Join Eric Wexler for an in-depth discussion in this video Evaluation of gel images, part of Photoshop CS3 Extended: Research Methods and Workflows.
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In this movie, the first thing we're going to do is evaluate this image of the gel. If you're working along with me, open the western 8.10 TIF from the Chapter 1 exercise files. We have the name of the file, we have that we're viewing it at 50%, and that this is a grayscale image so there's a single channel of intensities and this is an 8-bit image, meaning that there's between zero and 255 or a total of 256 levels of intensity. We can move down below and see that the document is 1. 2 MB, but most importantly we're going to evaluate the overall intensity found in this image and we're going to do that first by looking at the click on this warning button to make sure that it's not working with cached data and here we can see the range of intensities found in the entire image. We want to open this panel up to be able to see more data. We can click on this icon and select the expanded view and if in your Histogram you're unable to see this data, you need to go back and make sure you have Show Statistics on.
Histogram panel. In the upper right-hand corner I'll select on the Histogram tab and we'll Now in this case the mean and standard deviation is not that important. What's more important is to know the range of intensities in the image compared to the entire dynamic range. We can move our cursor to the left edge of the Histogram. We'll hold our button down and then we're going to sweep it across until we hit the right-hand edge. Keeping the button down, we can now see that the levels of intensities range from 16 to 230 and that's 100% of the intensities in this image. This tells me several things. Since no part of the Histogram is at the extreme left edge or the extreme right edge, we're not clipping the data so we're not underexposing this and we're not overexposing this.
The other thing is that if it did extend for the entire dynamic range it would go from zero to 255 but instead, it only uses 219 of the intensities. So we're missing about 10% of the sensitivity that we could get. If we needed that extra 10% So in no part of the image is it too dark or too bright for us to see differences. In this case, this image is very suitable for analysis. we could change the lighting or the exposure to be able to increase the way the image is captured to use more the dynamic range. The next thing we need to check is to see if there's any unevenness of intensity in the background of the image and we can do this using Photoshop and the analysis tools. In this way, if there is any severe shading artifact we would be able to compensate for it. We'll go to Analysis and in this case, we're just going to be making these measurements using the default calibration, which is in pixels. More importantly, we need to select the correct data points.
Analysis, select data points, custom and currently all the data points are on, but we don't need all of them. In fact, I'll turn off the date and time. We don't need scale units or scale factor, we can keep count on, area. Perimeter we do not need, circularity we do not need. We'll keep height and width and then most importantly, the gray values or basically intensity values. We wanted to collect minimum, maximum, the mean, the median and the integrated destiny or the sum total of all the intensities of all the pixels in a region of interest.
We don't need to collect the Histogram and we're not going to be using the ruler tool or the count tool. So these are the important data points. We're going to save this as a preset so we're able to return to this collection of points at any time. Click Save Preset and now we'll just title this Western. Hit OK and hit OK. Now we're going to do some analysis on the background of the image. We're going to open up the measurement log.
Go to window and pull down the measurement log. And here Photoshop will display all the numbers that we collect. We're going to open that up so we're able to see most of the numbers and now we're going to create a measurement box and in this case I want to actually design the box I want to use. So I will make sure the Ruler tool is selected and I am going to look at the third row down and create a box that's going to fit this particular band. If it fits this band it will fit all other bands. So we hold down our and the height of the band, we can grab that handle and move it down, is 0.54. And we are going to construct a fixed sized box so that we will be able to place this however we want to in any area of the image.
In order to do that we select the Rectangular Marque and we go to Style, Shift key and we can see width of the band is 1.05 as seen in the Options bar up top, Fixed Size and we're going to change the width to 105, and we will change the height to 54 and now when we click, we always get the same size box anywhere we want and we will use this to both analyze the background of the image and analyze the individual bands.
We can move this up top, and making sure that our Analysis, Select Data Points, we have the Western selected, we can simply record the measurements and we are going to look at the median grey value. 187 and the other thing we are going to do is we are going to keep a record of where we took this measurement, and we can do that by hitting Control-click or right-click on your button and we going to stroke the selection.
We only need two pixels, this is grayscale, the color is black, location of the border will be outside, blending mode Normal and opacity 100%, and we'll hit OK. Now we'll place the selection in a new area to measure that background. We moved it over, we can hit Record Measurements, we see that it's 187, we again want to record the fact that this is where we took the measurement so we right-click or Control-click, hit Stroke, hit okay and we will again reposition the region of interest. Now I will stay away from that obvious artifact and then I can just hit Record Measurements again, right-click or Control-click, Stroke. Hit OK and I will just move this around in a clockwise pattern.
Again remembering to stroke this region of interest every time, Record Measurements, Stroke, OK, and then our last position, in the bottom, the bottom left-hand corner. We'll record the measurements and then we will stroke the border. Hit OK. So now we have our gray values, from our six different positions. The one other thing I do need to do is at this time I'll label each of these boxes so they match with each of the measurements.
I select the Text tool. I click, I see this is way too large. I reduce its size. I can just say, type in one, space, use the Move tool to position that. And again I will just be marking each of the boxes. So I can, so I can relate the result to the correct region of interest.
And again I am just repositioning this so I have room to actually enter the data in the center of the box. 5. I'll move it up to the corner. And with the Smart Guides on, I know that my numbers are aligned. Six.
Position that. So we have our six data points and our six regions of interest. The one last thing I will do, and this is just to keep track of everything as I can actually type in the median grey value. 187. 187.
186. 181. And we'll have to scoot down to make sure we can see the 186 and the 181. 186. And lastly 181, and so now we have a marking for each of the intensities and we can see it ranges from 181 to 187 and with that minor type of gradation I'm not going to have to worry about trying to compensate for an unevenness in the background.
Now we can save this. Go to File, Save As, and in this case we'll save it as a Photoshop file. So I'll keep all the layers separate so we're able to modify this if need be. So say, Western 8 background. Hit Save. Now we've analyzed the background of the image and we've identified six different regions and we have the data. We'll export this into a spreadsheet program so we can place all the data together and if we need to do more analysis. In order to do that, we'll select all the data points by moving down to the measurement log and click on the bracketed ruler and then we'll export it by clicking on the ruler with the arrow and we'll give this a name and we'll save this on the desktop. And we'll just say Western background, hit Save and we want to be able to copy this picture and place it into the spreadsheet file.
We'll need to flatten this image, Layer, Flatten Image and we're now going to save this on the desktop as a JPEG. File, Save As, move down to a JPEG selection and we will save this also on the desktop. Hit save. We'll keep us as a large file, hit okay, and now we're going to bring the numbers in the pictures into Excel.
So open up Microsoft Excel, I'm going to open the text file. Desktop, Western background and here this is delimited by tabs, and we also say Next for tabs and we can finish this and here's our data and we can resize the columns. This is not an Excel course, but the next thing we'll want to do is insert the picture.
Insert Picture, From File, go to desktop and double-click on the Western. And we have our Western image. The last thing we have to do is add another column. Insert column and when Photoshop exports the measurement log, it doesn't export the number associated with the region of interest count. So you have to manually add that, 123456, and now we have each measurement and from each region of interest it was collected from. We go to File, Save As, we want to save this not as a text file, because that wouldn't keep the picture, but instead as the last version of Excel, 97 to 2004 workbook.
We'll save this onto the desktop, and hit Save. And now we have the images and the data associated with analyzing the evenness of the background and in then the next movie, we're going to go on and actually analyze the bands in this Western.
- Conducting gel analysis
- Comparing analysis of bands
- Determining an affected area
- Outputting data
- Understanding a penetration model
- Combining images from different acquisition systems
- Recording measurements from samples
- Creating and applying a standard curve
- Determining experimental activity