Scientific Imaging Image Analysis worksheet Page 1 of 8. Image Analysis

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1 Scientific Imaging Image Analysis worksheet Page 1 of 8 Image Analysis Part 1 - Data Analysis: We will Photoshop software to determine the area of leaves. Option 1: Open the file provided by your instructor. Go to step 5. Option 2: 1. Place a leaf on the scanner with a ruler close to it. 2. Open Adobe Photoshop and use the File:Import menu item to start the scanner. 3. Scan the leaf as a grayscale image. 4. Crop the resulting image as small as possible, leaving a portion of the ruler visible (be sure you can see part of the metric scale). 5. Use the analysis:set measurement:custom menu to bring up the Measurement Scale Dialog: 6. In the dialog, click and drag on the image of the ruler to mark off one centimeter. As you can see in the image above, in this case 1 centimeter = 92 pixels. WRITE THIS NUMBER DOWN! 7. Calibration target: With the ruler on the screen, draw a black square exactly 1cm on a side (use the rectangle tool set to square). Move the square to a convenient blank area (a target may already be provided, in which case you can skip this step). 8. Remove the ruler by cropping. If you are using the image provided, use the image:adjustments:desaturate menu option to convert the image to grayscale and then crop off the ruler. Flatten the image so the calibration target is on the same layer as the leaf. 9. Use the image:adjustments:threshold menu item. Adjust the slider (see below) in the dialog box until the leaf appears solid black, the background white, and the holes clearly delineated. This is as much art as science.

2 Scientific Imaging Image Analysis worksheet Page 2 of The leaf is now black and white. Use the brush tool to draw a black line to close off any insect holes that reach the edge of the leaf. Try to draw the line to approximate where the edge of the leaf would be. 11. The next step is to delete the background. Use the Layer:Duplicate Layer menu to make a copy of the background. Then, select the background layer and delete it (right)

3 Scientific Imaging Image Analysis worksheet Page 3 of Now, use the Magic wand tool. Set the tolerance to 0 and be sure the contiguous box is checked, and that the single selection mode(leftmost icon next to tolerance box) is selected. Click once on the background to select it, then press the delete key to remove the background. It should look like this: 13. Check calibration: Using the magic wand, click once in your calibration target to select it. Use the Analysis:Set Measurement Scale:Custom menu to confirm that your scale is still set to the value you found in step 6. Use the Analysis:Record Measurements menu to open up a box which will look like this: Note that the area is approximately 1 cm 2 and the width and height are both very close to 1cm. Also note that the minimum and maximum gray values are 0, which demonstrates that you have chosen black instead of white (which would read 255). 14. At this point, use the eraser tool to erase the calibration target(s). 15. Finally, you are ready to analyze the image. Go back to the Magic Wand and UNCHECK the contiguous box. Now, click on one of the white spaces (where the insects fed). 16. Use the Analysis:Set Measurement Scale:Custom menu to confirm that your scale is still set to the value you found in step With all of the white areas thus selected, use the Analysis:Record Measurements menu to open up a box which will look like this: 18. The top line of the box should say something like Measurement 1 (2, 3, whatever). This gives the sum total of all the white areas on the leaf where the insect has been feeding. The remaining lines give the data for each individual white spot on the leaf. In the example above, there were 204 such spots, most of them very tiny. Go into Excel and record the area of the total line (the first line) (see image below).

4 Scientific Imaging Image Analysis worksheet Page 4 of Use the trash can icon on the measurement table to delete all the measurements. Use the magic wand again to select the BLACK areas this time, and again record the measurements (note: if you use a supplied image you may have to first erase any calibration shapes outside the leaf). The table now gives you the leaf area. Record that number in Excel as well. 20. When you are done, add columns to your excel file to calculate total leaf area and percent eaten: Species: Maple Feeding area Leaf area Total Leaf Area Percent Eaten Leaf Leaf Leaf Leaf Leaf Leaf Leaf Leaf Leaf Leaf Totals Average Species: Oak Feeding area Leaf area Total Leaf Area Percent Eaten Leaf Leaf Leaf Leaf Leaf Leaf Leaf Leaf Leaf Leaf Totals Average

5 Scientific Imaging Image Analysis worksheet Page 5 of 8 Part 2 measuring length, quantifying cell area. In this section you will measure cells and quantify their area. 1. Open the file micro.jpg; this file has a number of algal cells and a scale bar: 2. Follow the earlier instructions to determine how many pixels there are in 50µ (note: to draw a straight line hold down the shift key). 3. Use the Analysis:Ruler tool to verify the length of the scale bar. 4. Now use the ruler tool to measure 3 healthy cells. 5. Next, quantify the healthy cell area. To do this, use the select:color range tool from the menu. It will open a dialog like the one below: Set the selection to White Matte. Use the dropper tool to select an area of healthy green cell. Next, use the Fuzziness slider in the dialog box to adjust the selection by eye. With the selection made, click OK. 6. Now record measurements. You can sort the measurement table by clicking on the titles at the top of the columns. Sort by area; this allows you to ignore smaller values. The measurement log can be exported to a text file that can be opened in Excel (or you can cut-and-paste). Note: this technique can also be used to sort cells.

6 Scientific Imaging Image Analysis worksheet Page 6 of 8 Part 3 measuring intensity of bands on a gel. In this section you will learn the basics of analyzing a gel using two tools, Photoshop and ImageJ 1. You might need to install ImageJ go to the NIH website: and download either the platform independent or windows version (note: most computers on campus already have JAVA installed, so you can go with the Platform Independent version. 2. Double-click on the zip file that downloads and install the software anywhere on the computer. 3. Find the resulting imagej.exe file in the folder you extracted the files to and double-click on it to start it. 4. Open the file gel.gif. 5. Use the rectangular selection tool to draw a rectangle over the first lane with dark bands and press 1. This selects lane 1 (see image right). 6. Move the box over the next lane and press2. Repeat for the 3 rd lane. 7. Use the Analyze:Gels:Gel Analyzer Options menu item and be sure the Label with Percentages and Invert Peaks boxes are checked. 8. Use the Analyze:Gels:Plot Lanes menu item to plot the 3 lanes. 9. Use the straight line tool to draw a straight line across the bottom of the 3 major peaks from lane 1 (below). Next, use the wand tool to click once each inside each of the peaks bounded on the bottom by the straight line. Now use the Analyze:Gels:Label Peaks menu item to place relative percentages in each of the peaks.

7 Scientific Imaging Image Analysis worksheet Page 7 of These percentages allow you to compare the relative amount of protein, DNA, or whatever was on the gel. In this case, the top peak had about 1.75 times as much protein as the band below it. In the image below, the measurements were taken across the top bands and they were compared to each other. The first 2 are roughly equal, the 3 rd is somewhat smaller, and the 4 th band has only about 1/3 the density of the other Now, let s use Photoshop to do some analysis. Open the file gel.gif in Photoshop. 12. Use Image:Adjustments:Invert to invert the image. This will make more intense bands have higher values, which is more intuitive. 13. Use the line tool to draw black lines between the bands. 14. At the top of the lanes add a lane number using the text tool. Number the lanes 1-5 starting with the 2 nd lane (the leftmost one with 3 bright bands. 15. In the leftmost lane (unnumbered) label the 3 bright bands, starting with A at the bottom and C at the top. 16. Select the magic wand tool. Set the tolerance to 32 and have only the contiguous box checked.

8 Scientific Imaging Image Analysis worksheet Page 8 of For each band: a. Click with the magic wand tool in the middle of the band b. Use the analysis:record measurements item to analyze the band c. Record the Lane, band, area, mean gray value and integrated density in the table below for the 3 bright bands in each of the 5 lanes. d. If the magic wand selects more than a band, try again. For some of the fainter bands you will have to make a freehand selection using the lasso tool. Lane Band Area Mean Gray Integrated Density 1 A 1 B 1 C 2 A 2 B 2 C 3 A 3 B 3 C 4 A 4 B 4 C 5 A 5 B 5 C Part 4 measuring length of structures In this section you will learn the basics of measuring structures given an image with an object of known size. 1. Open the file Beetle_2718.PSD 2. Since it is a psd file, you can manipulate the opacity of the lines overlay layer so that you can see the items you need to measure as well as where to make the measurement. 3. Use what you have learned to calibrate the ruler and measure the indicated structures. Record your measurements on the board.