Experimental design

General Considerations

  1. Imaging media

    • Choose to minimise background fluorescence

      1. Avoid FBS and Phenol Red if possible

      2. Avoid Methylene Blue for IR imaging

      3. Some slide/well coatings are fluorescent

    • Will depend on metabolites required for cells, e.g.

      1. EBSS + 4mM Gln, 1mM NaPyruvate, 2mM CaCl2, 1 mM MgSO4 (Anca and Sean)

      2. HBSS (Chris) with additional HEPES if needed

      3. Invitrogen Fluorobrite (George)

  2. CO2 control required?

    • Required for some imaging media

    • Often possible to avoid CO2 if using appropriate buffered media (e.g. HEPES)

  3. Temperature control

    • Experiment at RT or 37°C?

    • Turn on incubator previous evening, heating stage several hours before

    • Use objective heater collar if using immersion fluid

    • Equilibrate dishes after media changes in incubator

    • Must allow temperature to equilibrate after positioning dish on microscope (10-15mins)

    • Monitor room temperature (USB temperature monitor available)

  4. Humidity

    • Consider evaporation during long term imaging

    • Humidify image chamber if possible

    • Seal plates if possible (but may lead to pH change if CO2 required)

    • Use layer of mineral oil on well

  5. Stimulation

    • Ideally use flow chamber

    • Control temperature of stimulation media

    • Consider diffusion effects if adding small volumes

    • Check for mechanical disturbance after adding stimulant

    • Always make control measurements with vehicle only, e.g. DMSO to check for flat baseline

    • Check for fluorescence from stimulants

  6. Imaging Substrate

    • Make sure you know the properties/thickness of coverslip/well bottom

    • Generally use No 1.5 coverslips

    • Clean coverslips with ethanol before use

Multi-well plate experiment design

Consider including the following control/calibration wells in your plate

  1. Suitable controls (will depend on sample), consider

    • Donor alone

    • Positive control (e.g. constitutively active protein)

    • Negative control (non-binding partner)

    • Donor with free acceptor

  2. Empty well with same imaging media

    • Treat the same as the other wells during plate setup during media change

  3. Untransfected cells

  4. Reference dye

Other issues to consider include

  • Plastic well plates are fluorescent in blue/green

    • Glass bottom plates preferable optically, but not compatible with some cells lines

  • Edge effects

  • Pipetting strategy

Choosing a reference dye

  • Reference dye should be a bright, mono-exponential fluorophore

  • Suggestions for common imaging channels

    • CFP channel: Green chromaslide

    • YFP channel: Green chromaslide

    • GFP channel: Green chromaslide

    • RFP channel: Rhodamine 6G

  • Chromaslides are good when monoexponential as no anisotropy artefacts

  • Reference dyes should be dissolved directly in water

    • Mixed solvents can lead to multi-exponential decay

  • Use low concentration, 10uM generally good

    • Avoid inner filter, re-absorbtion