Single Particle Tracking
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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:
- Serum starve cells for 30min-24hrs in DMEM or HPMI, 37oC.
- 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
- Dilute antibodies into HPMI + 4% donkey serum, as per table below
- Treat cells with inhibitors for 10-30min at 37oC
- Block cells for 10min with 4% donkey serum, on ice or RT.
- Without washing, add primary antibody, 10min on ice or RT.
- Wash 2-3X with HPMI.
- Add secondary antibody, 10min on ice or RT.
- Wash 2-3X with HPMI.
- If using Q-dots wash with HBSS or PBS (no biotin!)
- If using Q-dots wash with HBSS or PBS (no biotin!)
- 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.
- If using Q-dots, add 1:10,000 Q-dots, ice cold, 4min
- Wash 2-3X with HPMI.
- Add inhibitors to cells, in HPMI, keep on ice or at RT until imaging.
Imaging:
- 30 min prior to imaging turn on Bert and both cameras & warm PBS+inhibitors to 37oC
- Camera power supplies are on top shelf.
- Camera power supplies are on top shelf.
- Set EPI to max, no lasers are needed
- Start velocity in 3DM mode:
- Open new file
- Start Video Preview:
- Select lower of two camera under Video -> Source
- Video -> Show Saturation
- Select lower of two camera under Video -> Source
- Set Capture to Maximum Speed
- Open Acquisition Panel
- Capture wCy3 channel only
- 30-50 timepoints (do not use points per second)
- Max Sample Protection
- Capture wCy3 channel only
- 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)
- If using Q-dots use Q-dot channel (button beside wCy3 button)
- Open new file
- Check that ND filter at back of scope is 100%. If not, ND's are stored in the wooden box behind the chair.
- Set path to Eye and focus on sample, find cell using DIC
- Set path to Bottom Port, using lower of the port-switching buttons
- Focus cell using DIC, focus on edge of cell, avoiding cell body; capture a snapshot
- 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
- Click mouse off of air table, or on frame of air table, to avoid motion
- Capture 5-10 cells per condition
Image Processing:
- Move all DIC images into a sub-folder
- Move all time sequences into a second fold; then duplicate this folder
- Crop the duplicates using the freehand crop tool and Action -> Crop to Selection
- Split image volume into individual images within a folder, using Image -> Split Volume
- 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
- Export as regular TIFF
- Transfer to server