Scientific Imaging: Visual Data and Ethics
By Jerry Sedgewick
Further Division of Areas into Categories
Images can be divided further into categories, depending on the intent of the image and the imaging system used for acquisition. Each group demands its own particular acquisition and post-processing treatment.
For example, if the visual data is intended for measurement of OD/I, changes to images acquired from that imaging system must be kept as close to the original image as possible, with only a few “allowable” post-processing methods and only the necessary methods for conformance to outputs (assuming the original was acquired on an imaging system that results in a linear distribution of grayscale or color values).
On the other hand, if the image is intended for visualization (in this sense, a conscious enhancement of visual data to draw attention to experimental phenomena, such as an image destined for a publication’s cover or a cartoon model) or any subsequent quantification (except OD/I), many more changes are allowable. In the case of quantification, these changes are absolutely necessary for separating measured features from the surrounding areas, often referred to as “background.”
The following categories broadly separate the intent and/or the means for acquiring the image:
- OD/I from flatbed scanners. Images made for measurement of brightness/darkness levels. These include electrophoretic samples done in the field of molecular biology (below,left).
- OD/I from camera and scanned beam systems. Images made for measurement of brightness/darkness levels, or for the measurement of color, not acquired from flatbed scanners. These include samples imaged via microscopes, confocal instruments, electron microscopy, x-ray devices, magnetic resonance imaging, and so on.
- Representation. Images made from any imaging device for purposes of creating an accurate representation of what was once seen (bemow, middle).
- Quantification/Visualization. Images made through conscious alteration of data using pseudocoloring, binarizing, and other techniques to separate relevant visual information from the background (below, right).
Following specific guidelines for acquisition, post-processing, and conformance ensures accurate representation of what was once seen by eye. Because part of the aim of research is to report or publish, the “gold standard” for ethical guidelines is found in the author guidelines from major scientific publications.
But the division of visual data into areas and categories will not be found in author guidelines from major scientific publications, at least to date. General guidelines are set forth in scientific publications to varying degrees of detail, but often these lack specifics for when and where potential for misrepresentation may exist. That lack of clarity leads to varying degrees of interpretation from one investigator to another and inconsistent responses from journal reviewers and editors.
Using Standards and References
In addition to when and where potential for misrepresentation exists, there is also the question of how: How can representative images be made while preserving a consistent approach to imaging?
The answer isn’t simple. In the best of situations, grayscale and color values can be objectively determined by fitting the imaging system (or the image, or the data derived from the image) to an external standard. Ideally, that standard is a calibrated object with known values. The imaging system can then be calibrated to the standard, and presto, all images from that system are also fitted to the known values. This is typical for situations in which OD/I measurements are derived from images acquired with self-calibrating, scanner systems.
However, other situations present difficulties. A calibrated standard may not be available, as in fluorescence imaging. Calibrated standards may work in an ideal world but not in the real world, such as in colors that can be defined by standards but do not exist with the same purity in a sample. The actual specimen may change in grayscale or color value as a result of preparation techniques and inherent factors, making a calibrated standard useless. Labeling of specimens may vary in intensity, making it more important to calibrate to an internal reference that is part of the specimen versus a calibrated standard. For these reasons and others, the use of an external, calibration standard is not always the answer for a consistent approach to imaging, which is what is desired for reproducibility and correct representations.
A consistent external reference—instead of a standard—may need to be substituted for a calibrated standard to provide a predictable reference value against which colors can be corrected or to which energy source intensities or exposure consistencies can be tracked over time. As in the calibrated standard, the external reference can be included with the specimen or taken at the beginning or end of an imaging session.
The reference can be internal: Specimens may have intrinsic values that can be ratioed against each other, or a consistent grayscale or color value may be found within the specimen, eliminating the need for either a calibration standard or external reference.
When no standards or references are available, distributions of grayscale or color values (histograms) can be matched to either a “perfect,” reference image, or images can be fit to the dynamic range of the imaging instrument (when acquiring images) or fit to a common histogram (post-processing). In that manner, all images are uniform.
As long as a reasoned approach is chosen for the type of specimen and the intent of the image, representative images can be produced, and imaging procedures can be duplicated. A summary of the approach to consistent imaging through the use of calibrated standards, internal references, or situations in which no standards or references exist is shown below.
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Excerpted from Scientific Imaging with Photoshop: Methods, Measurement, and Output by Jerry Sedgewick. Copyright © 2008. Used with permission of Pearson Education, Inc. and New Riders. All rights reserved.