Scanning Transmission
Electron Microscopy Negatives

Scanning Transmission Electron Microscopy Negatives

A Procedure From the Center for Microscopic Imaging
University of Illinois College of Veterinary Medicine

Specimen Requirements:
Size:
__ The negative(s) need to fit onto the transparency tray loading portion of the scanner. This is allows 8.25X10.5 inches as acceptable dimensions for the negative(s).

Cleanliness:
__ The scanner transparency tray itself should be checked for smudges and lint before scanning. Clean off the surface with a damp Kimwipe. Do not use alcohol or window cleaners.

Loading the Scanner :
The negative should be scanned with the scanner's tray all of the way closed. The film is held in the largest AGFA film holder square that the Microscopy Center owns.

The Procedure:
  1. Turn on the Scanner First.
    The scanner is a SCSI device, it is important to turn this on before the computer.

  2. Turn on the computer.

  3. Open the Adobe PhotoShop application.

  4. Load the negatives(s) onto the transparency tray using the largest square film holder.

  5. Under the "File" Header Menu, click and drag to select Import ---> FotoLook.

  6. The window that pops up will be like:

    Change any necessary settings so that the pop up window matches the settings on the image shown above.

    • Resolution can be increased by increasing the ppi value in settings.
      Note: This will increase your file size and scan time.

    • Resolution can also be increased by telling the scanner to scale to 200% or more and later decreasing the size, but increasing the pixels per inch using "image size.
      Note: This will increase your file size and scan time.

    • Sharpness can also be adjusted. What is required varies image to image.

    • For Final images larger than actual size, the *Scale To* option can be
      changed to 200%,300% etc.

  7. Select and click on the "Preview" button. Wait for the process to finish.

  8. Next, select the area to scan.

    This is done by carefully placing the curser over one of the lines in the white outlined box to be found in the right side screen where the image appears. The mouse is used to click and drag each of the 4 lines of the square to the desired location. The line is very thin, so this takes careful click & dragging. The curser icon should turn into a double headed arrow when the curser is in the proper position to click and drag.

  9. Once the area is selected, press the SCAN button. Wait for the process to finish. This may take 1-6 minutes depending upon your settings.

  10. Change the size of the image.

    ** It may be tempting to change the looks of the image right away.
    But, if you do, every adjustment you make will take a long time, so change
    the image size and pixel value 1st before doing anything else.

    What to change the size to???

    For Printing:

    For printing on a Dye-Sub or Epson Jet Ink printer, a very good quality is desired.

    1. Open the image size window by selecting Image-->Image Size.

    2. Set the image size to the paper limitations ( or smaller) on the Dye-Sub printer.
      8.5 X 12 paper: 7.7 X 9.8 inches
      9 X 14 paper: 8.4 X 11.9 inches

    3. Set the pixel value per inch to 600 for Dye-Sub printer or up to 1420dpi for the Epson printer

    4. Make sure that the final file size is less than 48 MB, which is the RAM Memory Contained in the Printer.

    For Web use, or screen viewing only:

    Less Resolution is needed in this case.
    The actual size can be much smaller than what appears on the screen.

    1. Open the image size window by selecting Image-->Image Size.

    2. For example, a height of 0.08 and a pixel resolution of 9999 can give a good resolution image that takes up 2/3 of the screen height

    3. A good starting point is to set the height to 0.08 with a Pixel Resolution of 9999. If this does not look good, select Edit-->Undo and resize the image.

    4. Width will automatically follow if the bottom box on the image size window for restrain proportions is checked.

    5. Try setting the pixel dpi to 9999, if the file is still within a reasonable size. Otherwise, cut down the Pixel Resolution.

    6. A reasonable file size is 700-900 KB for viewing, 100-200 KB for Web pages.

  11. Change the Gamma:

    ** It is very important that most of the image's adjustment be made with the GAMMA control, not the
    BRIGHTNESS / CONTRAST controls.

    The GAMMA allows you to take pixels that are not assigned and use them in the image. Changing the Gamma will not change the number of pixels used in the image, it will change what each pixel reads. BRIGHTNESS / CONTRAST does cause a loss of data. This is why much of the image adjustment should first be made with the GAMMA control.

    Examples of Images before the Gamma is Changed:

    1. Select Image--> Adjust--> Levels. A window to adjust the GAMMA histiogram should pop up.

    2. Move the LEFT most of the 3 arrows under the diagram so that it just hits the beginning of the curve that starts the graphed image from the left side. This will use up pixals not being used.

    3. Move the RIGHT most of the 3 arrows under the diagram so that it just hits the beginning of the curve that starts the graphed image from the right side. This will use up pixals not being used.

    4. Viewing the image at the same time as the GAMMA histiogram, adjust the CENTER arrow, this can move the image from extreme paleness to almost black.

      Select a setting that gives the best intensity, probably just a little darker than the desired brightness of the final image.

    5. Click OK to return to the adjusted image.

  12. Change the Brightness / Contrast:

    Examples of Images after Gamma & Before Brightness / Contrast:

    1. Select Image--> Adjust--> Brightness / Contrast.

    2. A window to adjust the BRIGHTNESS / CONTRAST histiogram should pop up. It will have 2 "numberline" like objects on it. The values of BRIGHTNESS or CONTRAST can be changed by moving the arrow on the line or by entering a number in the associated box.

      Changes can be seen in the actual image as the controls are moved.

    3. Change the values to fit personal preferences.

      ***If you are going to print on the Dye-Sub printer, the darkness needed depends upon how your monitor appears in relationship to how the Dye-Sub prints.

      **Ask a CMI staff member for test files and prints to aid you with this adjustment.

    4. Click OK when adjusted.

    5. If this does not look good, select Edit-->Undo. Readjust the BRIGHTNESS / Contrast again if needed.

  13. Sharpen the Image if necessary:

    • Look at the image closely, it may be a little soft. As a final touch, it is sometimes helpful to sharpen the image.

    • Select Filter--> Sharpen--> Sharpen. ( There are several flavors of sharpen, use the plain Sharpen.)
      The image should sharpen after the mouse button is released.

    • If this does not look good, select Edit-->Undo. Readjust the Sharpness again if needed.

    Examples of Final Images:

  14. Save the Image:

    1. Select File--> Save. A Save Window should pop up.

    2. Use the Box at the top of the window with the wedge arrow in it to select a path to the directory you wish to save your file in.

    3. At the Bottom of the window is box with a wedge arrow that allows you to click and drag to a file type you wish to save to .

      Almost always save the first and main copy of the image as a TIFF file. On the Macintosh, you are given the option of saving as a Mac or PC TIFF.

      TIF files convert well, and cross platforms better than other forms. Also, it allows the user to go back and make changes, a GIFF file will not always allow the user to make changes once saved.

    4. Then, do a "SAVE AS", similar to the save, and save Black and White images as Compuserve GIF files. Color pictures will need to be converted to GIF files with a shareware converter program. We use a Mac shareware program called "GIF CONVERTER".

      How To:

      • Open the GIF CONVERTER application.

      • Select File--> Open. An Open Window should pop up.

      • Use the Box at the top with the wedge arrow to locate the file.

      • When Found, Click OK.

    5. Naming the file ____ Type a File Name in the box labeled for this:

      The way a file is named will depend upon the format of the computer the file will be stored on. Mac's have much more freedom over the length of the file. For UNIX, DOS and even Windows 95, it is best to restrict the name to 8 characters.

  15. Print the File:

    • Printing on the Kodak Dye-Sub Printer:

      1. You must have the Kodak Driver and 2 ColorSync preference files to print from your terminal.

        ____ See the CMI staff for assistance.

      2. Select Chooser from the Apple Menu and select the Kodak PS8650 icon. Close the Chooser.

      3. Select File--> Page Setup. A Window should pop up. Choose the page format, and the paper size____ Click OK.

      4. Select File--> Print. A print Window should pop up.

        • Choose Black.

        • Choose Paper type, usualy PHOTOGRAPHIC paper.

        • Uncheck the anti-aliasing box at the lower left.

        • Check the number of Copies to be printed, and from what page to what page if needed.

    • Printing on the Epson Style 800 Jet Ink Printer:

      1. You must be working at the CMI Macintosh Work Station to use this printer.

        ____ See the CMI staff for assistance.

      2. Select Chooser from the Apple Menu and select the Epson Style 800 icon. Close the Chooser.

      3. Select File--> Page Setup. A Window should pop up. Choose the page format, and the paper size____ Click OK.

      4. Select File--> Print. A print Window should pop up.

        • Choose Black & White.

        • Choose Paper type, usualy PHOTOGRAPHIC paper (Jet Ink, not Dye-Sub).

        • Check the number of Copies to be printed, and from what page to what page if needed.

[ Return to Main Scanning Page ]

May 29th, 1997
email to: lamiller@illinois.edu