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ImagingSetup.md

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Imaging setup

Mount sample in flow chamber

All components should be rinsed in nuclease-free water and dried before assembly.

  1. Place the plastic chamber top upside down on the bench
  2. Fit the rubber gasket over the two metal aqueduct ports
  3. Place the glass aqueduct chamber on the gasket, aligning the holes with the metal aqueduct ports. The aqueduct wells cut in the glass should be face up.
  4. Place the aqueduct gasket on top of the glass aqueduct,to form a canal between the aqueduct wells.
  5. Wick off excess buffer from the coverslip and place sample side down ontop of the aqueduct gasket. The sample should align with the canal.
  6. Place the metal part of the chamber on top of the coverslip by aligning the clamps and holes (it only fits one way).
  7. Clasp the plastic chamber top against the metal chamber bottom to hold everything together, then invert the chamber.
  8. Move the metal clamps into place, and tighten using the metal collar.

Assembly

Prepare imaging buffer

Reagents

  • Glucose Oxidase powder
  • Catalase
  • 20x SSC buffer
  • 50% Glucose Solution
    • 15 g Glucose + 30 mL water,
    • heat up to 60C and Nutate overnight to dissolve,
    • or microwave 15s and vortex.

Mixing instructions

  • ( Recipe for 40 mL, rescale appropriately, the enzymes are expensive, Buffer lasts 72 hours at RT under 1" of mineral oil)
  • In a 1.5 uL tube measure out 10 mg Glucose Oxidase (yellow powder, stored at -20C) and dissolve it in 500 uL 2x SSC by vortexing
  • Add 100 uL dissolved Catalase solution (20 mg/mL stock) (yellow brown liquid, sediment free). Mix gently. Do Not vortex.
  • In a 50 mL flask, add 4 mL 20x SSC
  • Add 8 mL of 50% Glucose Solution (stored at RT, filter sterilize)
  • Add all 520 uL of enzyme mix
  • Gently fill to 10 mL with ddH2O.
    • (avoid mixing in extra air, the point of this buffer is to remove disolved oxygen from the buffer).
  • overlay solution with ~1 in (2-3 mL) of mineral oil.
    • (this prevents oxygen from the air dissolving back into the buffer)

Prepare readout probes

Reagents

  • Ethylene carbonate
  • 20x SSC
  • Nuclease-free water
  • universal readout probe (200 uM stock)
  • adapter-probes (10 uM stock)
  • toehold-probes (10 uM stock)

Setup

  1. Prepare labeling buffer mix with a final composition of:
    • 2X SSC
    • 0.1 uM universal-readout probe (0.5 uL of 200 uM stock per mL of hybridization buffer)
    • 25% ethylene carbonate (EC)
      • Note: heat pure EC to >42C in water bath to liquify
      • Add the 25% EC to the diluted SSC to avoid crystallization.
    • Prepare enough for 600 uL per hybridization
  2. Add 600 uL of labelling buffer mix to every well
    • negative controls (hybridizations/wells that don't get an adapter probe) don't need universal-readout probe (which is expensive).
  3. Add desired adapters at 0.1 uM final concentration (6 uL of 10 uM working stock in 600 uL of buffer)
  4. Add the toehold probes for each adapter to the following well at 0.3 uM final concentration.

Microscope Setup

Power up

  • turn on microscope main-power switch (if necessary)
  • turn on lasers (if necessary). Allow 5 min to heat up before activating
    • open the MPB GUIs to activate the 560 and 647 lasers
    • it may be necessary to select the serial-port icon and search COM ports for the GUI to connect to the laser.
    • turn on the IR focus lock laser (if necessary)
  • Power on the fluidics system (if necessary)
  • Open the Hal-software (controls the microscope camera)
    • Load parameter files into Hal
    • currently these are Bleach_Pars.xml and ConvZscan_Pars.xml
  • Open the Kilroy-software (controls the fluidics system)
    • Load desired fluidics protocol into Kilroy.

setup

  1. Create an Experiment folder on the data drive using the naming convention:
    • YYYY-MM-DD_SampleType_ProbeType
    • Create a Settings folder inside the data folder to save your parameter settings, recipes, and other run-documentation.
  2. Add oil to objective and mount flow-chamber with sample firmly on microscope stage.
    • center the objective in the middle of the flow chamber, or aligned with the sample if you can see it.
  3. Connect the fluidics tubes to the flow chamber.
    • check flow connections by flowing 2X SSC (Bleach buffer), using Kilroy
  4. Mount the 96-well plate of readout probes on the flow system
  5. Fill up the Bleaching Buffer (2X SSC) and Wash buffer reservoirs (30% formamide in 2X SSC).
    • Wash Buffer should be made fresh every 1 - 2 weeks.
  6. In Hal, get the sample in focus, and activate the focus lock.
    • this is a good time to check your sample staining.
    • also a good time to calibrate the laser intensities.
  7. Open the Steve-software and record a mosaic around the sample
    • this can be done in brightfield or using the fluorescence
    • "run-shutters" in hal needs to be unchecked to use the fluorescence, since the camera's first frame fires before the lasers do.
  8. Select and save positions.
    • Save your positions in the Settings folder.
    • Save the mosaic in a Mosaic folder in your data folder.
  9. Write the "Dave Recipe"
    • it is recommended you copy a previous recipe into your Settings folder and modify that.
    • Check that the save directory paths are updated
    • Check that the hybridization valve commands are correct.
    • Check that the offset value in the movie parameters matches the center of focus of your sample.
    • Generate all the empty folders ti save your hybridization data in.
    • There is a little matlab script to autocreate the folders and hybridization commands, which can speed things up over copy-paste.
  10. Launch Dave
    • open the Dave software
    • drag you Dave recipe into Dave to load it
    • select your saved position file
    • save the Dave XML that saves a record of Dave's actions (we typically call it "Run.xml").
    • Run the validate button
      • this will make sure the parameter files are loaded and that the target folders exist
  11. Final checks
    • make sure the Laser intensities are set as desired for each parameter / movie type
    • make sure run-shutters is checked for each parameter type.
    • make sure the focus lock is set to Z-scan and is "Locked"
    • Make sure all the fluidics wells are properly aligned.
  12. Start!
    • when all is ready, select "Start" in Dave.