Choosing Appropriate Fluorescent Proteins and Filters

Optimal results with fluorescence microscopy depend on a high signal to noise ratio. This can be achieved either by increasing the specific signal or by decreasing the background noise. To maximize the signal, a strong promoter should be chosen to drive fluorescent protein expression. Using fluorescent proteins with a high relative brightness also enhances signal intensity. Table 1 lists some of the properties of the four fluorescent proteins described in this article and the filter sets used to analyze them. The three versions of GFP, namely EGFP (here called GFP), ECFP (here, CFP), and enhanced yellow fluorescent protein, (EYFP, here, YFP) are currently the most widely used because of their spectral properties, brightness, and relatively low propensity for bleaching. The blue variant of GFP, BFP, is of limited use as it bleaches very rapidly and therefore was not included in our study. DsRedTl, the particular DsRed protein that we have used for our studies, was originally developed for rapid folding and high signal intensity (Bevis and Glick, 2002). It is here termed red fluorescent protein (RFP). The seven filter sets listed in Table 1 were purchased from a collection of filters from a single company (Chroma Technologies), although similar filters are available from other sources as well (such as Omega Optical, www.omegafilters.com; Semrock, www. semrock.com). The Cyan GFP, Endow GFP (developed by Dr. Sharyn Endow), and Yellow GFP filters are recommended by Chroma for visualization of CFP, GFP, and YFP, respectively. The TRITC and Texas Red filters were developed for visualization

Cyan GFP Endow GFP JP1 Yellow GFP JP2 TR ITC

Fig. 3. Phenol red in the culture medium increases the level of background fluorescence. Images of cell culture medium (in Iscove's modification of Dulbecco's medium without phenol red) with and without phenol red (2 mL of medium in 35-mm plastic culture dishes) were acquired with the indicated filters. The relative background fluorescence was calculated as the difference (percentage) between the values obtained with and without phenol red.

Cyan GFP Endow GFP JP1 Yellow GFP JP2 TR ITC

Fig. 3. Phenol red in the culture medium increases the level of background fluorescence. Images of cell culture medium (in Iscove's modification of Dulbecco's medium without phenol red) with and without phenol red (2 mL of medium in 35-mm plastic culture dishes) were acquired with the indicated filters. The relative background fluorescence was calculated as the difference (percentage) between the values obtained with and without phenol red.

of proteins labeled with rhodamine/DiI and Texas Red, but they can also be used for RFP. It has to be noted that the TRITC filter we used here (Chroma 41002) picks up light of slightly shorter wavelengths than other available TRITC filters, leading to a relatively strong signal with YFP expressing cells (see Subheading 2.5.). Finally, the JP1 and JP2 filter sets were developed by Dr. Jonathon Pines to visualize GFP and YFP in double labeling experiments (see ref. 15; Note 6).

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