Drummond:Fluorescence

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