DJC 8/27/2001

 

Scanning EM negatives

Using Polaroid SprintScan 45

 

Preliminaries

 

1.      Turn on SprintScan 45. Power switch is on lower right rear.

 

2.      Log into “Shaft”. Go to “Start”à “Programs” à “Polaroid Scanning” à “PolaColor Insight”.

 

3.      If an error message says that it doesn’t recognize the scanner, even though the scanner is turned on, this probably means that “Shaft” was turned off sometime after the last time the scanner was used. Rebooting the computer should solve the problem.

 

 

Inserting EM negative into Scanner

 

1.      Always handle EM negatives while wearing a glove, to avoid putting fingerprints on them.

 

2.      Lift open the clear plastic door on the front of the scanner, and pull the black mounting tray straight out with both hands. Be careful to pull it out flat, so as not to touch the negative (in case there’s a negative left in the scanner) against the metal protrusion on the top center of the “mouth” of the scanner.

 

3.      The mounting tray is unlocked by turning the thumbscrew, either by hand or (much easier) with a screwdriver. It’s best to use a short or stubby screwdriver, to eliminate the possibility of slipping and damaging the negative.

 

4.      If there is a negative in the scanner, lift the white cardboard, remove the negative with a gloved hand, and place it in its envelope.

 

5.      Take the EM negative you wish to scan, and align it on top of the black plastic so that the entire hole is covered by tissue image. Be consistent in your orientation. For example, always orient slide face up, with label towards front of scanner. Also, take note of how much of the label is sticking out above the black plastic (e.g., 1.5 lines of text), so you can position all future negatives at the same height.

 

6.      Close white cardboard on top of negative, and push both white cardboard and black plastic as far to the left as possible, so they are secure in the same spot with every scan.

 

7.      Close the mounting tray, taking care not to move any of the contents, and turn screw into “lock” position.

 

8.      Insert mounting tray into scanner, being sure to push all the way back, and close front door.

 

 

Initial Preview / Settings

 

1.      In the PolaColor Insight program, under the “Preview” tab, click on the “Preview” button.  If you have a settings file, you can use it by choosing “File” à “Import Settings”, or the first button on the left of the toolbar, after the Preview is complete.

 

2.      The scan settings should meet these parameters:

·        Rotate 90 degrees CW.

·        Input: B&W Negative

·        Display: Monitor, PC

·        Scan area approximately 3500 x 3000 pixels. It should cover as much of the tissue image as possible, leaving some room for error at the borders. The scan area can be adjusted in the “Preview” tab. It can be moved or resized as you would a window.

·        If the analysis is to be done blind, as is usually the case, the scan area should not include the EM label. You should omit a bit more than necessary, to take into account possible shifting between the preview and final scan, as well as imprecise placement of subsequent negatives.

·        The file size should be around 10 MB. If not, go to the “Color” tab, and drag the saturation level all the way to the “minus” end, which is B&W coloring.

 

 

Fine Tuning

 

To get a sense of what the image should look like, it’s best to refer to some other images from previous work.  Basically, you want to use a broad spectrum of grayscale, but without entering pure black or white (except where unavoidable, such as tissue breaks, beam burns, etc.).  All major structures should be sharply visible, unless there is reason to believe the tissue was badly damaged.  Glance at some other negatives to see if this is the case.  If not, use the following procedures to optimize the image:

 

1.      In the “Tone” tab, click the “Auto Exposure” button.

 

2.      If the image still has severe visibility problems, go to “Curves”.  The graph on the far right has a diagonal line by default. Click on the upper right endpoint and drag it to the left, reducing the X value, but keeping Y at a maximum.  Experiment until you find a relatively optimal location.

 

3.      Click on the toolbar’s Histogram button (the one with a blue waveform).  This will show a brightness distribution of the grayscale image.  By adjusting the brightness and contrast (under “Tone”), and the “Curves” parameter, modify the histogram so that it has the following qualities:

·        The waveform looks somewhat Gaussian, with a high peak, and fairly broad range.

·        The waveform is as far to the right as possible, without actually hitting the higher extreme. There may still be spikes at the ends due to some white cardboard, tissue breaks, etc.

·        The waveform is fairly solid, though there may be spikes due to many similar structures having the same luminosity.

 

4.      Increasing the brightness shifts the waveform to the right.  Increasing the contrast broadens the waveform. Ideally, you would like to maximize brightness and minimize contrast without compromising the waveform parameters or the visibility of smaller structures. This is not as difficult as it sounds, and there is much room for error.  It is better to make things a little darker and grayer than optimal, rather than too bright and black & white. This allows later users to adjust the brightness and contrast, rather than risk losing visual information.

 

 

Scanning a Series

 

1.      When a desirable group of settings is achieved, go to “File”à “Export Settings As”, and save your settings as a *.bsf file.  If later in the series you find it necessary to adjust the settings, you might wish to save these as additional files, specifying in the file name which sections used these settings.

 

2.      Go to “Scan”, and scan the image at 1000 dpi, 100% scaling. A scan should take about three minutes, so it’s useful to have something else to do during this time.

 

3.      File names must follow a rigid format. If the series name is ABCD, and the section number is 37, then the file name must be “_ABCD.37”.  The calibration grid should be named as section zero.

 

4.      The program will usually add a .bmp extension to this file name.  You must rename the file to remove this extension. (Make sure you are not “hiding” file extensions. See “View” à “Options” in Windows Explorer.)

 

5.      Run SEM Align from “Programs” Start menu.

 

6.      Go to “Series” à “Open” to open your series.

 

7.      Go to “Section à “Open” to open the section just scanned.

 

8.      Examine the quality of the scan, and the borders of the print area. If anything is significantly wrong with image quality, do a preview and readjust the settings before scanning again. If the negative is shifted, remove the tray from the scanner, and reposition the negative.  Then, depending on your confidence in how well you’ve positioned the negative, do a preview or scan again directly.

 

9.      When the scan is adequate, remove the scanner tray, taking care to keep it flat and not bump the negative against the metal above the “mouth” of the scanner. Unlock the tray and replace the negative deep in its envelope.

 

10.  Repeat this procedure for the rest of the series.  It is not necessary to do a preview each time.  Examining each image in SEM Align lets you know whether your settings need to be readjusted, or the negative needs to be repositioned.

 

11.  If your work is interrupted in mid-series, and you need to log off (always remember to turn off the scanner), you can continue later by proceeding directly to a preview of the next unscanned section using the settings file you saved.