Drummond:Fluorescence
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Introduction
Goal: to prepare yeast cells with RFP/TFP fluorescence for imaging on the LSM510.
Protocol
- Cell preparation
- day before imaging: prepare yeast precultures in 2 mL YPsugar (YPD, YPsuc/raf)
- 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) - 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 - Induce cultures with 2% galactose (for 4 mL culture, use 444.4 mL 20% galactose)
- Incubate induced cultures at 30°C for 3 hours
- Remove 1 mL culture to 1.5 mL centrifuge tube
- Spin samples for 12 seconds at highest speed
- Pour off supernatant
- Resuspend cells in 1 mL H2O
- Spin samples for 12 seconds at highest speed
- Resuspend cells in 500 μL PBS
- LSM510 Imaging
- Pipet 15 μL of sample onto standard glass slide (slide preparation ahead of time will allow for the settling of yeast cells)
- Cover with # 1 1/2 slide coverslip
- 543 nm laser > ON, Argon laser (in standby) > once ready, slowly increase laser power to 60%
- Config > Channel mode > multi-track > TFP/mCherry configuration
- Set 458 nm excitation level at 5% and increase as necessary
- Set 543 nm excitation level at 50% and increase as necessary
- Locate and focus on yeast cells using bright field and
- Adjust pinhole, gain, offset, etc. as necessary
Materials
- yeast growth media (YPD or YPsuc/raf)
- yeast colony
- water
- PBS
- 20% galactose