FRET - Microsope Set Up
That ends the introduction. We now describe the practical aspects of measuring FRET in yeast and begin with the microscope. Let's face it, yeast are small and signal strength is often a problem for visualization of CFP and YFP tagged proteins. The microscope should be the best you can get your hands on. For guidance we describe our set up for CFP and YFP FRET. We use the DeltaVision System from Applied Precision.
- Microscope Base: Olympus IX 70 set up for Differential Interference Contrast (DIC) Observation
- Objectives: UPlanApo/100X 1.35 Oil; UPlanApo/60X 1.40 Oil
- Immersion Oil: Applied Precision, N=1.514
- Camera: Photometrics CoolSNAP HQ
- Light Source: OSRAM HBO 100W/2, Mercury Short Arc Lamp. We replace the lamp when it has more than 100-150 hours of usage or the YFP photosensor values are less than 550,000.
- Excitation Filters: XF1071 (440AF21); XF1068 (500AF25)
- Emission Filters: XF3075 (480AF30); XF3074 (545AF35)
- Dichroic: XF2065 (436-510DBDR)
- All filters from Omega Optical
- Temperature Controlled Growth: Bioptechs Objective Heater and Controller Model FCS2, Closed Chamber System Controller, Model FCS3. Typically live cells are kept at room temperature. The Bioptechs controllers are used for temperature-sensitive mutants where temperature needs to be tightly regulated. We use the 60X objective with the protective sleeve removed for this purpose.