An Epifluorescent Digital Photographic System Suitable for Undergraduate Classroom Investigation of Expression of Red-Shifted Green Fluorescent Protein

Baker, Stokes, Cleo Vidican, Haittam Greib, and Christine Jarocki

An inexpensive epifluorescent attachment for digital cameras has been developed to photograph red-shifted Green Fluorescent Protein (sGFP) expression in whole Arabidopsis plants. The apparatus was designed to support quantitative inquiry instruction. Formal assessments of undergraduates that used transgenic Arabidopsis plants that contained an environmentally regulated GUS reporter gene indicated that students increased their quantitative analytical skills after completing inquiry investigations. However, the GUS reporter gene is not ideal for teaching laboratories because of the time-lag to detect gene expression and the qualitative nature of histochemical stains. Fluorescence in transgenic Arabidopsis expressing chimeric sGFP genes (CaMV 35S promoter and Arabidopsis ADH promoter) has been photographed with a Canon EOS 30D digital camera with an attached filter cube constructed with commercially available dichroic filters (magenta for excitation, yellow plus cyan for barrier, and 45o blue for beam splitting) that was illuminated with a 5 watt Luxeon V Star 440-460 nm blue light emitting diode. Tiff files were quantified with NIH ImageJ. Experiments with purified eGFP (BioVision) produced a linear camera response with protein concentration (r2 = 0.986) and exposure time (r2 = 0.981) when mean pixel intensity was below 35100 counts. eGFP concentrations as low as 1.33 ng/μl produced a signal 3.2 fold above background. A 10 sec. exposure time produced the optimum signal to noise ratio. Chlorophyll fluorescence with the cyan filter removed produced curvilinear relationships for both concentration (r2 = 0.944) and exposure time (r2 = 0.984).