SEM Magnification Calibration and Verification

SEM Magnification Calibration and Verification


Hello, and welcome to another McCrone Group webinar. My name is Charles Zona, and today we welcome Mak Koten. Mak is
going to talk to us about SEM Magnification Calibration and
Verification: Building Confidence in Your Scale Bar. Before we get started, I would
like to give you a bit of Mak’s background. Mak is part of McCrone Associates’ (MA)
electron optics group where he specializes in materials
characterization and phase identification via a combination of
X-ray diffraction, scanning electron microscopy, energy-dispersive X-ray
spectroscopy, and transmission electron microscopy. He designs and conducts
experiments that will help clients better understand or solve specific
problems such as product contamination, product specifications failure, and
unknown manufacturing and processing issues. He is also one of the instructors
for our scanning electron microscopy course here at Hooke College of Applied
Sciences, a member of The McCrone Group. Mak will field questions from the audience immediately following
today’s presentation and this webinar is being recorded and will be available on
The McCrone Group website under the webinars tab. And now I will hand the
program over to Mak. Thanks, Chuck, for that warm introduction, and thank you all for joining us this afternoon for our webinar! We are very pleased to have you. The topic I would like to discuss today is one of great importance to an SEM operator, and and that is how to ensure that measurements made from SEM images are accurate. In any project I’ve done where measurements are
made, this question always gets asked by the client and it is an important
question so we wanted to address it in some detail here today. When you ask yourself if the measurements you are making are accurate or not, what you’re
really asking yourself is, “Has this SEM been properly calibrated?” Some labs have
protocols in place where this is checked at regular intervals. Here at MA, for
example, we check this annually, or after a major service call. You may think that the SEM service engineer has calibrated the instrument so you don’t have to
worry about it, but they don’t always use traceable standards for that calibration.
Who usually checks the magnification calibration in academia? It is usually
the microscope specialist or lab manager, or an experienced user that is concerned
about calibration. In an industrial lab, there may be a particular operator
appointed to checking the calibrations. it may also be required for accredited
laboratories, such as if you have an ISO 17025 accreditation. So to begin this discussion, I will
introduce some of the relevant concepts concerning calibration and verification,
and I will begin with the concept of magnification. SEM magnification is
defined as the ratio of a length measured from the SEM monitor—Lm—to the same length measured on the sample—Ls. M equals Lm over Ls. The length measured can be anything from the side of a single pixel all the way up to the
entire horizontal or vertical field of view. The ratio of each length should be
the same; that is, they should yield the same magnification. If we look at this
equation, we see that the monitor length is fixed. So if the magnification, M, is to change, that means that the value of Ls must change. The SEM takes care of this
by changing the distance the beam is moved over the sample. Shorter distances
result in higher magnification images. In almost all SEM images, you will
see a data bar somewhere in the image like the one shown here. It’s usually
somewhere at the bottom. Something it nearly always includes is a nominal
magnification. I say nominal because it only applies to images that are viewed
on the microscope PC. The one shown here has a value of 35,000X, but it would not
be accurate if the same image is viewed on any other screen. In these three examples. Lm-1 is supposed
to represent the original image being viewed on the microscope PC. So, this is
the microscope PC. Lm2, then, is the same image being viewed on a laptop computer,
and Lm3 would be yet a third length obtained by the image that is projected
onto a projection screen, say, if you’re giving a PowerPoint presentation. For these three copies of the same image,
we have one value of Ls corresponding to the actual horizontal field of view of the image, for example, in microns. But we see that Lm is always changing unless we measure from the microscope PC. therefore, the actual magnifications are
different in these three cases. I’m calling this the magnification paradox
because as soon as the image is opened up on a different computer than the
microscope PC, the magnification burned onto the image is wrong. Now I will give
some examples of magnification calculations, except here, I’m going to
actually calculate the value of Ls that would be obtained from a specific
magnification. The first column in this table shows the magnification
range typical for an SEM: about 10X to 1,000,000X. In example one, I’m using the
horizontal field of view of the image— that is, the entire width of the image—for my value of Ls and Lm. You can see in the second column that the values
of Lm1 stay constant—24 centimeters is what we measured on our on our
microscope PC. However, the values for Ls1 scale according to the magnification.
This illustrates how the field of view has changed in an SEM just by
lengthening or shortening the raster distance. Similarly, in example two, I have the width of a single pixel in the image
as my length in the calculation. By dividing the value for Lm in example one by the number of pixels in the image, 1280, we see that each pixel is being
displayed on the microscope PC as being one hundred and ninety microns a side,
which stays constant. At each magnification, the value of Ls2 then
is calculated, and we see how short the pixel-to-pixel raster distance must be
for the SEM to achieve this magnification. At 1,000,000X, the
distance between two pixels is on the order of .2 nanometers. A side
note about vertical field of view is that this data bar at the bottom can
sometimes change this value. In our example, we had in the SEM microscope
settings, the value listed for the image resolution was 1280 x 960 pixels, but this data bar actually adds another 64 pixels to the image, so it changes the
overall image resolution to be 1024 x 1280. The reason this matters is, if
you try to repeat example two on your microscope for the vertical field of
view instead of the horizontal, then you would have to select the corresponding
image resolution to either include or exclude the data bar. Next, I’m going to
talk about SEM image formation. If you are an SEM user, you probably know that
the SEM is not like other microscopes, namely, that the process by which images
are formed is different. You cannot draw a ray diagram for the SEM, for example,
like you can for an optical microscope or a TEM. This is because an image is not projected onto a camera after passing
through the optical system, but rather the beam of electrons is being precisely
controlled by a scan generator, which deflects the beam over the sample in a
grid pattern. The detector collects electrons—both secondary and backscatter
electronsfor each beam position in x and y space, and saves the number of
electrons detected at each beam position. The example below shows the intensity
plot for a single line in the image. Finally, the computer is able to map this
information onto a file with a readable format that can display the electron
intensity measured at each beam position on the computer screen as a digital
image. It is here that we arrive at the crux of the calibration discussion for
an SEM. What is actually being calibrated in the SEM is the distance the beam
moves in both the x and y directions. Both the precision and the accuracy of
the scan generator are important for image quality and measurements. Because
the beam positions will be mapped to a square grid in the image,—that is to say,
the square periodic pixels that make up an image—the actual motion of the beam
must be square. If this is not the case, distortions in the image will arise.
Similarly, if the distances between the beam positions are not accurately known,
then measurements made from the images will be poor. In the diagrams below, the
one on the left shows a certain kind of distortion/ The pixels are stretched in
the horizontal direction. This will cause the object in the image to appear
horizontally compressed. The example on the right is the opposite effect, and
it will result in vertical compression. Distortions such as these are a
nightmare for an electron microscopist because they are very hard to see when
viewing your samples. Later on, I will show you the type of sample you can use
to evaluate your scan generator in terms of the image distortions. At this point, I’ve introduced the digital resolution in the form of pixel size and its
relationship to the scan generator, but what else goes into forming an SEM image? The image resolution is not always the same as your digital resolution. This is
particularly true at higher magnifications. Such factors as probe
size, interaction volume, and even the realistic shape of the beam play
important roles in the actual image resolution. The diagram on the right
shows the idealized picture of a focused beam of electrons scanning across a
sample. The grid below is meant to represent the beam positions that make
up the pixels in the final image. The beam is placed at each position, but
these other factors can sometimes cause considerable overlap from position to
position. The interaction volume depends on voltage, and increases with beam energy. The beam is not perfectly collimated, but rather it has a Gaussian
shape, meaning some electrons land on your sample outside of the expected probe size, which is often quoted as the full width half
max (FWHM) of the Gaussian profile. The updated diagram shows the Gaussian profile
extending outside a single pixel into neighboring pixels. Spurious signal
can be detected outside of the confined region of each position. We have already
calculated the range of pixel sizes for typical SEM images, and it is interesting
to note that—especially for thermionic sources—the probe size can be
significantly larger than the pixel size. This reduces your image resolution
compared to the digital resolution, particularly at higher magnifications. Next, I would like to review some of the advantages you get when you do save an image with a scale bar versus when you don’t save
the image with the scale bar. If you only have the nominal magnification, then you
don’t have any value of Ls from the magnification equation from before to
help you in making measurements from the image, or in recalculating the
magnification. If you are interested in doing that, you can always calculate—or
measure—the value of Lm from the screen that you’re using, and but the saved magnification is no longer valid, so features in your image can’t be measured
at a later date on a different PC, and if you want to measure something you
have to go back to the microscope PC, which you might not always have access
to, so it’s inconvenient. If you have the scale bar, then you always have a
measurement of Ls that’s embedded in the image and travels with the image—saved
in the image, so any number of calculations and measurements can be
made using that. You can always measure the value of Lm if you wish to calculate
the new magnification of your image, and you can make measurements of sample
features at a later date on any PC, as long as you have the software that
can open up your image. And something that’s undervalued, I think, is that
another person can much more easily look at your image and see what the size of
the features are. In the example on the right, here, I have two images, and you
know they’re the same image, but one has a scale bar one doesn’t. In the top image, if you just glance at it, you can see that the magnification is 1600X, but that doesn’t really give you a good idea of how large that that
window is. Whereas, in the image below, you can see the 10 micrometer scale bar,
and that does give you a much better, clearer indication of how large the
features are in the image. We say that including the scale bar is a good practice, and excluding it is not a good practice. Like I mentioned on the last slide, one of the biggest advantages of having the scale bar saved on the
image is that it allows you to import a pixel-to-length unit conversion factor
into most image analysis software for advanced analysis
techniques. Advanced imaging analysis is often done in third-party software, such
as ImageJ or Image-Pro, which can be used to make complete, complex
measurements of samples. These include thresholding for particle sizing or segmentation, and can extend to development of procedures that are
automated and can be run on image stacks or a batch of files. These measurements
often require the software to know the length of your scale bar in both pixels
and real length dimensions. From your scale bar you can obtain a pix/nm or µm conversion factor. Just measure the length of your
scale bar and pixels, and divide that by the length in real units. There is
usually a set scale function that allows for the software to apply this ratio to
the image or image stack. In this image, you can see the software knows the image
resolution in pixels, which is 1280 x 1024. The software has arbitrarily
assigned a real dimension of inches to the image. This is what I want to see
change, so I will measure my scale bar, which has a known distance of 500
nanometers, and I will import the lengths in both pixels and nanometers, leave the
aspect ratio as 1.0, and ImageJ computes the conversion factor, down here, at 0.35 pix/nm. Then it changes the dimensions on the image so
they’re in nanometers. What we did, was, we used the native resolution in pixels
and a conversion ratio to obtain the actual distance swept out by the
electron beam in nanometers. Now we’ll discuss the type of sample and specific
sample features that make for a good image calibration tool. The first thing
we need is for the sample to be made of something that is both chemically and
structurally very stable at ambient conditions. We also want the sample to
have sharp edges or transitions between features of known sizes, at the highest
possible magnifications. Since SEMS can reach up to 1,000,000X, this is not a
small request. For calibrating lengths, something periodic is ideal, but because
of the large mag range in an SEM, we need this periodic structure to be
recognizable at very high and very low magnifications. This means it would have
to span about four orders of magnitude and the pitch has to be very accurate
and constant over that entire range, also not a simple request. The image to the
right is ideal for calibrating images, whereas the images below are not. TEM
grids are not suitable because they are only to be viewed at lower
magnifications. The smallest feature fills the screen at 3000X. Their edges
are not very sharp, either, which makes it hard to measure distances accurately.
Another bad example is using a human hair. Even if you do know the width of
the hair accurately, it is not small enough for higher magnifications. The
best sources for calibration verification are known as Standard
Reference Materials, or SRMs, and they come from National Metrology
Institutes all over the world. A National Metrology Institute (NMI) is a
national laboratory that is tasked with the realization maintenance, improvement,
and dissemination of the SI units via traceable calibration and measurement
services based on their calibration and measurement capabilities. In the US, our
NMI is NIST; the National Institute for Standard Design Technology. The NIST
website lists all of the NMI labs for each country. Some of the others include
the UK’s NPL, National Physical Laboratory; France’s LNE, and Germany’s
PTB. I won’t try to pronounce the actual names of the last two. The key to making
an SRM is traceability. You want to choose an SRM that is traceable back to
an NMI like NIST. This means that an unbroken chain of validation from
processing to measurement exists for the material. Sample to sample uncertainties
are known and acceptable, which accounts for errors in manufacturing. Measurement
uncertainties are known and acceptable. These are the requirements to satisfy
category I traceability for an ISO 17025 accreditation body. Some good examples
are the MetroChip, which is made by MetroBoost. This is what we currently
use for our SEM calibration and verification. NIST has RM 8820, which is a
similar artifact for calibration to the MetroChip. Another option for SEM mag calibration is the MRS-6 reference standard that
is also made by NIST. And finally, for TEM image and diffraction calibration, a
great sample is the MAG*I*CAL x-section. The MAG*I*CAL calibration sample
is directly traceable to the lattice constant of silicon. This constant can be
measured directly on the MAG*I*CAL sample, providing unbroken traceability to a
fundamental physical constant in nature. All of these samples are available from
Ted Pella, except for RM 8820, which is sold by NIST. As I mentioned on the last
slide, our current SRM of choice is the MetroChip. To understand the
traceability of this reference standard, one has to begin with the micro
fabrication process that is used to manufacture it. It is manufactured using
a type of advanced semiconductor processing known as photolithography. In
this method the thin film heterostructure is grown on a pure
single crystal silicon wafer; on top of that is a native oxide layer about five
nanometers thick, and deposited on top of that is a polycrystalline silicon film
150 nanometers thick. This is then coated with a photoresist, and then covered by a
photo mask. The top surface is exposed to light, which develops the resist in the
unmasked regions. The developed resist is selectively washed away, leaving the
undeveloped resist and exposed polysilicon film. the wafer is then exposed
to a plasma, which sputters away at the polysilicon in the exposed regions
until it reaches the oxide layer. The undeveloped photoresist is then cleaned
off the surface, leaving an etched surface that mirrors the original mask. This micro fabrication technique is optimized to yield very accurate
periodicity at the expense of variations in feature sizes. The kind of periodic
structures found in this sample are illustrated here. There is a periodic
pattern of lines and spaces; together they add up to what is called the pitch for that sample. This is important to know, since pitch is the distance that
should be measured and not the line or space width. The green and blue patterns
shown here clearly have different line widths, but when they are overlapped we see that they have the same pitch. MetroBoost
guarantees pitch rather than line width, which is why is it is important to know
the difference and measure the right one. Finally, this image shows measurements of
line and space width rather than pitch. These are not the right way to measure
for image calibration. You might be wondering how NIST certified the MetroChip. They first determined the reliability of pitch from sample to
sample, showing that the deviation is very low—on the order of 2ppm. So the reliability is very high for pitch. Next, NIST used their line
scale interferometer to certify the accuracy of the pitch on a single MetroChip surface. This was done by overlapping an interference pattern and
counting the fringes on the actual surface of the MetroChip. This means
that the pitch for all MetroChips is both accurate and reliable if made from
the same mask NIDT certified. MetroBoost only uses that mask for fabrication of MetroChips. This does not mean that other features within the
pitch of the micro rulers are validated. They are not. For example, height, linewidth, and sidewall angle were not certified by NIST. These features can
vary up to +/- 10%. Now that we know what to use for the
calibration procedure, how do we calibrate our microscope? The short
answer is, in most cases, we don’t, but a vendor service engineer might. Most SEM
vendors perform annual preventative maintenance activities. You want to take
advantage of these visits to make sure that your SEM magnification is properly
calibrated. How do the vendors actually change the
calibration? They have to change the pixel-to-pixel raster distance. Older
SEMS have scan generators that can be adjusted via potentiometers by the
service engineer. Newer SEMS are corrected digitally during the mapping
process within the SEM operation software. Some vendors will perform
calibrations with traceable SRMs to a recognized authority, but others
will not. You may be able to ask them to use yours if they aren’t using one. It’s
a good idea to perform your verification while the service engineer is still on
site, since you will need their help if the verification fails . The best we
can do is check that the microscope has been properly calibrated by the vendor. How do we do that? Well, our general procedure is to first set up your
instrument for SEM imaging with the MetroChip installed, then locate the
calibration rulings on the MetroChip for both the x and y orientations. Capture images of both the x- and y-oriented rulings without rotating your
scan or stage, then calculate the percent error with the formula: percent error
=100 × the absolute value of the known value – the measured value
divided by the known value. Make sure that the distance is measured in both
the x and y directions meet your acceptance criteria—5% is what we use
here at McCrone Associates. This will verify that both x and y are accurate
and not distorted, which is important for reasons I mentioned
earlier during the discussion of image formation. In the example shown here, I
have taken four measurements, each with a different number of periods. This was
done on the smallest ruler on the MetroChip, which has a pitch of 250 nm.
I calculated the percent error for the four measurements, and you can see that
as the periods increase, the percent error decreases. This is something to
keep in mind at very high magnifications. Best
practices dictate that you take the longest measurement you can in the image.
Also note that the magnification here is 100,000X, so that is much
better verification than measuring a hair, or TEM grid at 1000X. Here are some
of the other features available on the MetroChip that are useful for SEM image
validation. We have mainly been talking about the linear micro skills, of which
there are two on the MetroChip that were NIST certified. They’re orthogonal
to one another so that you can measure both the x and y direction separately.
The smallest pitch available on the MetroChip is 250 nm, but they
also offer 300 and 400 nm. Some other reference standards go as low as
80 nm in pitch, which allows you to reliably test even higher
magnifications, but it will cost you. These are more pricey. Scatteronomy targets are also available for use on the MetroChip. These can also be used
for magnification calibration, however, these features were not tested directly
by NIST. Finally, there are many distortion targets that can be used for
calibrating a wide range of fields of view. These can come in handy if you are
concerned about your image being squeezed or stretched in the x or y direction. It is also important to keep your reference standard clean, but if you
are using a device made from advanced micro fabrication techniques, then you
will want to know what to do and what not to do to clean it. Let’s start with
the what not to do. Things to avoid include: wiping the sample with a cloth;—this will destroy the features; spraying the sample with gases that leave residue;
immersing the sample in liquids that leave residue; using cleaning solutions
that attack silicon dioxide—avoid plasma treatments that attack silicon dioxide
(these include gas mixtures containing fluorinated gases). You also want to avoid harsher plasma treatments, such as those using
chlorinated gases; these will etch the polysilicon layer. Some good cleaning
procedures include: for large particle removal, nitrogen gases and sprays can be
used (such as Dust-off); you can also rinse the sample in DI water. For cleaning
fingerprints, chemical baths that remove photoresist, if available, are effective;
and plasma sample cleaners with pure oxygen or forming gas can be used safely
to remove fingerprints and hydrocarbon residues. Finally, I will briefly describe
the MAG*I*CAL reference standard for TEM magnification calibration. One of the challenges in calibrating TEM images is in finding a sample you can use for the
entire mag range. The MAG*I*CAL sample was designed specifically for this purpose. It is a cross-sectional TEM sample with alternating layers of silicon germanium
alloy and silicon . It was grown via molecular beam epitaxy (MBE) on thedirection of a single crystal silicon wafer. Four sets of five alternating
layers make for a wide range of image magnifications. From top to bottom, this
feature is 5.21 µm long, and it is broken up into 1.2 µm sections. The silicon germanium layers are about 10 nm thin—overall, 100 nm, so you’re getting some of the idea of the the range
designed into the sample. Additionally, since the heterostructure is epitaxial, phase contrast imaging can also be used for high resolution TEM calibration at
the highest magnifications, and in diffraction mode, you can also use it for
the camera length calibration. To conclude this webinar, I would like to
highlight some of the more important points made throughout the presentation.
SEM image formation depends on the accuracy and precision with which the
scanning device moves the beam over the sample. Remember, you cannot draw a ray
diagram for the SEM. The pixel-to-pixel raster distance is responsible for
changing the magnification in an SEM, and if that distance is accurately known, it
is also responsible for image calibration. We are trying to make sure
there is a 1:1 correlation between the distance between adjacent
beam positions and adjacent pixels in the image to properly calibrate an SEM. A
traceable standard reference material must be used. We have listed a few
examples here, including the MetroChip and MRS-6 SRMs, This means a National Metrology Institute has evaluated the production
line used in manufacturing the SRM and has deemed the measurement uncertainties acceptable for the measurement in question. These aspects of SEM image
calibration and verification have been discussed in detail here for the MetroChip SRM, which is what we use here at McCrone Associates for our quality
assurance program. That concludes the webinar presentation. Thank you for joining us this afternoon. We hope that this discussion
highlighting the traceability of the MetroChip has successfully Illustrated
the details that go into selecting and using an SRM for magnification
calibration and verification. (Charles Zona) Okay, interesting stuff there Mak. Thanks for a
great webinar there, and thanks to everybody for attending today’s webinar. If you have any questions, please go ahead and type them into the questions
field. While you were giving your webinar, we we did receive a question
from Bill: “How much does the MetroChip cost?” (MAK) Oh, that’s a good question, Bill. All of the reference standards that I mentioned are available through Ted
Pella, which is one of the leading vendors in electron microscopy
accessories and supplies, and the MetroChip is one of the more moderately-priced reference standards; it costs about $750.00. (Charles) Alright. We have a question from
Christine: “Are the scale bars accurately sized to the value listed in the image?”
(Mak) That’s another good question. The short answer is yes; however, there are
two types of scale bars: the first is one that most the vendors currently use,
which is where they adjust the scale bar length to a whole number with a
reasonable length for the space provided. Other SEM vendors will give a fixed
scale bar length in pixels, and just compute the length at the magnification
used. Both are equally accurate, and it is an easy thing to test. Just make a few linear measurements using the microscope software and save
them in the image. Then open the image up in a third-party program, and set the image scale using the scale bar. You can then trace
your linear measurements from before and remeasure them to see how measurements derived from your scale bar compare to measurements made directly in the
microscope software. They should agree to within less than one percent error. (Charles) Okay, this question is from Jane: “Does the MetroChip need to be coded to view
without charging?” (Mak) Actually, no, it doesn’t need to be coded because it’s made from
a silicon wafer, which is a semiconducting material and does not
charge in the SEM, so its features are also visible eclectically and can be
used to calibrate optical microscopes. Coding it may interfere with this
capability. You don’t have to code it, but it’s a good idea to keep it clean as was
previously discussed. (Charles) Okay, Mak. Thanks. Thanks everyone for the questions. I
think that’ll do it for the questions. If any others roll in, Mak, we’ll be happy
to answer those offline. Be sure to check out our list of upcoming webinars
that will be posted shortly under the Webinars tab on The McCrone Group
website for 2019, and we hope to see you then. Thanks.

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