3 ibroblasts, which were prepared as follows: Cells were cultured at 37°C in 5% CO$ _2 $ in standard 100 mm x 20 mm cell culture dishes (Corning) in a medium referred to as DMEM++. This consists of DMEM (Cellgro) supplemented with 10% fetal bovine serum (FBS - from Invitrogen) and 1% penicillin-streptomycin (Invitrogen). The day prior to the microrheology experiments, fibroblasts were plated on 35 mm glass-bottom cell culture dishes (MatTek). On the day of the experiments, the cell confluency should reach about 60%. 1 μm diameter orange fluorescent microspheres (Molecular Probes) were mixed with the growth medium (at a concentration of 5 x 10^5</math> beads/mL) and added to the plated cells for a period of 12 to 24 hours for bead endocytosis. Choose cells with 3 or 4 particles embedded in them and take a movie as before. Take movies of about 3-5 cells. Now treat the cell with the cytoskeleton-modifying chemical cytochalasin D (CytoD). Pipet out the buffer, add 1 mL CytoD solution at 10 μM (pre-mixed for you) to the dish, and wait for 20 min. It's a good idea to check on your cells after 20 min.: sometimes they are in bad shape at that point but sometimes they still look very healthy. Wash and replace with buffer twice with 2 mL pre-warmed DMEM++. Repeat the particle tracking measurements again for 3-5 cells as quickly as you are able, since their physiology has now been significantly disrupted and they will die within a couple of hours. It's very unlikely that you'll be able to find the exact same cells you've already tracked; however it's very much advisable to use the same dish for the "before" and "after" so you're aren't also comparing between different cell populations.

References