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The Basics of Image Integrity

WHY CARE ABOUT IMAGE INTEGRITY?

When publishing, an increasing number of journals are paying greater attention to images. Some journals are actively checking images for image manipulation through the use of software.

Furthermore, from an internal report at the Office of Research Integrity (ORI) by John Krueger, the number of incidences of ORI cases involving falsified images have increased to an approximate 80% of all cases since 1990.

When publishing--and when your lab has published--authors are responsible for incorporating Good Laboratory Practices into all aspects of their research, including the acquisition and processing of images. This practice is generally known as "image integrity," and it involves keeping the published image as close to the original image as possible--and to what was once seen by eye--without arbitrarily enhancing parts of the image to make experimental evidence stand out from everything else.

  scientific misconduct: increase in cases involving images


THE MAJOR AREAS OF IMAGE INTEGRITY

Remember Ra Ra!

Record what you've done
Archive original images
Report what you've done

and then...


Apply any changes to the image in post-processing equally and globally to related images.

EXCEPTIONS

Electrophoretic samples (gels, blots, etc.): do NOT change tones to these sample in post-processing to avoid potential accusations of scientific misconduct down the road.

Samples meant for densitometry: do NOT change tones to these images either.

 

Ra Ra: Record, Archive, Report, Apply



RECORD WHAT YOUVE DONE

If you have newer versions of software, and you save images in the manufacturer's format, often MetaData is included with the image that includes all your microscope and camera settings.

Otherwise, take extensive notes on microscope and camera settings.

Post Processing

In Photoshop, a History Log can be recorded in versions of Photoshop more recent than version CS (vs. 10).

You can see what was done to an image under File > File Info in the History tab. The record of what was done to your image is saved to both the image and to a text file when you select Both for Edit Log Items in the History Log.

  Record what you do to images in Photoshop
File Info: History Log

ARCHIVE ORIGINAL (RAW) IMAGES

Do NOT save over original images:

Save in manufacturer's format (if it exists)

Save to a DVD/CD that is NOT rewrite-able. Instead, save on a DVD- (minus) or DVD + (plus) or combination minus/plus.

Do NOT store in Powerpoint, or run the risk of having the image's pixel resolution reduced.

Provide some means in post-processing software for eliminating this possibility (go to link).

 

  Archive images, but not in Powerpoint

REPORT WHAT YOU'VE DONE

Always report all post-processing steps; either in Methods, or in ancillary material provided to both reviewers and to publications.

If steps do not involve enhancement that includes over-brightening experimental evidence to max values (saturation), minimizing non-specific or undesired parts of speciment that could be mistaken for evidence, and changes are made in post-processing solely to improve visualization of hidden or obscured details, then the statement to the right can be appropriate (if true).

Tonal Adjustments

If experimental images are described as "brighter" or "darker" than control images, then tones should not be adjusted, except to match exposures linearly over several microscope sessions to the same "baseline."

Gamut and Colors/Tones that Don't Reproduce in Press

The range of colors and tones that can be reproduced by a device is described as the gamut. As seen in the image on the right, the range of colors and tones that can be reproduced differs by the reproduction device.

It is then appropriate to adjust tones and colors to fit the reproduction device...

UNLESS specifically forbidden by the publisher.

Color and tones that do not always reproduce comprise both pure colors (all red, all blue, etc.) and darkest/lightest tones.

For good reproduction, images can be adjusted to "fit" the gamut of the reproduction device.

Be sure to REPORT that you have adjusted to fit to the gamut of the output device.

  Report what you've done     
gamut of a printing press

Colors and tones that generally do not reproduce on a printing press

APPLY CHANGES TO IMAGES GLOBALLY AND EQUALLY TO ALL RELATED IMAGES

In post-processing, be sure to apply changes to the whole image, and not to parts of it.

Apply any changes to one image equally to all related images.

An extensive discussion of changes that can be applied according to current accepted standards in the research community can be found at this site (click here for link) or at other sites (click here for links).

Do NOT apply tonal changes to electrophoretic samples, even when the publication does not specifically prohibit it. Your lab will potentially be at risk for accusations of scientific misconduct. Do NOT acquire images with pure white backgrounds.

 

Apply changes to scientific and microscopy images equally

 


COMMON MISTAKES WHEN TAKING PICTURES (ACQUIRING IMAGES)

The list to the right lists common mistakes. Some of these can be "repaired" in post-processing, but the following result in un-usable and un-publishable images:

Use of Automatic Exposure with images in which densitometry will be done

No flatfield correction with images in which densitometry will be done (unless measuring from the same location)

Oversaturation of pixels because detail is permanently obscured

 

common mistakes when acquiring images

 


Common Mistake #1: Poor Display of Images on Computer Screen and in Software

Images should be displayed on a computer monitor that shows all the tones. If the monitor is in a well-lit room or next to windowlight, then likelihood of seeing the tones in the image are drastically reduced.

If the monitor suffers from a limited viewing angle (see more by clicking this link), then both colors and tones will be misinterpreted.

All the tones on the image to the right should be visible. If you can see all the tones when moving your head in relation to the screen, the viewing angle is too small, or you should be viewing further away.

The numbers under the square tones indicate the tonal value on a scale of 0 (pure black) to 255 (pure white).

You can more effectively test your screen by going to this link.

Calibration

Computer monitors should be calibrated with a hardware calibrator device.

  white patches to test monitor reproduction of tones
black square for monitor test

Common Mistake #2: Incorrect Display of Images on Monitor

For the best of reasons, imaging and analysis software may display images in such a way that these are auto-brightened. Sometimes these can also be displayed so that no discrete pixels can be seen even at the highest zoom.

The best of reasons lies in making it easier for users to visualize images.

However, in NOT seeing the actual tones of the image on the screen, and whether or not the image appears pixelated at reasonable zooms (e.g., 100% zoom), prevents users from seeing how images may reproduce.

Furthermore, it eliminates a standard viewing environment in workgroups and when images are shared with other workgroups.

The figure on the right shows how images are displayed in Image J and in Photoshop. If using other software for your work, determine whether your images are auto-scaled, or interpolated to eliminate a pixelated appearance.

 

 

Comparison of Image J and Photoshop viewing of images

The raw image display (leftmost image) viewed in Photoshop shows an image saved with 12-bits of tonal information (0 - 4095 potential tonal values) on an x-axis of 16-bits (0 - 65,535 tonal values). Note that 12-bits comprises only 1/16th of the 16-bit range,so the 12-bit image is displayed appropriately in Photoshop. After scaling to 16-bits (middle image) the tones are displayed as these would reproduce. In Image J, using default "Appearance" settings, the image (rightmost image) is autoscaled for display and appears to be at optimal brightness when it isn't inherently as bright. Note that the display can be changed in Image J to prevent autoscaling: Edit > Options > Appearance: select x-axis for "16-bit range" to match tonal range in image.


Common Mistake #3: No Calibrated Slide Taken at Microscopy Sessions

Some method for determining that microscope and camera settings are the same one day as they are the next must be implemented.

Otherwise, it is difficult to determine that conditions are the same. This is especially important when troubleshooting unusual results at a microscopy session.

Calibration absolutely must be done when measuring optical intensities and densities, unless ratioing within images against a constant, such as what is done with calcium ion imaging.

Calibration can be against known standards, or reference standards. A reference standard provides an average tonal value at the first microscopy session, and at subsequent sessions, the same value is acheived (by adjusting light levels) at the beginning of the session (see pdf file ..........)

  calibration tools

Common Mistake #4: No Koehler Illumination (for Brightfield images)

Under the stage, on microscopes intended for brightfield viewing and imaging, a condenser (lens) can be adjusted to widen or narrow the cone of light that strikes the sample.

The ideal cone of light to provide the highest resolution is referred to as "Koehler" Illumination (for info on how to set, go to link).

Often the condenser is used to reduce the brightness of the light going to the eyepiece or camera, or to increase contrast. If the cone of light is narrowed too far, false structures can be created in the image (see figure to right).

  kohler or koehler illumination set correctly and incorrectly

Common Mistake #5: No White Balancing, or Poor White Balance

When taking images of Color Brightfield samples (e.g., H&E stained samples), the camera must be white balanced at the beginning of the session, or colors will be incorrectly interpreted by the camera.

Generally, the camera is white balanced by clicking a button AFTER the sample is moved away from the imaging area.

It is critical that any automatic white balancing is then turned off, or each image will be slightly different in color.

Check with the microscope salesperson to understand how to white balance, or with a colleague who knows the software.

  White balancing color microscope images

Common Mistake #6: No Frame Averaging

When a sample is static (e.g. a fixed sample on a microscope slide), and when fluorescence is low, take more than one picture and average these together (frame averaging).

This method will reduce the noise that commonly accompanies the long exposures and high gain necessary to image dim samples.

In confocal imaging, the most common method for frame averaging is known as Kalman Averaging.

Note that pixelation can also result from taking pictures without frame averaging, especially when collected on a confocal.

  Frame averaging to reduce noise

Common Mistake #7: Saving as a High Compression JPEG

When saving from many software acquisition programs as a JPEG image, a high compression is applied. In so doing, visual data is thrown away and replaced with a "blocky" appearance (see images on right).

JPEG compression is known as "LOSSY" compression. The word "Lossy" implies what the compression does: it loses visual data.

Note that in Photoshop, JPEG compression can be accomplished with low compression. Low compression (a value of 12 in Photoshop) throws away data that is imperceptible to the eye.

  Don't save as a JPEG image to avoid loss of visual information

Common Mistake #8: Gamma Settings at Values Other than 1 (one)

Gamma settings at a value of 1 retain linear gray tones, assuming that the acquisition device records tones linearly (not always true, especially when noise interferes close to max and min tonal values).

When stating that one tone is brighter or darker than another, and when performing densitometry on images, a gamma of 1 is crucial.

However, when samples contain a limited number of tones, a change in the gamma is necessary to collect the image as it is seen by eye. Conversely, when the dynamic range of tones in the sample is greater than the dynamic range of the camera, a gamma change is essential to collect all the tonal information.

In these instances, it is useful to change the gamma. Be sure to report this change in Methods, or in ancillary material when publishing.

Gamma is applied to the image according to the methods shown on the bottom slide to the right.

Note that a gamma calculation is often more complex than what is described.

 

Linear and non-linear values as a result of gamma not at 1

gamma and linear non-linear tonal values

 

Gamma explained


Common Mistake #9: Taking Pictures that Exceed the Dynamic Range of the Camera

When a picture is acquired, and parts of the image are at max (pure white) and min (pure black) tones, you over- and under-expose these areas. The result is a permanent loss of detail.

It is generally accepted that a small portion of significant tones are at max and min values (about 1 percent), but any more than that results in loss of detail in structures.

Note that artifact (debris, detritus) will often be at max or min values, but these are not significant parts of the image.

Many image acquisition systems in the sciences have a way to overlay a graphic interface on your image, called a LUT (Look Up Table). The LUT will notify you that you have under- and over-exposed parts of your image, as in the image on the right.

You may also be able to bring up a histogram. By looking at the x-axis, you can see when you have spread the histogram to either the right (max) or left (min) end. If you have, you are under- and over-exposing your image.

If the image is over-exposed, you can decrease the dwell time (also found as Shutter or Exposure), gain, aperture, etc.

If the image is under-exposed, you can increase the Black Level (also found as Contrast).

 

 

saturation and clipping when acquiring images

LUT overlays to prevent oversaturation or clipping

 


Common Mistake #10: Not Correcting for Uneven Illumination

Microscope images, especially at lower magnification (10x, 4x, 2x, etc) suffer from uneven illumination from edge to edge.

Uneven illumination can be corrected by taking a Flatfield image (also known as a Blank Field or Shading image). The flatfield image is divided into the original image to create an evenly illuminated image.

Flatfield correction must be done when measuring optical intensity or density, unless measuring from the same location on the sample, or when measuring the entire field, assuming the uneven illumination pattern is the same for all images.

Flatfield correction is useful when montaging (stitching) images together so that a brigher side of images do not border darker sides of the next image.

Many camera acquisition software packages include a means to flatfield correct. This also involves taking a background image, which is an image of the sample with the light source turned off.

Find out from a colleague or microscope salesperson how to flatfield correct when acquiring images.

Note that uneven illumination can result in false densitometric measurements, depending on how the baseline is set.

 

 

uneven illumination in microscope microscopy images

Measurement errors when uneven illumination is present in images


Common Mistake #11: Using Automatic Exposure

Generally, when at a microscopy session, the first picture is taken of a representative sample with experimental evidence, the more the better.

To make it easier, you may use an automated method to determine exposure, generally by clicking an "Auto Exposure" button.

Then, unclick or disable the Automatic exposure, and manually set the exposure while looking at a LUT overlay or a histogram until the brightest significant part of the sample is near the max value.

Keep the same manual setting for the remainder of your pictures.

Otherwise, if you have artifacts in the image, the exposure will change from image to image.

Manual exposure is crucial when reading intensities or densities of images.

  Using Auto exposure

Common Mistake #12: Post Processing in Photoshop and Using the Image Size Function

The Image Size function in Photoshop is often used incorrectly.

Part of that has to do with a misunderstanding of the dialog box itself:

1. The Pixel Dimensions show the (Pixel) Resolution of your image. This is the spatial resolution of your image.

2. The box marked "Resolution: is NOT the resolution of your image. This is the resolution of your image were it to be printed. In other words, this is the Print Resolution of your image, and it depends upon the dimensions you set.

3. The box marked "Resample Image" is what you check when you want to change the number of pixels that make up your image into less pixels or more pixels (so each pixel is really a sample of that part of your specimen: resampling means that part of your specimen will be re-made into more or less pixels).

The Resample box is normally left UNchecked, EXCEPT when you need to resample for publication.

When going to Powerpoint, Illustrator, Word, etc., only the Width and Height are set to "tell" that software its dimensions. Ignore Resolution.

When going to publication, attempt to set the Width and Height to the column dimensions and the Resolution to 300 or 400 (depending on publication). If the Width and Height don't fit the publication dimensions, check Resample and repeat.

As a general rule, resample figures to smaller dimensions versus larger.

 

Image Size dialog box in Photoshop

 

 

 


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