EDITORIAL SUMMARYThis protocol describes how to estimate and spatially resolve the concentration and copy number of fluorescently tagged proteins in live cells using fluorescence imaging and fluorescence correlation spectroscopy (FCS).TWEETDetermining protein concentrations and copy numbers in live cells using fluorescence correlation spectroscopy (FCS)-calibrated imaging.COVER TEASER Map protein concentrations with FCS-calibrated imagingUp to four primary research articles where the protocol has been used and/or developed:Walther, N., Hossain, M. J., Politi, A. Z., Koch, B., Kueblbeck, M., Oedegaard-Fougner, O., Lampe, M. and J. Ellenberg (2018). A quantitative map of human Condensins provides new insights into mitotic chromosome architecture. bioRxiv, 237834. https://doi.org/10.1101/2378342.Cai, Y., Hossain, M. J., Heriche, J.-K., Politi, A. Z., Walther, N., Koch, B., Wachsmuth, M., Nijmeijer, B., Kueblbeck, M., Martinic, M., Ladurner, R., Peters, J.M. and J. Ellenberg (2017). An experimental and computational framework to build a dynamic protein atlas of human cell division. bioRxiv, 227751 https://doi.org/10.1101/227751Germier, T., Kocanova, S., Walther, N., Bancaud, A., Shaban, H.A., Sellou, H., Politi, A.Z., Ellenberg, J., Gallardo, F. and K. Bystricky (2017). Real-Time Imaging of a Single Gene Reveals Transcription-Initiated Local Confinement. Biophysical Journal, 113(7), 1383-1394, https://doi.org/10.1016/j.bpj.2017.08.014.Cuylen, S., Blaukopf, C., Politi, A. Z., Muller-Reichert, T., Neumann, B., Poser, I., Ellenberg, J., Hyman, A.A., and D.W. Gerlich (2016). Ki-67 acts as a biological surfactant to disperse mitotic chromosomes. Nature, 535(7611), 308–312. http://doi.org/10.1038/nature18610.AbstractThe ability to tag a protein at its endogenous locus with a fluorescent protein (FP) enables the quantitative understanding of protein dynamics at the physiological level. Genome editing technology has now made this powerful approach routinely applicable to mammalian cells and many other model systems, opening up the possibility to systematically and quantitatively map the cellular proteome in four dimensions. 3D time-lapse confocal microscopy (4D imaging) is an essential tool to investigate spatial and temporal protein dynamics, however it lacks the required quantitative power to make absolute and comparable measurements required for systems analysis. Fluorescence correlation spectroscopy (FCS) on the other hand provides quantitative proteomic and biophysical parameters such as protein concentration, hydrodynamic radius and oligomerization but lacks the ability for high-throughput application in 4D spatial and temporal imaging. Here, we present an automated experimental and computational workflow that integrates both methods and delivers quantitative 4D imaging data in high-throughput. These data is processed to yield a calibration curve relating the fluorescence intensities of image voxels to absolute protein abundance. The calibration curve allows the conversion of the arbitrary fluorescence intensities to protein amounts for all voxels of 4D imaging stacks. With our workflow the users can acquire and analyze hundreds of FCS-calibrated image series to map their proteins of interest in four dimensions. Compared to other protocols, the current protocol does not require additional calibration standards and provides an automated acquisition pipeline for FCS and imaging data. The protocol can be completed in 1 day.
Strengths and Weaknesses of Recently Engineered Red Fluorescent Proteins Evaluated in Live Cells Using Fluorescence Correlation Spectroscopy