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Figure 3 | BMC Biotechnology

Figure 3

From: Unmixing of fluorescence spectra to resolve quantitative time-series measurements of gene expression in plate readers

Figure 3

Estimating the mean fluorescence per cell requires several preliminary steps. a) Correcting autofluorescence using the wild-type fluorescence per cell, following the method of de Jong et al.[8] developed for bacteria, can lead to negative estimates in yeast. Correction using the bacterial method is shown in red; correction using spectral unmixing is shown in blue. Data are for cells grown in 2% raffinose and then grown further in 2% raffinose in the plate reader. Raffinose does not induce GAL expression. b) Emissions spectra for autofluorescence and enhanced GFP (EGFP) following excitation at 485 nm. Cells were grown in 2% raffinose and then in 2% galactose for the time indicated. Data for purified EGFP were obtained from the website of the R. Tsien laboratory at U.C. San Diego. c) The OD correction curve: relative cell density obtained from diluting a culture by known factors plotted against the measured OD of the diluted cultures. The blue line shows a regression and the shading gives 95% confidence limits (plus and minus twice the standard deviation of the posterior distribution found using a Gaussian process). d) Estimating r a : the ratio of fluorescence emitted at 585 nm to 525 nm plotted versus OD. The blue line shows a regression and the shading gives 95% confidence limits (found using a Gaussian process). Cells were grown in 2% raffinose and transferred to 1% galactose in the plate reader.

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