Measurement of green fluorescent protein concentration in single cells by image analysis Agnelo Furtado and Robert Henry * Cooperative Research Centre for Molecular Plant Breeding, Centre for Plant Conservation, Genetics, Southern Cross University, Military Road, Lismore, NSW 2480, Australia Received 21 May 2002 Abstract The gene encoding the green fluorescent protein (GFP) has been widely used in studies of gene expression. The GFP can be detected nondestructively in living cells or tissues by the green fluorescence of the protein under blue light. Solutions of enhanced GFP (EGFP) of known concentration were filled in glass capillaries and used to calibrate a method for quantitative determination of EGFP or GFP-S65T in plant cells. Images captured by a digital camera were analyzed to determine the linear range for mea- surement of EGFP expression. The value of the method was illustrated by analysis of the relative levels of GFP expression under control of different promoters in aleurone cells of barley. Ó 2002 Elsevier Science (USA). All rights reserved. Keywords: Image analysis; GFP; Fluorescence; Promoter analysis; Aleurone Studies on signal transduction, promoter activity, and gene expression can be carried out in various ways, one of which is using an in vivo reporter system. In such a system, a reporter gene is linked to a regulatory DNA sequence (promoter/enhancer), and changes in reporter gene activity reflect changes in the regulatory biological processes. Several reporter genes such as uidA [1] encoding b- glucuronidase, LacZ encoding b-galactosidase [2], cat encoding chloramphenicol acetyltransferase, (CAT) [3] and luc encoding luciferase [4,5] are commonly used to study gene expression and promoter activity in eukary- otic cells. The activities of such reporters can be detected by measuring the product of an enzymatic reaction. Although these reporter enzymes have several advanta- ges including high sensitivity, accuracy, and precision, disadvantages include cytotoxic assay, destructive sam- pling of tissue, and expensive substrates, and detection systems [1,6,7]. The green fluorescent protein (GFP) 1 from the jelly- fish Aequorea victoria is widely used as a reporter gene to study gene expression and protein localization in mammalian, bacterial, and plant systems [8–10]. The gfp gene encodes the GFP, which emits green fluorescence (509 nm) when exposed to blue light (488 nm), without the addition of substrate or cofactor, both in vivo and in vitro [11–13]. The protein requires the presence of oxy- gen [14] to emit light, and the fluorescence of GFP is independent of cell type or location and resistant to photo bleaching and remains stable under a wide variety of conditions [13,15,16]. The GFP fluorescence activity can be detected with minimal handling and, in particu- lar, no preparation of lysate, using a fluorescence mi- croscope, a fluorometer, a fluorescence-activated cell sorting machine, or an imaging microplate reader. The detection of GFP fluorescence can be enhanced using a charge-coupled device (CCD)-based system [17]. Several variants of the original wild-type GFP (wt- GFP) have been engineered with mutations affecting the Analytical Biochemistry 310 (2002) 84–92 www.academicpress.com ANALYTICAL BIOCHEMISTRY * Corresponding author. Fax: +61-2-6622-2080. E-mail address: rhenry@scu.edu.au (R. Henry). 1 Abbreviations used: GFP, green fluorescent protein; wt-GFP, wild- type GFP; EGFP, enhanced GFP; ABA, abscisic acid; CCD, charge- coupled device; GA, gibberellic acid; BS, background subtracted. 0003-2697/02/$ - see front matter Ó 2002 Elsevier Science (USA). All rights reserved. PII:S0003-2697(02)00281-6