Baker, Stokes S. .
Quantitative detection of GFP expression with digital SLR cameras.
Synthetic versions of green fluorescent protein (GFP) that shift the fluorescent spectrum towards red are ideal for in situ expression assays because the organism can be illuminated with visible blue light. Studies have shown that the level of GFP leaf surface fluorescence is directly proportional to the GFP soluble protein concentration. However, instruments that measure GFP surface fluorescence are expensive; thus, not readily available to many laboratories. The goal of this investigation was to develop techniques with consumer digital cameras to measure GFP fluorescence in transgenic plants. An inexpensive filter cube containing machine vision dichroic filters and illuminated with a blue light emitting diode (LED) was developed. The device can be attached to camera macro lenses or dissecting microscopes. Spectrum analysis showed that the apparatus illuminates specimens with wavelengths between ~450 nm and ~500 nm and detects fluorescence between ~510 nm and ~595 nm. Epifluorescent photographs taken with a single-lens reflex (SLR) digital camera were able to detect red-shifted GFP fluorescence in leaves and cotyledons of GFP expressing Arabidopsis thaliana. GFP fluorescence was detected in the green channel of the digital file. Titration studies with purified GFP were conducted with a complementary metal–oxide–semiconductor (CMOS) SLR camera. The raw image files responded linearly to protein surface density and exposure time (Ho: Β = 0 ng•s/mm2, r2 = 0.994, p = 1.75 x 10-29). Epifluorescent digital photographs taken with CMOS or CCD SLR cameras can be used to make quantitative measurements of red-shifted GFP surface fluorescence.
This research lowers the cost barrier associated with specialized equipment used to quantify in planta GFP expression. The price of constructing the apparatus is less than $175. As a result, undergraduates can now conduct sophisticated experiments on GFP expressing transgenic plants. The goals stated in the National Research Council's publication Biology 2010: Transforming Undergraduate Education for Future Research Biologists are advanced because students use the same image analysis software as research scientists to make quantitative measurements of GFP expression. Additionally, because of the quantitative nature of the data, statistical data analysis can be incorporated into inquiry undergraduate curriculum.
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1 - University of Detroit Mercy, Biology Department, 4001 W. McNichols Rd., Detroit, MI, 48221, USA
green fluorescent protein
Single lens reflex camera.
Presentation Type: Oral Paper:Papers for Sections
Location: 556A/Convention Center
Date: Monday, August 2nd, 2010
Time: 9:45 AM