TheJournalofCellBiology
The Journal of Cell Biology, Volume 166, Number 1, July 5, 2004 11–15
http://www.jcb.org/cgi/doi/10.1083/jcb.200406019
JCBFeature
11
What’s in a picture?
The temptation of image manipulation
Mike Rossner1
and Kenneth M. Yamada2
1
Managing Editor, The Journal of Cell Biology
2
Editor, The Journal of Cell Biology, and the National Institute of Dental and Craniofacial Research, National Institutes of Health
It’s all so easy with Photoshop1
. In the
days before imaging software became
so widely available, making adjustments
to image data in the darkroom
required considerable effort and/or expertise.
It is now very simple, and thus
tempting, to adjust or modify digital
image files. Many such manipulations,
however, constitute inappropriate
changes to your original data, and
making such changes can be classified
as scientific misconduct. Skilled editorial
staff can spot such manipulations
using features in the imaging software,
so manipulation is also a risky
proposition.
Good science requires reliable data.
Consequently, to protect the integrity
of research, the scientific community
takes strong action against perceived
scientific misconduct. In the current
definition provided by the U.S. government:
“Research misconduct is defined
as fabrication, falsification, or
plagiarism in proposing, performing,
or reviewing research, or in reporting
research results.” For example, showing
a figure in which part of the image was
either selectively altered or reconstructed
to show something that did
not exist originally (for example, adding
or modifying a band in a polyacrylamide
gel image) can represent falsification
or fabrication.
Being accused of misconduct initiates
a painful process that can disrupt
one’s research and career. To avoid
such a situation, it is important to understand
where the ethical lines are
drawn between acceptable and unacceptable
image adjustment.
Here we present some general guidelines
for the proper handling of digital
image data and provide some specific
examples to illustrate pitfalls and inappropriate
practices. There are different
degrees of severity of a manipulation,
depending on whether the alteration
deliberately changes the interpretation
of the data. That is, creating a result is
worse than making weak data look better.
Nevertheless, any manipulation
that violates these guidelines is a misrepresentation
of the original data and
is a form of misconduct. All of the examples
we will show here have been
created by us using Photoshop; although
they may appear bizarre, it is
remarkable that they are actually based
on real cases of digital manipulation
discovered by a careful examination of
digital images in a sample of papers
submitted (or even accepted) for publication
in a journal.
Why is it wrong to “touch up”
images?
If you misrepresent your data, you are
deceiving your colleagues, who expect
and assume basic scientific honesty—
that is, that each image you present is
an accurate representation of what you
actually observed. In addition, an image
usually carries information beyond
the specific point being made. The
quality of an image has implications
about the care with which it was obtained,
and a frequent assumption
(though not necessarily true) is that in
order to obtain a presentation-quality
image, you had to carefully repeat an
experiment multiple times.
Manipulating images to make figures
more simple and more convincing may
also deprive you and your colleagues of
seeing other information that is often
hidden in a picture or other primary
data. Well-known examples include evidence
of low quantities of other molecules,
variations in the pattern of
localization, and interactions or
cooperativity.
Journal guidelines
It is surprising that many journals say
little or nothing in their “Instructions
to Authors” about which types of digital
manipulations are acceptable and
which are not. The following journals
provide some guidelines, but they vary
widely in comprehensiveness.
Molecular and Cellular Biology. “Since
the contents of computer-generated
images can be manipulated for better
clarity, the Publications Board at its
May 1992 meeting decreed that a description
of the software/hardware used
should be put in the figure legend(s).”
Journal of Cell Science. “Image enhancement
with computer software is
acceptable practice, but there is a danger
that it can result in the presentation
of quite unrepresentative data as well as
in the loss of real and meaningful signals.
During manipulation of images, a
Reprinted with permission from The NIH Catalyst.
Address correspondence to Mike Rossner, Journal
of Cell Biology, Rockefeller University Press,
1114 1st Ave., 3rd fl., New York, NY 10021.
Tel.: (212) 327-8881. Fax: (212) 327-8576.
email: rossner@rockefeller.edu
1
The general principles presented here apply to
the manipulation of images using any powerful
image-processing software; however, because of
the popularity of Photoshop, we refer to several
specific functions in this application.
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12 The Journal of Cell Biology | Volume 166, Number 1, 2004
positive relationship between the original
data and the resulting electronic
image must be maintained. If a figure
has been subjected to significant electronic
manipulation, the specific nature
of the enhancements must be
noted in the legend or in the Materials
and Methods.”
The Journal of Cell Biology. “No specific
feature within an image may be
enhanced, obscured, moved, removed,
or introduced. The grouping of images
from different parts of the same gel, or
from different gels, fields, or exposures
must be made explicit by the arrangement
of the figure (e.g., using dividing
lines) and in the text of the figure legend.
Adjustments of brightness, contrast,
or color balance are acceptable if
they are applied to the whole image
and as long as they do not obscure or
eliminate any information present in
the original. Nonlinear adjustments
(e.g., changes to gamma settings) must
be disclosed in the figure legend.”
Because the last set of guidelines is
by far the most comprehensive we have
found to date (full disclosure: we wrote
them), we will continually refer back to
them in the following discussions
of the use and misuse of digital
manipulations.
Blots and gels
Gross misrepresentation
The simplest examples of inappropriate
manipulation are show in Fig. 1. Deleting
a band from a blot, even if you
believe it to be an irrelevant background
band, is a misrepresentation of
your data (Fig. 1 A). Similarly, adding
a band to a blot, even if you are only
covering the fact that you loaded the
wrong sample, and you know for sure
that such a protein or DNA fragment
or RNA is present in your sample, is a
misrepresentation of your data. In the
example shown in Fig. 1 B, the additional
band in lane 3 has been generated
by simply duplicating the band in
lane 2.
Another example of using Photoshop
inappropriately to create data is
illustrated in Fig. 2, in which a whole
single panel has been replicated (arrows)
and presented as the loading
controls for two separate experiments.
More subtle manipulations
Brightness/contrast adjustments. Adjusting
the intensity of a single band in a
blot constitutes a violation of the
widely accepted guideline that “No
specific feature within an image may be
enhanced, obscured, moved, removed,
or introduced.” In the manipulated image
in Fig. 3 A, the arrow indicates a
single band whose intensity was reduced
to produce an impression of
more regular fractionation. Although
this manipulation may not alter the
overall interpretation of the data, it still
constitutes misconduct.
While it is acceptable practice to adjust
the overall brightness and contrast
of a whole image, such adjustments
should “not obscure or eliminate any
information present in the original”
Figure 1. Gross manipulation of blots. (A) Example of a band deleted from the original
data (lane 3). (B) Example of a band added to the original data (lane 3).
Figure 2. Gross manipulation of blots. Example of a duplicated panel (arrows).
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What’s in a picture? | Rossner and Yamada 13
(Fig. 3 B). When you scan a blot, no
matter how strong the bands, there will
invariably be some gray background.
While it is technically within the
guidelines to adjust the brightness and
contrast of a whole image, if you overadjust
the contrast so that the background
completely drops out (Fig. 3 B,
part 2 vs. part 3), this should raise suspicions
among reviewers and editors
that other information (especially faint
bands) may have dropped out as well.
It may be argued that this guideline
is stricter than in the days before Photoshop,
when multiple exposures could
be used to perfect the presentation of
the data. Perhaps it is, but this is just
one of the advantages of the digital age
to the reviewer and editor, who can
now spot these manipulations when in
the past an author would have taken
the time to do another exposure.
Think about this when you are doing
the experiment and perform multiple
exposures to get the bands at the density
you want, without having to overadjust
digitally the brightness and contrast
of the scanned image.
Cleaning up background. It is very
tempting to use the tool variously
known as “Rubber Stamp” or “Clone
Stamp” in Photoshop to clean up unwanted
background in an image (Fig.
4). Don’t do it. This kind of manipulation
can usually be detected by someone
looking carefully at the image file
because it leaves telltale signs. Moreover,
what may seem to be a background
band or contamination may
actually be real and biologically
important and could be recognized as
such by another scientist.
Splicing lanes together. It is clearly
inappropriate manipulation to take a
band from one part of a gel and move
it to another part, even if you do not
change its size. But it is within usual
guidelines to remove a complete lane
from a gel and splice the remaining
lanes together. This alteration should
be clearly indicated, however, by leaving
a thin white or black line between
the gel pieces that have been juxtaposed.
Again, it could be argued that
this guideline is stricter than in the
days before Photoshop when paper
photographs of a gel were cut up and
pieces were glued next to each other.
This practice, however, usually left a
black line indicating to the reader what
had been done.
As it was with gel photographs, it is
unacceptable to juxtapose pieces from
different gels to compare the levels of
proteins or nucleic acids. Rerun all of
the samples on the same gel!
Figure 3. Manipulation of blots: brightness and contrast adjustments. (A) Adjusting the intensity
of a single band (arrow). B) Adjustments of contrast. Images 1, 2, and 3 show sequentially
more severe adjustments of contrast. Although the adjustment from 1 to 2 is acceptable
because it does not obscure any of the bands, the adjustment from 2 to 3 is
unacceptable because several bands are eliminated. Cutting out a strip of a blot with the
contrast adjusted provides the false impression of a very clean result (image 4 was derived
from a heavily adjusted version of the left lane of image 1). For a more detailed discussion
of “gel slicing and dicing,” see Nature Cell Biology editorial (2).
Figure 4. Manipulation of blots: cleaning
up background. The Photoshop “Rubber
Stamp” tool has been used in the manipulated
image to clean up the background in
the original data. Close inspection of the image
reveals a repeating pattern in the left
lane of the manipulated image, indicating
that such a tool has been used.
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14 The Journal of Cell Biology | Volume 166, Number 1, 2004
Micrographs
Enhancing a specific feature. An example
of manipulation by enhancement is
shown in Fig. 5, in which the intensity
of the gold particles has been enhanced
by manually filling them in with black
color using Photoshop. This type of
manipulation misrepresents your original
data and is thus misconduct. There
are acceptable ways to highlight a feature
such as gold particles, which include
arrows or pseudocoloring. If
pseudocoloring is done with the “Colorize”
function of Photoshop, it does
not alter the brightness of individual
pixels, but pseudo-coloring should always
be disclosed in the figure legend.
Other examples of misconduct include
adjusting the brightness of only a
specific part of an image or erasing
spots. Using the “Brightness” adjustment
in Photoshop is considered to be
a linear alteration (see below), which
must be made to the entire image.
Linear vs. nonlinear adjustments. Linear
adjustments, such as those for
“Brightness” or “Contrast” in Photoshop,
are those in which the same
change is made to each pixel according
to a linear function. It is acceptable
(within limits noted above) to apply
linear adjustments to a whole image.
There are other adjustments in Photoshop
that can be applied to a whole image,
but the same change is not made
to each pixel. For example, adjustments
of gamma output (“Color Settings”
in Photoshop) alter the intensity
of each pixel according to a nonlinear
function. Adjustments of “Curves” or
“Levels” in Photoshop alter the tonal
range and color balance of an image by
adjusting the brightness of only those
pixels at particular intensities and colors.
Such nonlinear changes are sometimes
required to reveal important features
of an image; however, the fact
that they have been used should be disclosed
in the figure legend.
Digitally altering brightness or contrast
levels can be misleading with fluorescence
micrographs. Some authors
mistakenly change the contrast of an
experimental compared with a control
photo, or change individual panels in a
time course, or use different contrast
levels when making merged images
compared with the original images. All
of these changes in individual pictures
used for comparisons can be misrepresentations.
On the other hand, certain
adjustments such as background subtraction
or using a filter or digital mask
may be needed to extract information
accurately from complex images. Reporting
the details and logic of such
manipulations that are applied to images
as a whole should resolve concerns
about their use. Standards and guidelines
in the field will continue to
evolve, but full disclosure will always
be the safest course.
Misrepresentation of a microscope
field. The reader assumes that a single
micrograph presented in a figure represents
a single microscope field. Combining
images from separate microscope
fields into a single micrograph
constitutes a misrepresentation of your
original data. In the manipulated image
in Fig. 6 (top panel), cells have
been combined from several microscope
fields into a single micrograph.
This manipulation becomes visible
when the contrast of the image is adjusted
so that the inserted images become
visible (bottom panel). You may
want to combine images from several
fields into a single micrograph to save
space, but this assembly should be
clearly indicated by thin lines between
the different pieces.
Resolution
A pixel is a square (or dot) of uniform
color in an image. The size of a pixel
can vary, and the resolution of an image
is the number of pixels per unit
area. Although resolution is defined by
area, it is often described using a linear
measurement—dots per inch (dpi).
Thus, 300 dpi indicates a resolution of
300 pixels per inch by 300 pixels per
inch, which equals 90,000 pixels per
square inch (1).
High-resolution digital cameras (in
2004) can acquire an image that is 6
megapixels in size. This can generate
an image of approximately 2400 ϫ
2400 pixels, or 8 inches ϫ 8 inches at
Figure 5. Misrepresentation of immunogold data. The gold particles, which were actually
present in the original (left), have been enhanced in the manipulated image (right). Note also
that the background dot in the original data has been removed in the manipulated image.
Figure 6. Misrepresentation of image data.
Cells from various fields have been juxtaposed
in a single image, giving the impression
that they were present in the same microscope
field. A manipulated panel is
shown at the top. The same panel, with the
contrast adjusted by us to reveal the manipulation,
is shown at the bottom.
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What’s in a picture? | Rossner and Yamada 15
300 dpi. Note that, with the right settings
in Photoshop, physical size and
resolution can be traded off against
each other without a gain or loss in the
amount of information—that is, you
can resize an image without altering
the total number of pixels.
You should be aware of the resolution
at which the image was acquired
by the digital camera on your microscope.
When that file is opened in Photoshop,
you have the option of setting
the size and resolution of the image.
You should not set the total number of
pixels to be greater than that in the
original image; otherwise, the computer
must create data for you that
were not present in the original, and
the resulting image is a misrepresentation
of the original data—that is, the
dpi of an image can only be increased
if the size of the image is reduced
proportionately.
It is acceptable to reduce the number
of pixels in an image, which may be
necessary if you have a large image at
high resolution and want to create a
small figure out of it. Reducing the resolution
of an image is done in Photoshop
by sampling the pixels in an area
and creating a new pixel that is an average
of the color and brightness of the
sampled ones. Although this does alter
your original data, you are not creating
something that was not there in the
first place; you are presenting an
average.
Other data-management issues
It is crucially important to keep your
original digital or analog data exactly as
they were acquired and to record your
instrument settings. This primary rule
of good scientific practice will allow
you or others to return to your original
data to see whether any information
was lost by the adjustments made to
the images. In fact, some journal reviewers
or editors request access to such
primary data to ensure accuracy.
There are other important issues
concerning data handling that we have
not addressed by focusing on manipulations
of existing data. Examples include
selective acquisition of data by
adjusting the settings on your microscope
or imager, selecting and reporting
a very unusual result as being representative
of the data, or hiding negative
results that may contradict your conclusions.
Any type of misrepresentation
of experimental data undermines scientific
research and should be avoided.
Conclusion
Data must be reported directly, not
through a filter based on what you think
they “should” illustrate to your audience.
For every adjustment that you make to a
digital image, it is important to ask yourself,
“Is the image that results from this
adjustment still an accurate representation
of the original data?” If the answer to
this question is “no,” your actions may be
construed as misconduct.
Some adjustments are currently considered
to be acceptable (such as
pseudocoloring or changes to gamma
settings) but should be disclosed to
your audience. You should, however,
always be able to justify these adjustments
as necessary to reveal a feature
already present in the original data.
We hope that by listing guidelines
and publicizing examples of transgressions,
all of us can become more vigilant,
particularly in guiding junior colleagues
and students away from the
tempting dangers of digital manipulation.
Just because the tools exist to
clean up sloppy work digitally, that is
no excuse to do sloppy work.
If you would have redone an experiment
to generate a presentation-quality
image in the days before the digital age,
you should probably redo it now.
References
1. Rossner, M. and R. O’Donnell. 2004. The JCB
will let your data shine in RGB. J. Cell. Biol. 164:
11.
2. 2004. Gel slicing and dicing: a recipe for disaster.
Nat. Cell Biol. 6:275.
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