Fundamentals of MetaMorph® Exercises for Day 1



Download 211 Kb.
Date11.07.2018
Size211 Kb.

Fundamentals of MetaMorph®

Exercises for Day 1

MetaMorph Hands-on:
Image display


Purpose of exercises:

  • Learn how LUTs, scaling can be used to change the display of the image without altering the image’s original data. You can use the Histogram on the side of the image to verify the image’s data during exercises 1 & 2.


Exercise – Scaling


  1. Open the yfp-4.tif image.




  1. On the Image Window Toolbar, click the Scale tool and select Scale Image…. The dialog box opens.




  1. Uncheck the Auto scale checkbox. Note that the Low scale value is 238 and High scale value is 3741.




  1. Grab the wedges in either the Image Window Toolbar or in the Scale Image dialog box. As you move the low scale wedge, what is happening in the image? As you move the high scale wedge, what is happening in the image? Which slider helps you create more contrast to see the structure in the image?



  1. Click on the Scale tool on the Image Window Toolbar and select Auto scale again. You can adjust the low and high scale wedges again and see the contrast change in the image. What is the benefit of having Auto scale versus just using the scale wedges to specific values?



  1. Extra Credit: What is the other way to open the Scale Image dialog besides the Scale tool on the Image Window Toolbar?



  1. Draw a simple graph of the relationship between the intensities in the image and the relative display intensity in an image with scaling.



Exercise – Lookup tables (LUTs)


  1. Open the dapi1.tif image.




  1. On the image window toolbar, click the Display icon (rainbow circle). Note: You will see a list of different lookup tables. Monochrome should be selected.




  1. Select Pseudocolor, and then some of the other lookup tables.




  1. Adjust the Scaling with the low and high wedges. What is happening on the image and in the Image Window Toolbar when the Pseudocolor LUT is selected?



  1. With the Pseudocolor LUT and adjusting the Scaling on the dapi1.tif image, two “artifacts” can be made visible in the image. What are they?



  1. What are the most likely causes of the artifacts in the dapi1.tif image and how could have we confirmed that on the microscope, assuming that we still had it setup?


Calibrate Distance
Purpose of exercise:

  • Use the sample calibration slide image to calibrate it from pixels to microns.


Exercise:


  1. Open the Calibrate Distances function from the Measure menu.




  1. Press the Add (“+”) button to add a new spatial calibration to the list.




  1. Double click in the Calibration Name box and edit the name to be more meaningful, i.e.“20x”.




  1. You can now click in the X, Y, & Units cells in this table to enter the calibration values if you like. Note that usually the values are the same for X & Y.




  1. Open the 20XMicrometer.tif image




  1. Press the “Calibrate by Region…” button in Calibrate Distances window.




  1. Follow the 4 steps in the Calibrate by Region window to calibrate this image to the 10x objective. Note the smallest spacing between the calibration lines is 20um. When you are finished the 4 steps, press OK and close the Calibrate by Region window.




  1. Select the 20XMicrometer image in the Calibrate Distances window and press the Apply button. Note the distance calibration has been applied to this window and can be applied to other open images as well.




  1. Repeat the process with the 40xmicrometer image.



Multi-Dimensional Acquisition and Review
Purpose of exercise:

  • Use the emulated hardware to gain familiarity with acquiring and reviewing multi-dimensional experiments.


Exercise – Timelapse


    1. Configure MDA to acquire a timelapse of a single wavelength, no stage or z movement.




    1. Set the timelapse for 5 time points, 10 seconds per time point, for a duration of 40 seconds




    1. Set the exposure time for 100ms




    1. Acquire the timelapse




    1. Review the timelapse with the Review Multi Dimensional Data



Exercise – Multiple Wavelengths


  1. As in the previous exercise, but use two wavelengths, DAPI and FITC. Set the DAPI exposure to 50ms and the FITC to 20ms.




  1. Acquire the experiment




  1. Review the experiment.

Extra: Use the Color Combine option in Review MDA to combine the DAPI and FITC into a blue/green composite image.


Exercise – Multiple Dimensions



  1. As in the previous exercise, but enable Z series.




  1. Set the Z series to “range around current” with a total of 3 z positions being acquired




  1. Acquire the experiment, with 5 time points, 2 wavelengths, and 3 z positions.




  1. Review the experiment


Exercise – Using the xy stage


  1. As in the previous exercise, but enable the XY Stage




  1. Define 3 stage positions




  1. Acquire the experiment, with 5 time points, 2 wavelengths, 3 z positions, and 3 stage positions




  1. Extra: Set the exposure for the DAPI channel to 500ms but only for Stage Position 2




  1. Review the experiment



Working with Stacks
Purpose of exercise:

  • Familiarity with using and manipulating stacks


Exercise – Stacks, Montage and Keep Planes


  1. Open the “pollen.stk” file




  1. Play through the stack as a movie




  1. Move to plane # 35




  1. Draw a rectangular region around the top pollen grain




  1. With the region active, open the Montage dialog box




  1. Make the montage


Exercise – Stacks, Montage and Keep Planes (Continued…)



  1. Return to the Montage dialog box and select to draw a separator frame around each image, and to number each image. With the region active on the pollen stack, and the pollen stack selected, Make the montage




  1. Notice that frames 1 thru 28 have either “junk” or nothing, but frames 29-51 contain the different slices of the pollen grain.




  1. Using Keep Planes, select planes 29-51 of the pollen stack. Then Delete unselected planes.




  1. Now on the first plane of the pollen stack, draw the rectangular region around the top pollen grain




  1. Make the montage

Filtering
Exercise – Median Filter


    1. Open the NoisyNuclei.tif image.




    1. Zoom into a small area of the image .




    1. Now go to the Process menu and select Basic Filters, and Median filter to the image. Try a 5 x 5 kernel size and hit Apply. Note the difference in the image?




    1. Try some of the other processing filters and see how the results compare.



Exercise – Detect Edges


  1. Open DetectEdges.tif




  1. Under the Process menu, choose Detect Edges.




  1. Process the image using each of the 6 filter choices.




  1. Note the differences in the result images and connect these with the kernel choices:




    1. The Laplace choices only use a few neighboring pixels so struggle to distinguish grainy noise from true object edges. But the kernels are nicely symmetric and detect edges in any direction.

    2. The Horizontal and Vertical choices have directional bias, like a shadow, due to the orientation of the kernel, but they do a nice job combining 3 pixels in a row to verify an edge in a particular direction.

    3. The other choices are attempts to combine the best of “a” and “b” above: combine multiple passes of directional kernels at multiple orientations.

Extra: Try a “morphological” version: ProcessMorphology FiltersGradient with filter size 3 pixels. This detects an “edge” when there’s a contrast change between the neighborhood’s brightest value (Dilate result) and darkest value (Erode result). More on Morphology later…


Exercise – Morphology Filters: Dilate, Erode, Open


  1. Open Morphology.tif and go to ProcessMorphology Filters.




  1. Erode then Dilate the erode result, both times using Filter Shape “Circle, 25 pixels”—note that not all objects are restored; some are filtered out.




  1. Verify that the same result comes from using the Open operation (same filter shape) on the original image: Open performs erode then dilate, those objects where the filter doesn’t “fit” are filtered out.




  1. Check out the Reconstruction option: objects surviving the open filtering are identical to the originals. Without reconstruction, we see only where the filter “fit”, even in surviving objects. Sometimes you want to remove detail like the outgrowths on the bottom cell in the example, and sometimes you want to preserve them.


Exercise – Morphology Filters: Open and Top Hat


  1. More practice with the Open filter—use the Area option on Morphology.tif at sizes 200, then 1000, then 2000.




  1. Apply Top Hat using the same options as Step 1. The Top Hat result is precisely what the Open filter removed.




  1. Verify that applying an Open filter and subtracting the result from the original reproduces the Top Hat result. Top Hat saves you time when filtering for small objects.




  1. Apply Top Hat to the real life grayscale image Granules.tif. We’re looking for fluorescent granules. A good setting is Area 20 pixels.



Exercise – Morphology Filters: Combining steps


  1. In Morphology.tif, see if you can isolate just the 3 outgrowths in the bottom cell using only Morphology Filters.




  1. Can you get your procedure down to just 3 steps?



Segmentation

Purpose of exercise:

  • Gain familiarity with thresholding and regions of interest (ROIs). Threshold and ROIs are important as they are used by many MetaMorph tools to segment the image in preparation to making measurements.


Exercise - Thresholding


  1. Open the frogeggs.tif image.




  1. Select the ellipse tool and create a region to outline a nucleus. Imagine how painstaking it would be to use the ellipse tool to create a region around each nucleus on frogeggs.tif!




  1. Delete the ellipse region.




  1. Set the threshold by choosing Auto Threshold for Dark Objects with the Threshold Tool icon on the image window:




  1. You can use the blue wedges on the Image Window Toolbar to adjust the thresholding further.




  1. When you are happy with the thresholding, go to the Region menu and select Create Regions Around Objects to create regions around all the thresholded nuclei. What kind of regions did it create?




  1. The frogeggs.tif image is an easy example of how to use thresholding the segment the sample from the background. Close this image now.




  1. Open the Count_Nuclei.tif image




  1. Turn on thresholding and try to threshold all the nuclei in the image


Exercise – Thresholding (Continued…)


  1. Notice that this segmentation process can be more difficult if the background of the image is not uniform.




  1. Besides uneven background what else can cause problems with thresholding (hint: examine the NoisyNuclei.tif image).


Thresholding and Measuring Regions:

  • Understand how to obtain intensity measurements.

  • Learn how to log to Excel.

  • Gain more familiarity with ROIs and thresholding.


Exercise:


  1. Open the Nuclei.tif image.




  1. Threshold the image to identify the nuclei from background.




  1. Go to the Regions menu and select Create Regions Around Objects. This will create a region around all the thresholded nuclei.




  1. Go to the Measure menu and select Show Region Statistics. Selecting regions with your mouse will display some size and intensity measurements. Make sure the Entire Image checkbox is not checked.




  1. In the Show Region Statistics dialog box, click the Open Log button. The Open Data Log dialog box will open. Check the Dynamic Data Exchange (DDE) checkbox, click OK. The Export Data Log dialog box will open. Click OK and Excel will open.




  1. Select different regions with the mouse and for each press F9 to log measurements to Excel.




  1. Close the Show Region Statistics dialog.




  1. Go to the Measure menu and select Region Measurements. Select to include All Regions. Make sure you are viewing the Measurements table to see the measurements for all the Regions at once.




  1. You can select rows in the table with the mouse to find the corresponding region or select regions in the image and find the corresponding data in the table.




  1. You can log all the data in the Measurements table to Excel by pressing the F9:Log Data button in Region Measurements.




  1. Extra Credit: If you go to the Graph Tab, why does all the data graph along a vertical line? What is needed if you wanted to graph an intensity over time measurement?


Integrated Morphometry Analysis:


  • Understand how to measure the size and shape of objects in your images, as well as a number of other parameters.

  • Understand how to set classifiers to exclude/include certain objects from being measured.


Exercise – Measuring an Image with the IMA


  1. Open the frogeggs.tif image.




  1. Threshold the image with Auto Threshold for Dark Objects.




  1. Go to the Measure menu and select Integrated Morphometry Analysis.




  1. Go to the Measurements tab, select to Display the Area, Centroid X, Centroid Y, Average Intensity, and Length & Breadth measurement parameters.




  1. Click the Measure button. All the “frog eggs” turn green, indicating they were measured. A list of measurements appears.




  1. Look at the different data views: Object Data, Summary, and Histogram, and ScatterPlot displays. Don’t forget to select different measurement parameters (X drop-down) in the Histogram display.




  1. Select the Object Data tab to view the object measurements.




  1. Clicking on a row in the list of objects highlights the corresponding cell in yellow, and clicking a cell in the image highlights its corresponding row. You can also click in the Histogram (to find all the cells of a particular group).




  1. Go back to the Histogram display, you can set classifiers on the population of eggs using the red calipers (red lines) on the left and right of the graph. Once you have moved the calipers to exclude some of the eggs, click the Set Filters from Calipers button. This will exclude the eggs outside the calipers from being measured. Note that the excluded eggs are no longer green, but are the threshold color and they are not listed in the Objects table.




  1. You can double click on individual objects in the image to add or remove them from the current data set.




  1. Reset the filters by clicking the Reset Filters button. The Reset Filters button is on the Measurements tab when the list of possible measurements are configured. All the filters are set to their default values.



Integrated Morphometry Analysis:

  • The purpose of this exercise is to measure a group of nuclei in an image. Filter out the cell that’s dividing and the small stuff and then count the number of cells that are left.


Exercise - Analyzing the Measurements and Setting a Filter


  1. Open the acquired-3.tif image. Notice that there is one dividing cell in the image and its location.




  1. Set the threshold using Auto Threshold for Light Objects.




  1. Under the Measure menu, select Integrated Morphometry Analysis. The dialog box will open.



Exercise - Analyzing the Measurements and Setting a Filter


  1. Go to the Measurements tab, select to Display the Area, Centroid X, Centroid Y, Average Intensity, and Length & Breadth measurement parameters.




  1. Press the Measure button to measure the thresholded objects in the image.




  1. Go to the different data displays to view: each object’s measurements, the summary of measurements, and the Histogram of the different measurements.




  1. Go to the Summary Display and look in the Count column to see how many objects are in the measurement. Note that you can also find the Minimum, Maximum, total, etc. of each measurement parameter




  1. Go to the Object Data tab, click on the dividing cell to get the measurements for the dividing cell. Examine the measurements of that cell to determine how to filter it out of the measurement.




  1. Once you select a parameter to filter the object from step 7, go back to the Measurements tab and check the parameter to filter and set the range. Remember from exercise 1, that you can do the same thing by using the Histogram.




  1. Filter out the smaller objects and some of the objects touching the edge of the image based on their Area using either the parameters list & the lower/upper limits or the histogram.




  1. Go to the Summary data and look in the Count column to see how many objects are left in the measurements.




  1. Click the Reset Current button to clear the measurements. The measurements have been cleared: the data is gone and the green overlay is gone in the image.




  1. Click on the Reset Filters button to reset the Classifier parameters and ranges to the IMA default.




  1. Extra: Select a parameter to Teach the system the “good” objects to measure. Go to the Measurements tab, check the parameter then teach the IMA the “good stuff”. Go to the Preferences tab and make sure the Update Filter Ranges on Shift Click is checked. Hold down the shift key and click on objects in the image. You can go to the Measurements and the data tabs to watch the results as you continue to click on objects.




  1. Extra: Why might you setup the IMA to filter out objects that touch the edge of the image and how would you do it? There are at least 4 ways, you might be able to think of 3. (Hint: Preferences).


Color Separate & Color Combine:

  • Provide some familiarity with MetaMorph’s color separate and color combine functions. Users can color combine images for presentation purposes or, to visualize how the data in different images overlap.


Exercise – Color Separate & Color Combine:


  1. Close all open images.




  1. Open the dapi1.tif, fitc1.tif, & rhodamine1.tif images.




  1. Under the Display menu, select Color Combine.



Exercise – Color Separate & Color Combine (Continued…)


  1. In the Color Combine window, set the Red component to rhodamine1, set the Green component to fitc1, set the Blue component to dapi1 and press the Color Combine button. Observe the resultant image.




  1. Now, adjust the scaling in each of the dapi1, fitc1, & rhodamine1 images and after adjusting the scaling in each, create a new color combined result. Compare the new combined images to the first.




  1. The Color Combine function is making an assumption regarding how you want to combine images into a color result. What is the assumption (hint: think of the image used in exercise 1)?




  1. What kind of image is the result of combining the three individual images and what has happened to the original data? Is it appropriate to publish data from analysis done on these color images?




  1. Open the Color Separate function from the Display menu. Separate the color image back into monochrome components and see how the data compares to the original images.



MetaMorph Hands-on: Journals – First Exercise Set

Exercise 1 – Maximum





  1. Open a through-focus z-series stack, such as “nerve”.




  1. Start Recording (Journal menu)




  1. Stack Arithmetic (Process menu)




    1. Choose the “maximum” option




  1. Stop Recording (Journal menu)




  1. Save the journal as “maximum.jnl”




  1. Test the “maximum” journal with the “nerve”, “fitc_bin” and “pollen” stacks.




    1. Use Run Journal (Journal Menu) to do the testing




  1. Try using the Journal toolbar to Run Journal (instead of the menu item)



Exercise 2 – Taskbar





  1. Create Taskbar (Journal menu, Taskbars submenu)




  1. Drag the Taskbar to be 1 column wide by 5 buttons high




  1. Set the Category to Journal, then Drag the “maximum” journal to the first button




  1. Rename the Taskbar to be “Wednesday”




  1. Save the Taskbar as “Wednesday.jtb”




  1. When you Close the Taskbar editor, the Taskbar will close




  1. Choose Load Taskbar (Journal menu, Taskbars submenu)




  1. Load the “Wednesday” taskbar




  1. Open a through-focus z-series stack, such as “nerve”




  1. Test the “maximum” button




  1. Try using the keyboard shortcut, it is CTRL-1 for the first button


Exercise 3 – Best Focus


  1. Using the same procedure as Exercise 1, but…




    1. Use the “best focus” operation instead of “maximum”




    1. Save the journal as “best focus”




    1. Add the journal to the Taskbar – and test it with the stacks you used previously



MetaMorph Hands-on: Journals – Second Exercise Set


Exercise 4 – Sharpen and Best Focus


  1. Open the “nerve” stack




  1. Start Recording (Journal menu)




  1. Basic Filters (Process menu)




    1. “Sharpen” operation, “Low” parameter




  1. Stack Arithmetic (Process menu)




    1. “Best focus” operation




  1. Stop Recording (Journal menu)




  1. Save the journal as “sharpen and best focus.jnl”




  1. Add “sharpen and best focus” to the Taskbar




  1. Test “sharpen and best focus” with “nerve”, “fitc_bin”, “pollen”


Exercise 5 – Journal Editor


  1. Edit Journal (Journal menu) – or, hold down SHIFT and click on the “sharpen and best focus” in the Taskbar




  1. Highlight the Basic Filters command and Double-click it




  1. Within the Basic Filters dialog




    1. Change the parameter to “Medium”




  1. Save the journal




  1. Test the journal with the standard test stacks


Exercise 6 – Journal Editor


  1. Edit Journal (Journal menu) – or, hold down SHIFT and click on the “sharpen and best focus” in the Taskbar




  1. Find the “Close” command in the Builtin-Functions list, drag it into the journal, positioning it just above the “End of Journal” marker




  1. Within the Close dialog




    1. Set the Image Selector to be “1: Sharpen”




  1. Save the journal




  1. Test the journal with the standard test stacks




© 2011 Molecular Device, All Rights Reserved Page



Share with your friends:


The database is protected by copyright ©dentisty.org 2019
send message

    Main page