This page contains detailed protocols developed here at Wake Forest University for quantitative microscopy acquisition and analysis.

Fluorescence Recovery After Photobleaching

Technical Considerations

Acquisition Protocol

Excellent articles about performing FRAP include:

1) Methods in Cell Biology, Vol. 85, pp. 329-351, 2008 by J.G. McNally.
2) Biophysical Journal, Vol. 94, pp. 3323-3339, 2008 by F. Mueller et al.

Raster Image Correlation Spectroscopy

Excellent articles about performing RICS include:

1. Rossow MJ, Sasaki JM, Digman MA, and Gratton E. 2010. Raster image correlation spectroscopy in live cells. Nature Protocols 5:1761-1774
2. Brown CM, Dalal RB, Herbert B, Digman MA, Horwitz AR, and Gratton E. 2008. Raster image correlation spectroscopy (RICS) for measuring fast protein dynamics and concentrations with a commercial laser scanning confocal microscope. J. Microscopy 229:78-91.
3. Kolin DL and Wiseman PW. 2007. Advances in image correlation spectroscopy: measuring number densities, aggregation states, and dynamics of fluorescently labeled macromolecules in cells. Cell Biochem Biophys 49:141-164.

Colocalization Analysis

Acquisition Protocol

Analysis Protocol

Excellent articles about performing colocalization analysis include:

1) Journal of Microscopy, Vol. 224, pp. 213-232, 2006 by S. Bolte and F.P. Cordelieres
2) Biophysical Journal, Vol. 91, pp. 4611-4622, 2006 by J.W. Comeau et al.

Spectral Imaging and Analysis

Spectral protocol

Airyscan Imaging and Processing

Airyscan Handbook

Airyscan Processing