Film Photochemistry Effects

Chemiluminescent Westerns:

How film and photochemistry

affect experimental results

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

1. Introduction

Since the 1970s, enhanced chemiluminescence has been used to detect proteins on Western blots.1 Sec-

ondary antibodies are labeled with the horseradish peroxidase (HRP) enzyme, which oxidizes the luminol-

based chemiluminescent substrate and causes it to transiently produce light at ~428 nm (Fig. 1). This

sensitive, reliable detection chemistry is typically documented by exposure of the blot to x-ray film, which

darkens in response to the emitted light. Signal intensity is determined by the number of HRP molecules

reacting with substrate. Chemiluminescent blots are often analyzed by visual assessment of band intensi-

ties. This method is sufficient to confirm presence or absence of a signal, or to compare bands of substan-

tially different intensities. For more detailed analysis, film images may be digitized and relative band

intensities measured by densitometry.

Table of Contents Page

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1

2. Photochemistry of x-ray film. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2

2.1 The photographic emulsion. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2

2.2 The Reciprocity Law and reciprocity failure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3

Physics and statistics affect silver grain activation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3

Faint signals: low-intensity reciprocity failure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3

Strong signals: high-intensity reciprocity failure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4

2.3 Image clarity and resolution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5

“Blow-out” and spreading of strong signals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5

Film handling and processing artifacts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5

Parallax. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6

3. Types of x-ray film used for chemiluminescent Westerns . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6

Choosing an x-ray film . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6

4. Quantification of chemiluminescent Westerns . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6

4.1 Variables that affect quantification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7

Detection chemistry (enzyme/substrate). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7

Signal capture and densitometry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7

4.2 Densitometry. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8

Important factors in densitometry. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9

4.3 Examination of film response, using an LED light source . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9

5. Conclusions & 6. References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11

Figure 1. Enhanced chemiluminescence. Luminol is a widely used chemiluminescent reagent. Oxidation

of luminol by peroxide creates an excited-state product, 3-aminophthalate. Photons of light are tran-

siently produced when this product decays to a lower energy state.

Although most researchers have used film to document Western blots, many may be unfamiliar with the

photochemical process that creates a visible image on a sheet of x-ray film. Because this process affects

data output, it is important to understand how chemiluminescent signals are recorded by film – particu-

larly if the results will be quantified by densitometry.2 This paper describes the effects of photochemistry

on the response of film to both faint and strong signals, image quality, image clarity, and quantification

by densitometry.

2. Photochemistry of x-ray film

2.1 The photographic emulsion

Photographic emulsions were first introduced in the 1850s, and the basic principles remain unchanged.

The photographic film used to document chemiluminescent Western blots is coated with an emulsion that

contains light-sensitive silver halide crystals (also called “grains”).

• Photons of light activate the silver grains, converting some silver ions to silver atoms. This creates

a latent image.

• During film processing, the silver atoms in each activated grain catalyze the reduction of the entire

grain to black metallic silver. This creates the visible image on film.

• Unexposed silver crystals are dissolved and washed away during processing.

The concentration of metallic silver that remains on the film after development is called optical density (OD).

The degree of darkening is related to the intensity of light exposure. OD and densitometry are discussed

further in Section 4. The “characteristic curve” demonstrates the response of a film to the full range of pos-

sible exposures (Fig. 2).3 The sigmoidal, non-linear shape of this curve is caused by the physics and statis-

tics of silver grain activation, which are discussed in detail below.

The accuracy of densitometry depends on the sensitivity, linear response range, and exposure time of the film.

Figure 2. Characteristic curve of X-ray film (adapted from Kodak, 2007).

The “toe” region of the curve indicates very low exposures. The center or

“straight line” region is the approximate linear response range, where

OD is proportional to the log of the exposure. The “shoulder” region rep-

resents higher exposures and is relatively flat. The slope of the “straight

line” region indicates the film’s scale of contrast. A steeper curve indi-

cates a shorter scale of contrast and narrower dynamic range.

Chemiluminescent Westerns: How film and photochemistry affect experimental results – Page 2

LI-COR Biosciences www.licor.com

2.2 The reciprocity law and reciprocity failure:

Physics and statistics affect silver grain activation

The response of film to light is governed by the reciprocity law. This law states that film response is deter-

mined by total exposure, which is dependent on light intensity and exposure time:

light intensity x duration = total exposure

The reciprocity effect is an inverse relationship. Bright light delivered for a shorter duration can produce

the same response as dim light delivered for a longer duration. This holds true across a range of values,

but becomes inaccurate outside that range. When signals are very strong or very faint, the relationship

falls apart and film response is no longer proportional to light intensity and duration (Fig. 2). This is called

reciprocity failure. It occurs at both high and low intensities of light, but with different mechanisms.4

Faint signals: low-intensity reciprocity failure

At very low intensities of light, film is less responsive and is therefore disproportionately insensitive.

Although this property keeps background low, it also causes faint signals to be under-represented.4 The

response of film to light is affected by the rate at which photons are received. If the film receives 100 pho-

tons of light all at once, it will react and create a latent image; however, if 100 photons filter in slowly over

one hour, they will probably not be detected. This non-linear response is caused by the physics of silver

crystal activation (described below).2,4

A minimum threshold level of exposure is required to expose a silver halide crystal and create a latent

image. When a crystal is activated by one or two photons of light, it is unstable and rapidly reverts back

to its stable, inactive form. Multiple photons must be absorbed by the activated silver crystal before it re-

verts, to form a stable latent image that can be developed during film processing. When signals are faint

and require long exposures, a stable latent image is unlikely to form. As a result, faint signals are under-

estimated and appear to drop off rapidly on film. This is often observed in dilution series of samples, when

it seems to the eye that additional faint bands should be visible (Fig. 3). However, signals below a certain

intensity level simply cannot be detected, even with extremely long exposures.

Reciprocity failure effects can be reduced by pre-exposure of the film to an instantaneous flash of light.

This approach to hypersensitization of film was introduced in the mid-20th century.5 “Pre-flashing” by-

passes the reversible stage of latent image formation, such that multiple photons are not required to fully

activate each silver grain.4 This increases sensitivity and creates a more linear relationship between OD

and light exposure for low-intensity signals; however, pre-flashing is inconvenient and difficult to perform

reproducibly.

Figure 3. On film, faint signals are underestimated and drop off very quickly. ERK2 was de-

tected in serially diluted NIH/3T3 cell lysates. A) SuperSignal® West Pico substrate and 5-min

film exposure. B) Custom ECL substrate and 5-min film exposure. On both blots, signal

drops off abruptly even though it seems that additional bands in the dilution series should

be visible.



A) B)

Strong signals: high-intensity reciprocity failure

The non-linear response of film to strong signals is caused by the statistics of silver grain activation. Most

researchers are aware that strong signals cause saturation of film, a point at which all silver grains have

been activated. Once saturation is reached, no further signals can be recorded, regardless of their intensity.

However, it is also important to recognize that film response begins to plateau well before saturation is

reached. As the film becomes progressively more exposed and more silver grains are activated, each new

photon of light is statistically less likely to strike an unactivated grain.2,4 This under-represents strong sig-

nals, causing a logarithmic, non-linear response in darkening and OD prior to saturation (Fig. 4). As strong

signals approach the maximum possible density, they lose their ability to show tonal variations on the de-

veloped film. This “overexposure” causes high- and moderate-intensity bands to appear similarly dark and

dense. The non-linear response of film to strong signals contributes to its narrow linear range.4

The tendency of strong bands to blur and spread on film (“blow-out”) also makes quantification difficult.

Stronger bands do not have clear margins; they obscure adjacent bands and cannot be accurately sepa-

rated (Fig. 5). This is especially problematic when stronger bands are located near faint bands, because

the longer exposures required for detection of faint bands increase the spreading of stronger bands.

Figure 4. Strong signals plateau and become saturated. Akt was detected in serial dilutions of NIH-3T3 cell lysate,

using SuperSignal® West Dura substrate and 5-min film exposure. Strong signals are underestimated (boxed region);

they plateau and fail to show tonal variation. Densitometry clearly shows the lack of linear response at higher con-

centrations. The linear range in this experiment spans only 3-4 dilutions (4- to 8-fold).

Figure 5. On film, strong signals blur and

spread to obscure adjacent bands. ARNO

protein was detected in cell lysates, using

ECL Plus substrate and 90-sec film exposure.

Spreading of bands is especially problematic

when longer exposures are required to de-

tect faint bands (right side of blot).



The nuances of photochemistry introduce variability into Western blot analysis.

The nuances of photochemistry introduce variability into Western blot analysis. Optical density (OD) is not

a direct function of light generation by the chemiluminescent substrate. It is an indirect function that de-

pends on the response of the film to light.2 The accuracy of densitometry depends on the sensitivity, linear

response range, and exposure time of the film. Reciprocity failure introduces error and limits the ability of

film to present a complete picture of the data. To complicate matters further, reciprocity failure can occur

differently at different locations on the photographic emulsion.

2.3 Image clarity and resolution

When chemiluminescent Western blots are documented with film, band resolution and clarity are often

compromised. Several key factors are involved.

“Blow-out” and spreading of strong signals. On film, strong signals blur and spread – obscuring adjacent

bands (Fig. 5). If you are attempting to detect both strong and faint signals on the same blot, or several

bands in close proximity, blown-out bands can be a serious limitation (Fig. 6).

Blurring and spreading of bands is primarily caused by scattering of light. During film exposure, light is

simultaneously collected from all areas of the blot. As light scatters, it travels away from the site where it

originated and activates silver grains in the surrounding area. This creates large, diffuse bands that expand

in size and frequently obscure adjacent bands. Longer film exposures increase the severity of blurring and

spreading. In addition to light-scattering issues, the layer of liquid substrate, plastic wrap, and the film

itself can act as waveguides to encourage horizontal propagation of light. This phenomenon further pro-

motes spreading of bands.

Film handling and processing artifacts. Image quality is also affected by artifacts that occur during expo-

sure and processing. If the blot or film shifts during handling, blurry bands and “after-images” may occur,

especially if signals are strong. Chemiluminescent blots must be wrapped in plastic before exposure to

film, to contain the liquid substrate. Leakage of liquid during exposure will cause artifacts on the devel-

oped film, as will static electricity from the plastic wrap. During processing, scratching and mechanical

damage may occur (Fig. 7). If the sheet of film becomes caught inside the automatic developer, scratching

can be severe.

Figure 6. Bands that are close together may

not be clearly resolved.The ERK1/ERK2 dou-

blets in these A431 cell lysates cannot be sep-

arated and resolved at higher protein concen-

trations, even in this short, 15-second exposure.



Figure 7. Scratches and other artifacts affect image quality on

film. In this 30-sec exposure, scratches and static electricity

artifacts are observed.

Parallax. Most commercial X-ray film is double-emulsion (light-sensitive emulsion coatings are applied to

both sides of the film). This provides maximum sensitivity and ease of use, but reduces image clarity and

sharpness. When a Western blot is exposed to double-emulsion film, images are created on both emul-

sions (both sides of the film), then superimposed. This enhances sensitivity, but causes an unwanted paral-

lax effect. Because the two images have different path lengths, the image on the far side of the film is

slightly larger than the image on the near side. When the two images are superimposed, the parallax effect

causes blurring of the edges and reduces the sharpness of the resulting bands.6 If single-emulsion film is

used to avoid parallax, detection sensitivity will be reduced.

3. Types of X-ray film used for chemiluminescent Westerns

Many brands and varieties of X-ray film are available for documentation of chemiluminescent Westerns.

Commercially available X-ray films have many similarities. Most films:

• Use a polyester base to support the light-sensitive photographic emulsion, with an outer layer of

supercoat material to protect the emulsion from scratching, mechanical damage, and static.

• Are double-emulsion (the film is coated with photographic emulsion and anti-scratch material on

both sides). This increases film speed and sensitivity, but may cause blurring due to parallax.

• Are sensitive to blue and green light (orthochromatic). They are not sensitive to red light and are

compatible with red darkroom safelights, although safelights may cause fogging.7

The polyester support layer is typically grey or blue in color. Blue-tinted film has been used for many

years. The blue color is believed to reduce glare and eyestrain during transillumination, and may improve

visualization.7 Some newer, high-performance films are made with a clear base that is thought to improve

visualization of very faint bands.

Choosing an X-ray film. Proper film choice is determined by the speed (sensitivity) and resolution re-

quired. Speed and resolution are determined by the size of the silver halide crystals and thickness of the

photographic emulsion. Larger crystals and thicker emulsions enhance sensitivity, but at the expense of

resolution. Finer crystals and thinner layers provide greater detail. Maximum-resolution films are best

suited to lower energy β-emitters such as 33P and 14C. High-performance films such as BioMax Light and

Hyperfilm ECL are recommended for chemiluminescence and high-energy emitters such as 32P.

Film sensitivity varies widely, and high-performance films are considerably more expensive. Price varies

widely, ranging from ~ $0.50 to $10 per sheet. Individually-wrapped films are much more expensive than

those in standard, alternate-interleaved packaging. Blue-tinted film is typically more economical but some-

what less sensitive. For maximum performance, a more expensive grey- or clear-base film may be pre-

ferred. Because limited technical specifications are provided, it is very difficult to compare different film

products without side-by-side testing.

4. Quantification of chemiluminescent signals

Chemiluminescent Western blots are sometimes used for quantification. Densitometry of exposed films is

a common approach. The accuracy and dynamic range of this method are variable, and proper controls

are critical for data interpretation.



4.1 Variables that influence quantification

Two important variables affect accuracy when chemiluminescent Western blots are quantified.

Detection chemistry (enzyme/substrate kinetics). Chemiluminescence is chemical reaction that releases

energy in the form of light. Enzymatic amplification of signal provides high sensitivity, but signal intensity

is dynamic and transient. Intensity peaks shortly after substrate is applied, and then fades exponentially

with time as the chemical reaction slows. Persistence of signal depends on the chemiluminescent sub-

strate used. Extended-stability substrates are typically more expensive than substrates that fade quickly.

Substrate availability and exhaustion are also significant concerns.1 Substrate distribution and accessi-

bility are never uniform across the entire surface of the blot. Unevenly distributed substrate, pooled sub-

strate, and bubbles can affect substrate availability and cause localized variation in signal intensity (shown

in Fig. 8). Because enzyme/substrate kinetics govern the generation of signal, intensity is unlikely to be

proportional to the amount of target present, or its proportionality may change as the reaction proceeds.

Strong bands with high concentrations of HRP enzyme may cause rapid local depletion of the substrate,

generating central “white-outs” in strong bands where light can no longer be generated.

The limitations of enzymatic detection are intrinsic to chemiluminescent Westerns, regardless of the signal

capture method used. Non-enzymatic detection, such as fluorescent detection with fluorophore-labeled

secondary antibodies, eliminates this source of variability.

Signal capture and densitometry. Chemiluminescent signals are often captured by exposure to X-ray film.

This method is very sensitive, because long exposures can be used to detect low-intensity signals; how-

ever, the response of film to light intensity is non-linear. Because of reciprocity failure, strong and faint

signals may not be accurately captured and the range of linear response is quite narrow.4

Several film exposures of different lengths are typically required to capture the desired image. Exposure

time must be long enough to detect the desired target, but short enough to limit undesired background.

Figure 8. Substrate availability can affect signal intensity on chemiluminescent Westerns. The same pair of

samples was loaded three times on a single blot (10 μg or 5 μg of C32 cell lysate; circled areas). SuperSig-

nal West Pico substrate was used for detection, with 5 min film exposure. Non-uniform distribution of sub-

strate across the blot made some signals noticeably stronger than others (orange circle).



A single exposure can only provide optimal detection across a narrow range of signal intensities. If the

exposure is too long, background may begin to obscure the signal. Stronger bands merge and blur to-

gether, and “blown-out” bands make quantification difficult. Reciprocity failure causes faint signals and

strong signals to be under-represented (Fig. 9). Saturation prevents measurement of further change in

signal intensity, and prevents accurate comparison of band intensities.

4.2 Densitometry

After film exposure, densitometry is sometimes used to quantify protein levels on Western blots. This

method measures the optical density (OD), or degree of darkness, of photographic material. It is important

to remember that OD is not a direct function of light generation by the chemiluminescent substrate.2 It is

an indirect function that depends on the response of film to light, which is non-linear. The accuracy of den-

sitometry is therefore dependent on the sensitivity, linear response range, and exposure time of the film.

Densitometry measures the degree of darkness of the image as a function of light transmission; density

equals the logarithm of the reciprocal of transmittance. For example, if a region transmits one-tenth of the

incident light, its density is equal to 1 (Table I).10

Figure 9. Densitometry of film is limited by signal saturation. A) Purified AKT was detected on a Western blot

with chemiluminescence and exposure to film. B) Signals were quantified by densitometry of the exposed film.

The sigmoid curve indicates signal saturation and limited dynamic range using densitometry and film.

Reprinted from Wang et al, 2011.9



OD is not a direct function of light generation by the chemiluminescent substrate. It is an indirect function that

depends on the response of film to light, which is non-linear.

% Light Transmitted Optical Density (OD)

80% 0.1

60% 0.2

40% 0.4

20% 0.7

10% 1.0

5% 1.3

1% 2.0

Table I. Relationship between light transmittance and OD.10

A) B)

For Western blot analysis, the useful range of densities is approximately 0.2 – 2.0 (roughly 10-fold).2 When

unexposed film is processed, it has an OD of 0.15 - 0.20. This is called the gross fog density of the film, and

signals must exceed this density to be recorded. An OD of 1.0 indicates a medium grey tone (halfway be-

tween white and black). High ODs (above 2.0) appear black and indicate saturation; they cannot be discrim-

inated from one another. The overall response of film is non-linear, but approaches linearity over narrow

ranges (Fig. 9). A standard curve is strongly recommended to confirm the linear range of film

response for each experiment.

Important factors in densitometry

The variable nature of Western blot densitometry methods was examined in detail by Gassmann et al(2009).11 This study correlated plasma Epo (erythropoietin) values for various mammalian species, deter-

mined by radioimmunoassay (RIA) and Western blot densitometry. Densitometry was performed under

28 different conditions. Variables included method of image acquisition (office scanner or CCD system),

background correction, software, and definition of OD value. The comparison was based on the p-value

of the linear regression between RIA and Western blot values. Depending on the densitometry conditions

used, the p-value of the regression line ranged from 0.000013 to 0.76. The same data could be used to

make statistically valid, but completely contradictory statements about the correlation between RIA and

Western blot results.

This 2009 study describes two key pitfalls in densitometry.11

• First, variability can arise during digitization of films. Office scanners are frequently used, but may

provide limited dynamic range and uneven illumination of the scan area. More importantly, many

desktop scanners have automatic gain controls that make the OD of a band also a function of the

surrounding image structure. Automatic gain control resulted in 1.5- to 105-fold variation in the

measured OD of identical spots, due to differences in the structure of the rest of the image. Auto-

matic gain controls should not be used when scanning films for densitometry.

• The second pitfall involves the method of measurement. Important factors include the background

correction algorithm, the sample tool size relative to lane width, and the definition of OD value (the

integral or sum of all peak heights is recommended). The authors consider image acquisition and

software parameters to be an important aspect of Western blot densitometry and analysis.

4.3 Examination of film response using an LED light source

To examine signal capture issues and reciprocity failure in more detail, it is helpful to use a model system

that eliminates all variability contributed by detection chemistry. One approach is to replace the enzyme/

substrate reaction with a light-generating LED performance test target (PTT) manifold.12 The PTT emits

calibrated light across a wide range of intensities, using an LED backlight panel with regulated brightness

levels that decrease in two-fold steps (Fig. 10). There are four duplicate slots for each brightness level, and

a total of 18 brightness levels. The PTT is ideal for direct testing of film response because detection chem-

istry is eliminated, creating consistent and reproducible light intensities that are unaffected by timing.



The PTT manifold was used to generate a series of film exposures (Fig. 11) that were analyzed by densito-

metry.12 High-intensity reciprocity failure was clearly observed. As strong signals caused film to become

progressively more exposed, the optical density of bands began to plateau. Because each new photon of

light was statistically less likely to strike an unactivated silver grain, strong signals were under-represented

on film. Near the saturation point, bands no longer showed tonal variations on the developed film. “Over-

exposure” caused strong and moderate bands to appear similarly dark and dense (Fig. 11A).

Quantification of signals from the PTT manifold is shown in Fig. 12. All exposure times (1 sec - 15 min)

demonstrated plateau and saturation of strong signals – even when exposure time was limited to 1 sec.

Each exposure displays a narrow range of linear response (4 - 8-fold), but the boundaries of that range

are different for each exposure. This illustrates the importance of using an appropriate standard curve for

analysis of protein levels with film and densitometry.

Figure 10. Layout of the PTT light manifold.The

LED backlight panel emits regulated levels of

brightness, decreasing in two-fold steps. There

are four duplicate slots for each brightness level.

Each slot is 8 mm x 1 mm. Slots are spaced 1 mm

apart within each brightness level, and 1.5 mm

from one level to another.

Figure 11. Saturation of strong signals on film. A) 15-second film exposure of PTT light manifold. Strong and mod-

erate signals appeared similarly dark and dense. The strongest signals (top left) were saturated, and had begun to

blur and spread. Weaker signals (A, right) were not visible in this short film exposure. B) 1-minute film exposure.

All strong bands appeared dark and dense. The strongest signals (B left) showed significant blurring. Weaker sig-

nals (B, right) began to be detected. C) 15-minute film exposure. This long exposure allowed weaker signals to be

detected (C, right), but those began to saturate and blur at the top. Strong signals (B, left) were saturated and

blurred together completely.



One brightness level

5. Conclusions

Exposure to film is a sensitive and widely-accepted method for documentation of chemiluminescent West-

ern blots. However, the photochemistry of film has several inherent limitations that affect data output and

can introduce error. These include the non-linear response of film to faint and strong signals (reciprocity

failure), lack of image clarity caused by blurring and spreading of bands, and the narrow range of optical

densities that can be recorded by film.

These limitations may affect interpretation of Western blot data, but are not commonly acknowledged or

discussed. For experiments that require precise quantification of protein levels on Western blots, an alter-

native detection method may be appropriate.

6. References

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

clinical laboratory. Clin Chem. 25(9): 1531-46 (1979).

2. 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).

3. Kodak. The essential reference guide for filmmakers: Basic sensitometry and characteristics of film.

Kodak Educational Products, Code: H-845 CAT No. 145 6144. Eastman Kodak Company (2007).

4. Laskey, R.A. Efficient detection of biomolecules by autoradiography, fluorography or chemilumines-

cence. Methods of detecting biomolecules by autoradiography, fluorography and chemiluminescence.

Amersham Life Sci. Review 23:Part II (1993).

5. Bowen, IS and LT Clark. Hypersensitization and reciprocity failure of photographic plates. J Optical Society Amer. 30:508-10 (1940).

6. Kopans, DB. Breast imaging, 3rd edition. Lippincott Williams & Wilkins (2007).

7. Dickerson, RE. Discussion of fog standards for general-purpose radiographic film. Carestream Health.

www.carestream.com/Fog_Standards--General_Purpose_Radiographic_Films

Figure 12. Quantitative analysis demonstrates under-representation and saturation of strong signals. The PTT

manifold was used to perform a series of film exposures (1 sec, 5 sec, 15 sec, 1 min, and 15 min). Films were

analyzed by densitometry. At all exposure times, strong signals quickly reached a plateau. The log/log plot (right)

illustrates the narrow linear range of film detection (<10-fold). Light intensity values that fall within the linear

range are different for each exposure.



8. Tuan, TL, P Hwu, W Ho, P Yiu, R Chang, A Wysocki, and PD Benya. Adenoviral overexpression and small

interfering RNA suppression demonstrate that plasminogen activator inhibitor-1 produces elevated

collagen accumulation in normal and keloid fibroblasts. American Journal of Pathology 173(5):1311-25

(2008).

9. Wang, X, Y Dong, AJ Jiwani, Y Zou, J Pastor, M Kuro-o, AA Habib, M Ruan, DA Boothman, and

C-R Yang. Improved protein arrays for quantitative systems analysis of the dynamics of signaling

pathway interactions. Proteome Science 9:53 (2011).

10. Davis, R and FM Walters Jr. Sensitometry of photographic emulsions and a survey of the characteris-

tics of plates and films of American manufacture. Scientific Papers of the Bureau of Standards18:1-120 (1922).

11. Gassmann, M, B Grenacher, B Rohde, and J Vogel. Quantifying Western blots: pitfalls of densitometry.

Electrophoresis 30:1845-55 (2009).

12. Anderson, JP et al. Evaluation of signal capture methods with custom-built LED backlight manifold

(performance test target; PTT). LI-COR Biosciences, unpublished data (2012).

4647 Superior St. • P.O. Box 4000 • Lincoln, Nebraska 68504 LI-COR Biosciences North America: Sales Support: 888-645-2304 Order Support: 888-645-7242 • Technical Support: 800-645-4260www.licor.com/bio

LI-COR GmbH, Germany, Serving Europe, Middle East and Africa: +49 (0) 6172 17 17 771 LI-COR 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, and C-DiGit are trademarks or registeredtrademarks of LI-COR, Inc. in the United States and other countries. All other trademarks belong to their respective owners.For patent information, visit www.licor.com/patents.


Doc # 979-138060613

Date post:	20-Jul-2016
Category:	Documents
Upload:	16dev5038
View:	23 times
Download:	2 times

Film Photochemistry Effects

Documents