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The intensities of the identified objects were then collected for statistics. For the photobleaching data, the mean per-pixel intensity of each nucleoid was calculated independently. To compare the photostability of more than two FPs in the main figure, the total intensities of all identified objects were summed and divided by the total number of pixel for all found objects in the first image, in order to take the completely bleached objects at the later stage of the bleaching period into account. In the latter case, the sum fluorescence intensity of the FPs in individual objects was plotted only in the supplementary figures. The standard deviation values of the intensities were not shown in the main figures for the convenience of display, and are instead plotted in supplementary figures. The matlab scripts used for these measurements can be found at http://ceesdekkerlab.tudelft.nl/downloads/. The signal-to-noise ratio (SNR) of the cytosolic FPs is calculated using SNRcell = (Icell-Ibg)/SDbg. We use the standard deviation value of the cell-free region as a measure of background noise (SDbg). We use the difference between the mean intensity of a cell (Icell) and the mean background intensity (Ibg) as a measure of signal. The mean and SD values of the SNR calculated from all cells are used for plotting. Results A Bright Blue Fluorescent Protein for Multi-Color Imaging in Bacteria The blue variants of the fluorescent proteins (BFPs) have seen few applications in bacteria due to their low brightness and short excitation wavelength (see Table ?Table11). In principle, if a BFP would be sufficiently bright, it can be imaged with excitation light that is weak enough to avoid photodamage to the cells. It was recently reported that the purified monomeric mTagBFP (commercial name TagBFP) has a brightness that is similar to EGFP and 1.8 times that of EBFP2 (Subach et al., 2008). To examine its performance in bacteria, we first transformed a high-copy plasmid carrying the tagBFP gene under a T3 promoter into an E. coli strain, and indeed we observed bright blue fluorescence upon excitation through a customized filter set in a Nikon Intensilight. Furthermore, we engineered a new construct into the leuB locus in the E. coli genome, yielding strain FW1268, where the expression of tagBFP gene is driven by a synthetic constitutive promoter and a synthetic ribosome-binding site (RBS). The fluorescence signal of the Selleck Androgen Receptor Antagonist expressed TagBFP is indeed sufficient for full cell labeling of bacteria (Figure ?Figure1A1A), indicating that TagBFP may have the brightness and maturation rate suitable for labeling proteins expressed at their endogenous level. FIGURE 1 Bright blue fluorescent proteins for multi-color imaging in bacteria. (A) TagBFP is bright when expressed from a genomic copy under a constitutive promoter for cytosolic label in live Escherichia coli cell.