Single Particle Tracking

Notes

• For imaging, use a space ship with the inner surface painted flat black
• The dilution for the primary Fab' must be worked out for each antibody, and after each digest.
• Image capture should be done in phenol-red free media such as HBSS or PBS. Inhibitors should be added to imaging media unless inhibitor autofluorescence is an issue
• Antibody addition can be done at RT or on ice. Ice may alter cytoskeleton, particularity microtubules.
• Q-dots must be added on ice to prevent endocytosis of dots.

Protocol

Cell Preparation:

1. Serum starve cells for 30min-24hrs in DMEM or HPMI, 37oC.
2. Prepare antibodies & inhibitors:
   
   • Dilute antibodies into HPMI + 4% donkey serum, as per table below
     
   • Prepare 2 volumes of inhibitors in HPMI, and 1 volume inhibitors in HBSS
     
3. Treat cells with inhibitors for 10-30min at 37oC
4. Block cells for 10min with 4% donkey serum, on ice or RT.
5. Without washing, add primary antibody, 10min on ice or RT.
6. Wash 2-3X with HPMI.
7. Add secondary antibody, 10min on ice or RT.
8. Wash 2-3X with HPMI.
   
   • If using Q-dots wash with HBSS or PBS (no biotin!)
     
9. Add tertiary antibody, 10min on ice or RT.
   
   • If using Q-dots, add 1:10,000 Q-dots, ice cold, 4min
     
   • Incubate with ice-cold HPMI (has biotin) for 2-3min.
     
10. Wash 2-3X with HPMI.
    
11. Add inhibitors to cells, in HPMI, keep on ice or at RT until imaging.

Imaging:

1. 30 min prior to imaging turn on Bert and both cameras & warm PBS+inhibitors to 37oC
   • Camera power supplies are on top shelf.
     
2. Set EPI to max, no lasers are needed
3. Start velocity in 3DM mode:
   
   • Open new file
     
   • Start Video Preview:
     
     • Select lower of two camera under Video -> Source
       
     • Video -> Show Saturation
       
   • Set Capture to Maximum Speed
     
   • Open Acquisition Panel
     
     • Capture wCy3 channel only
       
     • 30-50 timepoints (do not use points per second)
       
     • Max Sample Protection
       
   • Set wCy3 (white Cy3 button) exposure to 100ms, sensitivity to max, gain to best Signal-to-Noise ratio
     
     • If using Q-dots use Q-dot channel (button beside wCy3 button)
       
4. Check that ND filter at back of scope is 100%. If not, ND's are stored in the wooden box behind the chair.
   
5. Set path to Eye and focus on sample, find cell using DIC
6. Set path to Bottom Port, using lower of the port-switching buttons
7. Focus cell using DIC, focus on edge of cell, avoiding cell body; capture a snapshot
8. Switch to wCy3, focus using autolevels, record time series as soon as dots are focused
   
   • Click mouse off of air table, or on frame of air table, to avoid motion
     
9. Capture 5-10 cells per condition

Image Processing:

1. Move all DIC images into a sub-folder
2. Move all time sequences into a second fold; then duplicate this folder
3. Crop the duplicates using the freehand crop tool and Action -> Crop to Selection
4. Split image volume into individual images within a folder, using Image -> Split Volume
5. Export as TIFF, each time lapse into its own folder
   
   • Export as regular TIFF
     
   • Number, pad with 3 zeros
     
   • Append library or item name, use underscores to connect name parts
     
6. Transfer to server