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Zeiss Scanner

dgmorgan 28-may-2004 Brandeis University

Zeiss Scanner


Using the Zeiss Scanner

Here is a step-by-step description of how to use the Zeiss scanner. This
document is intended to be a quick reference guide for using the Zeiss, and
should not replace learning to scan from a qualified person in the lab.

NOTE: It is generally not a good idea to use the scanner's
computer to do anything else while actively scanning. This action can lead to
errors when the scanner and the computer communicate, and can potentially cause
enough trouble that the system will need to be rebooted.

Step 1

Power on the Zeiss (switch at back left) and allow it to complete
the self-test. While the scanner warms up, start an entry in the
log book.

Step 2
Start the PhotoScan program by double clicking the
icon
on your desktop.
For future reference, once the program is running, the menu
bar across the top of the PhotoScan window looks like:
Step 3

Clean the three accessible surfaces of the plattens which hold
the negatives. Use a lint free cloth and the cleaning solution
near the scanner. The camel hair brush can be used to remove
dust from negatives and anything that falls onto the plattens
after this initial cleaning.

Step 4
Place negatives on glass platten emulsion side down. The
emulsion side of a negative will be duller than the side with
the plastic backing (ask for help if this description
is not clear). There
is at least one scratch on the lower glass platten (upper left
as one looks at the scanner), and this area should be avoided
for the best results. If additional scratches are observed,
please let the rest of us know about them! Lower the upper
platten slowly so as not to disturb the negatives.

Step 5
Collect an overview of everything to be scanned. This is
performed by clicking on "File" (in the
menu bar across the top of the
PhotoScan Window) and then "New scan." This starts a tabbed
window:

A)

The first tab (Scan Setup) looks like:

This page is used to set the area to scan (yellow area in the
figure above), the pixel
size (56 microns in the "Resolution" area of the figure), the mode
(color vs gray, set in the example to "Gray Scale"), where to save
the image collected during this scan (E:\temp\prescan.tif in this
example). etc. The scan area can be altered by using the
mouse (left button) to grab a black square in the corners or along
the sides of the yellow area and dragging it appropriately.

Note that the "co-ordinate system" in this image (i.e.,
the relationship of the scanned area to the platten area) is
"left/right reversed." In other words, the upper left corner of
any displayed scan is the upper right (relative to the rest of the
scanned area) of the real
platten as one looks at the scanner. This seems like a silly thing
for PhotoScan to do, and one way to make it seem a bit less silly
is to think of the images on the computer monitor as what one
would see looking from below and through the glass plattens.

Always save images as "TIFF Uncompressed." For this
overview scan, 56 micron pixels are fine. Any scans intended for
data processing should be done at the highest possible resolution
(7 microns) and binned appropriately at some later time. If one
choses not to scan using 7 micron pixels, note that
all available pixels sizes are multiples of 7 microns.
There should be preset parameter files (accessible through
the "Scan Setup" area and called
"dgmprescan.par" in this example) which will automatically set
these prescan parameters properly.

B)
The second tab (Film/Output) looks like:

This page is used to set the film and output polarity (positive
to negative in this example), the output data mode (set to density
here) and the upper/lower limits for scanning (set here using Film
Transmissivity from 0.01 to 1 or Film Density from 2 to 0). Film
Transmissivity and Film Density are related by the
following simple equation:
density = -log10( transmissivity )
and are coupled in the PhotoScan program (i.e., changing
the density changes the transmissivity and vice versa).
The PhotoScan program will not allow you to set improper limits
(e.g., upper limits below lower limits).

The scanner will

allow you to set a large Film Density range (e.g., 3 to 0),
but the Zeiss only behaves well over approximately 1 OD unit
(e.g., 1 to 0, 1.5 to 0.5, 2.0 to 1.0, etc.).
Follow this link to see
some data addressing this observation.

The "Film/Output Setup" area accesses stored parameter files
(here that file is called "dgmout.par").

We have recently begun to scan using density mode and
because we are using this mode, we also use either
positive to negative or negative to positive polarity settings.
The upper and lower limits will be reset for individual images
(see the section below which deals with
setting the histogram),
but the range used in this example is a good starting point.

Step 6
Start the overview scan by clicking the

button in the menu
across the top of the PhotoScan window.

NOTE: The scanner has recently begun to hang at this point,
and may require a reboot. If that
happens, turn the scanner off and back on, and allow the self-test
to complete. Reboot the scanner's computer only after this has
finished: the computer can be rebooted under conditions where it
doesn't seem to know about the scanner, and if that happens, the
machine must be rebooted again. Waiting until the self-test is
complete guarantees that this will not happen.

When a scan is done, a new window containing the scan appears.
The initial view of scanned windows is "1:1 image pixel:monitor
pixel" which means that one usually sees only a very small portion
of the scanned image. The image can automatically be fit to the
display region by first clicking on
the button in the
menu bar across the top of the PhotoScan window
and then clicking anywhere in the scan window. A typical overview
of a single scanned micrograph looks like:

Note that in this display, the clear area of the micrograph
which contains the date, magnification, etc. shows white
text on a dark background. This is a consequence of using the
postive to negative polarity settings mentioned earlier. This is
the proper contrast for image processing here, and no operations
that reverse this contrast are necessary (n.b., there may be
circumstances where reversing this contrast is warranted, but those
circumstances should be rare).

Step 7
Open the tabbed windows first seen in step 5
by clicking "File" then "Scan properties..." in the
menu bar across the top of the PhotoScan
window. There is now a fourth tab (Histogram) which shows a
histogram of the current scan:

Switching from "Count" to "Log(Count)" in the "Histogram
Type" box allows one to see the occupancy of infrequently used
gray levels. For most practical purposes, the "Count" display is
most informative. In the interests of truth in advertizing, I
must confess that I have no idea what the "Histogram Collection"
box means or does. The "Histogram Clip" area allows the user to
manipulate certain aspects of the histogram, but I do not know
what such manipulations do nor whether they are useful.

Since this is the histogram of an overview scan, there is
little to be gained by manipulating the scanner to change this:
as long as the user can clearly see what should be scanned at
higher resolution, the current scanner settings are fine.

Step 8
Use the stretchy box to select an area for a higher resolution
scan. This can be accomplished either by manipulating the yellow
scan area seen in step 5A above, or by
clicking the button
in the menu across the top of the PhotoScan
window and then drawing the stretchy box in the scan window
directly:

Also set the appropriate pixel size and both the file name and the
location to save the high resolution scan (options in the Scan
Setup tab). Save this information to a new parameter file ("Scan
Setup" area in step 5A above).

Step 9
At this point, it becomes important to set the upper and lower
limits of the scanner appropriately. In the example above, the
histogram of the overview indicates that
most of the image data is compressed into a very narrow range of
gray levels. A better image can be obtained by broadening the
range of gray levels which contain most of the image data.

Since there is generally no need to scan the text window of a
micrograph, it is possible to ignore the small peak at the left
of the histogram (those few gray levels that describe the white
text on a dark background) and to concentrate on spreading the more
centrally located peak across all available gray levels. This is
done by making adjustments to the "Film Transmissivity/Density"
area of the "Film/Output" window. In a case
like that illustrated above, it is necessary to bring the upper and
lower limts much closer together.
This is the high resolution scan after performing this
adjustment:

Here is the accompanying histogram, which is fully spread
across all 256 gray levels:

In order to spread the histogram, the "Film Transmissivity"
limits were changed from the initial 0.01 to 1.0 interval
to the values shown here:

The process of adjusting the limits can be time consuming and
will require practice to do well. The good news is that for a set
of similar micrographs, the limits are likely to be similar if not
identical.

Step 10
Changing the area to scan, the saved file's name and any
adjustments to the scan limits is iterated until all the
micrographs and areas have been scanned. At this point, power
off the
scanner and finish the entry in the log book. Remove negatives
from the scanner and leave the scanner with a lint free cloth
sandwiched between the glass plattens.
NOTE: The scanner occassionally has problems when the scan
spped is set too high. Control over scan speed is accessed by
clicking on "Tools" and then "Scan speed" in the
menu bar across the top of the PhotoScan
Window. This opens this dialog window:

The speed setting here (25) is lower than most people will use,
but is a good value if one is patient. Speeds as high as 70 can
be used, but will occassionally foul up the scanner (requiring at
least a scanner restart if not a system reboot).

Step 11
The remaining step is to convert the scanned TIF files into MRC
images. Start the conversion program by double clicking
the icon on your
desktop. This starts an easy to use program that looks like:

Set the amount of averaging/binning in the "Pixel Averaging"
area then drag all the images to be converted into the white panel
below the "Apply Averaging" button. Click on the "All" button.
This will start the conversion process, adding an "mrc" extension
to the scanner file names (replacing the "tif" extension if it is
used, or simply adding "mrc" to the file names if no extension was
specified).

During the conversion, the user will see the program toggle
between the input image and the (converted) output image. This
comparison is shown in the following image pair:

Note that there is no longer a contrast reversal between the
input and output images. This is correct: the images were scanned
in density mode and simply converted to MRC format (whereas
previously the images were scanned in transmittance mode and
transformed into density during the conversion to MRC fomrat).

That's the short course in using the Zeiss scanner here at Brandeis.

If this document needs clarification or some sort of update, please contact
the webmaster.
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Comments

DGM #1

From: David G Morgan
To: emcomp
Subject: scanner stuff --- read at your own risk :-)
Date: Wed, 7 Apr 2004 19:39:13 -0400 (EDT)

Hi,

Since I have talked to several people over the past few days about
some of the things I've learned concerning our Zeiss scanner, I thought I
would send a general note out (and invite people to talk to me directly if
things are not clear). I will also try to put some of the data I have
collected on the web site so anyone can take a look at it. This note will
be too wordy (one of my many failings) but complete enough to answer a
number of questions without (perhaps) raising too many more.

There are a couple of "bottom lines" in what I have learned. The
first is that the Zeiss is NOT capable of scanning a dynamic range greater
than about 1.2 OD. This is in agreement with my tests of the scanner in
Tom Walz's lab several years ago. Since our scanner is the "Windows 2000
version," I had been hoping Zeiss might have addressed this issue. The
good only news is that the 1.2 OD range the Zeiss can scan is "mobile."
By that, I mean that the user can set the scanner to scan from 0 to 1.2,
from 0.5 to 1.7, from 1.0 to 2+, etc. The major downside of this is that
it takes time and effort on the part of the person scanning the
micrographs to find the "best" range.

This isn't as good as it could be, and maybe not even as good as
it should be (people argue about what a scanner needs to do). However,
numerous people (including Matthias) have done relatively high resolution
structures using data obtained from a Zeiss, so this limitation isn't a
huge roadblock. On the other hand, it might prevent one from getting the
maximum possible information from a set of negatives (always a worry!).

If one's data happen to span a larger dynamic range (some
micrographs of negatively stained stuff have a larger range), too bad!
Most micrographs of ice embedded material have a much smaller dynamic
range, at least on the scale of what one sees. However, on the scale of
the film's grainularity, the dynamic range of even these micrographs is
probably on the order of 2 OD or more...

It is also possible that we can set our Zeiss to collect more grey
levels. We currently collect "8 bit data," but we think this particular
scanner can be set to collect more bits, and that _might_ increase the
dynamic range that can be scanned. If this is possible, it is probably
worth any additional effort it would require.

The second bottom line concerns what is likely to be the best way
to scan negatives. Here are the three most critical steps in the scanning
procedure:

1) place negatives on the scanner emulsion side UP.

This is different from the other Zeiss scanners I have used, but
it seems clear that the focal plane of the scanner's optics is set at a
position that is much closer to the upper surface of a negative than it is
to the bottom surface. If one happens to scan something that is thicker
or thinner than an EM negative, this will need to be investigated and the
"best" position determined. The focal plane may even change (i.e.,
someone could change it either by accident or on purpose), so it should
probably be checked every so often.

Aside: if the focal plane is set at the "lower surface" of the
negative, there is no influence from "negative thickness." This is why
all the other Zeiss scanners I have used have been set up to scan emulsion
side down...

2) scan using the "density" setting on the "Film/Output" page of
the "Scan Properties" setup stuff.

It does not seem to matter whether one uses the "positive ->
positive" or "negative -> negative" setting (i.e., the results appear to
be identical). This also seems true for the use of the "positive ->
negative" or "negative -> positive" settings. There _is_ a difference
between "positive -> positive" and "positive -> negative" in the sense
that the outputs will have reversed contrast: micrograph labels will
appear as black writing on a white background (pos->pos or neg->neg) vs
white writing on a black background (pos->neg or neg->pos). It is NOT
true that pos->pos is the same as neg->neg when using "transmittance"
mode.

NOTE: The contrast one sees in the scans is the same whether one
uses the "transmittance" or the "density" setting on the Zeiss. If one
scans pos->pos (or neg->neg), the micrograph label will be black writing
on a white background. If one scans pos->neg or neg->pos, the label will
be white writing on a black background.

If I knew what using the "density" option did, I would explain it
to anyone who asked. However, I don't really have a clue. What I do know
is that the "density" data appear better than the "transmittance" data,
especially after converting the transmittance data to density (which is
what one technically should use for EM reconstructions). I have several
reasons for saying this, and will try put those data on the web site so
people can decide.

3) convert the TIFF's the scanner generates into MRC files using
the program called "Noreens MRC program.exe" and do NOT use the "convert
to OD" option on the settings page.

The reason not to convert to OD is that the Zeiss has already
automagically done something similar to that OD conversion, and it would
be a bad thing to convert something that already resembles OD into OD.

This conversion to MRC format will NOT reverse the contrast of the
image, which likely means that the contrast of a micrograph scanned this
way will differ from that of prior scans. If this is the case, the best
way to keep the contrast consistent is to chaange the "pos->pos" setting
appropriately. There are also ways to use SPIDER, IMAGIC and label.exe to
reverse the contrast of existing files, but it's easier to do this at the
scanning stage.

One bit of bad news is that the only way to know for certain how a
negative was scanned is to examine the TIFF itself: the original scanner
files can be re-opened using the scanner's program, and one can see all
the gory details of what was done during the scan. However, since it's
possible to reverse the contrast of a micrograph at multiple places, it is
NOT possible to examine an image on the other computers and know for
certain what was done.

I apologize for the length of this note and also hope that both
reading and writing it have been useful. Please let me know if there are
things I can clarify, and I'll let people know when things get to the web
site.

--
David Gene Morgan
Brandeis University
Rosensteil Basic Medical Sciences
Research Center
781 736 2411 (office)
dgmorgan@brandeis.edu

DGM #2

From: David G Morgan
To: emcomp
Subject: yet another &@!*$?# scanner update
Date: Fri, 16 Apr 2004 13:07:35 -0400 (EDT)

Hi,

One more update to scanning issues, which I hope will be the last.
If there are any questions about this, please check with me.

After staring at various scans, looking at some CTF determinations
and general mayhem, the royal WE have decided that scans should be done
with the emulsion DOWN (which is what we thought in the first place, and
is in contrast to what I said in my last note). This has some rather bad
consequences for the scanner's ability to distinguish grey levels, but
that's probably OK (especially since all data from the Zeiss should be
binned by at least a factor of 2).

Since it does appear that scanning in density mode is the better
way to use the Zeiss, the program to comvert to MRC format has been
simplified to the point that the only option available is the amount of
binning. This means that there is no longer a "Settings" tab in the GUI
(i.e., the user does not have the option of converting to OD, binning at
the level of the the scan or of the output density, flipping left/right,
flipping up/down, etc.). In a further attempt to simplify life, there
should be a single "convert to MRC" icon/shortcut on the desktop: use it
and the conversion should be fine.

Another consequence of using the density mode to scan concerns
contrast issues. The original negatives all have black text in clear
regions, while "standard" scans after conversion to MRC format have had
white text on a black background. The way to keep new scans consistent
with such older ones is to use the "positive -> negative" setting on the
scanner. This will reverse the contrast of the scanner output (relative
to what has been seen in the past), but since the conversion to MRC will
not cause a contrast reversal (a consequence of the conversion to OD
option), this is correct. After converting to MRC format, the contrast
should be the SAME as the scanner output (and should be white text on a
black background).

As always, if there are questions, find me and I'll try to answer
them.

--
David Gene Morgan
Brandeis University
Rosensteil Basic Medical Sciences
Research Center
781 736 2411 (office)
dgmorgan@brandeis.edu