Field 150 Activity Instructions Pilot 3.pdf - The Field Museum

Field 150 Activity Instructions Pilot 3, Spring 2013

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Objectives Develop an understanding of using software to capture, measure, and analyze data

electronically. Perform measurements on a series of morphological characters from digital images. Record observations on a series of morphological characters from digital images. Discuss and analyze the variability of morphological characters.

Materials

Computer with Image J installed. Folder with digital images of selected species of early land plants. Instructions for Image J and laboratory.

Software Instructional Videos

Video instructions for Image J and Excel can be found here http://www.youtube.com/watch?v=bMU_Tqrti5o&feature=plcp Basic Excel Functions http://www.youtube.com/watch?v=6zuace-nmnQ&feature=context-cha Sorting data with Excel http://www.youtube.com/watch?v=4TDRUbdgCm4&feature=plcp Basic Stats and Excel http://www.youtube.com/watch?v=8_yuEemH7Qs&feature=plcp Setting the scale bar in ImageJ

Computer Shortcuts

To copy and paste: Cut = Ctrl C, Paste=Ctrl V To open image in ImageJ: File>Open>Select folder or file>click Open To set scale in Image J: Open image> Click line button on tool ribbon > trace scale bar >analyze > set scale > enter in known distance > enter in unit of length >click OK button To measure an image in ImageJ: Click line button on tool ribbon >draw line > analyze >measure To draw a straight line with ImageJ: Hold the shift key while drawing. To zoom in with ImageJ: Press Shift + “+”, Shift + “-“ to zoom out.


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Activity – Getting Started and Setting the Scale Each student should checkout a netbook (or use their own laptop) and a flash drive. Start up the computer and, once booted, insert and open the flash drive.

1) Find the excel file called, “Student Data Template Trial 3.” Double click to open this file. Go to File>Save As> and save this file using your name and section number. For example, I might save as my file as “CampbellTom 2.” Please save this file to the folder on your flash drive called, “Student Data.” Please do not alter or delete the original template file.

2) Please fill out the header information in the top of the spreadsheet (boxes F1-I7) including your name, date, section, student number, and email.

3) Open Image J. If using your own computer, Image J can be freely downloaded here http://rsbweb.nih.gov/ij/download.html

4) Open an image using the ImageJ menu. File>Open>select the image you wish to work with from the image folders. See chart below for your assigned images.

5) Setting the scale bar. For each image we measure using Image J, we will need to set a scale. Notice! Images MUST be opened from within the ImageJ program. - In ImageJ, go to File>Open> and select the image you wish to work with from the flash drive. Once that image opens, you should see a scale bar at the bottom of the photo. - Select the straight line drawing tool in Image J (5th button from the left) and click and drag to trace over the scale bar. A yellow line should appear over the scale bar of the image. You can hold down the shift button to help you make a straight line. - Next, go to Analyze>Set Scale> You will see a value in the “distance in pixels” box. This is the length of the line you just drew in pixels. We need to convert that number of pixels into a real measure, so in the next box titled, “Known Distance” type in the measure of the scale bar from the picture. In the unit of length box, type in the units (um for microns). - Click OK. - For each new image you open, you will need to repeat this procedure and reset the scale.


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Exercise 1 – Measuring Lobules Find your class on the table below. This will tell you which sets of images are assigned to your class. Day Time Room Sec. Instructor Images Mon 2:00 – 3:50pm BBH 356 1 Frankel A Wed 2:00 – 3:50pm BBH 356 2 Frankel A Fri 2:30 – 4:20pm BBH 356 3 Park B Tues 5:40 – 7:30pm BBH 356 4 Nuss B Thurs 5:40 – 7:30pm BBH 356 5 Mungre C Fri 10:00 – 11:50am BBH 356 6 Slate C

1) Find the appropriate image folder and photo set for your class on the flash drive and open the first Lobule image using ImageJ. Each section will measure two specimens of one species. For example, according to the chart above, Dr. Frankels 2PM Monday class will measure the lobules of Species A Individual 1 and Species A Individual 2. To open the files go to: >>My Computer>Select Flash Drive>Images>Select Image Set>Image Set >Image Set #-Lobules>Species A Individual 1.

2) Use the directions above to set the scale bar. In this image, we would be measuring how many pixels are in 100µm.

3) After setting the scale, our next task is to collect the actual data. To do this, we are going to collect the dimensions (length and width) of each complete lobule. For our activity, length will be defined as the longest axis and width as the shortest. We are only measuring lobules that are completely visible and will force our axis to be perpendicular. (In the above example, we would measure the length and width of nine lobules.) Take the length

Length

Width

Length

Width


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by bisecting the lobule (measure the length along the axis of bilateral symmetry).

4) To make the measurements, select the straight line tool and trace the line along one of the axis’s, similar to what you did to measure the scale bar. For example, select any lobule and measure it’s “length.” After drawing the line, press Ctrl-M to record the measure within ImageJ. This will open a small window where you will be able to see the measurement you just made.

5) Next measure the other axis in the same manner as you did in step 4. In this example, you would be measuring the “width” of the same lobule. Unfortunately, starting a new measure will delete the previous yellow measurement line from your screen. Keep your width measure perpendicular to the length measure and measure across the approximate middle of the cell even if there is a wider area. We are measuring the middle of the lobule, not the widest part. Press Ctrl-M to record the measurement to ImageJ.

6) After each set of measures, you will need to transfer this length and width data to your excel spreadsheet. You can do so by copying and pasting into the excel file under the appropriate heading. Columns on the spreadsheet are clearly labeled. Please place your measurements in the correct column. You will not use all columns and cells in your spreadsheet. In this example, the data would come from the ImageJ “Results” box (Analyze>Measure) under the column “Length.” This is the only column of data we are concerned with. Please do not round the data.

7) Continue until you have recorded the length and width of all lobules in the image.

8) After finishing species A, Individual 1, please continue in the same manner for Species, A, Individual 2. If time permits, you may continue to another species in the same manner.

9) Do not forget to periodically save your work in your excel spreadsheet on the flash drive. Tips: If you are having trouble remembering which lobules you have already measured or where your measurement lines are, you can mark each line and cell using the paintbrush tool (5th button from the left). If you do this, please do not save changes you make to the images. Remember that to draw a straight line, you can hold the shift key and to zoom in, you can press Shift + “+”.


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Exercise 2 – Measuring Cells 1) Find the appropriate image folder for your class on the flash drive according to the chart

above and open the first Cell image using ImageJ. For example, according to the chart above, Dr. Frankels 2PM Monday class will measure the cells of Species A Individual 1 and Species A Individual 2. A sample path might be, >>My Computer>Select Flash Drive>Images>Select Species>Image Set #>Image Set #-Median Cells>Species A Individual 1 Median Cells.

2) Use the directions above to set the scale bar. In this case we are measuring how many pixels are in 20µm.

3) After setting the scale, our next task is to collect the data. Here we would like to measure cell dimensions. Similar to what we did for lobule measurements, we will consider the longest axis of the cell the “length” and the shortest axis of the cell the “width.” We will measure at 90° angles down the “center” of the cells. (See example above). If the “length” and “width” of the cell appear to be the same, you will have to use your judgment to decide which is which. Measure from the inside lumen edge of one cell to the outside lumen edge of the next as shown in the image above. (see accompanying handout for cell anatomy) We will only measure cells that are completely visible. In the example below, there are 11 cells totally visible.

Width

Length


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4) To make the measurements, select the straight line tool and trace the line along one of the axis’s, similar to what you did to measure the scale bar and lobules. For example, select any cell and measure its “length.” After drawing the line, press Ctrl-M to record the measure within ImageJ. This will open a small window where you will be able to see the measurement you just made.

5) Next measure the other axis in the same manner as you did in step 4. In this example, you would be measuring the “width” of the same cell. Unfortunately, starting a new measure will delete the previous yellow measurement line from your screen. Try to keep your width measure perpendicular to the length measure and measure across the approximate middle of the cell. Press Ctrl-M to record the measurement to ImageJ.

6) After each set of measures, you will need to transfer this length and width data to your excel spreadsheet. You can do so by copying and pasting into excel. In this example, the data would come from the ImageJ “Results” box (Analyze>Measure) under the column “Length.” This is the only column of data we are concerned with. ImageJ will record everything as “Length” even if you are measuring the width. You can “Save As” your results to create a new excel file to copy and paste from, you can copy and paste to a worksheet within the workbook, or you can retype the data. Please do not round the data. Unfortunately, you cannot copy single cells from ImageJ.

7) Continue until you have recorded the length and width of all visible cells in the image.

8) After finishing your species, continue onto Individual 2. If time permits, you may continue to the other species.

9) Do not forget to periodically save your work in your excel spreadsheet on the flash drive. Tips: If you are having trouble remembering which cells you have already measured or where your measurement lines are, you can mark each line and cell using the paintbrush tool (5th button from the left). If you do this, please do not save changes you make to the images. Remember that to draw a straight line, you can hold the shift key and to zoom in, you can press Shift + “+”.

Student Survey 1) Now that you have completed this portion of the project, please find the student survey folder, “Student Survey” on the flash drive. Open the file called “Student Survey” 2) Please answer each question on the survey by simply typing on the survey form. Please do not include any personally identifiable information in your survey. 3) Save your survey by going to File>Save As to rename the survey. Please make the name anonymous.


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Your Data and Project Wrap Up - Email the excel data file to yourself so that you can use it for class analysis. - Close all files and programs that you were working with and safely eject the flash drive. - Return the computer to the cart and return your flash drive to your instructor. - Thank you for participating!

Documents

Field 150 Activity Instructions Pilot 3.pdf - The Field Museum