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Scanning Electron Micrographs with the
Nikon Super Coolscan 8000ED PDF

Joshua Kaufman, December 2005

Procedures (Click on each thumbnail for a larger image. Click on the large image to close.):
  1. Power on and log into the PC. The username and password are both "nikon". Passwords in Windows are not case sensitive.
  2. Power on the Nikon scanner (Fig. 1). The power button is on the top right corner on the front of the scanner. A green light, along the left edge, will flash for 2-3 minutes. Wait for the light to stop flashing prior to starting the Nikon Scan application.
  3. Launch the "Nikon Scan" program. This can be accomplished either by clicking on the shortcut on the PC desktop or by finding the executable file nested away on the C drive. The path for the program is C:\Program Files\Nikon\NkScan4\Nikon Scan.exe.
  4. Remove the micrograph holder from its plastic case and clean the glass surfaces. The holder contains two plates of anti-Newton glass that are used to hold the micrographs flat (Fig. 2). Handle the glass carefully and only at the edges. Do not touch the glass with bare fingers. Use canned air and the anti-static brush to remove dust particles. When using canned air be sure to hold the can upright since tipping it will cause propellant to be blown onto the item you are trying to clean. This could ruin a micrograph. Use lens paper to carefully remove any remaining dust particles from the glass surfaces. Do not use force on the glass or you could crack it.
  5. Clean the micrograph carefully. Use cotton gloves to handle it since fingerprints can be nearly permanent. Handle it by the very edge or by the info box that has the micrograph number. Use the anti-static brush and canned air to clean the micrograph (Fig. 3). Any dust can affect the data and the density histogram.
  6. Insert the micrograph between the glass plates of the holder, emulsion side down. The emulsion side is the duller side of the micrograph and it is also the side facing you when the micrograph is held like that shown in Figure 4. It is generally best to avoid scanning any of the text info area of the negative since the density values in that area will affect the histogram. Always align the film against frame of the holder with the info area furthest away from the hinge of the glass plates. This will minimize the unscanned area of the film. Remember that the scanner can not scan the full area. Our films are 3.25x4 inches (~8x10 cm) but the opening in the holder is only ~6x9 cm. You may wish to tweak the positioning to maximize scanning the desired area of the micrograph.
  7. Feed the micrograph carriage into the scanner bay. Make sure that the sliding cover in the scanner is fully open. Look for the arrow printed at the end of the holder opposite the film end. It goes in the scanner first. Do not force it. Load it slowly and gently until it stops. If it makes a clicking noise, gently nudge the holder forward until the scanner grabs it. The holder will shuttle back and forth a few times until the holder type is sensed. The holder will not go fully into the scanner at this point.
  8. Tell the scanner which opening in the film holder to use by clicking the thumbnail drawer that is just to the right of the Settings menu (Fig. 5). This will open a window containing three frames. Click on the frame labeled "2." The holder will now be fully drawn into the scanner to the proper position.
  9. Do a preview scan by clicking on the blue "Preview" button in the lower left corner of the window. Wait for the progress meter to show that the preview has been acquired (Fig. 6). This should take about a minute.
  10. Click on the purple "Tools" button and select "Tool Palette 1".
  11. Adjust the histogram. It can be found under the "Curves" section of the Tool Palette. Adjust the black-and-white, point, slider arrows to stretch the contrast. The sliders should be set just to the edges of the histogram. Moving the arrows any closer together could result in changing the density values of important data (Fig. 7).
  12. Scan the image by clicking the "Scan" button. Do not bump or lean on the bench top during the scanning process.
  13. Create a directory for yourself on Drive "D" and save your files there. This should not be considered a permanent storage area and the files will be deleted if the drive becomes full.
  14. Exit the program when you're finished scanning. Eject the holder from the scanner, remove the film, and power down the scanner. Put the film holder back into the protective case in the drawer. After each session transfer your data to your home computer and clean up the disk space for the next user. Micrographs recorded for the Baker group are stored together in the micrograph storage cabinets. Please return the micrographs there.
File Conversion:

The Nikon software outputs the scans as TIFF files. In the Baker lab our reconstruction programs use files in the PIF format. To convert these files so that particle images can be boxed with ROBEM, a program called UNICONVERT is used. These two programs are installed on the lab's Linux boxes. Follow these procedures to convert your images. Remember that what you type is case sensitive:
  1. Type the command "uniconv". A main menu will appear.
  2. You will see the prompt "Enter the letter of the type of conversion you would like:" Select choice: "G) TIFF(Nikon)->PIF".
  3. "Enter Tiff filename ?". Enter the filename. Make sure that you enter the ".tif" extension. Wait until the next prompt. The program will say "Reading tiff data."
  4. "Bin pixels by integer factor of 1,2,3,...>". Choose a bin factor. This will depend on where you are in the reconstruction process. Projects in the early stages usually have images binned with a factor of "2" to increase processing speed. Otherwise images are usually not binned (factor of 1). This will take awhile and then you will see "Writing PIF".
  5. "Do you want to invert contrast? (y/N) -". Type "y". It will calculate for a few seconds and output the converted .PIF file into the current directory. You can then exit the program or convert another image.
How do I find out the pixel size of my images?

Nikon scanner
Fig. 1. Front panel of the Nikon scanner.

Film holder
Fig. 2. Modified film holder with anti-Newton glass.

Cleaning the negative
Fig. 3. Cleaning the micrograph

Emulsion
Fig. 4. Identifying the emulsion side of a micrograph

Nikonscreen1
Fig. 5. Loading the settings file. The film holder selection drawer is in the fully open position. The inset shows the drawer in the partially open position.

Nikonscreen2
Fig. 6. The Progress Window during a preview scan.

Nikonscreen3
Fig. 7. Stretching the histogram contrast.

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