Abstract

Flatbed scanner densitometers can be operated under various illumination and recording exposure levels. In this work, we show that optical density measurement accuracy, sensitivity, and stability of stained polyacrylamide electrophoresis gel densitometry are crucially dependent on these two factors (brightness and exposure level), notwithstanding that the source is monochromatic, spatially uniform, and the measurements are made using an accurately calibrated step wedge in tandem. We further outline a method to accommodate the intensity deviations over a range of illumination and exposure levels in order to maintain sensitivity and repeatability in the computed optical densities. Comparisons were also made with results from a commercial densitometer.

© 2010 Optical Society of America

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References

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  1. N. C. Kendrick, J. J. Johansen, P. R. Lee, and D. A. Santek, “Optimization of an HP Scanjet for quantification of protein electrophoresis gels,” Anal. Biochem. 219, 297-304 (1994).
    [CrossRef] [PubMed]
  2. S. G. Vincent, P. R. Cunningham, N. L. Stephens, A. J. Halayko, and J. T. Fisher, “Quantitative densitometry of proteins stained with Coomassie blue using a Hewlett Packard Scanjet scanner and Scanplot software,” Electrophoresis 18, 67-71 (1997).
    [CrossRef] [PubMed]
  3. H. Y. Tan, T. W. Ng, and O. W. Liew, “Light spectrum in flatbed scanner densitometry of stained polyacrylamide electrophoresis gels,” Biotech. 42, 474-478 (2007).
    [CrossRef]
  4. H. Y. Tan, T. W. Ng, and O. W. Liew, “Adapted LCD backlighting unit for densitometry of stained polyacrylamide electrophoresis gels,” Electrophoresis 30, 987-990 (2009).
    [CrossRef] [PubMed]
  5. H. Y. Tan and T. W. Ng, “Accurate step wedge calibration for densitometry of electrophoresis gels,” Opt. Commun. 281, 3013-3017 (2008).
    [CrossRef]
  6. J. Cabello, A. Bailey, I. Kitchen, M. Prydderch, A. Clark, R. Turchetta, and K. Wells, “Digital autoradiography using room temperature CCD and CMOS imaging technology,” Phys. Med. Biol. 52, 4993-5011 (2007).
    [CrossRef] [PubMed]
  7. A. Ferrero, J. Campos, and A. Pons, “Correction of photoresponse nonuniformity for matrix detectors based on prior compensation for their nonlinear behavior,” Appl. Opt. 45, 2422-2427 (2006).
    [CrossRef] [PubMed]
  8. M. Gassmann, B. Grenacher, B. Rohde, and J. Vogel, “Quantifying Western blots: pitfalls of densitometry,” Electrophoresis 30, 1845-1855 (2009).
    [CrossRef] [PubMed]
  9. U. K. Laemmli, “Cleavage of structural proteins during the assembly of the head of bacteriophage T4,” Nature 227, 680-685 (1970).
    [CrossRef] [PubMed]

2009 (2)

H. Y. Tan, T. W. Ng, and O. W. Liew, “Adapted LCD backlighting unit for densitometry of stained polyacrylamide electrophoresis gels,” Electrophoresis 30, 987-990 (2009).
[CrossRef] [PubMed]

M. Gassmann, B. Grenacher, B. Rohde, and J. Vogel, “Quantifying Western blots: pitfalls of densitometry,” Electrophoresis 30, 1845-1855 (2009).
[CrossRef] [PubMed]

2008 (1)

H. Y. Tan and T. W. Ng, “Accurate step wedge calibration for densitometry of electrophoresis gels,” Opt. Commun. 281, 3013-3017 (2008).
[CrossRef]

2007 (2)

J. Cabello, A. Bailey, I. Kitchen, M. Prydderch, A. Clark, R. Turchetta, and K. Wells, “Digital autoradiography using room temperature CCD and CMOS imaging technology,” Phys. Med. Biol. 52, 4993-5011 (2007).
[CrossRef] [PubMed]

H. Y. Tan, T. W. Ng, and O. W. Liew, “Light spectrum in flatbed scanner densitometry of stained polyacrylamide electrophoresis gels,” Biotech. 42, 474-478 (2007).
[CrossRef]

2006 (1)

1997 (1)

S. G. Vincent, P. R. Cunningham, N. L. Stephens, A. J. Halayko, and J. T. Fisher, “Quantitative densitometry of proteins stained with Coomassie blue using a Hewlett Packard Scanjet scanner and Scanplot software,” Electrophoresis 18, 67-71 (1997).
[CrossRef] [PubMed]

1994 (1)

N. C. Kendrick, J. J. Johansen, P. R. Lee, and D. A. Santek, “Optimization of an HP Scanjet for quantification of protein electrophoresis gels,” Anal. Biochem. 219, 297-304 (1994).
[CrossRef] [PubMed]

1970 (1)

U. K. Laemmli, “Cleavage of structural proteins during the assembly of the head of bacteriophage T4,” Nature 227, 680-685 (1970).
[CrossRef] [PubMed]

Bailey, A.

J. Cabello, A. Bailey, I. Kitchen, M. Prydderch, A. Clark, R. Turchetta, and K. Wells, “Digital autoradiography using room temperature CCD and CMOS imaging technology,” Phys. Med. Biol. 52, 4993-5011 (2007).
[CrossRef] [PubMed]

Cabello, J.

J. Cabello, A. Bailey, I. Kitchen, M. Prydderch, A. Clark, R. Turchetta, and K. Wells, “Digital autoradiography using room temperature CCD and CMOS imaging technology,” Phys. Med. Biol. 52, 4993-5011 (2007).
[CrossRef] [PubMed]

Campos, J.

Clark, A.

J. Cabello, A. Bailey, I. Kitchen, M. Prydderch, A. Clark, R. Turchetta, and K. Wells, “Digital autoradiography using room temperature CCD and CMOS imaging technology,” Phys. Med. Biol. 52, 4993-5011 (2007).
[CrossRef] [PubMed]

Cunningham, P. R.

S. G. Vincent, P. R. Cunningham, N. L. Stephens, A. J. Halayko, and J. T. Fisher, “Quantitative densitometry of proteins stained with Coomassie blue using a Hewlett Packard Scanjet scanner and Scanplot software,” Electrophoresis 18, 67-71 (1997).
[CrossRef] [PubMed]

Ferrero, A.

Fisher, J. T.

S. G. Vincent, P. R. Cunningham, N. L. Stephens, A. J. Halayko, and J. T. Fisher, “Quantitative densitometry of proteins stained with Coomassie blue using a Hewlett Packard Scanjet scanner and Scanplot software,” Electrophoresis 18, 67-71 (1997).
[CrossRef] [PubMed]

Gassmann, M.

M. Gassmann, B. Grenacher, B. Rohde, and J. Vogel, “Quantifying Western blots: pitfalls of densitometry,” Electrophoresis 30, 1845-1855 (2009).
[CrossRef] [PubMed]

Grenacher, B.

M. Gassmann, B. Grenacher, B. Rohde, and J. Vogel, “Quantifying Western blots: pitfalls of densitometry,” Electrophoresis 30, 1845-1855 (2009).
[CrossRef] [PubMed]

Halayko, A. J.

S. G. Vincent, P. R. Cunningham, N. L. Stephens, A. J. Halayko, and J. T. Fisher, “Quantitative densitometry of proteins stained with Coomassie blue using a Hewlett Packard Scanjet scanner and Scanplot software,” Electrophoresis 18, 67-71 (1997).
[CrossRef] [PubMed]

Johansen, J. J.

N. C. Kendrick, J. J. Johansen, P. R. Lee, and D. A. Santek, “Optimization of an HP Scanjet for quantification of protein electrophoresis gels,” Anal. Biochem. 219, 297-304 (1994).
[CrossRef] [PubMed]

Kendrick, N. C.

N. C. Kendrick, J. J. Johansen, P. R. Lee, and D. A. Santek, “Optimization of an HP Scanjet for quantification of protein electrophoresis gels,” Anal. Biochem. 219, 297-304 (1994).
[CrossRef] [PubMed]

Kitchen, I.

J. Cabello, A. Bailey, I. Kitchen, M. Prydderch, A. Clark, R. Turchetta, and K. Wells, “Digital autoradiography using room temperature CCD and CMOS imaging technology,” Phys. Med. Biol. 52, 4993-5011 (2007).
[CrossRef] [PubMed]

Laemmli, U. K.

U. K. Laemmli, “Cleavage of structural proteins during the assembly of the head of bacteriophage T4,” Nature 227, 680-685 (1970).
[CrossRef] [PubMed]

Lee, P. R.

N. C. Kendrick, J. J. Johansen, P. R. Lee, and D. A. Santek, “Optimization of an HP Scanjet for quantification of protein electrophoresis gels,” Anal. Biochem. 219, 297-304 (1994).
[CrossRef] [PubMed]

Liew, O. W.

H. Y. Tan, T. W. Ng, and O. W. Liew, “Adapted LCD backlighting unit for densitometry of stained polyacrylamide electrophoresis gels,” Electrophoresis 30, 987-990 (2009).
[CrossRef] [PubMed]

H. Y. Tan, T. W. Ng, and O. W. Liew, “Light spectrum in flatbed scanner densitometry of stained polyacrylamide electrophoresis gels,” Biotech. 42, 474-478 (2007).
[CrossRef]

Ng, T. W.

H. Y. Tan, T. W. Ng, and O. W. Liew, “Adapted LCD backlighting unit for densitometry of stained polyacrylamide electrophoresis gels,” Electrophoresis 30, 987-990 (2009).
[CrossRef] [PubMed]

H. Y. Tan and T. W. Ng, “Accurate step wedge calibration for densitometry of electrophoresis gels,” Opt. Commun. 281, 3013-3017 (2008).
[CrossRef]

H. Y. Tan, T. W. Ng, and O. W. Liew, “Light spectrum in flatbed scanner densitometry of stained polyacrylamide electrophoresis gels,” Biotech. 42, 474-478 (2007).
[CrossRef]

Pons, A.

Prydderch, M.

J. Cabello, A. Bailey, I. Kitchen, M. Prydderch, A. Clark, R. Turchetta, and K. Wells, “Digital autoradiography using room temperature CCD and CMOS imaging technology,” Phys. Med. Biol. 52, 4993-5011 (2007).
[CrossRef] [PubMed]

Rohde, B.

M. Gassmann, B. Grenacher, B. Rohde, and J. Vogel, “Quantifying Western blots: pitfalls of densitometry,” Electrophoresis 30, 1845-1855 (2009).
[CrossRef] [PubMed]

Santek, D. A.

N. C. Kendrick, J. J. Johansen, P. R. Lee, and D. A. Santek, “Optimization of an HP Scanjet for quantification of protein electrophoresis gels,” Anal. Biochem. 219, 297-304 (1994).
[CrossRef] [PubMed]

Stephens, N. L.

S. G. Vincent, P. R. Cunningham, N. L. Stephens, A. J. Halayko, and J. T. Fisher, “Quantitative densitometry of proteins stained with Coomassie blue using a Hewlett Packard Scanjet scanner and Scanplot software,” Electrophoresis 18, 67-71 (1997).
[CrossRef] [PubMed]

Tan, H. Y.

H. Y. Tan, T. W. Ng, and O. W. Liew, “Adapted LCD backlighting unit for densitometry of stained polyacrylamide electrophoresis gels,” Electrophoresis 30, 987-990 (2009).
[CrossRef] [PubMed]

H. Y. Tan and T. W. Ng, “Accurate step wedge calibration for densitometry of electrophoresis gels,” Opt. Commun. 281, 3013-3017 (2008).
[CrossRef]

H. Y. Tan, T. W. Ng, and O. W. Liew, “Light spectrum in flatbed scanner densitometry of stained polyacrylamide electrophoresis gels,” Biotech. 42, 474-478 (2007).
[CrossRef]

Turchetta, R.

J. Cabello, A. Bailey, I. Kitchen, M. Prydderch, A. Clark, R. Turchetta, and K. Wells, “Digital autoradiography using room temperature CCD and CMOS imaging technology,” Phys. Med. Biol. 52, 4993-5011 (2007).
[CrossRef] [PubMed]

Vincent, S. G.

S. G. Vincent, P. R. Cunningham, N. L. Stephens, A. J. Halayko, and J. T. Fisher, “Quantitative densitometry of proteins stained with Coomassie blue using a Hewlett Packard Scanjet scanner and Scanplot software,” Electrophoresis 18, 67-71 (1997).
[CrossRef] [PubMed]

Vogel, J.

M. Gassmann, B. Grenacher, B. Rohde, and J. Vogel, “Quantifying Western blots: pitfalls of densitometry,” Electrophoresis 30, 1845-1855 (2009).
[CrossRef] [PubMed]

Wells, K.

J. Cabello, A. Bailey, I. Kitchen, M. Prydderch, A. Clark, R. Turchetta, and K. Wells, “Digital autoradiography using room temperature CCD and CMOS imaging technology,” Phys. Med. Biol. 52, 4993-5011 (2007).
[CrossRef] [PubMed]

Anal. Biochem. (1)

N. C. Kendrick, J. J. Johansen, P. R. Lee, and D. A. Santek, “Optimization of an HP Scanjet for quantification of protein electrophoresis gels,” Anal. Biochem. 219, 297-304 (1994).
[CrossRef] [PubMed]

Appl. Opt. (1)

Biotech. (1)

H. Y. Tan, T. W. Ng, and O. W. Liew, “Light spectrum in flatbed scanner densitometry of stained polyacrylamide electrophoresis gels,” Biotech. 42, 474-478 (2007).
[CrossRef]

Electrophoresis (3)

H. Y. Tan, T. W. Ng, and O. W. Liew, “Adapted LCD backlighting unit for densitometry of stained polyacrylamide electrophoresis gels,” Electrophoresis 30, 987-990 (2009).
[CrossRef] [PubMed]

M. Gassmann, B. Grenacher, B. Rohde, and J. Vogel, “Quantifying Western blots: pitfalls of densitometry,” Electrophoresis 30, 1845-1855 (2009).
[CrossRef] [PubMed]

S. G. Vincent, P. R. Cunningham, N. L. Stephens, A. J. Halayko, and J. T. Fisher, “Quantitative densitometry of proteins stained with Coomassie blue using a Hewlett Packard Scanjet scanner and Scanplot software,” Electrophoresis 18, 67-71 (1997).
[CrossRef] [PubMed]

Nature (1)

U. K. Laemmli, “Cleavage of structural proteins during the assembly of the head of bacteriophage T4,” Nature 227, 680-685 (1970).
[CrossRef] [PubMed]

Opt. Commun. (1)

H. Y. Tan and T. W. Ng, “Accurate step wedge calibration for densitometry of electrophoresis gels,” Opt. Commun. 281, 3013-3017 (2008).
[CrossRef]

Phys. Med. Biol. (1)

J. Cabello, A. Bailey, I. Kitchen, M. Prydderch, A. Clark, R. Turchetta, and K. Wells, “Digital autoradiography using room temperature CCD and CMOS imaging technology,” Phys. Med. Biol. 52, 4993-5011 (2007).
[CrossRef] [PubMed]

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Figures (7)

Fig. 1
Fig. 1

(a) Schematic description of experiment to determine the illumination level emitted from adapted LCD backlighting using a lux meter as the distance of the fluorescent lamp to light guide was varied. (b) Values of illumination recorded as the distance between the fluorescent lamp and light guide (x) was varied. Inset in (b) depicts brightness from the time the lamp was switched on.

Fig. 2
Fig. 2

Flatbed scanner image recorded of the calibration wedge: (a) without and (b) with marking. (c) The optical densities measured at each wedge set from both images do not show any differences.

Fig. 3
Fig. 3

Laser calibrated OD values versus intensity levels (as determined by Quantity One) of the wedge recorded at brightness 265 lux , exposure level 6, brightness 265 lux , exposure level 14, brightness 460 lux , exposure level 6, and brightness 460 lux , exposure level 14.

Fig. 4
Fig. 4

Plots of r (coefficient of variation) against protein amounts for measurements (a) in the 150 to 460 lux and 46 to 460 lux ranges for all exposure levels as well as (b) in the 0 to 14 and 6 to 14 ranges for brightness values between 150 to 460 lux .

Fig. 5
Fig. 5

Linear regression coefficient R 2 value distribution at specific brightness and exposure levels for protein quantity ranges of (a)  10 ng to 200 ng , (b)  50 ng to 600 ng , and (c)  200 ng to 1000 ng .

Fig. 6
Fig. 6

Plots of intensity levels (as depicted by Quantity One) against amount of proteins on the stained gel at brightness 460, exposure level 14 (circles), brightness 460, exposure level 6 (asterisks), and brightness 206, exposure level 14 (triangles).

Fig. 7
Fig. 7

Plots of OD computed for different (a) brightness (at constant exposure level of 14), and (b) exposure levels (at constant brightness of 460 lux ) against amount of proteins on the stained gel using the flatbed scanner. Included in both sets of plots is the measurement result obtained using the AlphaImager, in which recording was done at 3710 lux .

Tables (1)

Tables Icon

Table 1 Percentage of Negative Optical Density Values (Which Denote Error) at Each Brightness Level Taken from Seven Defined Exposure Levels (2 to 14) as Well as at Each Exposure Level Taken from Eight Defined Brightness Levels ( 46 Lux to 460 Lux )

Equations (4)

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N N o = R ( λ ) E ( λ ) t exp ,
N = R 0 ( λ ) r L N ( N ) E ( λ ) t exp + N o .
OD ( λ ) = log 10 ( N i N t ) .
r = σ ( f ) f ,

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