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Veteran pharmaceutical research scientist and member of Adobe's Biomedical Image Advisory Group, Eric J. Wexler shares his experience creating detailed biomedical imaging in Photoshop CS3 Extended for Biomedical Research. Eric shows how to use Photoshop CS3's selection, analysis, and editing tools to evaluate an image's color composition, modify images for research, optimize exposure with levels and curves, transform images with layers, and compensate for acquisition problems and limitations. Eric also explains how to add reference information to images, annotate and optimize DICOM animations, and share finished images with colleagues. Exercise files accompany the tutorials.
NOTE: Actual biological research images are used for this title's examples. Some of these images, including those of internal organs and dissected animals, may be considered graphic or offensive to some viewers. Viewer discretion is strongly advised.
In this video, I am going to show a way to threshold an image and that's to simplify the image and get to a point that we can binarize it, have either a positive, which would be black in this case, or a negative which would be white. That way we can apply image analysis to it. What we see here is the picrosirius red-stained heart and what I want to do is to simplify the image so that we can easily move it in and measure the red using either Photoshop now or a different image analysis program.
First I am going to correct the image, I will bring up the Curves command with the Command or Ctrl, on the PC, +M and I am going to set a white point by clicking on Set White Point eyedropper and moving it to an area which has no tissue. Then we will hit OK and next, and I did show this previously, we are change the way we look at this image from RGB, and we're changing to the mode to CMYK. Then we did remember that magenta was the most appropriate single channel to look at just the pure intensity levels of the stains we are interested in. So what I can do is I can remove the other channels by right-clicking, Delete, right-click on yellow, Delete, and right- click on black and Delete. Now I have a channel with a single intensity.
Now we want to convert this to a gray scale image. So we go to Image > Mode > Grayscale and that converts the single intensity, removing the color of just what we can see, in this case the black matching the stain. To make this into a one-bit image, we need to threshold it and again we go to Image > Adjustments and down near the bottom is Threshold. Now in this case, we would be testing out different thresholds making sure that the most appropriate setting for analysis and that would be done by doing multiple experiments and testing this out on some sort of standards, a way to validate our method.
In this case, I am just going to move the threshold up to 146 and I will make sure that every subsequent image researched would the same settings so that they are comparable. We hit OK. Now I convert this directly into bitmap, Mode > Bitmap. The resolution of the original image is 300 and we want to keep that. In this case, we can go 50% threshold, hit OK and now we can analysis this sample knowing that the black represents the positive stain and the white is just tissue that has not been stained.
There is the one last item I wanted to show you about how Photoshop can be used to adjust images for additional work. In the next set of videos, I will be going over the concept of overcoming some of the acquisition limitations.
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