And X-RAY Microanalysis: Elect

EL ECTRON MIC
ING RO
N SC
AN O
C PY
S
and X-RAY
MICROANALYSIS
Dick Briggs
John Brady
Bob Newton
'2000
1
TABLE OF CONTENTS
Section 1 Scanning Electron Microscope Operation

Specimen Exchange...........................................................4
Generation of the Electron Probe............................................6
New Alignment Procedure ...................................................8
Viewing the Specimen ........................................................9
Screen Text................................................................... 10
Photography ................................................................. 11
Finishing Up................................................................. 13
Section 2 Energy Dispersive X-Ray Microanalysis

Theory ........................................................................ 15
Oxford Link ISIS............................................................ 21
Introduction.............................................................. 21
Microscope Setup....................................................... 21
ISIS Startup ............................................................. 22
Image Acquisition....................................................... 22
Beam Current Setup .................................................... 23
X-ray Spectrum Acquisition........................................... 23
Identifying the Elements Present...................................... 24
Quantitative Analysis ................................................... 25
Quant Calibration ....................................................... 25
Advanced Features...................................................... 26
Section 3 Appendices
Appendix A: Sputter Coater ............................................... 28
Appendix B: Condenser Lens Setting.................................... 29
Appendix C: Resolution vs. Magnification.............................. 31
Appendix D: Specimen Preparation ...................................... 32
Appendix E: Vacuum Evaporator......................................... 34
Appendix F: Image Manipulations and Labelling
Using Photoshop........................................... 38
Appendix G: Using Photoshop 4.0 to Label Scanning
Electron Micrographs ...................................... 42
2
Section 1
Scanning Electron Microscope

Operation
JEOL JSM 6400
3
SPECIMEN EXCHANGE
It is easiest and most convenient to insert your specimen into the microscope as the very
first step. Once it is in, you can generate the beam, make the appropriate adjustments, and
begin viewing. When you are finished, you must remove the specimen as part of shutting
down for the day.
SPECIMEN INSERTION
1. Insert the specimen, mounted on a 12 mm. diameter support stub, into the cylinder of
the specimen holder. There is a height adjustment screw in the bottom of the holder to
adjust the specimen surface flush with the top of the cylinder. Fix the specimen in
place with the set screw. Wear gloves at this step to avoid contamination of the brass
holder.
2. Make sure the filament emission is off (knob at right on 2nd control panel fully
counterclockwise) and the accelerating voltage is turned off. To do this, push and
release the ACCEL VOLTAGE button on the same control panel (the button lamp goes
out when it is off).
Working Distance
Selector
Secondary
Electron
Detector
Stage Controls:
Tilt
Rotate
X axis
Y axis
Vacuum Control
Button
Specimen Exchange
Exchange Chamber Isolation
Chamber Valve
Specimen Exchange
Rod
Working Distance
Indicator Lamp
General View of the Column
3. Make sure the stage controls are set as follows:

X control 25 mm
Y control 35 mm
Tilt control 0
Rotation control 0°
WD control 39 mm
This will insure that the entering specimen holder will be aligned with the specimen
stage.
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JEOL JSM 6400
4. Attach the specimen holder to the specimen exchange knob, if not already done. This is
accomplished by screwing the rod into the threaded hole in the side of the holder.
5. Pull the exchange rod back so the rod is caught in the detent of the glass disk. Then
place the whole assembly into the exchange chamber, hold it firmly in place, and press
the vacuum control button; this will open the valve and start the evacuation of the
chamber. The lamp in the control button will go out when the evacuation is complete -
about one minute.
6. Then open the specimen exchange chamber isolation valve by turning it fully (90°)
counterclockwise (toward you) and pulling it fully to the right. The light inside the
column will go on. While viewing the chamber through the glass disk on the exchange
rod, fit the specimen holder into the dovetail slide of the mount on the specimen stage
by pushing the specimen holder with the rod. Then unscrew the rod and fully retract it.
THE ROD MUST BE SLID IN AND OUT CAREFULLY. IF THERE IS

MUCH LATERAL TENSION APPLIED TO IT, THERE WILL BE A
LEAK OF AIR THROUGH THE "O" RING IN THE GLASS DISK AND
THE VACUUM WILL BE DUMPED. PERFORM THESE STEPS
CAREFULLY.
7. Once the rod is retracted fully, close the isolation valve by pushing it in fully and
locking it by turning the knob clockwise (away from you)90°. While supporting the
rod with your left hand near the glass disk, again depress the vacuum control button to
vent the exchange chamber. The vacuum will be broken, the rod will come loose in
your hand, and the button will light up. Remove the rod and place it on the stand.
SPECIMEN REMOVAL
This is basically the same as insertion, so only a skeletonized procedure is given.
1. Make sure the FILAMENT EMISSION is off (knob at right end of 2nd control panel,
fully counterclockwise, SLOWLY) and the ACCELerating VOLTAGE button is off
(button light out). Make sure the stage controls are set as follows:
X control 25 mm
Y control 35 mm
Tilt control 0
WD control 39 mm
2. Pull the exchange rod back so the rod is caught in the detent of the glass disk. Then
place the whole assembly into the exchange chamber, hold it firmly in place, and press
the vacuum control button; this will open the valve and start the evacuation of the
chamber. The lamp in the control button will go out when the evacuation is complete -
about one minute. Then open the specimen exchange chamber isolation valve by
turning it fully (90°) counterclockwise (toward you) and pulling it fully to the right.
The light inside the column will go on. While viewing the chamber through the glass
disk on the exchange rod, carefully slide the rod into the chamber, directing the tip into
the hole in the side of the specimen holder. Attach the rod to the specimen holder by
screwing it into the hole. [If step 1 above has not been followed, the rod and the hole
will not line up. Do not try to line them up by twisting the rod to one side or the other -
adjust the positions instead.] Fully withdraw the rod and attached holder.
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JEOL JSM 6400
3. Close the isolation valve and lock it, vent the chamber by pressing the red button, while
firmly supporting the specimen exchange rod, and remove the rod and holder from the
chamber when it comes to atmosphere.
4. Place the rod in its rack and remove your specimen by undoing the set screw.
GENERATION OF THE ELECTRON PROBE

1. Be sure there is a specimen in the chamber.
2. Turn up fully the brightness and contrast knobs of the right and left viewing CRTs
(each has two knobs under the ledge beneath them, turn these fully clockwise). There
may not be anything on the left screen.
3. Go to the keyboard and press ESC; this will put the cursor on the upper left corner of
the right viewing screen. Then press BREAK; this will display information on the right
hand screen.
There are now two different approaches to setting the operating parameters: electronic
and manual. Choose one or the other.
PROCEDURE #1: ELECTRONIC

4. On the keyboard, press PF-5. Use the arrows to move the cursor around until
STARTUP is highlited. Then press the letter L. This automatically sets the start-up
parameters. Then press PF-2 followed by the number 1; this gives you a screen titled
EOS-1 showing most of the important operating parameters.
PROCEDURE #2: MANUAL

5. There should be a list of information on the right screen, titled EOS-1. If it reads
something else, on the keyboard press PF2, then hit 1. This should give you a list on
the right of a number of operating parameters and their settings. You will need to make
some adjustments:
ACC VOLT Set this to 5 kv; this is done by turning the knob
found immediately to the right of the ACCEL
VOLTAGE button.
CL COARSE This should be set to 7; it is changed by turning

the probe current knob on control panel 1.
MAG Set this to around 100 with the

magnificationknob on panel 1.
SEI (two readings)
CONTrast Set to around 225, using the contrast knob on
panel 1
BRIGHTness Set to around 225 with the brightness knob on

panel 1.
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JEOL JSM 6400
6. Return to panel 1 and, in the SCAN box on the left, press the MODE button. You
should get a bright scan line across the left hand CRT. If not, press it again, and
perhaps again. This line should be positioned about midscreen and can be adjusted
vertically with the brightness knob.
7. Press the ACCEL VOLTAGE button; the light should light up. SLOWLY turn up the
EMISSION knob. You are heading for a setting where no more turning of the knob
will increase the number of electrons emitted; this position is known as
SATURATION. As you turn up the knob, the scan line on the screen should become
uneven, with peaks and valleys. Additionally, it should move vertically on the viewing
screen. As you turn the knob clockwise, the line will move up a bit, drop down and
then move back up and plateau (saturation). You should leave the EMISSION knob set
just at this plateau (saturation) position. See the drawing below for a graphic
representation of this. If the line moves to the top of the screen while increasing the
emission, simply lower the line using the BRIGHTNESS knob and continue with the
emission increase. The overall position of the line on the screen is a function of
BRIGHTNESS, the peaks themselves are very bright, the valleys are dark areas. The
distance from the top of the peaks to the bottom of the valleys represents CONTRAST.
SATURATION
BRIGHTNESS or
VERTICAL POSITION OF
LINE ON SCREEN
DIST ANCE KNOB IS T URNED
8. Then go to the GUN ALIGNMENT region of the panel, just below and to the right of
the right viewing screen. Make sure the SHIFT button light is NOT lit; if it is, press
the button and it will go out. Using the two knobs on either side of this button, make
sure the scan line is at its maximum height (i.e. saturation) on the left screen.
9. Using the FOCUS knob, adjust the scan line for maximum detail. Then adjust the
amplitude (contrast) of the line so that it is about 2-3 cm., using the CONTRAST knob.
Position the line on the screen using the BRIGHTNESS knob so that it is about
midscreen.
10. On control panel 1, under SCAN, press the PIC button. This should give you a full
screen (left) image of your specimen. You will want to adjust the brightness and
contrast for your viewing pleasure.
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JEOL JSM 6400
NEW ALIGNMENT PROCEDURE FOR SEM

October 10, 1994
The recent installation of the BBD (Beam Blanking Device) has necessitated a more thorough
and rigorous beam alignment procedure. Previously, the beam was coming through a large
aperture at the top of the column, leaving lots of room for error, but now there is a 50 micra
aperture at the very top of the column, and so to get the beam directed straight down this
narrow channel is trickier. If not properly done, it makes saturation tougher, including finding
the beam when starting up, and the screen sometimes goes blank when changing probe current.
Remember, it also needs to be checked when changing the KV.
1. Be sure the instrument is in MODE or SEM-1 position. To do this, either press MODE on
the EOS panel (under the left monitor) or go to PF-2 (EOS-? then page 2; toggle cursor
over to SEM-1 and hit enter (return). This shuts off the upper deflection coils, the ones
that allow you to electronically shift the image around.
2. Turn on the KV and saturate the filament, either by eye in the PICT mode, using the
traditional horizontal line scan in MODE, or simply watching the beam current meter.
3. Turn PROBE CURRENT up to 13, and maximize brightness using the TILT controls
(SHIFT lamp UNLIT).
4. Go to PICT mode, if not already there, and stepwise drop PROBE CURRENT to 1; if you
lose brightness, bring back with SHIFTs (SHIFT light ON).
5. Obtain maximum signal with SHIFTs at PROBE CURRENT 1(again, be sure SHIFT light
is on).
6. Return PROBE CURRENT to 13 (SHIFT light OFF), and maximize signal using TILT
Recheck saturation; if it is off, resaturate and then repeat steps 2 through 6.
Check what you have done by focusing on some small object at 5000x, press MODE to get the
cross-hairs on the screen and superimpose them on the object; also be sure to have the scale
marker (micron bar) showing. As you run through the PROBE CURRENT settings from 13
to 1, the image shift should be less than one micron. If the shift is greater than that, the beam
alignment is off and steps 1 through 6 should be repeated more carefully.
When finished, return the EOS panel button to SEM; this returns the image shift capability if
you want it.
When you change accelerating voltage, the above procedure must be repeated.
8
JEOL JSM 6400
VIEWING THE SPECIMEN

At this time, it may be desirable to make a number of adjustments to the image on the left
screen. These are presented in no particular order.
1. You should adjust the viewing CRT contrast and brightness to suit your needs.
2. You will need to focus the image. Use the FOCUS knob; note too that there is a FINE
button adjacent to it. Depress this button (it lights up) to allow fine focus.
3. A fine focusing must be accompanied by a correction of the astigmatism. If the image

shows a directional blurring during focusing, and the direction of blurring changes
when going from underfocus to overfocus (the directions will be at right angles to each
other), then there is astigmatism present in the electron probe system. When there is no
astigmatism, the image blurs by defocusing, but there is no directionality to the
blurring. Under conditions of astigmatism, it is impossible to get a good clear image.
Astigmatism is quite unobtrusive at magnifications less than 1000x. To correct
astigmatism, get the image in focus and check for astigmatism, i.e., directional blurring
as you pass from over to under focus and back. If astigmatism is present, adjust the
STIGMATOR X and Y knobs to obtain the sharpest possible image. Repeat the test for
astigmatism and recorrect if necessary.
4. You can move the specimen in a number of ways. You can translocate left to right and
front to back using the X and Y knobs on the front of the specimen chamber. You can
ROTATE the specimen or TILT it, again with the knobs on the specimen chamber.
Additionally you can change the working distance by turning the big knob on top of the
specimen chamber door. You can read the working distance in mm from the right
screen or in the information under your picture or from the number on the knob. The
image must be in focus for the information on the screens to be accurate.
5. To properly adjust the SEI (signal) brightness and contrast, press the waveform
monitor (WFM) button on panel 1. You should see a series of 6 horizontal, parallel
lines positioned on the screen and superimposed on the line scan. The top line
represents full white, the bottom is full black; the third line is a medium gray shade. In
other words, they represent reference lines to enable the user to reproducible set the
same contrast and brightness. This is especially important for taking pictures. If you
adjust the line scan - again by using the BRIGHTNESS and CONTRAST knobs on
panel 1, you see that you can change it with reference to these lines. For taking
pictures, try to get the peaks (i.e., the bright portions of the specimen) up to the second
line from the top, and the valleys (i.e., the dark areas of the specimen) about halfway
between the second and third lines from the bottom. This may require some judgement
and adjustment, depending on the specimen. Look at the image of the specimen on the
viewing CRT carefully before you adjust the WFM. Determine which part of the image
should be in medium gray. Set that part roughly over the third line. When the
specimen has a relatively flat smooth surface with small detail, the signal from the flat
"background" should be set at gray. Thus there may in this case be no signal reaching
the black level.
6. You may want to change the accelerating voltage. To do this, first desaturate the
filament by turning down the EMISSION knob. Do this SLOWLY, all the way. Then
select another voltage, using the knob to the right of the ACCEL VOLTAGE button and
reading the setting on the right viewing screen (ACC VOLT). Then resaturate the
filament, using the line scan image, etc. Turn up the EMISSION SLOWLY.
9
JEOL JSM 6400
7. As you change magnification, you may want to change the diameter of the beam or
probe. This is accomplished by the PROBE CURRENT knob on panel 1, and the
probe current can be read on the right viewing CRT on the CL COARSE line. As you
increase the probe current, you are decreasing the diameter of the probe by changing the
amount of demagnification carried out by the condenser lenses. A smaller probe (a
higher probe current setting) increases resolution but decreases the amount of signal
generated. This necessitates amplification, which introduces noise, seen as sparkles on
the viewing screen. To diminish this, slow down the scan speed.
8. You will notice that the bottom line of the EOS - 1 screen has OPTimum APERTure
written on it. This is followed by a number. This is the instrument recommendation,
based on accelerating voltage and probe current, for the proper diameter final aperture.
This is selected on the right side of the column. Changing apertures requires a
recentering of the aperture. Do not undertake this unless you have been
shown how to do it. Ask for assistance if you think this is necessary.
9. You may want to change specimens. To do this, follow the instructions in the section
on specimen exchange. Start with the section on specimen removal, then specimen
insertion. Be sure to have the beam off and the stage positioned properly.
SCREEN TEXT
1. To move the cursor:
a. ESC on the keyboard toggles the cursor back and forth between screens.
b. Use the arrows on the keyboard to move the cursor around on the screen.
2. To change the existing text at the bottom of the image:

a. JEOL
1. Place cursor on JEOL
2. INS on the keyboard
3. Type text - up to six characters (if you change your mind, CLS will remove
the INS effect)
4. Press the ENTER key - curved arrow, lower right corner of keyboard
b. Typing numbers in the other blocks will change the operating parameters of the
instrument. The magnification and accelerating voltage may be changed this way,
but changing the WD simply changes the focal length of the final lens, throwing the
image out of focus. Changing the millimicron number will change the
magnification).
3. To delete or return text at bottom of screen:

a. Place cursor on the block of text, press ENTER key (curved arrow, lower right) and
it will disappear.
b. To return the text, place cursor on area and press RETURN.
4. To change the position of the text at the bottom of the screen (as a whole block), put the
cursor on it and move it up or down with the arrows.
5. To write additional material on the screen:

a. Get the cursor on the left screen, using ESC, then hit the BREAK key. This
toggles the big cursor on and off with the small cursor used for text input.
b. Move this cursor with the arrows to the desired location, type in the text.
10
JEOL JSM 6400
c. Use the CLS key to erase the text.

d. If you want your text plus the information across the bottom of the screen, enter
your text, then hit the BREAK key.
PHOTOGRAPHY
INSTRUMENT SETUP
1. Find and center the portion of the specimen you want to photograph. Focus the image
carefully by going to a higher magnification and, if desired, view it on a reduced
screen. To obtain the latter, press the SCAN PIC button one or more times. This gives
you a smaller, brighter image. The instrument is the equivalent of parfocal on the way
down: if you are in focus at a high magnification, you will be in focus at a lower
magnification - IF YOU DON'T CHANGE ANYTHING ELSE. Be sure to check for
and accommodate any astigmatism that may be present.
2. Return to the appropriate magnification. Press the D-MAG button within the DISPLAY
square on the control panel 1. This reduced the image to the actual, staten
magnification, the same size as it will appear on the photograph. (The regular viewing
mode is twice the indicated magnification.) Then press the WFM button immediately to
its left. This displays the the waveform of the video signal and also the six parallel
horizontal lines. Adjust the contrast and brightness on the control panel so that the
peaks are at the second line down from the top and the valleys are between the second
and third lines up from the bottom. See VIEWING section for further details.
3. Turn off the WFM, check to see that the image is what you want. Insert the film into
the camera (see below), and in the PHOTO area, press LEFT. This will begin the
super slow scan for taking the picture.
PHOTOGRAPHY
1. Follow the instructions above regarding focus, astigmatism, setting up brightness and
contrast.
2. The camera we use with this instrument uses two different types of Polaroid film, 4
inches x 5 inches. Both are fine grain, panchromatic films. Type 52 delivers a
positive print only; Type 55 delivers both the positive and the negative. This latter
type is generally more useful, and only marginally more expensive, as it gives a
negative suitable for further enlargement in the darkroom.
3. Handling the film is not difficult. In both cases, to make an exposure you proceed as
follows:
a. Make sure the lever on the front of the Model 545 film back is pushed to the
right (L) position. This separates two rollers inside so the film can be slipped in
between.
b. Examine the film to be sure which side is up - or which side should face the
lens. Slide the film between the lips on the front edge of the camera, the end
with the metal strip in first, pushing it all the way in until a sharp "click" is
heard Do not squeeze the film in the marked area. Then pull the cardboard
cover back out as far as it will go, leaving the film exposed inside the camera.
c. Press the LEFT button. Sit very quietly while the exposure is being made.
Vibration of the column at this point will show up dramatically on your image.
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JEOL JSM 6400
d. The recording is finished when the viewing CRT goes back to its rapid scan
mode. Then push the cardboard sleeve back into the camera - ALL THE WAY.
Pull the lever to the left, and then very smoothly and evenly pull the film out of
the camera.
4. The processing differs with the two different film types:
TYPE 52
a. Be sure the f-stop setting is at f 16.
b. After you remove the film from the camera, allow it to develop for 15 seconds,
then peel off the paper covers. Timing starts as soon as the film leaves the
camera.
c. The print should be coated as soon as possible. Use the coaters supplied with
the film. Inside the cylinder is a plastic holder with a pink sponge attached; this
is saturated with acetic acid and other materials. Coat the picture using several
strokes, covering the entire surface. Failure to do this will result in faded
pictures. The coating material smells, and it can burn sensitive skin. Handle
carefully and wash any off your skin. Do this on a paper towel, not on the
console of the instrument!
c. NOTE: Dispose of the film pack and the used up coaters properly. They can
stain clothes, etc. so be cautious.
TYPE 55
a. Be sure the f-stop setting is at f 5.6.
b. Allow the film to develop for 20 seconds.
c. Open the pack and separate the print from the negative carefully and quickly.
1. Remove the tabs from both ends of the negative by folding them over,
creasing and then carefully tearing them away. Discard the tabs. BE
CAREFUL NOT TO TOUCH THE SURFACE OF THE NEGATIVE.
Handle the negative carefully for the emulsion is soft and easily damaged.
2. Swirl it gently in an 18% solution of sodium sulfite to clear the dye and to
cause the developer layer (i.e., slime) to drop off. Rinse at least five
minutes in running water, dip in Photoflo and hang up to dry.
3. Coat the print with the special coaters provided. Do this on a paper towel,
not on the console of the instrument!
4. Dispose of the film pack and used up coaters properly.
Detailed instructions accompany each film pack.
IMPORTANT NOTE
IF YOU ARE PROCESSING NEGATIVES IN THE SINK, BE VERY CAREFUL
ABOUT THE SULFITE SOLUTION. IT IS RATHER INSIDIOUS AND SEEMS TO BE
ABLE TO GET EVERYWHERE - INCLUDING FLOOR, LIGHT SWITCHES,
DOORKNOBS AND ON THE INSTRUMENT. WHEN IT DRIES, IT TURNS INTO A
WHITE POWDER WHICH FLAKES OFF AND MIGRATES EVEN FURTHER.
WHEN YOU ARE FINISHED, RINSE EVERYTHING WITH LOTS OF WATER:
TANKS, BOTTLES, SINK, COUNTER, HANDS, ETC. DRY THE SINK AND
COUNTER WITH PAPER TOWELS. CHECK THE FLOORS FOR DRIPS.
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JEOL JSM 6400
FINISHING UP
1. When you are finished viewing for any period of time, there are several steps to take
prior to leaving the instrument.
a. SLOWLY turn the filament emission down, all the way. Push the ACCEL
VOLTAGE button and release; the light will go out, indicating it is off.
b. Be sure the stage controls are as follows:
X control 25 mm
Y control 35 mm
Tilt control 0°
Working distance 39 mm
2. Be sure to return the electronics of the microscope to the proper shut-down positions.
There are two ways to do this: electronic and manual. Choose one or the other.
PROCEDURE #1: ELECTRONIC

3. Go to the keyboard and press PF-5. move the cursor down to highlight SHUTDOWN.
Then press the letter L.
PROCEDURE #2: MANUAL

4. Go to the keyboard and press PF-2 and then 1. The screen should now read EOS- 1.
Set the following parameters, reading their valuse on the right screen:
a. Accelerating voltage at 5 kv
b. Return the probe current to 7
c. Turn the MAG up to 300,000x.
d. Turn down the contrast and brightness to about 100
5. Remove your specimen from the microscope following the instructions given in the
Specimen Exchange section.
6. At the keyboard, press PF-1. Then press BREAK which makes the information
disappear and the cursor go to the upper left corner of the right screen. Then press
ESC and the cursor should disappear. If there is information on the left screen, the
cursor will be there; press break and the information will disappear.
3. Sign out in the log book, recording all the requested data.
4. Be sure to leave the room clean and tidy. Take all your stuff and put it away. If you
have been fixing negatives in the sink with the sulfite solution, be sure to rinse and dry
the sink and counter area; look for spills and drops on the floor.
13
Oxford Link ISIS 300
Section 2
Energy Dispersive X-Ray

Microanalysis
14
Energy Dispersive X-ray Microanalysis
Theory
Fundamental Concepts
X-rays are generated as a result of the ejection of an inner level electron (low energy) by
an energetic electron from an electron column. The ejected electron is replaced by an electron
from a higher energy shell. The energy lost as it moves from a high energy shell to a low
energy shell is released in the form of x-rays.
Each element has many energy levels and therefore many potential vacancy-filling
mechanisms. As a consequence even pure elements emit x-rays at many energies. Because the
atomic structure of each element is different, it follows that each element will emit a different
pattern of x-rays.
hc 12.4
λ= λ=
Planck's equation Ε or Ε
where λ = wavelength of the radiation in angstroms
c = speed of light
h = Planck's constant
E = Energy of the radiation measured in kilo-electron volts
These x-rays can be analyzed either by wavelength dispersive methods or energy

dispersive methods. The Oxford system uses the energy dispersive method. Energy
dispersive systems use a semiconductor detector first developed in the 1960's. Basically it is a
single 3mm thick by 15 mm2 crystal of silicon which has been treated with lithium (lithium-
drifted silicon).
An X-ray photon first creates a charge pulse in a semiconductor detector; the charge pulse
is then converted into a voltage pulse whose amplitude reflects the energy level of the detected
x-ray. This voltage pulse is converted into a digital signal which causes one count to be added
to the corresponding voltage channel of a multichannel analyzer. Counting over some time,
results in an x-ray spectrum.
Characteristic X-Ray Emission

Electron-Sample Interactions
Electrons are accelerated through the microscope column, acquiring kinetic energy
which is transferred to the sample. The dissipation of this enegy yields a variety of
signals.
X-Ray Continuum (bremsstrahlung)

The primary electron may be scattered inelastically by the coulomb field of an atomic
nucleus, thus giving up some or all of its energy (Figure 2.1). This energy may be
emitted in the form of x-radiation called bremsstrahlung (German for "braking radiation").
Since the primary electron can give up any amount of its energy, the energy distribution of
the emitted x-rays is continuous, thus the term x-ray continuum.
15
Bremsstrahlung
e Elastically scattered
electron
e
Inelastically scattered
electron
Figure 2.1. The energy released due to inelastic scattering of the primary electron beam as it interacts with
atoms in the sample results in the continuum radiation or Bremsstrahlung
Characteristic x-ray
High-energy
secondary
electron
Inelastically scattered
electron
Auger electron
Figure 2.2 The incident electron beam can eject an electron from an inner shell. The vacancy created may be
filled by an electron from a higher energy shell. The energy released is given off in the form of
characteristic x-rays or it causes the ejection of another electron (Auger electron).
16
Secondary Electrons
The primary electron may interact with an electron in the sample, ejecting it with some
amount of kinetic energy. If the ejected electron was weakly bound, it typically emerges
with only a few eVs (<50) of energy and is called a secondary electron. Since they have
little energy, they can only escape from the sample if they are created near the surface.
Characteristic X-Rays
When an electron is ejected from an inner atomic shell by interaction with a high-energy
electron beam, the result is an ion in an excited state (Figure 2.2). Through a relaxation
process this excited ion gives up energy to return to a normal ground state. The most
likely process in most cases is a series of transformations in each of which an electron
from an outer shell "drops" into a vacancy in an inner shell. Each drop results in the loss
of a specific amount of energy, namely, the (quantum) difference in energy between the
vacant shell and the shell contributing the electron. For the high energy inner shells, this
energy is given off in the form of x-rays. The energy of the radiation uniquely indicates
the element from which it came, hence the term characteristic x-rays. X-ray lines are
referred to using a nomenclature shown in Figure 2.3. The position of the initial vacancy
determines the first letter and the source of the vacancy filling electron determines the
second term. For instance, if a collision with a column electron causes an initial vacancy
in the K shell and that vacancy is filled from the L shell than the x-ray line is referred to as
a Kα peak.
K lines
γ
L lines
α β
β
α
K L M N
α
M line
Nomenclature
Figure 2.3 Origin of the nomenclature associated with the x-ray lines
The intensity of the characteristic radiation under given excitation conditions is influenced
by three factors:
17
1. The atomic number of both the emitting atom and the average atomic number of the
bulk sample. Emitting atom atomic number determines both the ionization cross
section and the fluorescent yield. The average atomic number of the sample affects
the energy lost due to other scattering processes.
2. The probability that the emitted characteristic x-rays will be adsorbed before they
emerge from the sample.
3. Secondary fluorescence, which may be one result of absorption. For example, a
high-energy x-ray characteristic of element A may be absorbed by an atom of
element B, thus stimulating a lower-energy emission characteristic of the second
element. The presence of elements A and B in the same sample will therefore
increase the intensity of characteristic emission from element B and decrease it from
A (matrix effect).
Backscattered Electrons
If the primary electron interacts with the nucleus of a sample atom, it may be scattered
in any direction with little loss of energy. Some are directed back out of the
sample,allowing them to be detected. These backscattered electrons are much more
energetic than secondary electrons and so may escape from a greater depth within the
sample. They don't carry the topographic information of the secondary electrons but the
main influence on the strength of the backscattered signal is the mean atomic number
within the interaction volume. The higher the atomic number of an atom the more protons
present and the higher the backscattered signal.
Escape Peaks
Emitted x-rays are measured by the lithium-drifted silicon detector. Basically an x-ray
enters the crystal disrupting the electron structure which in turn causes a temporary
increase in its electrical conductivity. The total charge conducted (integrate with respect to
time) is directly proportional to the energy of the absorbed x-ray. It is also possible for
the incoming x-ray to stimulate the emission of an x-ray characteristic of silicon. This x-
ray could escape from the crystal without adding to the charge pulse in the detector. The
energy measured for the detected x-ray will therefore be less than the actual x-ray energy.
The amount of this difference will be 1.74 keV, the energy of the silicon Kα x-ray.
Therefore as counts accumulate in an x-ray peak for any major constituent of the sample,
an escape peak can be expected to appear at an energy 1.74 keV below that of the parent
peak.
Effect of Accelerating Voltage

The accelerating voltage used in the electron column of the microscope influences both
the spatial resolution of the x-ray signal and the efficiency with which characteristic x-rays
are excited from the sample. Higher voltages produce higher energy electrons that
penetrate more deeply into the sample and spread more widely than low-energy electrons.
The result is degradation in resolution on the one hand, but more efficient excitation on the
other. It is generally accepted that this trade-off is optimized at an overvoltage (the ratio of
the accelerating voltage to the energy of the excited line) of 2.5 to 3.
18
Relative peak intensities will change as the accelerating voltage is changed. For
example at 10 KV the copper K lines at 8.04 and 8.91 keV are not efficiently excited in
contrast to the copper L lines at just below 1 keV. However at 20 KV the ratio of the line
intensities changes dramatically as the K lines become more intense than the L lines. The
standard operating voltage for EDS analysis on the Oxford is 20 kV.
Statistical Considerations
The x-ray spectrum, which is created by counting a number of x-ray photons of various
energies, is the information that we use to determine the elemental composition of the
sample. There are a number of statistical considerations that are important to obtaining
good chemical analyses. In the creation of the spectrum there are errors associated with
the assignment of an energy level to any detected x-ray photon. The first type of error is
classified as systematic error, which includes instrumental factors such as errors in
calibration. Systematic error is somewhat controllable and can be minimized. The second
type of error is random error, which is not controllable, but its magnitude can be estimated
from theoretical considerations.
The breadth of each peak in an x-ray spectrum shows that the energy of an individual x-
ray cannot be measured exactly. The amount of charge the x-ray generates in the detector
is vulnerable to random variations and the electronic circuitry inevitably contributes noise
to the signal. Consequently, a series of energy measurements of x-ray energy E will form
a distribution about a mean value, which we hope is very close to E. This energy
distribution can be assumed to be an example of a normal (Gaussian) distribution with the
breadth of the distribution indicated by its standard deviation (σ). In a normal distribution
68 percent of all measurements lie within ± 1σ and 95 percent lie within ± 2σ. It is also
possible to determine from a group of means the standard deviation of the mean which is
also given by the formula:
σN = σ
N
We can use this to evaluate how close a single measured value of the mean is to the "true"
value of E. If a spectral peak has a standard deviation of 100 eV and is the result of
detecting 10,000 individual counts we can take σ as 100 and N as 10,000 and σN is only
1eV. This gives us confidence that the mean of the peak is very close to the true energy of
the x-ray.
Counting the number of x-ray photons hitting the detector over some time period is
critical in determining how much of an element is present in the sample. Counting errors
affect how well we can measure the rate at which x-ray photons are detected by the
detector. The statistics of this process are governed by the Poisson distribution, which
has a standard deviation determined by:
σ= n
n
where is the mean number of x-ray photons detected
19
For large values of n , the Poisson distribution can be considered Gaussian, so 95% of all
measurements of n lie between ± 2σ. Therefore, the larger the value of n, the narrower
the distribution relative to the mean. This can be shown by the relative standard deviation:
ε= 1
n
Thus, if we have a spectral peak representing a single element which has 100 counts then we
can say with 95 percent confidence that the counting error is no greater than 20%. If on the
other hand our peak had 10,000 counts then we would be 95 % sure our counting error would
be only 2%. Clearly it is better to have more counts!
20
Oxford Link ISIS

Introduction
The Oxford Link ISIS EDS system consists principally of a liquid-nitrogen-cooled
detector crystal mounted in an SEM port, interface electronics mounted in a blue box, and a PC
running Windows 98 and Link ISIS software. The computer communicates with the detector
and with the SEM via the blue box. To use this system, you will need to be able to run the
SEM, you will need to learn about the ISIS software, and you will need some familiarity with
Windows 98.
You can learn about the ISIS system by following the tutorial provided by Oxford, The
Oxford Guide to X-ray Microanalysis, which resides on 3 CD’s in the SEM lab. Volumes 1
and 2 contain general information about x-ray microanalysis. Volume 3 is packed with detailed
information about the ISIS software. By viewing and listening to Volume 3, you can learn
many of the important features of ISIS. There is also a 3-volume set of manuals, The Link
ISIS Operator’s Guide, which is a good reference source not designed for cover to cover
reading. In addition, there are help files included with the ISIS software that can be viewed on
screen. Because there are so many good sources of information, this manual will focus on the
daily operating details.
Microscope Setup
The basic principal of many analytical systems is to stimulate the sample in some way, to
measure the response of the sample, and to compare the sample’s response with the responses
of a set of standards of known composition. If the responses are to be comparable (giving
good analyses), the experimental conditions must be identical and the sample and standards
must be similar. Many of the steps described in the following paragraphs are designed to
ensure that the analytical conditions are identical each time the Oxford EDS is used.
To setup the microscope for EDS work,
Load a carbon-coated sample in the microscope.

Wait for the vacuum to drop below 10-5 torr.
Set the working distance to 15 mm using the large knob on the top of the sample
chamber.
Check to see that Aperture 2 is selected.
Load the STARTUP =20kV= conditions from the PF5 window on the JEOL
6400. These conditions should include:
CL COARSE – 7
CL FINE - 75
OL COARSE – 124
OL FINE – 544
Saturate the beam in the normal way.
Focus on the sample using the Z-axis knob, not the electronic focus knob.
Adjust the stigmators.
For important quantitative work, wait approximately one hour for the instrument to
stabilize. You can use this time to explore your sample, to start ISIS, and to collect
images.
21
ISIS Startup
ISIS can be loaded by selecting “Link ISIS” from the
Start menu. After a message about “Resetting Hardware”, the
Welcome Window will appear. Now you must choose an
operator, which will ordinarily be you. If your name is not
on the list, it can be added from the Edit menu. Next, click on
the blue Labbook button to get the Labbook screen.
Now you must choose a job. It will be

simplest to keep each project as a separate
job. ISIS will remember what job you were
working on the last time. However, if you
want to change jobs or need to define a new
job, you must click on the Jobs button.
The most important thing about choosing a

job is to be sure it uses AC2 (Analytical
Configuration – 2, SiLi detector, 1024 channels,
10 keV range). The ISIS software will use or
create a folder for the job in the AC2 folder with
the path C:\ISIS\AC2\J#, where the # is an
integer assigned to the job when it is created.
Having chosen an operator and a job, ISIS is
ready to start collecting data.
Image Aquisition
Once the beam has stabilized (as demonstrated
by little change with time of the Faraday Cup
readings), you can begin x-ray analysis. To do so,
select the Autobeam button. This will open a
window like the one at the left (but blank). Clicking
on the round Start button on the upper left of this
window will cause the ISIS software to acquire the
current SEM image (SEI or BEI, depending on the
SEM settings). This is a digital image (like the right
SEM screen) and will improve by averaging over
time. The size and detail of the image (# of pixels),
as well as the speed of acquisition, are determined by
the Image Setup choices in the Edit menu. Normal
22
operation will use Fine Resolution (256x192 pixels) and Medium Acquisition Speed.
If you want a high quality image for printing, choose, High Resolution (1024x768 pixels)
and Slow Acquisition Speed. When you are satisfied with the image, press the square
Stop button. You can adjust the brightness and contract of the image using the Contrast
Enhancement button. The image can be printed, saved, or exported as a tiff file now using
commands in the File menu.
Beam Current Setup

Once you have acquired an image, left-mouse-click at a spot on the image that you wish to
analyze. A cross ⊕ will appear marking the spot. The image on the left screen of the SEM
will blank because the electron beam is now a tiny spot. (Note: A right-mouse-click on
the image will return control to the SEM.) Now you must make set the beam current.
Insert the Faraday Cup by pressing the blue PCD button to the right of the SEM
screen. You should hear a click on the column and see a (negative) current reading
on the Keithey Meter.
You want this current to read -.6000 ±.0025 nA. This current gives the best result
for the Oxford EDS system and is the current that is used to collect standard data. If
the current is not -.6000, you will need to adjust it as follows:
Press PF2 to get the EOS-1. OPTICS window on the right SEM screen.
Make a coarse adjustment using the Probe Current knob on the left JEOL keyboard,
which changes the COARSE CL setting, normally to 11.
To make a fine adjustment, move the cursor on the right SEM screen to illuminate the
FINE CL value. (Use the ESC key to move the cursor from the left SEM screen to
the right SEM screen if necessary.)
Use the + and – keys on the upper right of the right SEM keyboard to make the
FINE CL adjustment.
When a beam current of -.6000 is achieved, remove the Faraday cup.
You may find it convenient to keep the SEM at a beam current of –.6000 for the rest of your
work. The images will be more grainy and the brightness and contrast settings will have to be
changed, especially for BEI. However, if you can navigate under these conditions, there is
less setup for each analysis.
X-ray Spectrum Acquisition

To collect the first x-ray spectrum, return to
the Labbook window (maximize it) and select the
X-ray Analysis button. This will open a blank
spectrum window. Press the round Start
Acquisition button on the upper left of this
window to begin counting the x-ray photons. A
graph should appear, such as the one on the next
page, with peaks for energies corresponding to the
x-rays emitted in the 0-10 keV range by the
sample. As the spectrum appears, it is important
to check the count rate and deadtime.
23
When the Start Acquisition button is pressed, the Acquisition Status window appears.
Check to see that the % Deadtime reading is 30-40% and that the Acquisition Rate is
2.02.5 kcps. If the readings are different, there is a problem with your setup. A common
problem is that you forgot to remove the Faraday Cup.
Another common problem is that you did not set the beam
current properly. It is also possible that the X-ray Acquisition
Setup (an Edit menu item) was previously set for Optimum
Acquisition Rate rather than Best Resolution. The ISIS
software is set to collect data for a preset time of 50 seconds.
This is “livetime”. Because 30-40% of the time is deadtime,
the actual analysis will take 80-90 seconds. You do not have to
wait for the full time if you are only doing qualitative work and want to try other points. You
can press the square Stop button, or simply use the mouse to select a new point on your image
(which will restart the preset time clock).
Identifying the Elements Present

You can easily identify
most of the common elements in
geological samples by first
pressing the Identify Peaks
“?” button. This will open the
Identify Peaks window. A
right-click of the mouse on
the peak of interest will bring up
a list of possible peaks in the
Identify Peaks window. You
can label individual peaks, or all
24
the peaks for an element, by pressing the Label Peak and Label Series buttons. You can also
paint a window for an element (for x-ray mapping) using the Paint Window button.
Quantitative Analysis
To quantify your analysis, press the Quantitative Analysis “%” button on the X-ray
Analysis window to open the SEMQuant Window. Select the elements present in your sample
to analyze using the periodic table that appears when you press the Select Elements button.
For many elements you can choose the standard to be used by pressing the
Configure button. Be sure the elements you wish to analyze have a green bar over them in
the periodic table. When the elements have been selected, choose the Processing Options. For
minerals, these will ordinarily be Combined element by Stoichiometry, with Oxygen as
the combined element. If you want to see a mineral formula
calculated from the analysis, select the Nos. of ions
Calculation from the Edit menu and enter the appropriate
value. Finally, press the Quantify button on the SEMQuant
window. An analysis will appear in an SEMQuant Results
window that includes Weight % (Element %) and Atomic % of
the elements, Weight % (Compound %) of the oxides, and the
number of ions in the formula. The standards used are also listed. You can print a copy of the
analysis by selecting Print from the File menu. You can also save the results to another file.
Choose Copy Text from the Edit menu to paste the analysis into a Microsoft Word document.
Choose Copy Table from the edit menu to paste the final table into an Excel document,
which will make later data manipulation easier.
Quant Calibration
To ensure that you have the best analyses, you must periodically (every hour or two)
perform a Quant Calibration. To do so, you must analyze the Cobalt Standard, which is
located in a corner of the thin section and one-inch disk sample holders. Setup for the analysis
as described above and acquire a spectrum. |Operating conditions must be the same for the
25
Cobalt as for your unknowns. Note the deadtime will be higher (40-50%) for the Cobalt than
for most minerals (30-40%) for the –.6000 nA beam current. When the spectrum is acquired,
select Quant Calibration from the Options menu of the X-ray Analysis window. The Gain
Calibration window will open. Press the Calibrate button. Enter the new calibration values
in the Excel spreadsheet. C:\Oxford Instruction Files\Quant Calibration Record.xls. The Quant
Calibration data are saved with each spectrum so that data collected under different conditions
can be compared.
Advanced Features
The Oxford ISIS system has a number of other features that may be of use. One that
many geologists will use is x-ray mapping. This feature will allow the user to collect x-ray
intensity maps for specified elements. To collect an x-ray map, Acquisition Setup must be
changed to Optimum Acquisition Rate using the Edit menu of the X-ray analysis window. A
test spectrum must be acquired. Windows for the elements of interest must be painted using
the button in the Identify Peaks window. The Mapping button is then selected in the X-ray
Analysis window. Maps can take minutes to hours to collect, depending on the resolution
required. Use the manuals to learn more about mapping and other advanced features.
26
Section 3
Appendices
27
Sputter Coater
Appendix A
SPUTTER COATER
1. Lift the lid off the vacuum chamber. Carefully remove the glass cylinder and lay it on
its side where it won't roll off the counter. This prevents messing up the vacuum
grease on the top and bottom rims. Place your specimens on the central area of the
pedestal, not the raised ring around the periphery. Replace the glass cylinder and close
the lid.
2. CLOSE the vent toggle valve (down) and CLOSE the increase/decrease needle valve
(turn to full decrease). Do not overtighten this needle valve. Be sure the high voltage
control switch is OFF and the high voltage dial selector is at 0.
3. TURN ON the main power switch. Wait until the vacuum reaches 45 millitorr on the
vacuum gauge. This will take anywhere from a few minutes to perhaps fifteen. Then
OPEN (turn toward increase) the increase/decrease needle valve to bring the pressure
up to (and stabilized at) 50 millitorr. This necessitates a considerable number of turns
of the valve stem.
4. You are now ready to begin coating:

a. High voltage switch should be OFF; set high voltage dial to 9
b. Press ON the high voltage switch
c. Allow about 10 seconds for the instrument to stabilize and the vacuum chamber
to outgas; then ADJUST the current so that the current meter reads 10
milliamps.
>> if the current is too high, turn the needle valve toward decrease
>> if the current is too low, turn the needle valve toward increase
d. Coat your specimens for 2 to 3 minutes; you may need to touch up the current
flow occasionally to keep the meter at 10 milliamps.
5. After coating, turn high voltage switch OFF, dial to 0, main power OFF, and
immediately OPEN the air inlet toggle valve (lift). Open the lid, remove the glass
cylinder (lay it on its side where it won't roll), and remove your specimens to a safe
storage container. CLEAN THE CYLINDER inside with a paper towel wet with
absolute EtOH or acetone. Check that the vacuum seals are clean and greased and
reassemble the machine. Do not pump it down; leave it under atmospheric pressure.
28
Condenser Lens Setting
Appendix B
CONDENSER LENS SETTING

(PROBE DIAMETER)
This description is taken with some modification

from an old AMR publication.
The proper condenser lens setting, which determines the probe or beam diameter or
spot size, for the scanning electron microscope depends on the degree of resolution desired in
the image, for resolution is dependent to a large degree on the area covered by the beam or
probe as it is focussed on the specimen by the final (objective) lens. Resolution too is linked to
magnification, as outlined in the next section. In theory, the smaller to spot size the greater the
resolution and the sharper the image. However, a smaller spot size necessitates increased
amplification of the signal and this results in increased noise or graininess of the image. The
message is that you want a smaller spot size at the higher magnifications to give you the
resolution you may need, but the smaller spot size results in a need for more amplification of
the signal and thus noise (or graininess) is introduced into the image. This graininess does not
show up on the pictures; it can also be reduced on the viewing screen by slowing down the
scan speed.
The SEM has many operating parameters which can be adjusted over wide ranges to
accommodate a large variety of samples. The optimum condenser setting must be determined
by the actual operating conditions in use at the time. When you turn on the instrument, it will
start up with the magnification set at 100x and the condenser setting at 7; this is a medium spot
size, chosen as a middle point to initially obtain a usable image; it may or may not be the proper
setting for further specimen examination. This setting can be changed by turning the probe
current knob. In order to know when it is appropriate to change the spot size, you need a
feeling for what happens in the electron-optical system when you change it. This instrument
uses two condenser lenses to demagnify the relatively large beam source that is formed in the
electron gun. This is sort of like looking through the wrong end of a telescope - the image
formed of the object is smaller than the object itself - hence demagnification. There is also a
final lens to focus this demagnified or reduced beam onto the surface of the specimen. The
final lens contributes very little to the demagnification. The final spot size can be adjusted by
changing the strength of the condenser lenses; this is accomplished with the probe current
knob, as mentioned above. When one increases the probe current setting by turning the knob
clockwise, the condenser lens current in increases and the focal length of the lenses is
decreased. The beam spot is made smaller and the resolution of the image is increased.
However, the beam diverges more after it leaves the stronger lenses, and a smaller percentage
of the electrons in the beam pass through the aperture below the condenser lenses. This means
there are fewer primary electrons forming the beam and thus there will be fewer secondary
electrons generated at the specimen. The signal is reduced - due to smaller spot size and fewer
primary electrons. More electronic amplification (contrast setting) is needed to view the image,
and the noise level or graininess increases. If carried too far, the signal to noise ratio could
become so small that the image is too noisy to be useful, i.e., the noise level becomes so large
that the signal can no longer be recognized. To obtain a large signal to noise ratio for easy
viewing of the image, one must increase the signal level. Then not as much amplification is
necessary and there is a reduction in the noise level. The signal level can be increased if a
larger percentage of the electrons leaving the gun were to reach the specimen. This can happen
if one increases the focal length of the condenser lenses by turning the probe current knob
counterclockwise, thereby reducing the beam divergence beyond the aperture angle. But this
allows the spot to become larger and reduces the resolution. If carried too far, images will not
be sharp enough to be useful. The other option to increasing the signal output without
increasing the probe diameter is to slow down the scan speed so that the probe spends more
time in each position in its raster.
29
Condenser Lens Setting
When might you want to change the condenser settings, that is, move away from the
initial setting of 7? Once you have obtained an initial image, you may want to go to a lower
magnification to orient yourself and/or perhaps search for some particular feature. When you
turn down the magnification, the image may look grainy. The spot size resulting from the 7
setting of the probe current may not be big enough (have enough electrons in it) to yield a
signal to noise ration suitable for clear, low noise viewing at this rapid scan speed. So you
decrease the condenser lens setting by one or two (or more) positions. This increases the focal
length of the condenser lenses, reduces beam divergence, increases the diameter of the spot,
and increases the percentage of the beam that reaches the specimen, thus increasing the signal.
The result, after some fiddling with contrast, brightness and focus, is a clear, low noise image.
Remember that the spot size is bigger so the resolution will suffered a bit. However, this is not
noticeable at lower magnifications because the resolution if the viewing screen and your eye
become the limiting factors.
When you have found some particular feature, you may want to examine it at a higher
magnification, so you increase the magnification, focus and correct the astigmatism, and touch
up the contrast and brightness, but the image is still not sharp. Then you must increase the
probe current or strength of the condenser lenses. As outlined above, this decreases the
number of primary electrons reaching the specimen, as well as reduces the spot diameter. Thus
resolution is increased, as is the noise. You can try now to reduce the scan speed.
30
Resolution vs. Magnification
Appendix C
Resolution vs. Magnification

In the final analysis, the resolution of the scanning electron microscope, operated in secondary
electron mode, is determined by the diameter of the electron beam (probe) as it is focussed on
the surface of the specimen. As implied in the preceding section, one typically varies the beam
diameter as one changes the magnification, so as to provide optimum viewing conditions for
specific magnifications. In other words, one must select the appropriate beam size to permit
maximum sharpness and detail (i.e., resolution) in the image, either on the viewing CRT or in
the micrograph, and this optimum beam diameter varies with the magnification.
One needs to keep in mind that the average human eye can has the ability for spatial resolution
on the order of 0.2 mm. This means that in order for the detail in the specimen to be
appreciated clearly, it must be enlarged to at least this size. In the table below, one finds the
actual size of the smallest feature capable of being resolved by the eye (0.2 mm) at a number of
different magnifications; in other words, the true size of a 0.2 mm. feature found in the image.
The magnification refers to the final magnification of the micrograph and includes any
enlargement done during printing.
IMAGE FEATURE SIZE vs. FINAL MAGNIFICATION

Real size of 0.2mm
Magnification feature in image**
100,000X 2 nm.
50,000X 4 nm.
28,570X 7 nm.
20,000X 10 nm.
10,000X 20 nm.
5,000X 40 nm.
2,000X 100 nm.
500X 400 nm.
200X 1000 nm. (1 µm.)
** instrument resolution
This table contains some rather interesting information. For example, in order for a final image
to appear with maximum sharpness to the eye at 100,000X magnification, the instrument and
its camera, etc. must be resolving at 2 nm. or better. This is an unattainable goal, for on a very
good day, with the instrument working at its best, with a real good specimen, and at 8 mm.
working distance, the optimum resolution attainable is about 3.5 nm. This means that any
magnification above around 50,000 is truly “empty magnification”; it merely makes small and
fuzzy portions of the image appear larger and fuzzier. For the average user, working at the
typical distance of 39 mm. to maximize depth of field, the best attainable resolution is 10 nm.,
so 20,000X is pretty much the upper limit of useful magnification. One might wonder why
they built this instrument with a maximum magnification of 300,000X!!
Much of our work is done at magnifications less than 5,000x. At these lower magnifications,
it is not necessary to pursue those very small beam diameters, with all the attending problems
of decreased signal, increased amplification and accompanying noise, etc., if the eye cannot see
anything smaller in specimen dimensions than 40 nm. Don’t use a 10 nm. probe if you can’t
visualize the extra resolution because you haven’t magnified the image sufficiently. In other
words, match the probe diameter and the magnification. This is easily accomplished by using
the condenser lens current knob. Turn up the current to further demagnify the probe, making
the spot smaller; turn it down for a larger spot.
This description is taken with some modification from an old, AMR publication.
31
Specimen Preparation
Appendix D
SPECIMEN PREPARATION
A wide variety of specimens may be viewed with the scanning electron microscope. Their
preparative techniques may be equally diverse. This section intends only to outline on very
general terms some of the procedures more commonly used for viewing specimens in the
secondary electron mode.
Type of specimen
a. Those materials which are already hard and dry, such as mineral specimens, bone, hair,
scales, metal, etc.
b. Those which may not be dry, but are fairly rigid and resistant to distortions which
might result from desiccation. This group might include some hard bodied insects
(beetles, flies), seeds, pollen grains, wood, etc.
c. Those samples which are soft and wet; this includes most biological specimens. These
need to be dried carefully so as to avoid distortion. They are likewise in need of
chemical fixation in order to preserve structure.
Fixation
This applies especially to those samples represented in group c. above. Fixation stabilizes and
cross-links the proteins and to some degree the lipids of the specimen, thus preserving the
morphology in a life-like state, hopefully, and making the specimen more resistant to
subsequent treatments. Most commonly, one fixes in a buffered aldehyde solution, for
example 2% glutaraldehyde in 0.1 M cacodylate buffer, pH 7.4. This will stabilize the tissue,
helping it to resist collapse during drying, and it also may be advantageous because it hardens
the tissue so that it can be cut to expose fresh, internal surfaces and structures. A postfixation
is also desirable, and OsO4 is most frequently used. This helps stabilize the lipids and adds a
potential source of secondary electrons to the specimen. In this latter regard, investigators may
follow postfixation with an incubation in thiocarbohydrazide, and then more OsO4 in an effort
to get more metal into the sample.
The methods of applying the fixative are varied as well. Often simple immersion fixation will
work, especially if the sample is small or if only exposed surfaces are going to be examined. If
one wants to visualize the internal structures, immersion fixation may well be unsatisfactory,
for the interior may be poorly fixed. Perfusion fixation must be used; here the fixative is
injected, under gentle pressure, into the circulatory system. The disadvantage is that perfusion
is more difficult and time consuming.
Dehydration
For SEM, dehydration is an essential process; it eliminates water from the specimen, typically
by slowly replacing it with an organic solvent with a low surface tension, usually either ethanol
or acetone. This process would apply to those specimens in groups b. before they are air dried
and helps to reduce shrinkage and minimize collapse and distortion. It is also done prior to
critical point drying of those specimens in group c., where it provides a solvent more amenable
to exchange with the critical point drying solvent (liquid CO2).
Drying
Air drying is useful for those specimens that can stand it, such as those from group b. The
specimens are simply dumped out on a piece of lint free paper and the solvent is allowed to
evaporate.
32
Specimen Preparation
A more elaborate method must be used with more delicate specimens. It is known as critical
point drying and is applied to cells and tissues and other specimens that are likely to undergo
distortion and shrinkage as a result of drying. The procedure uses a specifically designed
machine, and the operational details can be found in the instruction manual in the lab. The
procedure is designed to remove the solvent from the specimen without damage from the
surface tension forces which would cause it to collapse as the fluid interface recedes through
the specimen. The water in the tissue is replaced by acetone or ethanol, and this in turn is
replaced by liquid CO2 under pressure and at 0°. This takes place in a special chamber, and
following the exchange, the chamber is sealed and the temperature, and thus the pressure too,
is elevated above the critical point for CO2. Then the pressure is slowly reduced while the
temperature is maintained, allowing all the CO2 to escape the chamber as a gas. The sample is
left behind, completely dry.
There are a number of solvents which have extremely low surface tensions. These too are now
finding their way into the specimen preparation procedures. Peldri II® and
hexamethyldisilazane are two examples.
Coating
This step is not necessarily essential, and indeed for some work it may be undesirable, but
typically specimens (from groups a., b., and c.) are coated with a thin layer of gold and
palladium before viewing. This surface layer provides an ample supply of secondary electrons
for the signal. Coating can be done in a vacuum evaporator where the metal is evaporated
under a high vacuum. The metal molecules leave the source and move in essentially straight
lines until they strike any surface, including the specimen. Then they condense out onto the
surface, forming a film. Because of this directionality, the specimen must be rotated to try to
get an even coat.
A better procedure uses a sputter coater. Here the metal molecules are physically removed
from the target source by ion bombardment. since they are in a poor vacuum, they are
deflected around inside the chamber. They too condense onto the surface of the specimen, but
since they lack directionality and are moving in random directions, they serve to coat all the
surfaces evenly. See Appendix A for operational instructions.
33
Vacuum Evaporator
Appendix E
VACUUM EVAPORATOR
Start-Up Procedure
NOTE: Observe the pump-down times; if they differ very much from those suggested
in these instructions, notify Judith Wopereis, Dick Briggs, or John Brady.
1. Turn ON cooling water to diffusion pump and check for adequate flow rate. The
valve is located in the sink in adjoining room.
2. Turn Main Power Switch ON. This switch is located on the right side of the
instrument (see above diagram).
3. Check to be sure that the Backing-Roughing Valve is in the MIDPOSITION, the

MAIN VALVE is CLOSED, and the CHAMBER VENT VALVE is CLOSED.
4. Turn the Rotary Pump ON. After about 3 minutes or less, the reading on the
Pirani gauge should be less than 2x10-2 mbar.
34
Vacuum Evaporator
5. Turn the Backing-Roughing Valve to BACKING.
6. Turn Diffusion Pump ON. Wait 20 minutes to allow the diffusion pump to warm-
up. During this time one can vent the bell jar, insert specimens and set up
electrodes (see following section for instructions on this), or, after the 20 minutes,
follow the pump-down procedures on the next page .
Sample Insertion or Change Procedure
1. Check that the Penning Gauge is OFF.
2. CLOSE the MAIN VALVE, if not already closed.
3. OPEN the CHAMBER VENT VALVE.
4. Remove cage and Bell Jar; be sure to keep gasket clean. Do not allow to roll onto
the floor - place it bottom up in the box provided!
5. Samples should be placed on white paper on the support platform to help you see
the thickness of the coat and to help keep the sample holder platform clean. They
should be lined up directly under the slot in the electrode assembly.
Electrode Replacement (to be performed after each firing)
If you are finished using the insturment, proceed through step 3, then reassemble the
electrode holder without a new, sharpened electode. If you are continuing, follow
through step 5. If you are just starting up, you will need to insert a new electrode,
starting with step 4.
1. Unscrew the power cable and remove the electrode assembly by first loosening the
set screw at the top of the assembly
35
Vacuum Evaporator
2. Loosen set screw B and remove the flat-ended electrode; use fine sand paper to
smooth the flat surface and then replace in the holder.
3. Loosen set screw A; remove the used, sharpened electrode.
4. If you are going to immediately use the coater, replace the sharpened electrode with
one of the appropriate size. Then pull back the end assembly C to apply spring
tension to the sharpened carbon rod and tighten set screw A. This spring tension
will hold the two electrodes together as the tip of the sharpened electrode is burned
off.
5. IT IS ABSOLUTELY CRITICAL THAT THERE IS A GAP BETWEEN THE

METAL COVERS HOLDING THE TWO ELECTRODES. IF THESE TWO
PARTS ARE TOUCHING THERE IS SHORT CIRCUIT WHICH CAN
SEVERELY DAMAGE THE INSTRUMENT (see above figure). If necessary,
adjust the set screws on each cover.
Pump-Down Procedure
NOTE: Observe the pump-down times; if they vary very much from those
suggested in these instructions, notify Judith Wopereis or Dick Briggs.
1. CLOSE Chamber Vent and replace Bell Jar and Cage, if not already done.
2. Turn Backing-Roughing Valve to ROUGHING
3. When Pirani Gauge reads less than (to the right of) 1x10-1 mbar (less than 1
minute) turn Backing-Roughing Valve to BACKING.
4. OPEN the Main Valve.
5. Turn Penning Gauge ON when vacuum reaches 6x10-2 mbar. The instrument
should reach 10-4 mbar within 5 minutes, maximum.
Coating
1. Wait until vacuum reaches less than (to the left of) 1x10-4 mbar on the Penning
gauge.
2. Turn Electrode Selector to position 1.
3. Turn Electrode Power ON.
4. Turn the Evaporation Control Toggle Switch to PULSE.
5. Turn Electrode Voltage to 8. Use position 7 for a “thick” electrode
36
Vacuum Evaporator
6. Apply coat by pressing the PULSE button in approximately one second intervals.
Check Penning between pulses; it should come down to less than 1x10-4 mbar.
Approximately six pulses will generally apply a good coat. Watch that the current
does not reach 100.
7. On completion of coating: turn Electrode Voltage to 0: Turn Evaporation Control

Toggle Switch to OFF: Turn Electrode Power OFF: Turn Penning Gauge OFF.
Follow Sample Insertion or Change procedures (above) to retrieve samples. If
more samples are to be coated, insert new samples during this time, then follow
instructions for Electrode Replacement and Pump Down.
Shut-Down
1. Prior to shut-down, the Bell Jar and Sample Holding Assembly should be
thoroughly cleaned and a new electrode left in the Electrode Assembly but with no
spring tension applied.
2. The system must be completely pumped-down and left under high vacuum (Follow
Pump-Down Procedure).
3. Once a high vacuum has been achieved (less than 1x10-4 mbar), turn OFF the
Penning Gauge.
4. CLOSE the Main Valve.
5. Turn Diffusion Pump OFF. Leave cooling water on for at least 20 minutes and
continue to back the Diffusion Pump (Backing-Roughing Valve in BACKING
position).
6. After 20 minutes or more, turn Backing-Roughing Valve to MID position. Turn

cooling water OFF. Turn Rotary Pump OFF; pull the plug as well. Turn main
power switch OFF.
7. Log out in Log-Book.
EMERGENCIES:
IN CASE OF FIRE, FIRE DRILL, OR POWER FAILURE, IF THERE IS

TIME, TURN OFF THE PENNING GAUGE, CLOSE THE MAIN VALVE,
AND TURN OFF THE DIFFUSION PUMP. IN CASE OF A REAL DISASTER,
PULL THE PLUG AND RUN!!
LET SOMEONE KNOW WHAT HAPPENED (notify Judith Wopereis, Dick

Briggs, John Brady, or Bob Newton).
37
Image Manipulation Using Photoshop
Appendix F
IMAGE MANIPULATIONS AND LABELLING USING PHOTOSHOP

by Tina (Weatherby) Carvalho
Biological EM Facility, University of Hawaii
Text scanned from Microscopy Today, #97-10, December, 1997
Ready or not, digital imaging in the microscope lab is here to stay, While I am one of those
fogies who insist that digital images are nowhere near as good as photographic prints, I've also
bowed to the inevitable, and am beginning to find ways to assist the clients in our multi-user
facility with their digital imaging needs. We can acquire digital images from our FESEM, from
our light microscope with a CCD, and from a flatbed scanner. Clients use their images for
image analysis, transport to others scientists via the Internet or World Wide Web, for reports,
slides, overhead transparencies, and publication. Generally, these images need to be
manipulated for use, just as one would manipulate a photographic print to correct any
deficiencies in exposure or area of view. Adobe PhotoShop is the software of choice for image
adjustment, accounting for more than 80% of the digital image-editing software market.
Photoshop is extremely powerful, and the ethics of using it to manipulate visual data is being
hotly debated. I strongly feel that any manipulation beyond what is outlined here would be
unacceptable, and I'm sure there are some who will feel that even this is too much, but that is
the subject for another article! The learning curve for Photoshop is steep, and the majority of
the available books deal at length with color management and effects. We, as scientists,
however are more interested in faithful reproduction and presentation of our visual data. To this
end, I've tried to distill the contents of many books and a two-day workshop down to the basic
techniques of interest to microscopists. The following is a step-by-step mini-tutorial designed
to lead you through the rudiments of Photoshop, taken from a manuscript I am preparing,
tentatively titled 'Photoshop Distilled: Basic Black and White Image Adjustment and Layout for
Scientists'. If you have any corrections, comments, or contributions, please feel free to e-mail
me at tina@pbrc.hawaii.edu.
Image Adjustment
1. Start up AdobePhotoshop 3.0 or 4.0.
2. Select File-Acquire to obtain an image with a scanner or other plug-in, or.
3. Select File-Open, then find and select the filename for an existing image, such as one
obtained with NIH Image. If your file doesn't have an extension recognizable to
Photoshop, click on Show All Files to find and then rename it.
4. Crop the image if necessary.

a. In 3.0 or 4.0 you may discard a few pixels around an image by selecting Image-
Canvas Size, selecting a slightly smaller size than your image, and then placing
your image in the center or to one side.
b. For more cropping, use the Crop Tool. In 4.0 press C to select it or hold the
mouse button down over the rectangle selection tool in the Toolbox to get a pop-up
menu of other selection tools, and select the Crop Tool. In 3.0 click on the Crop
Tool in the toolbox.
.
c. Drag the crop marquee around the portion of the image you want to keep, then
double-click inside it (4.0) or click once with the scissors cursor (3.0) to crop.
d. In 4.0 the Crop Tool may also be resized and rotated before cropping. In 3.0 you
must Select All of the image, then use Image.
38
5. Adjust image tones:

a. Select Image-Adjust-Levels and get the gray levels histogram
b. Adjust input levels:

1) Move the left, black arrow under the histogram to the right to select the
darkest input pixels, or enter a value in the left most Input Levels option box
(e.g.,20).
2) Move the right, white arrow to the left to select the lightest pixels, or enter
value in the rightmost option box (e.g., 235).
3) For midtone, or gamma correction, move the middle, gray arrow to the
left, or enter a value in the middle option box (e.g., 1.8). Be aware that in
order to get a good print you will need to adjust your on-screen image until
it shows the full range of grays but overall looks too light! This is due to the
fact that the image you see on your monitor has already had gamma values
applied to it that will not be sent to your printer.
c. Click on OK. Check image, and re-do gamma correction again, if needed.
6 Determine if image needs sharpening. Check your ethics barometer; will this
change your data?
a. View image in the size it will be printed (in 4.0 by selecting View-Print Size).
b. Resampling or resizing up (not a good idea) or down changes the apparent

sharpness. Best sharpening is accomplished by Filter-Unsharp Mask:
1) Set a value in the dialog box (e.g., 144%). Values less than about 25 will be
subtle and more than about 300 will be too dramatic.
2) Select a radius of perhaps 1 to 4 pixels.
3) Select a threshold of perhaps 0 to 16 levels.
4) Click OK.
7. Save your file using Save As... and select a name, format and location for it.
a. For greatest fidelity and transferability between computer platforms, select
TIFFwith no compression.
b. TIFF with LZW compression will compress the file size somewhat with no loss of
information, but may not be accepted by some computers.
c. The best compression is performed by selecting JPEG and then the highest level of
10 (4.0) or maximum setting (3.0). JPEG is a lossy compression scheme, meaning
that some information is lost, but often this loss is unnoticeable.
39
Applying Magnification Bars
Applying a magnification bar in Photoshop makes sense because then it is always available
with your image, no matter what its ultimate fate, such as reduction in print or as a slide. Other
labeling with text and arrows can be done either with Photoshop (the subject of the next
section) or, perhaps more effectively, with additional programs such as PowerPoint,
PageMaker, Quark Express, etc.
1. With your image file open, go to Window-Show Rulers. You may set the units to
centimeters, inches, or picas in File-Preferences -Units.
2. Select the color you want your bar as your foreground color. To get true black or white,
click on the tiny black and white rectangles at the bottom of the Toolbox; black will be the
foreground color. White can be brought to the foreground by clicking on the curved arrow
pointing between the two.
3. Double-click on the Line Tool to get the options palette. Select Anti-aliased, Normal,
and 100% opacity. Enter a line width (e.g., 4 pixels) and make sure that Arrowheads
Start or End are not selected.
4. Use the Line Tool cursor lines at the top and side rulers to position and measure the length
of the line that you draw by pressing the mouse button and dragging on your image.
5. If you made a mistake or don't like your line, use Edit-Undo before you do anything
else.
6. Make sure you remember what this magnification bar represents. You mayappend notes to
the image by selecting File-File Info. This information will be available to you each time
you open the image.
Labeling images in Photoshop
Addition of type to digital images is best done with software designed for that purpose, such as
QuarkExpress, Adobe PageMaker, or Microsoft PowerPoint. With Photoshop, type is treated
as another bitmapped image and, as such, will exhibit pixelation at low resolutions. However,
at high resolutions the jaggedness is not so apparent. Check the anti-aliasing box in the Text
options palette if you wish to have PhotoShop soften the edges of the type. Type applied to an
image will have the same resolution as that image.
1. To apply text to an image, click on the Type Tool (T) in the Toolbox. Yourcursor will
become an I-beam. Position this cursor over the image approximatelywhere you would like
the type and click. You will get a dialog box.
a. Select Font, Size, Style(s) and Alignment.
b. Type your label in the box, then click OK.
c. Your text will appear in the foreground color and as a selection with marching
ants (3.0) or as its own layer (4.0). To turn of the marching ants in 3.0 so that you
can more readily see the text, select Select-Hide Edges, but note that it still
remains a selection until you click elsewhere or otherwise deselect it.
40
d. While selected or in its own layer, text can be moved with the Move Tool or with
the keyboard arrows. If you make a mistake, the type, while still selected, is in its
own layer (4.0) or as a floating layer (3.0) and can still be deleted with the delete
key (3.0) or thrown in the trash in the layers palette (3.0 and 4.0).
e. Deselect to paste the label down in 3.0. If this is a mistake, you can Edit-Undo if
it is the last thing you did. In 4.0 you can delete this layer now or later.
f. In 4.0 text always appears as a new layer. Non-text additions, such as arrows,
cannot be applied to text layers.
2. To apply arrows to an image, double-click on the Line Tool in the Toolbox to see the
Line Tool Options palette.
a. Select Line Width in pixels (e.g., 4), Anti-aliasing, Normal, and 100% Opacity.
b. Select Arrowheads Start or End, depending on whether you want the arrowhead
to start where you begin to drag your cursor, or end where you stop your cursor.
c. Select Shape-Width (e.g., 400%), Length (e.g., 600%), and Concavity (e.g.,
16%), the amounts of which are related to the Line Width. Click on OK.
d. The arrow will be in your foreground color, so check it now.
e. Your best bet is to place arrows in their own layer. Go to Window-Show

Layers palette and click on the little folded paper icon at the bottom, or to the right-
pointing black triangle on the top right and select New Layer from the pop-up
menu. This also works for 3.0.
f. Drag the mouse across your image to make an arrow.
g. In 3.0 you may hide the marching ants but maintain the selection by going to
Select-Hide Edges, allowing you to better position the arrow.
h. In 3.0 you may move the arrow around with the Move Tool or keyboard arrows.
In 4,0 you must use the Move Tool, and it will move the entire layer.
I. If you don't like your arrow, go to Edit-Undo while it's still the last thing you did
(3.0, 4.0) or throw away the layer (4.0).
j. Save As a new filename so that the labeled image stays separate from your
unlabeled one. Trust me, you may want your unlabeled image back someday!
41
Using Photoshop 4.0 to Label Scanning Electron Micropgraphs
Appendix G
USING PHOTOSHOP 4.0 TO LABEL SCANNING ELECTRON

MICROGRAPHS
by John Minter; from the Microscopy Listserver
This is the recipe that I use in Photoshop 4.0 to put Black on White scale markers, text, and
symbols onto micrographs. The results look just like the rub-on transfers that I used to use.
1. Create a layer (Photoshop 4.0 does this automatically with text tool). You must use
Photoshop image mode for the layer option.
2. In that layer in the font and font size that you want, type the text. Add a black line at an
appropriate length and width and any other text, symbols, arrows, etc. that you want to put
on the micrograph. By using the layer, you preserve the original micrograph in the
background layer. you can use the info window to draw lines to particular lengths. If
other layers are created when new text is added, merge those layers. Don't merge them
with the background layer!
3. Select all (ctrl-A in the PC) The marquee will be around the whole layer.
4. You have to move the selected region up then down with an arrow key. (This is done in
the PC with the Ctrl-shift-arrow key in the PC). What this does is to select all of the
objects in the layer individually. A Marquee should be around each object.
5. Select the foreground color as white. (You are going to write a white border around each
Marquee.)
6. Go to Edit-Stroke and select the width of the white line you want (Width) and select the
Outside option. For 300 dpi images at about 4" x 5", I suggest a font size of about 14
(Arial) with a Width of about 3-4 pixels for the stroke width. This will write a white
border 4 pixels wide around all of the selected black features.
7. Deselect (ctrl-D)
8. If you want to save this as image in another format such as TIF or BMP, then you have to
Merge the layers and save the image in that mode.
Note: you should have anti-aliasing selected for all this.
This technique works very well for me. You can make them look like real Rub-ons with
another technique and offsetting the white a little, but that is another story.
We use Adobe Photoshop. In version 4.0, be sure to select the outlined text tool so that the
letters are surrounded by marquees when you are done typing. Type your text on the image.
Before you de-select the text, go to the pallette region of the toolbar and exchange foreground
(usually black) and background (usually white) colors. Then go to the "Edit" menu and select
"Stroke" chose a 2-3 pixel stroke width "outside" the letter. This makes an outlined text in any
font on your system.
John Minter
email: minter@kodak.com
42

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EL ECTRON MIC

Section 1 Scanning Electron Microscope Operation

Section 2 Energy Dispersive X-Ray Microanalysis

Scanning Electron Microscope

JEOL JSM 6400

General View of the Column

3. Make sure the stage controls are set as follows:

THE ROD MUST BE SLID IN AND OUT CAREFULLY. IF THERE IS

GENERATION OF THE ELECTRON PROBE

PROCEDURE #1: ELECTRONIC

PROCEDURE #2: MANUAL

CL COARSE This should be set to 7; it is changed by turning

MAG Set this to around 100 with the

BRIGHTness Set to around 225 with the brightness knob on

DIST ANCE KNOB IS T URNED

NEW ALIGNMENT PROCEDURE FOR SEM

Recheck saturation; if it is off, resaturate and then repeat steps 2 through 6.

VIEWING THE SPECIMEN

3. A fine focusing must be accompanied by a correction of the astigmatism. If the image

2. To change the existing text at the bottom of the image:

3. To delete or return text at bottom of screen:

5. To write additional material on the screen:

c. Use the CLS key to erase the text.

4. The processing differs with the two different film types:

4. Dispose of the film pack and used up coaters properly.

Detailed instructions accompany each film pack.

PROCEDURE #1: ELECTRONIC

PROCEDURE #2: MANUAL

Energy Dispersive X-Ray

These x-rays can be analyzed either by wavelength dispersive methods or energy

Characteristic X-Ray Emission

X-Ray Continuum (bremsstrahlung)

Effect of Accelerating Voltage

Oxford Link ISIS

Load a carbon-coated sample in the microscope.

Now you must choose a job. It will be

The most important thing about choosing a

Beam Current Setup

X-ray Spectrum Acquisition

Identifying the Elements Present

4. You are now ready to begin coating:

CONDENSER LENS SETTING

This description is taken with some modification

Resolution vs. Magnification

IMAGE FEATURE SIZE vs. FINAL MAGNIFICATION

3. Check to be sure that the Backing-Roughing Valve is in the MIDPOSITION, the

5. Turn the Backing-Roughing Valve to BACKING.

Sample Insertion or Change Procedure

1. Check that the Penning Gauge is OFF.

2. CLOSE the MAIN VALVE, if not already closed.

3. OPEN the CHAMBER VENT VALVE.

Electrode Replacement (to be performed after each firing)

3. Loosen set screw A; remove the used, sharpened electrode.

5. IT IS ABSOLUTELY CRITICAL THAT THERE IS A GAP BETWEEN THE

2. Turn Backing-Roughing Valve to ROUGHING

4. OPEN the Main Valve.

2. Turn Electrode Selector to position 1.

3. Turn Electrode Power ON.

4. Turn the Evaporation Control Toggle Switch to PULSE.

5. Turn Electrode Voltage to 8. Use position 7 for a “thick” electrode

7. On completion of coating: turn Electrode Voltage to 0: Turn Evaporation Control

4. CLOSE the Main Valve.