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to expand/contract the menu item or on the link to go directly to the page.
Scanning Electron Micrographs with the
Joshua Kaufman, December 2005
Procedures (Click on each thumbnail for a larger image. Click on the large image to close.):
- Power on and log into the PC. The username and password
are both "nikon". Passwords in Windows are not case sensitive.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- Click on the purple "Tools" button and select "Tool Palette 1".
- 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).
- Scan the image by clicking the "Scan" button. Do
not bump or lean on the bench top during the scanning process.
- 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.
- 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.
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:
How do I find out the pixel size of my images?
- Type the command "uniconv". A main menu will
- You will see the prompt "Enter the letter of
the type of conversion you would like:" Select choice: "G)
- "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."
- "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
- "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.
Fig. 1. Front panel of the Nikon scanner.
Fig. 2. Modified film holder with anti-Newton
Fig. 3. Cleaning the micrograph
Fig. 4. Identifying the emulsion side of a micrograph
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.
Fig. 6. The Progress Window during a preview
Fig. 7. Stretching the histogram contrast.