Image Selection

The program should start in 10 sec or less, displaying a practice image of earthworm hemoglobin and TMV from the nk7375 folder. If you did not copy the practice folder, you will get an error message. You can select any other image using the "File" pull-down menu (top left of the screen) and selecting STEM_Image. An open-file window should appear showing the contents of the current folder. Navigation is the same as a standard Windows program. Left-clicking on the "Look-in" panel at the upper left of the window shows the directory tree. Navigate by clicking on the folder you want. Click on the file you want to select to place it in the "File Name" panel near the bottom of the window. Click "Open" to load it (or double click on the file name). Frequently one wants to step through sequential files. Advancing to the next file can be done by striking the <1> key. Go back one file by striking the <-> key. The same thing happens if you click with the mouse on the "hot spots" at the bottom center of the display labeled "Next File" and "Prev File". To skip forward or back exactly 64 files, use the "Next Tape" and "Prev Tape" hot spots. Note, that hitting "Next File" when on file 64 goes on to file 1 of the next tape. Similarly in reverse. If the selected image is not present on your a disk, an error message box will appear, (You may need to click on OK several times to get this to go away.)

Thumbnail Galleries

Normally we collect data in units of six or 64 images. It is useful to know what is in the remainder of the folder. PCMass27 incorporates the old 64-on-a-page program supplied with PCMass15. When you move the cursor to the right of the centerline and the program is otherwise idle, it scans through the entire current folder (block of 64 files) making thumbnail images and checking the headers for magnification, new specimen number and presence of previous measurements on each file. The thumbnails are displayed on the right panel of the image whenever the mouse pointer is in the right portion of the screen. The header information for the first image of a new specimen is given at the bottom of that panel, separated from the previous specimen data by a horizontal black line. A bar to the left of the file numbers indicates the scan size of the image (magnification increasing to the right). The image being viewed has a black or partially black bar. Images with measurements already in the "MassMeas" folder have purple bars and the remainder are green. Moving the mouse pointer over the thumbnail images or up and down in the lower right panel causes one file to be selected as denoted by a white box around its thumbnail image in the upper right panel. The full header of the selected file is displayed in the bottom center. Clicking the left mouse button causes the highlighted file to be read in. This mode is useful for a quick survey to see similarities and differences among specimens in an experiment.

SA and LA Image Pair

Prior to image read-in the program draws several curves (see below) for data linearization. Then it writes the channel 0 image (usually large angle annular detector, LA) to the left panel and the channel 1 image (usually SA) to the right panel and displays the header under the left image. If the contrast is not suitable, run the background as described below, place the mouse pointer on an object you wish to be white and strike the 'k' key. The background program provides a reliable "black" level and removes the effects of sloping background. This can also be done with Photoshop.

Linearizing Detector Response

Linearization of the detector signals is more important for thicker specimens. The bright field (BF) signal obeys Beer's law falling monotonically to zero exponentially with a characteristic mean free path. The LA and SA signals are more complicated, initially increasing linearly, peaking and then falling back to zero for very thick specimens. This can be described by the equations: IBF=I0*exp(-t/1100) ISA=I0*(1-exp(-t/2500))*exp(-t/2000) ILA=I0*(1-exp(-t/1964))*exp(-t/10000) where I0 is the incident current, the incident voltage is 40keV, IBF, ISA and ILA are the measured currents on the bright field, small angle and large angle detectors, respectively and t is the total specimen thickness in A (assumed to have density = 1) along the direction of the beam. For thin specimens, the exponentials can be approximated, giving a linear relationship between annular detector current and specimen thickness. At any point in the thickness curve the combination of signals giving the best S/N can be calculated from counting statistics. The STEM1 computer system now in use records 2 signals simultaneously. For thin specimens, LA and SA are recorded after normalizing by the sum of LA+SA+BF. For thicker specimens, LA and BF are recorded, either normalized or direct. For thin specimens, LA & SA detector preamps are set to a gain of 10 to make best use of the 256 available recorded gray scales. For thicker specimens, gains of 5, 2 and 1 may be used.

Look-up Tables

PCMass corrects for deviation from a perfect linear relationship by using a look-up table to read in the LA, SA and BF signals. After starting the image read-in procedure, it first plots the above curves appropriate for the detector gains (see below) with LA in purple and SA in yellow. Also plotted is the look-up table to be used with the input data value along the X-axis and the "corrected" value in the Y direction. For a specimen like earthworm hemoglobin, the correction at the thickest portion of the molecule is roughly 5% for the LA detector and 11% for SA.

Changing Detector Gain

If any STEM detector gains or pixel size are not correct, they can be changed by clicking on the appropriate hot-spot at the bottom left of the display. Normally the read-in program picks up the pixel size from the image header (scan size). The detector gains are normally 10 for LA and SA. However for thick specimens lower gains (5,5 or 2,2) are used and these must be entered. The STEM mass calibration is adjusted for the detector gain and pixel size and is normally stable over time (115.0 Dalton/intensity unit). If the preamp gain was not 10 when the image was recorded, this should become obvious as soon as you start measuring TMV M/L. Instead of 13.1 kDa/A, you will get 6.6 (if G = 5) or 2.6 (if G = 2).

Negative Stain Exceptions

In the case of negatively stained specimens, we normally record the BF signal on Ch1, since the SA signal is relatively flat around 1,000 A apparent thickness typical of good stain areas and therefore contains little information. PCMass searches the header for terms such as uranyl or vanadate indicative of negative stain. If it finds one, it assumes that Ch1 contains the BF signal and Ch0 is the LA signal, setting the preamp gains to LA=2, SA=0 and BF=1. The maximum information will then be in the difference BF-LA which it places in the Ch0 display. Such an image is useful for viewing or comparison to models but not for mass measurement, except in special circumstances.

Verify Pixel Size

The size of a pixel is read by the program from the image header and displayed along the bottom left of the image. A 0.512u scan with 512 pixels has a pixel size of 10 A or 1 nm. If this is incorrect for some reason, enter the correct value by clicking the mouse on the appropriate area. Note that the pixel size is an important parameter in mass measurement.

Length Measurements

Simple length measurements can be made on the upper left image as follows. Place the mouse cursor on the starting point and hold down the right button while dragging the pointer to the ending point. This will draw a line on the screen and print the end-to-end distance (temporarily). The X,Y coordinate of the mouse pointer and the values of ch0 and ch1 for that pixel are displayed near the bottom center of the screen. The profile along the line (plus 10% beyond the endpoints) will be displayed below the image. A pair of parallel lines superimposed on the image encloses the pixels contributing to the profile (change width of the band with t/y). A power spectrum of frequencies along the central line defined above and within the indicated region appears below the profile. The most prominent periodicity is identified and printed below the spectrum. Note that the starting and ending points should have approximately the same intensity, otherwise the signal will have a sloping baseline and the longest period component will be dominant. If the endpoints of a periodic object are not at equivalent points in the waveform, the intensity will be split among nearby frequencies. Therefore it is useful to move the endpoint controlled by the mouse and observe the changes in power spectrum to get the highest intensity in a single order.

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