Imaging Chemiluminescence by Scanning

Imaging Chemiluminescence by Scanning
LI-COR® Biosciences • 4647 Superior Street, Lincoln, NE 68504 • 402-467-0700 • 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 impossible:
line-read scanning of chemiluminescent signals.1 Film-quality Western images are digitally captured in as
little as 6 minutes, and no changes are required in detection protocols or reagents. The low-noise sensor
array and short optical working distance maximize the efficiency of light collection and enable rapid scanning. The sensitivity, dynamic range, and affordability of the C-DiGit Blot Scanner make it a true digital
replacement for film in Western blotting.
Outline:
Page
1. Introduction to chemiluminescence. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
Conventional imaging methods for chemiluminescent Westerns . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
2. The C-DiGit approach . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
2.1 Maximizing light collection – short working distance and high numerical aperture . . . . . . . . . . . . . 3
2.2 Affordable low-noise sensors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
3. Imaging chemiluminescence by scanning. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
3.1 Very short scan times . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
3.2 Multi-scan, multi-exposure imaging and wide dynamic range . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
4. Summary and References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
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 enzyme causes oxidation of the luminol-based chemiluminescent substrate, creating an excited state product. As this product decays to a lower energy state, it transiently produces light (Fig. 1).2 Unlike fluorescent
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.
O
O
NH
H2O2 +
HRP
NH
NH2
O
NH2
O
O
*
O–
O–
O–
O–
NH2
+
LIGHT
@ 425nm
O
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.
Imaging Chemiluminescence by Scanning – Page 2
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
collected 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 generated 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. They provide a greater linear dynamic range
than film, do not require darkroom facilities, and generate a digital image for archiving and analysis.
However, CCD systems have their own limitations. To achieve the low noise level that is required, highend 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 consequences. To accommodate the long
optical path, the physical size of the imager must increase. The increased 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. 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.
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 integration times to capture sufficient signal.
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Imaging Chemiluminescence by Scanning – Page 3
2. The C-DiGit 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.
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
captured very close to the surface of the blot, preventing loss of photons (Fig. 3 & 4). The linear array
of imaging sensors performs a fast-exposure line read to quickly capture data across the entire blot.
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 can be wrapped in plastic, if desired, but
it isn’t necessary.
Because of the short working distance and specialized optics of the C-DiGit 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 signal to be obtained with very short exposures.
Obtaining similar sensitivity with a CCD area imager would require long integration times. As a result, it is
now possible to accurately image chemiluminescent signals by line scanning.
Efficiency of light collection
Digital Image
CCD
Film
C-DiGit
Reduced
High
High
High
No
Yes
Figure 4. The C-DiGit Scanner combines high-efficiency light collection with digital image output.
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Imaging Chemiluminescence by Scanning – Page 4
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 footprint 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, exposure times are orders of magnitude
shorter than a wide-area imager (a fraction of a second, 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 combine
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 sensor array’s dramatic increase in light collection efficiency makes the exposure time for each line so short that accurate line-scan
imaging is now possible. The C-DiGit 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 substrate. 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 pixel 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 increased
by more than 10,000%. This allows comparable signals to be obtained with exposure times
that are dramatically shorter than a CCD imager, and the sensor array can move quickly
across the blot to capture data. As discussed, very short exposure times allow the sensors
to be used without temperature cooling.
•
Multiple linear sensor arrays. To cover the blot area quickly, an array of 16 linear sensors
is mechanically scanned across the blot (Figs. 5 & 6). The sensors are spatially staggered
for complete coverage of the scan area, and all sensors collect data simultaneously (Fig. 6).
Because each individual sensor must scan only a fraction of the total area (~2.5 cm), this design
innovation dramatically reduces scan time. The resulting data are assembled to generate an
image of the entire blot area.
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Imaging Chemiluminescence by Scanning – Page 5
Figure 5. C-DiGit Blot Scanner design. The Western blot is placed facedown in the 8.5 x 10 cm scan window on the glass scan surface. Blots
can be wrapped in plastic if desired. When the lid is closed, light baffles
in the lid ensure that ambient light does not reach the sensors.
Ligh
Sca
Figure 6. CCD sensor array. A staggered array of
16 linear sensors is mechanically scanned back and
forth to capture the image. The sensors are organized
into four scan heads. Each scan head contains four
linear sensors, which move together as a unit. Use
of multiple 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 window,
and moves down into the scan window during image
capture.
Scan Head
PLACE SAMPLE
FACE DOWN
Figure 7. C-DiGit scan time is very short, relative to time-dependent change of most
chemiluminescent substrates. Signal stability of various substrates was measured over
time, using the Odyssey® Fc Imager. Yellow
bar indicates a 6-min scan window.
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Imaging Chemiluminescence by Scanning – Page 6
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. Figure 7 shows light emission over time
for several commercial substrates. The rate of substrate decay during the scan window is very small; however, 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 (as shown in Fig. 7). Quantification clearly
shows that signals were very accurately documented (Fig. 8B; R2 = 0.998). This confirms the reliability and
precision of C-DiGit 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.
A.
B.
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 applied, 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 enzyme and signal intensity.
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 available: standard sensitivity (6-minute scan) and high sensitivity (12-minute scan). “Standard” sensitivity is
suitable for most Western blot experiments, and “high” is recommended 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.
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Imaging Chemiluminescence by Scanning – Page 7
A) Standard
B) High
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 imaged
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.
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 (~ 4-10 orders of magnitude6) and Western blots often include both
strong and faint samples.
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 different “exposures”, short or long. Image display settings do not alter or affect the raw
data or signal intensities; they affect visual display only.
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Imaging Chemiluminescence by Scanning – Page 8
A) Film
B) C-DiGit
Figure 11. Strong and faint bands can be clearly viewed in the same
C-DiGit Blot Scanner 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).
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.
Figure 12. A single C-DiGit image can be adjusted to replicate a traditional film exposure, and generate additional exposures. All C-DiGit images (A-E) were rendered from a single image file by adjusting image display
settings in ImageStudio 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.
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Imaging Chemiluminescence by Scanning – Page 9
4. Summary
The C-DiGit Chemiluminescent Western Blot Scanner is the first true digital replacement for film. This innovative technology makes it possible to quickly and accurately image chemiluminescent signals by 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 chemiluminescent
signals on Western blots can now be achieved by 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 science 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 Cytochemistry
41(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).
LI-COR is an ISO 9001 registered company. ©2013 LI-COR, Inc. LI-COR, Odyssey, and C-DiGit are trademarks or registered
trademarks of LI-COR, Inc. in the United States and other countries. All other trademarks belong to their respective owners.
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Doc # 979-13541
01/13