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Microscopy
http://www.nuance.northwestern.edu/
2015 Edition
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Basic Photoshop for Electron Microscopy
By Eric Jay Miller
Introduction
This paper will discuss several simple procedures that can help to punch your
micrographs up a notch. These instructions are written in such a way that just about
anybody should be able to pick them and start using them without any previous
experience with Photoshop (although that certainly wont hurt either).
This is not intended to be (nor does the author claim it to be) the end-all and be-
all of Photoshop instructions. There are often multiple ways of doing the same operation
in Photoshop. These are just a few that work pretty well for this particular application of
electron micrographs. There might be other procedures that work better for specific
applications (or work just plain better). The reason for the omission of any of those
techniques is due to the author not having been smart enough to have thought of them
prior to writing this.
The program used for this paper was Photoshop CS4 (aka the full version),
though most older versions of Photoshop will work perfectly fine with most of these
techniques. Smaller versions of Photoshop, like Photoshop Elements, will be very
limited when it comes to performing most of these procedures.
In electron microscopy there are often concerns about the authenticity of an
image once it has been processed through Photoshop. These can be valid concerns as
there are a lot of really crazy things one can do with this high-powered program. The
main purpose with the techniques discussed here is to either a) make the information in
the image easier to see, thus yielding more usable information, or b) to make the image
more aesthetically pleasing. These instructions are geared towards image processing
and not image analysis.
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Basic Photoshop for Electron Microscopy Section I Levels
By Eric Jay Miller
I. Levels
The most basic thing one can do to improve the overall look of a micrograph is to adjust
the Brightness and Contrast levels. With Adobe Photoshop, this is a simple, quick and
very accurate procedure.
or Ctrl + O
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Section I Levels Basic Photoshop for Electron Microscopy
By Eric Jay Miller
or Ctrl + L
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Basic Photoshop for Electron Microscopy Section I Levels
By Eric Jay Miller
NOTE: By saving your image, youll make these changes we made PERMANENT! You
might not want to do that. A good idea is to always keep your original, unprocessed
image, in case you want to do something different to it later on. So once we have
processed an image, perform Save As to save your file as a new file.
Or Shift + Ctrl + S
Then when you close your original file, PS will ask you if you want to save it, SAY NO!
You should end up with 2 files, one is the original, the second is the one you did stuff to.
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Section I Levels Basic Photoshop for Electron Microscopy
By Eric Jay Miller
Here is the image before processing. It is not horrible, but it is a little dull.
This is the image after the simple Levels procedure. It now has much more definition
and is much snappier and appealing to the eye.
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Basic Photoshop for Electron Microscopy Section II Resizing
By Eric Jay Miller
Rule 1: When we resize images, we want to do it evenly if we can. Ex. Take an image
that is 1000x1000 pixels and enlarge it to 2000x2000 or reduce it to 500x500. Its best if
you can cut things evenly in halves (or quarters, eighths, etc.) or double them (or
quadruple, etc.).
However, the final image size we need is not always going to correspond to such sizes.
At some point well have to enlarge or reduce an image to some weird size, like 81% of
its original size, or enlarge a 1000x1000 pixel image to 2365x2365. So if we cannot
abide by Rule 1, we move to:
Rule 2: Dont ever resize an image twice. Do it once and youre done.
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Section II Resizing Basic Photoshop for Electron Microscopy
By Eric Jay Miller
or Alt + Ctrl + I
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Basic Photoshop for Electron Microscopy Section II Resizing
By Eric Jay Miller
5. Depending on your image, different resampling methods might make your image look
blurry, while reducing the noise in the image, while others might make your image look
sharper, while increasing the noise in the image. Some methods will help, while others
will hurt. It depends on your image, how much you are enlarging or reducing and how
you want your image to look when its done.
The differences may appear subtle on the screen, but if you plan to blow up an image to
print on a huge poster, small differences become very noticeable.
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Section III Simple Color Table Basic Photoshop for Electron Microscopy
By Eric Jay Miller
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Basic Photoshop for Electron Microscopy Section III Simple Color Table
By Eric Jay Miller
NOTE: If you picked the light color first when you should have picked the dark color first
[or vice versa] it will be obvious pretty quickly as your image will look like a negative
(with areas that should be dark being light, etc.). If this happens, just hit cancel on the
Color Table and start over from Step 4.
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Section III Simple Color Table Basic Photoshop for Electron Microscopy
By Eric Jay Miller
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Basic Photoshop for Electron Microscopy Section III Simple Color Table
By Eric Jay Miller
Here is the image once the pseudo color has been applied. There are many different
ways to use the Color Table, but this is the simplest and quickest.
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Section IV Simple Gradient Map Basic Photoshop for Electron Microscopy
By Eric Jay Miller
or Ctrl + L
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Basic Photoshop for Electron Microscopy Section IV Simple Gradient Map
By Eric Jay Miller
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Section IV Simple Gradient Map Basic Photoshop for Electron Microscopy
By Eric Jay Miller
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Basic Photoshop for Electron Microscopy Section IV Simple Gradient Map
By Eric Jay Miller
NOTE: There are a few other ways we can play with our image. One of them is by
selecting one of the more complex presets.
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Section IV Simple Gradient Map Basic Photoshop for Electron Microscopy
By Eric Jay Miller
Or Ctrl + S
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Basic Photoshop for Electron Microscopy Section IV Simple Gradient Map
By Eric Jay Miller
What we got in the end is a reasonable 2 color image. The important part is that it was
FAST!
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Section V Complex Color Table Basic Photoshop for Electron Microscopy
By Eric Jay Miller
or Ctrl + L
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Basic Photoshop for Electron Microscopy Section V Complex Color Table
By Eric Jay Miller
NOTE: If you wanted to get REALLY fancy, you could use the Gradient Map on this
step instead. (See Section III.)
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Section V Complex Color Table Basic Photoshop for Electron Microscopy
By Eric Jay Miller
Now we need to decide which areas of which images we need to cut out.
The sky in the blue image seems to have the largest surface area, so Im going to
designate it as the background image. So all Ill need to do is cut the sun and the rays
out of the yellow image and the ground out of the green image and then paste them on
top of the blue image.
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Basic Photoshop for Electron Microscopy Section V Complex Color Table
By Eric Jay Miller
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Section V Complex Color Table Basic Photoshop for Electron Microscopy
By Eric Jay Miller
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Basic Photoshop for Electron Microscopy Section V Complex Color Table
By Eric Jay Miller
or Shift + Ctrl + I
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Section V Complex Color Table Basic Photoshop for Electron Microscopy
By Eric Jay Miller
or Shift + Ctrl + I
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Basic Photoshop for Electron Microscopy Section V Complex Color Table
By Eric Jay Miller
or Ctrl + V.
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Section V Complex Color Table Basic Photoshop for Electron Microscopy
By Eric Jay Miller
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Basic Photoshop for Electron Microscopy Section V Complex Color Table
By Eric Jay Miller
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Section V Complex Color Table Basic Photoshop for Electron Microscopy
By Eric Jay Miller
26. The image on the left has the yellow layer on top with the green layer behind. The
image on the right has the green layer on top with the yellow layer behind.
The right image looks the smoothest, so Ill stick with that arrangement.
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Basic Photoshop for Electron Microscopy Section V Complex Color Table
By Eric Jay Miller
Or Ctrl + S
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Section V Complex Color Table Basic Photoshop for Electron Microscopy
By Eric Jay Miller
In the end, we have ourselves this nice colorful image that really kind of looks like
something! (The colors dont always need to be this happy, thats just how I ended up
making them.)
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Basic Photoshop for Electron Microscopy Section V Addendum
By Eric Jay Miller
So I mentioned at the beginning of Section IV that there are a number of ways that we
can go about grabbing different areas of our images to throw together later into a final
colorized image. Here we will discuss some of those other ways. They probably wont
be better than using the Quick Mask, but they may end up being a little faster.
TO START: Well follow the instructions in Section IV up through Step 7. Now instead
of using the Quick Mask, well look at the use the Quick Selection Tool (Section A)
and the Lasso Tool (Section B). Well start off with the Quick Selection Tool.
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Section V Addendum Basic Photoshop for Electron Microscopy
By Eric Jay Miller
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Basic Photoshop for Electron Microscopy Section V Addendum
By Eric Jay Miller
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Section V Addendum Basic Photoshop for Electron Microscopy
By Eric Jay Miller
B. Lasso Tool
B1. Now well look at the use of the Lasso
Tool. This will probably be the fastest
technique and can give pretty decent results.
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Basic Photoshop for Electron Microscopy Section V Addendum
By Eric Jay Miller
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Section V Addendum Basic Photoshop for Electron Microscopy
By Eric Jay Miller
or Ctrl + Shift + I
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Basic Photoshop for Electron Microscopy Section V Addendum
By Eric Jay Miller
Results
This is our first image that we
combined using the Quick
Mask. All the edges look nice
and smooth and pretty.
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Section V Addendum Basic Photoshop for Electron Microscopy
By Eric Jay Miller
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Basic Photoshop for Electron Microscopy Section VI Easy Pseudo Color
By Eric Jay Miller
or Ctrl + L
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Section VI Easy Pseudo-Color Basic Photoshop for Electron Microscopy
By Eric Jay Miller
or Ctrl + Shift + N
Click OK.
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Basic Photoshop for Electron Microscopy Section VI Easy Pseudo Color
By Eric Jay Miller
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Section VI Easy Pseudo-Color Basic Photoshop for Electron Microscopy
By Eric Jay Miller
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Basic Photoshop for Electron Microscopy Section VI Easy Pseudo Color
By Eric Jay Miller
OPTIONAL
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Section VI Easy Pseudo-Color Basic Photoshop for Electron Microscopy
By Eric Jay Miller
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Basic Photoshop for Electron Microscopy Section VI Easy Pseudo Color
By Eric Jay Miller
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Section VI Easy Pseudo-Color Basic Photoshop for Electron Microscopy
By Eric Jay Miller
Our image with 2 colorized elements and one light color over everything.
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Basic Photoshop for Electron Microscopy Section VII Calculations
By Eric Jay Miller
VII. Calculations
This process is solely for images that have uneven illumination. This is a common
problem in many TEM micrographs and can also be encountered in low mag SEM
images. The results of this technique can be very dramatic in repairing otherwise
unsalvageable images.
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Section VII Calculations Basic Photoshop for Electron Microscopy
By Eric Jay Miller
5. Click:
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Basic Photoshop for Electron Microscopy Section VII Calculations
By Eric Jay Miller
NOTE: This is a subjective, trial-and-error process. You may have to switch the images
from Source 1 to Source 2 and go through all of the Blending options several times
before you find the best result. You can even switch which image is inverted. (This
rarely works though, but give it a try anyway. As a rule, the image that you performed
the Median on should be inverted.)
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Section VII Calculations Basic Photoshop for Electron Microscopy
By Eric Jay Miller
Here is the original image. There is a large amount of vignetting around the bottom of
the image, nearly omitting information.
Here is the image after Calculations. The illumination is now much more even across
the whole image.
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Basic Photoshop for Electron Microscopy Section VIII Color Maps
By Eric Jay Miller
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Section VIII Color Maps Basic Photoshop for Electron Microscopy
By Eric Jay Miller
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Basic Photoshop for Electron Microscopy Section VIII Color Maps
By Eric Jay Miller
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Section VIII Color Maps Basic Photoshop for Electron Microscopy
By Eric Jay Miller
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Basic Photoshop for Electron Microscopy Section VIII Color Maps
By Eric Jay Miller
or Ctrl + V
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Section VIII Color Maps Basic Photoshop for Electron Microscopy
By Eric Jay Miller
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Basic Photoshop for Electron Microscopy Section VIII Color Maps
By Eric Jay Miller
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Section IX Multi-Detector Color Basic Photoshop for Electron Microscopy
By Eric Jay Miller
If you only have 1 SE detector, this procedure IS STILL POSSIBLE! Check out the
Tips, Tricks and Suggestions section at the end of this section.
ATTENTION: This may look like a long and complicated procedure at first, but its really
quite simple and once youre familiar with it, can be done very quickly. The time
consuming part is really taking the images.
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Basic Photoshop for Electron Microscopy Section IX Multi-Detector Color
By Eric Jay Miller
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Section IX Multi-Detector Color Basic Photoshop for Electron Microscopy
By Eric Jay Miller
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Basic Photoshop for Electron Microscopy Section IX Multi-Detector Color
By Eric Jay Miller
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Section IX Multi-Detector Color Basic Photoshop for Electron Microscopy
By Eric Jay Miller
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Basic Photoshop for Electron Microscopy Section IX Multi-Detector Color
By Eric Jay Miller
Click OK.
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Section IX Multi-Detector Color Basic Photoshop for Electron Microscopy
By Eric Jay Miller
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Basic Photoshop for Electron Microscopy Section IX Multi-Detector Color
By Eric Jay Miller
or Shift + Ctrl + S
Or Ctrl + O
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Section IX Multi-Detector Color Basic Photoshop for Electron Microscopy
By Eric Jay Miller
or Ctrl + L
or Shift + Ctrl + B
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Basic Photoshop for Electron Microscopy Section IX Multi-Detector Color
By Eric Jay Miller
We started off with 4 regular old B/W micrographs taken of the exact same area, but
with different detectors.
What were left with is this rather colorful, if not slightly unusual, image.
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Section IX Tips for Multi-Detector Color Basic Photoshop for Electron Microscopy
By Eric Jay Miller
A. Opacity
Here are some examples of how, by changing the opacity on the B/W layer in Step 22,
we can really control how subtle or in your face the colors are.
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Basic Photoshop for Electron Microscopy Section IX Tips for Multi-Detector Color
By Eric Jay Miller
First off, if we take a look at the setup for the detector I used, it
actually has 5 segments. There is one extra segment (labeled E)
that is off-axis and will give a little shadow to the BSE image
(which, as you may know, gives a rather flat-looking image, since
the detector is directly above the sample).
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Section IX Tips for Multi-Detector Color Basic Photoshop for Electron Microscopy
By Eric Jay Miller
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Basic Photoshop for Electron Microscopy Section IX Tips for Multi-Detector Color
By Eric Jay Miller
Our second major option (if your SEM has it) is to use the TOPO
mode. This turns 2 segments positive (marked in red as +) and
the other 2 are turned negative (marked in blue as -). So what
does that do?
Well, positive means on, and we know what that is. Signal we get
shows up as white, so the more signal we get from a certain area,
the lighter colored it is.
If a segment is turned negative, it does the opposite of that.
Signal then shows up as black instead of white.
The images above were each taken with the single segment POSITIVE and the other 3
OFF. The lower 3 images were taken in TOPO mode, with 2 segments POSITIVE and
the other 2 NEGATIVE.
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Section IX Tips for Multi-Detector Color Basic Photoshop for Electron Microscopy
By Eric Jay Miller
So how do these 2 different techniques affect the final result? If we work on our
samples exactly the way we described in the instructions through Step 17, this is what
we have:
OK, heres the same image, but with the B/W master image
on top at 50% opacity.
The coloring still seems dramatic, but the colors are much
more muted. Again, this is something you can tinker with.
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Basic Photoshop for Electron Microscopy Section IX Tips for Multi-Detector Color
By Eric Jay Miller
You may notice that still, given all the many little things we can do from start to finish in
this process, you still will end up with a generally similar image. They have their
differences, but all the examples we just looked at are not WILDLY different. Now Im
not saying that small details are not important (surely they can be), but as with the other
techniques weve discussed, more time and effort = more better results. (Or more
gooder results, whatever.)
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Section IX Tips for Multi-Detector Color Basic Photoshop for Electron Microscopy
By Eric Jay Miller
If your microscope isnt SUPER old or SUPER cheap, you will probably have some kind
of eucentric rotation option somewhere in the software to rotate the stage around the
area youre currently looking at.
First, this wont work if youre using an upper (through the lens) SE detector. Its
always looking straight down on your sample, so rotating your sample around isnt
going to do anything. This will only work with a lower (in-chamber) detector.
Second, find an area on your sample that you think will be visible as you rotate your
sample around 360. (That might not be as easy as it sounds. Dont be surprised if after
youve taken 1 or 2 images, you have to start over in a new area because you lost what
you were looking at.)
Fourth, we need to decide how many images were going to take, and therefore, how
much to rotate the stage by in between images. 4 images would probably be nice, so
we can have 2 or 3 color images and 1 master image, like we have in the BSE images.
(If youre using an SEM that has a through the lens detector, take your master image
with that.) So we would just rotate the stage by 90 each time after taking our images. In
the example Ive cooked up, I took 3 images, so I rotated the stage by 120.
At this juncture, we must ask ourselves two very important questions about our SEM.
1. Does my SEM have a software controlled stage?
2. Does my SEM have a Raster Rotation in the control software (and not a
wobbly knob) that I can control the exact number of degrees my image is
rotated?
If the answer to both questions is YES, then youre in luck and this will be pretty easy if
you just follow the instructions under Option A.
If you answered NO to either of those questions, youre going to need to follow the
steps under Option B for this to work properly.
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Basic Photoshop for Electron Microscopy Section IX Tips for Multi-Detector Color
By Eric Jay Miller
You should end up with a group of images that look kind of like this:
To make the color image happen, we can follow the instructions as before up through
Step 12 of Section VIII and then continue with the following instructions:
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Section IX Tips for Multi-Detector Color Basic Photoshop for Electron Microscopy
By Eric Jay Miller
Ctrl + T
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Basic Photoshop for Electron Microscopy Section IX Tips for Multi-Detector Color
By Eric Jay Miller
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Section IX Tips for Multi-Detector Color Basic Photoshop for Electron Microscopy
By Eric Jay Miller
or Shift + Ctrl + B
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Basic Photoshop for Electron Microscopy Section IX Tips for Multi-Detector Color
By Eric Jay Miller
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Section IX Tips for Multi-Detector Color Basic Photoshop for Electron Microscopy
By Eric Jay Miller
Thats it.
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Basic Photoshop for Electron Microscopy Section IX Tips for Multi-Detector Color
By Eric Jay Miller
In conclusion, the fantastic thing about this technique is not only the ease and speed
you can put these images together, but also the fact that all the tiny details in your
image are not lost, like they can be when using something like the Gradient Map.
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Section X Mag Line Marker Basic Photoshop for Electron Microscopy
By Eric Jay Miller
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Basic Photoshop for Electron Microscopy Section X Mag Line Marker
By Eric Jay Miller
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Section X Mag Line Marker Basic Photoshop for Electron Microscopy
By Eric Jay Miller
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Basic Photoshop for Electron Microscopy Section X Mag Line Marker
By Eric Jay Miller
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Section X Mag Line Marker Basic Photoshop for Electron Microscopy
By Eric Jay Miller
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Basic Photoshop for Electron Microscopy Section XI Mag Line Marker (Cheating)
By Eric Jay Miller
EXTRA CAUTION: This only works if your image HAS NOT been resized. (Unless you
know by how much. i.e. If its exactly half the size, reduce your line 50%.)
SUPER CAUTION: This will only work with .TIF, .JPG, .BMP and .PNG files. It WILL
NOT WORK with a .GIF file. You have been warned.
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Section XI Mag Line Marker (Cheating) Basic Photoshop for Electron Microscopy
By Eric Jay Miller
or Ctrl + R
Or F8
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Basic Photoshop for Electron Microscopy Section XI Mag Line Marker (Cheating)
By Eric Jay Miller
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Section XI Mag Line Marker (Cheating) Basic Photoshop for Electron Microscopy
By Eric Jay Miller
Ive tried this with numerous micrographs from several different microscope
manufacturers (and even on actual photographic micrographs). It didnt matter what
size or resolution they were, as long as it was the original non-resized image, not
a .GIF, and done in Photoshop, this procedure works. Just dont ask me why.
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Basic Photoshop for Electron Microscopy Appendix I
By Eric Jay Miller
Appendix I
File Formats
PHOTOSHOP (.PSD or .PDD) This is the native file format for Adobe Photoshop. While
you cant really use this file format for display purposes, its great for saving files that
you are currently processing. For example, if I cant finish fixing up my image in one
sitting, I might save it as a .PSD so that I can save all the layers and changes Ive made
to my image so I can pick up again right where I left off at a later time. Then when I was
finished fixing my image, I would Save As one of the actual file formats listed below.
This file is a good insurance policy to have, especially if you need to go back and tinker
with 1 little thing in your image, you wont have to completely redo it.
TIFF (.TIF or .TIFF) Uncompressed file format with a large file size. In one way you can
think of the image data being very pure, in that it is not compressed, with the large file
size being the only major downside. Good for scaling images up and down. This is my
preferred file type when saving images from a microscope. TIFF files can also save
layers, like a .PSD file.
BITMAP (.BMP) Uncompressed file format with large file size. Similar to .TIF in that it is
not compressed, and it is also easy to open and work with since it is a Microsoft file
format. Unfortunately it does not resize up or down very well.
GIF (.GIF) An uncompressed file format with a small file size. An 8-bit indexed color file,
which is limited to 256 colors. Good for grayscale images, not so good for color images.
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Appendix I Basic Photoshop for Electron Microscopy
By Eric Jay Miller
Appendix II
The Short Versions
I. Levels
Instructions Shortcuts
1 Open image Ctrl + O
2 Is image 8bit gray scale? Image > Mode
3 If no, change. Image > Mode
4 Open Levels Ctrl + L
5 Make sure Preview box is checked -
6 Adjust Levels -
7 Save Ctrl + S
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Basic Photoshop for Electron Microscopy Appendix I
By Eric Jay Miller
Instructions Shortcuts
1 Open image Ctrl + O
2 Is image 8bit gray scale? Image > Mode
3 If no, change. Image > Mode
4 Open Levels Ctrl + L
5 Make sure Preview box is checked -
6 Adjust Levels -
7 Save Ctrl + S
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Appendix I Basic Photoshop for Electron Microscopy
By Eric Jay Miller
Instructions Shortcuts
1 Open Image Ctrl + O
2 Duplicate Image Image > Duplicate
3 Close original image Ctrl + F4
4 Switch Image to Indexed Color Image > Mode > Indexed Color
5 Open the Color Table Image > Mode > Color Table
6 Click and drag cursor from top left box -
to bottom right
7 Select black in the Color Picker. OK -
8 Select the other color. OK -
9 Preview Image. OK -
10 Save Ctrl + S
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Basic Photoshop for Electron Microscopy Appendix I
By Eric Jay Miller
Instructions Shortcuts
1 Open Image Ctrl + O
2 Duplicate Image Image > Duplicate
3 Close original image Ctrl + F4
4 Switch Image to RGB Color Image > Mode > RGB Color
5 Open the Gradient Map Image > Adjustments >
Gradient Map
6 Click the color gradient bar -
7 Select a preset to start from -
8 Select a Color Stop -
9 Click Color box to open Color Picker -
10 Choose color. OK -
11 Repeat Steps 8, 9, 10 until all colors are -
as desired
12 Gradient Editor, OK -
13 Gradient Map, OK -
14 Levels Ctrl + L
15 Save Ctrl + S
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Appendix I Basic Photoshop for Electron Microscopy
By Eric Jay Miller
Instructions Shortcuts
1 Open image Ctrl + O
2 Duplicate image Image > Duplicate
3 Close Original Ctrl + F4
4 Adjust Levels Ctrl + L
5 Duplicate image until you have 1 image Image > Duplicate
for each color youll be using
6 Set all images to Indexed Color Mode Image > Mode > Indexed Color
7 Open the Color Table Image > Mode > Color Table
8 Click and drag cursor from top left box to -
bottom right
9 Select dark color in the Color Picker. OK -
10 Select light color. OK -
11 Preview Image. OK -
12 Repeat 7 thru 11 for each image -
13 Choose which images will have areas -
cut out and which image will be the
background
14 Enter Quick Mask Mode on any image Q
other than the background image
15 Select Pencil tool & brush diameter -
16 Draw over bulk areas, avoid edges -
17 Select Brush tool & brush diameter -
18 Draw over edges -
19 Exit Quick Mask mode Q
20 Invert selection Ctrl + Shift + I
21 Copy Ctrl + C
22 Switch Background image to RGB Color Image > Mode > RGB Color
23 Paste Ctrl + V
24 Repeat Steps 14 thru 23 on remaining -
images
25 Select background image with all layers -
copied into it
26 Select layer to align -
27 Set layer Opacity to 50% -
28 Select Move Tool V
29 Left click on layer to drag and align -
30 Use arrow keys for fine alignment -
31 Set layer Opacity to 100% -
32 Repeat Steps 26 thru 31 for remaining -
layers
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Basic Photoshop for Electron Microscopy Appendix I
By Eric Jay Miller
33 Arrange layer order -
34 Save Ctrl + S
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Appendix I Basic Photoshop for Electron Microscopy
By Eric Jay Miller
Instructions Shortcuts
1 Open image Ctrl + O
2 Duplicate image Image > Duplicate
3 Close Original Ctrl + F4
4 Adjust Levels Ctrl + L
5 Switch image to RGB Color Image > Mode > RGB Color
6 Create New Layer Ctrl + Shift + N
7 Choose Pencil or Brush B
8 Choose Color -
9 Paint areas to be colored -
10 Repeat 6 thru 9 if desired -
11 Create New Layer Ctrl + Shift + N
12 Choose Color -
13 Fill layer G
14 Adjust Opacity of colored layers -
15 Save Ctrl + S
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Basic Photoshop for Electron Microscopy Appendix I
By Eric Jay Miller
VII. Calculations
Instructions Shortcuts
1 Open Image Ctrl + O
2 Make 2 Duplicate images & close the original Image > Duplicate
3 Run a 100 pixel Median Filter on copy_2 Filter > Noise > Median
4 Open Calculations Image > Calculations
5 Set Source 1 as copy_2 and Invert -
6 Set Source 2 as the first copy -
7 Choose Blending option -
8 Set Result to New Document -
9 Adjust Levels Ctrl + L
10 Save Ctrl + S
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Appendix I Basic Photoshop for Electron Microscopy
By Eric Jay Miller
Instructions Shortcuts
1 Open all your color map images Ctrl + O
2 Select All of one of the images Ctrl + A
3 Copy it Ctrl + C
4 Create a New Image Ctrl + N
5 Set Foreground Color to black D
6 Use Paint Bucket to fill new image black -
7 Select one of your color map images -
8 Select Magic Wand and set Tolerance to 0 -
9 Use Magic Wand to select black areas of your image -
10 Invert the selection Ctrl + Shift + I
11 Copy the selection Ctrl + C
12 Paste it into your New Image Ctrl + P
13 Repeat 7-12 on your other color map images -
14 Arrange the layers in the New Image -
15 Save Ctrl + S
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Basic Photoshop for Electron Microscopy Appendix I
By Eric Jay Miller
Instructions Shortcuts
1 Open all your images Ctrl + O
2 Select All of one of your images Ctrl + A
3 Copy it Ctrl + C
4 Create a New image Ctrl + N
5 Make sure new image is set to RGB Color Image > Mode > RGB Color
6 Set Foreground Color to black D
7 Use Paint Bucket to fill new image black -
8 Paste copied image into New image Ctrl + V
9 Copy and Paste remaining images into new Ctrl + A, Ctrl + C, Ctrl + V
image
10 Make sure master image layer is at the -
bottom
11 Align other images to master image. (50% V
opacity, make 1 layer visible at a time )
12 Make all layers visible -
13 Right click a layer > Blending Options -
14 Uncheck 1 or 2 color Channels -
15 Repeat steps 12, 13 on other layers -
16 Drag master layer (not colored) to top layer -
17 Adjust master layer Opacity (lower = more -
color)
18 Save As (.JPG, .TIF, etc.) & discard layers Shift + Ctrl + S
19 Open just saved image Ctrl + O
20 Levels Ctrl + L
21 Auto Color (optional) Shift + Ctrl + B
22 Save Ctrl + S
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Appendix I Basic Photoshop for Electron Microscopy
By Eric Jay Miller
Instructions Shortcuts
1 Open your image Ctrl + O
2 Using the existing line as a guide, draw a -
rectangle the same length
3 To change the size, scale the Width Edit > Transform Path > Scale
4 Use Text Tool to type a label -
5 Use Move tool to position line and label V
6 Save As Shift + Ctrl + S
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Basic Photoshop for Electron Microscopy Appendix I
By Eric Jay Miller
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