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Introduction

Goal: to prepare yeast cells with RFP/TFP fluorescence for imaging on the LSM510.

Protocol

  • Cell preparation
  1. day before imaging: prepare yeast precultures in 2 mL YPsugar (YPD, YPsuc/raf)
  2. day of imaging (allow for 3 hour induction): measure optical density (OD) of preculture.
    Dilution may be necessary (1:20 dilution of preculture:growth media; 50 μL preculture/950 μL YPsugar)
  3. normalize OD of yeast culture to 0.4
    target absorbance (0.4)/original absorbance = ratio
    4000 μL (4 mL) * (ratio) = X μL preculture in 4 mL solution
  4. Induce cultures with 2% galactose (for 4 mL culture, use 444.4 mL 20% galactose)
  5. Incubate induced cultures at 30°C for 3 hours
  6. Remove 1 mL culture to 1.5 mL centrifuge tube
  7. Spin samples for 12 seconds at highest speed
  8. Pour off supernatant
  9. Resuspend cells in 1 mL H2O
  10. Spin samples for 12 seconds at highest speed
  11. Resuspend cells in 500 μL PBS
  • LSM510 Imaging
  1. Pipet 15 μL of sample onto standard glass slide (slide preparation ahead of time will allow for the settling of yeast cells)
  2. Cover with # 1 1/2 slide coverslip
  3. 543 nm laser > ON, Argon laser (in standby) > once ready, slowly increase laser power to 60%
  4. Config > Channel mode > multi-track > TFP/mCherry configuration
  5. Set 458 nm excitation level at 5% and increase as necessary
  6. Set 543 nm excitation level at 50% and increase as necessary
  7. Locate and focus on yeast cells using bright field and
  8. Adjust pinhole, gain, offset, etc. as necessary

Materials

  • yeast growth media (YPD or YPsuc/raf)
  • yeast colony
  • water
  • PBS
  • 20% galactose

References

  1. []