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 0.4target absorbance (0.4)/original absorbance = ratio4000 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