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Calibration of Imaging System Is the First Step toward Quantitative Image Analysis

Immunofluorescence image provides important spatial information for biomarkers and is an essential tool for cell biologists, especially with the development of digital imaging technology. However, it is still regarded as a semi-quantitative method because there are so many factors affecting your final image. Here we are working to fill the gap to make immunofluorescence imaging a quantitative method. The first step toward this goal is to calibrate the imaging system that includes light source, optical path (filter cube) and CCD camera. Dark current is an important parameter to gauge the quality of your camera and can be estimated by capturing an image with no light condition. Knowing the dark current of your camera could help you to compare two images with different exposure times. Next we found that you can use colored plastics bars to calibrate the intensity of the light source, attention has to be paid on the focal plane on the plastic bar and the uniformity of the plastic bar in order to compare day to day variation coming from the light source. Finally flat field correction needs to be performed for each filter cube because even with the best alignment, you could still get 20% difference in pixel intensity at the field of view. We will use antibody validation as an example to show how we quantitatively assess the quality of antibodies. All images were acquired using Zeiss Imager Z1 and processed using ImageJ (version 1.40J).

calibration, quantitative, flat field correction, dark current, light source

Full Name   
Zhengyu Pang

General Electric Company Global Research Center


Short Biography   


Pennsylvania State University, University Park, PA

Ph. D., 2003

Major in Chemical Engineering, special interest in biotransport

East China University of Science and Technology, Shanghai, China

M. S., 1996

Major in Biochemical Engineering, special interest in bioseparation

Nanjing University of Chemical Technology, Nanjing, China

B. S., 1993

Major in Chemical Engineering

Peer-reviewed publications (in chronological order)

1. Pang Z, Cawse JN, Yu L, Richards WD. Doubly stochastic Poisson distribution of platelet adhesion on material surfaces and its implication on fluorescence image analysis. J Biomed Mater Res A 2008.

2. Lavender MD, Pang Z, Wallace CS, Niklason LE, Truskey GA. A system for the direct co-culture of endothelium on smooth muscle cells. Biomaterials 2005;26(22):4642-53

3. Florian JA, Kosky JR, Ainslie K, Pang Z, Dull RO, Tarbell JM. Heparan sulfate proteoglycan is a mechanosensor on endothelial cells. Circ Res 2003;93(10):e136-42.

4. Pang Z, Antonetti DA, Tarbell JM. Shear stress regulates HUVEC hydraulic conductivity by occludin phosphorylation. Ann Biomed Eng 2005;33(11):1536-45.

5. Pang Z, Tarbell JM. In vitro study of Starling's hypothesis in a cultured monolayer of bovine aortic endothelial cells. J Vasc Res 2003;40(4):351-8.

6. Pang Z, Wu X, Cao X, Zhu J, Dai G. Affinity chromatography purification of urokinase with a polystyrene-based medium. J. East China Univ. Sci. & Tech. 1996;22(4):417-422.

7. Zhou X, Cao X, Pang Z, Tang M, Wu X, Dai G. Isolation of urokinase from human urine. Chinese J. Pharmaceuticals 1996;27(8):243-245

Patent Application

1. Gerdes MJ, Sood A, Montalto MC, Can A, Ginty F, Bresnahan MA, Filkins RJ, Pang Z; GENERAL ELECTRIC COMPANY, assignee. SEQUENTIAL ANALYSIS OF BIOLOGICAL SAMPLES. US patent 28118934. 2008 2007-09-28.

2. Larsen M, Sood A, Gerdes MJ, Montalto MC, Pang Z, Ginty F; GENERAL ELECTRIC COMPANY, assignee. SEQUENTIAL ANALYSIS OF BIOLOGICAL SAMPLES. US patent 28118944. 2008 2007-09-28.

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