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Scanning Transmission Electron Microscopy Facility


PCMass Manual: Tutorial 1

Tutorials >>  1 2 3 4 5 6 7

Manual Mass Measurement on Earthworm Hemoglobin and TMV Standard Any use of PCMass should begin with a careful inspection and manual mass measurement of selected particles or filaments, as described here. Once the quality and variability of the data is known, one can plan a strategy for automated analysis or other options.
  1. Start PCMassXX.exe and verify that image NK737537 is read in. The header should be displayed on a green background in the bottom left portion of the image. If the NK7375 folder is not in the PCMass directory, you can find it using the option which gives a standard File Manager window.
  2. Strike the <2> key or move the mouse slightly. Observe that all particles and a significant area of the background are masked out in blue. The 'picture background' is computed using the remaining pixels. The results are tabulated along the bottom of the image, showing mean, SD and num pts for each of 16 sub-areas for both large angle and small angle detectors. This test image has a relatively clean background, so there are no complications to deal with at this point.
  3. Move the mouse pointer. The screen should clear and three concentric circles should appear with a size specified by the default model number 35, earthworm hemoglobin face-on. Continue moving the mouse pointer until it is centered on one of the round, slightly hexagonal particles. After a brief delay, the particle of interest should appear in the zoom window below the image. Below and to the right of the zoom window should be a fitted model and immediately to the right of the zoom window should appear a difference image. Moving the mouse pointer on the lower portion of the screen (below the images) activates on-screen help, describing the item pointed to after a 1 sec delay.
  4. If the circle does not appear to be centered, move it left or right and up and down with the arrow keys.
  5. Strike the <t> and <y> keys to decrease or increase the circle radii. Note the effect on the measured mass, listed below the zoom window. Also note the radial mass profile displayed to the left of the zoom window. Strike the <TAB> key to view the numerical values, along with a tabulation of mass vs. radius of integration. The radius should be chosen approximately 2 units larger than the point where the mass plateaus. Strike the <CONTROL> key to view the actual pixel data from the large angle detector. Strike the <0> (zero) key to restore the image.
  6. Press the left mouse button, strike <ENTER> or hit the <=> key to enter a measurement. A single circle should be drawn with the sequential measurement number and the mass to the right of the particle.
  7. Repeat this for the remaining face-on particles that are separated from other objects. Working from right to left avoids obscuring particles by previous measurements. Stop after 10 if you find this tedious.
  8. Strike the <m> key and move the mouse to select a new model or use the pull-down <Model: Select>. Move the mouse until model number 21 (TMV) is highlighted and press the left mouse button. Note that models are displayed at the current magnification and contrast setting.
  9. Select TMV segments as above. The automatic model tune-up should align the box at the correct angle if it is fairly close to the centerline. If the circle markings are obscuring your view, strike the <0> (zero) key to restore the screen.
  10. Select a new model as above. Move the mouse until model number 49 (EHb side) is highlighted and press the left mouse button.
  11. Select side views as above. The automatic search should align the box at the correct angle if it is fairly close to the centerline. Note that the ends of the box are now closed and total mass is measured (instead of mass per unit length).
  12. Strike the <i> key to list the measurements to date. Strike it a second time if there are more than 25. Scroll using the up/down arrow keys. The particle corresponding to the first measurement in the list should be marked in the image with the circle or rectangle used for the measurement. Particles can be 'removed' by hitting the <delete> key, which changes the mass to a negative value, removing them from the averaging process. (A second hit on <delete> switches the mass back to positive.)
  13. Strike the <o> key to observe the results. Only the first three rows should be populated. The column headings identify the models. The first row is all measurements you entered, giving mean, SD in percent and number of measurements. The second row is a subset passing a test for clean background around the particle or along the sides of the segment. The third row summarizes particles or segments passing a pre-defined set of quality tests. Move the highlight with the arrow keys. A histogram for that class of measurements will appear on the right panel, along with scattergrams describing the population.
  14. Strike <ENTER> to mark all particles of the selected type. Hold down <SHIFT> and hit <ENTER> mark the particles and list masses. Strike the <0> (zero) key to restore the screen.
  15. Hold down the space bar and move the mouse to the right histogram panel. A pointer should appear under the large histogram. Particles in the bin above the pointer should be circled in white while the remainder should be circled in red.
  16. In the file menu, select save *.smm. This saves all current measurements, as well as the parameters of the models used. The same happens when you go to another file or shut down the program. With a text editor such as Notepad, open \\Mass_Meas\nk737537.smm and view the tabulated measurements. These are suitable for import into a plotting program such as SigmaPlot. Note that the masses are not scaled and are in kDa for particles, Da/A for mass per unit length and Da/A**2 for mass per unit area
  17. Add a scale bar to the current image by placing the cursor at the left end where you want the scale bar, hold down <CONTROL> and strike <s>. Hit <0> (zero) to restore the screen if you do not like the result and try again. To restore markings, strike <o> go to step 14.
  18. Go on to the next file by striking the <1> (one) key. Return to NK737537 by striking the <-> (minus) key. Strike <i> or <o> to verify that the measurements were read back in. Any subsequent measurements will be appended to this file. If any model was changed from the previous measurements, the program will ask if you wish to go back to the previous parameters.

 

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Last Modified: June 12, 2009
Please forward all questions about this site to: Kathy Folkers


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