Imaging Chemiluminescence by Scanning

Imaging Chemiluminescence by Scanning

LI-COR® Biosciences • 4647 Superior Street, Lincoln, NE 68504 • www.licor.com

The C-DiGit® Chemiluminescent Western Blot Scanner is a digital replacement for film, combining the

sensitivity of film with the convenience and flexibility of a CCD imager. To achieve this breakthrough at an

affordable price, LI-COR implemented a new imaging approach that was previously considered impossi-

ble: line-read scanning of chemiluminescent signals.1 Film-quality Western images are digitally captured

in as little as 6 minutes, and no changes are generally required in detection protocols or reagents (if

very high sensitivity is required, a high-performance substrate such as WesternSure™ PREMIUM or

SuperSignal® West Femto is recommended). The low-noise sensor array and short optical working dis-

tance maximize the efficiency of light collection and enable rapid line-scan imaging. The sensitivity, dy-

namic range, and affordability of the C-DiGit Blot Scanner make it a true digital replacement for film in

Western blotting.

1. Introduction to chemiluminescence

Enhanced chemiluminescence (ECL) is widely used for detection of target proteins on Western blots. For

detection, horseradish peroxidase (HRP) enzyme is typically conjugated to a secondary antibody. The en-

zyme causes oxidation of the luminol-based chemiluminescent substrate, creating an excited-state prod-

uct. As this product decays to a lower energy state, it transiently produces light (Fig. 1).2 Unlike fluor-

escent detection, chemiluminescence does not require excitation light. Light emission only occurs in

areas where the chemical reaction occurs, enabling low optical background and high detection sensitivity.

Outline: Page

1. Introduction to chemiluminescence. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1

Conventional methods for imaging chemiluminescent Westerns . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2

2. The C-DiGit Scanner: a new approach to chemiluminescence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3

2.1 Maximizing light collection – short working distance and high numerical aperture . . . . . . . . . . . . . 3

2.2 Affordable low-noise sensors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4

3. Imaging of chemiluminescence by scanning . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4

3.1 Very short scan times . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4

3.2 Multi-scan, multi-exposure imaging and wide dynamic range . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7

4. Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9

5. References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9

+ LIGHT@425nm

Figure 1. Chemiluminescent reaction. Luminol is a widely used chemiluminescent reagent. Oxidation of luminol

by peroxide creates an excited-state product, 3-aminophthalate. Photons of light are released when this product

decays to a lower energy state.

H2O2

Two important characteristics of chemiluminescent signals must be considered: they are weak, and they

change over time. Because signals are weak, high signal-to-noise detection methods (such as X-ray film

or digital imaging with high-efficiency imaging optics and low-noise detectors) are required. Because light

emission is non-constant, signals are typically recorded with stationary imaging methods rather than

scanning. Stationary imaging simultaneously collects signal from all light-emitting areas of the sample,

and allows signal to be integrated over an extended time period.

Conventional methods for imaging chemiluminescent Westerns

The most common method used to document chemiluminescence is photographic film. Film exposure

is very sensitive, because the film is placed directly onto the Western blot and photons are efficiently col-

lected as they are generated at the blot surface. However, film has several significant limitations. The

response of film to light is non-linear, with a limited dynamic range that is easily saturated.3 It requires

darkroom facilities, costly supplies, photochemical processing, and hazardous waste disposal. Data gen-

erated on film must typically be converted to digital format for analysis and publication. The limitations

of film for Western blot imaging are discussed in detail elsewhere.

CCD cameras are also used to image chemiluminescence. Like film, this is an “area imaging” approach

that requires continuous exposure of the entire blot. CCD systems provide a greater linear dynamic range

than film, do not require darkroom facilities, and generate a digital image for archiving and analysis. How-

ever, CCD systems have their own limitations. To achieve the low noise level that is required for extended

imaging of the entire blot area, high-end scientific CCD chips are traditionally needed. However, because

these chips are small (~1 cm2) and the Western blot is much larger, imaging optics are required to project

the sample area onto the CCD. A long optical path is therefore required, with several important conse-

quences. To accommodate the long optical path, the physical size of the imager must increase. The in-

creased optical path also decreases the collection efficiency of the lens, due to the inverse-square law

(Fig. 2). This requires special, high-end optics and longer integration times to achieve sufficient signal

intensity for area imaging. Long exposure times, in turn, require the CCD to be highly cooled for reduction

of dark noise.4 These interconnected factors account for the high cost and large size of CCD imaging systems.

Imaging Chemiluminescence by Scanning –

LI-COR Biosciences www.licor.com

Figure 2. Close proximity to the source increases signal intensity. The inverse-square law applies when light is

radiated outward from a point source. As emitted light moves farther from the source, it spreads out over a larger

area. The intensity of light at a given distance is inversely proportional to the square of the distance from the

source. Because a long optical path reduces the intensity of emitted light, a CCD area imager requires long inte-

gration times to capture sufficient signal.

2. The C-DiGit® Blot Scanner: a new approach to chemiluminescence

The LI-COR C-DiGit Western Blot Scanner takes a different approach. It combines the advantages of film

and CCD imagers to create a high-performance, highly-affordable chemiluminescence imager. C-DiGit

imaging combines the sensitivity and familiarity of film detection with the flexibility, dynamic range, and

digital data output of CCD imagers. This innovation is the first true digital replacement for x-ray film.

The C-DiGit approach maximizes light collection, using affordable low-noise sensors that do not require

cooling and revolutionary new line-scan imaging technology.

2.1 Maximizing light collection – short working distance and high numerical aperture

The sensitivity of film is boosted by its short working distance, with very close proximity to the Western

blot. In contrast, a CCD area imager has a working distance of hundreds of millimeters. This distance

greatly impacts the efficiency of light collection by the camera; as dictated by the inverse-square law,

light intensity drops off rapidly as distance from the source increases5 (Fig. 2). Like film, the C-DiGit

sensor array uses a very short working distance (~1 mm) to maximize light collection (Fig. 3). Light is cap-

tured very close to the surface of the blot, preventing loss of photons (Figs. 3 & 4). The linear array of im-

aging sensors performs a fast-exposure line read to quickly capture data across the entire blot, one line-

read at a time. More than 400 lines of image data are compiled to create the final image.

Because of the short working distance and specialized optics of the C-DiGit Blot Scanner, the working

numerical aperture of the optical system is at least 10 times higher than a typical CCD area imager. This

increases light collection by 100 - 400-fold, and allows comparable signals to be obtained with very short

exposures that enable line-scan imaging. During a scan, any given point on the membrane is only exposed

Figure 3. Like film, C-DiGit imaging uses a very short

working distance to maximize light collection.The

Western blot is placed face down, directly on the scan

surface. Blots must be kept wet during imaging.



Figure 4. The C-DiGit Scanner combines high-efficiency light collection with digital image output.

CCD Film C-DiGit

Efficiency of light collection Reduced High High

Digital Image Yes No Yes

to the detector for a few seconds, to generate that line-read. The detector then moves on to capture the

next line in the image. Hundreds of line-read scans are assembled to generate an image of the entire blot.

High-efficiency light collection is required to obtain results similar to continuous exposure of the entire

blot area. A CCD area imager would require long integration times, high-end optics, and a supercooled

CCD to collect similar signals across the entire blot area. The C-DiGit Blot Scanner provides sensitive, low-

noise digital imaging at an unprecedented low price.

2.2 Affordable low-noise sensors

Signal-to-noise ratio is a critical aspect of high-performance imaging. Control of electronic noise drives

the design and cost of CCD imagers. Because the long working distance of a CCD area imager decreases

signal intensity, long exposures are required to image chemiluminescent blots. Long exposures, in turn,

increase dark noise. CCDs must therefore be supercooled (typically to -30 °C or below) to suppress dark

noise,4 and supercooling dramatically increases the cost of the imager. There is a clear need for a more

cost-effective digital alternative to film for chemiluminescence imaging.

C-DiGit technology meets this need, offering high-sensitivity linear sensor imaging with a compact foot-

print and no need for cooling. Elimination of supercooling dramatically reduces the cost of the imager.

Because light collection efficiency is at least 100-fold higher, the exposure time for each line read is orders

of magnitude shorter than a wide-area imager (a few seconds, compared to several minutes or longer).

Short exposure times, in turn, produce less dark noise and enable use of low-noise linear sensors with no

cooling and reduced cost. Careful sensor choice, creative implementation, and short exposure times com-

bine to produce low noise and high sensitivity, at an unprecedented low price.

3. Imaging of chemiluminescence by scanning

Chemiluminescence has not typically been imaged by scanning, because long exposures are required and

signal brightness can change during an extended scan.1 But the C-DiGit Blot Scanner sensor array’s dra-

matic increase in light collection efficiency makes the exposure time for

each line read so short that accurate line-scan imaging is now possible.

The C-DiGit Blot Scanner’s proprietary technology provides reliable

detection of chemiluminescence with an enhanced dynamic range.

3.1 Very short scan times

The time required to image a Western blot is quite short, relative to

the temporal change in light emission of the chemiluminescent sub-

strate. The time scale of C-DiGit imaging is tightly compressed, with

very quick scan passes, to minimize temporal effects. Short scan

times are achieved in two ways:

• Very high light collection efficiency. Each line-read requires a very

short collection time (dwell time) for signal capture. Because the

C-DiGit Blot Scanner uses a numerical aperture at least 10 times

higher than a typical CCD imager, light collection efficiency is in-

creased by more than 10,000%. Comparable signals for each line-

read are obtained with only a few seconds of exposure, allowing

the sensor array to move quickly across the blot and capture hun-

dreds of individual lines of image data to compile the final image.

As discussed above, very short exposure times allow the sensors

to be used without temperature cooling.



Ligh

Sca

Figure 5. C-DiGit Blot Scanner design.

The Western blot is placed face-down

in the 8.5 x 10 cm scan window on the

glass scan surface. Blots must be kept

wet during imaging. When the Scanner

lid is closed, light baffles in the lid en-

sure that ambient light does not reach

the sensors.

• Multiple linear sensor arrays. To cover the blot area quickly, an array of 16 linear sensors is mechani

cally scanned across the blot (Figs. 5 & 6). The sensors are spatially staggered for complete coverage

of the scan area, and all sensors collect line-read data simultaneously (Fig. 6). Because each individual

sensor must scan only a fraction of the total area (~2.5 cm), this design innovation dramatically re-

duces scan time. The resulting data are assembled to generate an image of the entire blot area.

Figure 6. CCD sensor array. A staggered array of

16 linear sensors is mechanically scanned across

the blot to capture the image by line-read scan-

ning. A) The sensors are organized into four scan

heads. Each scan head contains four linear sen-

sors, which move together as a unit. Use of multi-

ple scan heads greatly increases the speed of

scanning. In this image, only three scan heads are

seen. The fourth head is just above the scan win-

dow, and moves down into the scan window dur-

ing image capture. B) Each linear sensor performs

line-scan imaging of one region of the blot. Line-

read data are assembled to generate the complete

image.



Figure 7. C-DiGit scan time is very short, rel-

ative to time-dependent change of most

chemiluminescent substrates. Signal stabil-

ity of various substrates was measured over

time, using the Odyssey® Fc Imager. Yellow

bar indicates a 6 min scan window.

A)

B)

The C-DiGit® scan time (< 6 min per pass) is very short, compared to the scale of time-dependent change

for most chemiluminescent substrates used for Western blotting. Fig. 7 shows light emission over time for

several commercial substrates. The rate of substrate decay during the scan window is very small. How-

ever, it is possible for time-dependent change in signal to affect data collection during this small window.

Sophisticated, proprietary algorithms are used to compensate for temporal effects.

The accuracy of C-DiGit imaging is illustrated in Figure 8. Serial dilutions of HRP conjugate were applied

to a dot blot (Fig. 8A). SuperSignal West Femto substrate was chosen for this experiment because of its

known time dependency and relatively fast decline in signal (shown in Fig. 7). Quantification clearly shows

that signals were very accurately documented (Fig. 8B; R2 = 0.998). This confirms the reliability and preci-

sion of C-DiGit line-scanning technology, even when the complexity of the experiment is increased by

placing comparable signals in opposing orientations and using a substrate with strong time dependency.

3.2 Multi-scan, multi-exposure imaging and wide dynamic range

C-DiGit technology uses a multi-scan, multi-exposure approach (Fig. 9). Two exposure options are avail-

able: standard sensitivity (6-minute scan) and high sensitivity (12-minute scan). “Standard” sensitivity is

suitable for Western blots that generate strong signals. “High” is recommended for blots with less intense

signals or for low-abundance protein targets that require longer film exposure. To perform a scan, the user

selects the desired exposure option from the Acquire tab, and clicks START to begin image capture. There

are no imaging parameters or settings to adjust.

Figure 8. C-DiGit scanning technology produces very accurate results. A) Two-fold serial dilutions of

HRP-conjugated secondary antibody were applied to a dot blot. Duplicate rows of dilutions were ap-

plied, in opposing orientations. The dot blot was detected with SuperSignal West Femto substrate.

B) Quantification shows a very accurate linear response between the relative concentration of en-

zyme and signal intensity.



A) B)

Because the dynamic range of the image is very wide (much wider than film), C-DiGit image data contain

the full range of possible “exposure times” for that Western blot (Fig. 10). Unlike film, C-DiGit detection

does not saturate easily when signals are strong. Both low-abundance and high-abundance protein targets

can clearly be viewed in the same image (Fig. 11). This is useful, because endogenous protein levels span

an extremely wide dynamic range in the cell (~ 4 - 10 orders of magnitude6) and Western blots often in-

clude both strong and faint samples.

Figure 9. “Standard sensitivity” and “high sensitivity” scan options

can be selected. ERK1/2 was detected in two-fold serial dilutions of

Jurkat cell lysates (beginning with 10 µg at left). SuperSignal West

Pico and the C-DiGit® Blot Scanner were used. A) The blot was im-

aged with “standard” sensitivity. B) The same blot was imaged again

immediately, with “high” sensitivity. This setting typically reveals ~1-2

additional bands in a two-fold dilution series.



A) Standard B) High

Figure 10. A C-DiGit image file captures the full range of chemiluminescent data for

that blot. Image display settings can be adjusted to render a wide range of differ-

ent “exposures”, short or long. Image display settings do not alter or affect the raw

data or signal intensities; they affect visual display only.

Image display settings can be adjusted to replicate any film exposure, short or long (Fig. 12). Adjustment

of image display settings does not alter or affect the raw data or quantification in any way; it affects visual

appearance only.



A) Film B) C-DiGit®

Figure 11. Strong and faint bands can be clearly viewed in the same

C-DiGit image. pEGFR was detected in A431 cells treated with EGF.

A) In a 15-sec film exposure, strong bands were “blown out” and

blurred together. Band margins could not be identified. B) In the

C-DiGit image of the same blot, image display adjustment was used

to resolve and separate the strong bands. Detection sensitivity was

equivalent (7 dilutions were visible in each image).

Figure 12. A single C-DiGit image can be adjusted to replicate a traditional film exposure, and generate addi-

tional exposures. All C-DiGit images (A-E) were rendered from a single image file, by adjusting image display

settings in Image Studio software. The image file captures the full range of chemiluminescent data for that

Western blot, and provides access to all image data that will be needed for that blot.

4. Summary

The C-DiGit® Chemiluminescent Western Blot Scanner is the first true digital replacement for film. This in-

novative technology makes it possible to quickly and accurately image chemiluminescent signals by line-

scanning. The short optical working distance and linear sensor array provide highly efficient collection of

light to enable rapid scanning and make supercooling unnecessary. Accurate detection of chemilumines-

cent signals on Western blots can now be achieved by line-read scanning.

This instrument was specifically engineered for affordability and quality. Detection protocols, reagents,

and incubations are unchanged for the easiest possible transition from film. The C-DiGit Blot Scanner sets

a new standard for simplicity and performance in Western blot imaging, and replaces film in the life sci-

ence laboratory.

5. References

1. Kodak. CCD Primer #KCP-001: Charge-coupled device (CCD) image sensors. Eastman Kodak Co.,

Microelectronics Technology Div., Rochester, NY, USA (1999).

2. Whitehead, TP, LJ Kricka, TJN Carter, and GHG Thorpe. Analytical luminescence: its potential in the

clinical laboratory. Clinical Chemistry. 25(9):1531-46 (1979).

3. Baskin, DG and WL Stahl. Fundamentals of quantitative autoradiography by computer densitometry

for in situ hybridization, with emphasis on 33P. Journal of Histochemistry and Cytochemistry41(12):1767-76 (1993).

4. Fellers, TJ and MW Davidson. Concepts in digital imaging - CCD noise sources and signal-to-noise

ratio. Hamamatsu Learning Center (2005).

5. Hamilton, CJ. Views of the solar system: guide to the inverse-square law. Jet Propulsion Laboratory(1997).

6. Mitchell, P. Proteomics re-trenches. Nature Biotechnology. 28(7):665-70 (2010).


4647 Superior Street • P.O. Box 4000 • Lincoln, Nebraska 68504 USATechnical Support: 800-645-4260 • North America: 800-645-4267International: 402-467-0700 • Fax: 402-467-0819 • www.licor.com/bio

LI-COR GmbH Germany, Serving Europe, Middle East, and Africa: +49 (0) 6172 17 17 771LI-COR UK Ltd. UK, Serving UK, Ireland, and Scandinavia: +44 (0) 1223 422104All other countries, contact LI-COR Biosciences or a local LI-COR distributor:http://www.licor.com/distributors

LI-COR is an ISO 9001 registered company. ©2013 LI-COR, Inc. LI-COR, Odyssey, WesternSure, and C-DiGit are trademarks orregistered trademarks of LI-COR, Inc. in the United States and other countries. All other trademarks belong to their respectiveowners. For patent information, visit www.licor.com/patents.

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Imaging Chemiluminescence by Scanning

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