Confocal microscopy
Lab/Group: Wolf Frommer lab (Carnegie Institution, Stanford)
Related Journal & Article Information
Journal: Nature
Introduction
To determine the subcellular localization of a protein of interest, the protein can be fused to a fluorescent protein, e.g. GFP. The localization can be analyzed by confocal microscopy.
Materials
Reagents
Equipment
For our equipment, an incident Argon (488 nm) ion laser (Coherent Inc.) beam was coupled to a modified Yokogawa spinning disc confocal scan head (Yokogawa Electric, Japan; and Solamere Technology, USA) via an acoustical optical tunable filter (NEOS, USA). The confocal head was mounted on an inverted microscope (DM IRE2, Leica, Germany) equipped with a 63x water immersion objective (n.a.-1.3 HCX PL APO 21°C, Leica, Germany) and a motorized Z-stage. Fluorescence images (525/50-nm for GFP) were acquired (500 msec acquisition time) with a cooled on-chip multiplication gain Cascade 512B digital camera (Roper Scientific, Tucson, AZ). Instrumentation was driven using Metamorph Version 6.3r3 software (Molecular Devices, Sunnyvale, CA).
Procedure
1) Grow yeast cells transformed with GFP-fusion on solid media.
2) Transfer cells to a microscope slide and cover with a coverslip.
3) Acquire Z-sections using a custom-made spinning disk confocal microscope as described above.
Troubleshooting
Critical Steps
Anticipated Results
References
Acknowledgements
Keywords
GFP, Confocal, Microscopy