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

1. Induce cultures with 2% galactose (for 4 mL culture, use 444.4 mL 20% galactose)
2. Incubate induced cultures at 30°C for 3 hours
3. Remove 1 mL culture to 1.5 mL centrifuge tube
4. Spin samples for 12 seconds at highest speed
5. Pour off supernatant
6. Resuspend cells in 1 mL H₂O
7. Spin samples for 12 seconds at highest speed
8. Resuspend cells in 500 μL PBS

• Imaging

1. Pipet 50 μL of sample onto standard glass slide
2. Cover with # 1 1/2 slide coverslip
3. 543 nm laser > ON, Argon laser (in standby) > slowly increase laser power to 60%
4. Config > Channel mod > 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

Materials

References