Zeiss EVO 50 Scanning Electron Microscope

Operating Instructions
A. TURNING ON THE INSTRUMENT:

1. Make sure the main nitrogen tank valve and the small
side tank valve are open. DO NOT adjust the pressure
regulator.

2. If the EVO is in the STANDBY position (YELLOW button lit on the front of SEM), then go to the ON
position by depressing the GREEN button.

3. Computer should boot up automatically. If it does not, depress the computer’s ‘on’ button located on the
front of the main CPU.

4. From the main desktop, double click on the Zeiss SmartSEM User Interface icon. An EM Server panel
will appear on the right-hand monitor with a progress bar shown on the right.

5. An EM Server panel will appear on the right-hand monitor

with a progress bar shown on the right-hand side of the panel.

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6. Once the progress bar reaches 100%, an EM Server Log On box will appear on the left-hand monitor.
For the User Name type in mmiller and for the Password type in miller

7. When EM Server is completely loaded, a black window will appear on the left-hand monitor. You may
need to maximize to fill the screen.

8. From the top menu tool bar on left-

hand monitor, select Vacuum

and then select Vacuum Status from the

drop-down menu.

A SEM Control panel should now appear on the right-hand monitor.

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9. From the top menu tool bar on the
left-hand monitor, select Stage

and then select Navigation from the

drop-down menu.

A Stage Navigation panel will

appear on the right-hand monitor.

B. INSERTING A SPECIMEN:

1. From the SEM Control panel (right-hand monitor), select the Vacuum tab and then select Vent. Select
Yes on the confirmation box.

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2. Wait until the SEM chamber has come to room pressure; this will take approx. 2-3 minutes.

3. Once the chamber is at room pressure, open the chamber by pulling the chamber door toward you.

4. Using forceps (NO HANDS/FINGERS) insert the pin of your support stub in to one of the eight holes on
the specimen holder.

5. Gently tighten the stage set screw. Caution: Do not over-tighten the screw. When it stops, you stop.

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6. Slowly and carefully close the chamber door.

7. Once you are sure the chamber door is closed, from the SEM
Control panel (right-hand monitor) select the Vacuum tab and
click on the Pump button.

Progress of the pump down process will be shown in the Vac

Status window on the Vacuum tab.

The first message displayed will read “Waiting Turbo Speed” ( turbo pump is coming up to speed)
The second message displayed will read “Waiting Penning” (high vacuum penning gauge is activated).
The third message displayed will read “Pumping”.

8. Chamber vacuum is displayed in the System Vac window on the Vacuum tab. At a vacuum reading of
approx. 1.0e-004 (i.e. 1.0 x 10-4) mbar a GREEN check mark (%) will appear next to the word Vac at the
bottom of right-hand monitor indicating that the vacuum is sufficient enough to turn on the electron beam.

Special note: Under certain circumstances you might also have a GREEN check mark (%) next to the word GUN and a RED X
next to the word EHT (i.e. Vac:% Gun:% EHT:X) or in some cases you might have two RED Xs next to the words GUN and
EHT (i.e. Vac:% Gun:X EHT:X).

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C. TURNING ON THE BEAM:

1. Click on the word Vac at the

bottom of the right-hand monitor and
select the Beam On option.

The beam will “run up” slowly as shown by an

indicator meter at the bottom of the screen.

2. Once the beam is on completely, an All: % button will appear in the lower right-hand corner of the right-
hand monitor.

D. GUN/BEAM ALIGNMENT (Gun Aperture Alignment and Beam Alignment):

1. From the SEM Control

panel (right-hand monitor),
select the Aperture tab and
click on the Emission button.
A GREEN target pattern (t)
will appear on the left-hand
monitor.

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3 Adjust contrast with the Contrast Knob so that a large white spot is visible in the middle of the screen.

4. Adjust position of the white spot with the TILT button. Using the left mouse button, click and hold on to
the small red ball (C). While holding down on the left mouse button and while watching the white spot,
move the ball around to center the white spot.

5. Click on the Normal button on the Aperture tab to

return to normal imaging.

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E. CHANGING VOLTAGE (EHT):

The accelerating voltage or EHT of the SEM is displayed in the white date zone strip located at the bottom
of the left-hand monitor. An EHT setting of 20kV is used most frequently the widest variety of sample
types. However, the EHT can be adjusted up or down depending on specific needs.

If you find it necessary to adjust the voltage (EHT), do the following:

1. Double click on the letters EHT shown on the white data zone strip located on the bottom of the left-
hand monitor. Type in the desired kV value. Hit OK and the new kV value will be displayed.

2. If you make a change to the EHT (kV), you may need to realign beam. Refer back to Section D:
Gun/Beam Alignment if you need help with this.

F. ADJUST WORKING DISTANCE: (Only Accurately Determined on a Focused Specimen)

1. Check to see that you are positioned on the stub/sample you want to image. This can be done by looking
at the display shown on the Stage Navigator panel.

If you need to reposition the stage, either use the large joystick on the stage controller box OR you can
double-click on the desired position on the Stage Navigator display. This will move the stage automatically.

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Once the general area of interest is visible on the monitor, you can move a specific area of the sample to the
center of the viewing screen by clicking on the Centre button located on the upper portion of the left-hand
monitor. When the Centre button is depressed, a Green \ symbol is displayed on the screen. Position the
Green \ symbol over the item you want to center and click on it. The item of interest will be moved
automatically to the center of the screen.

2. Adjust brightness and contrast in order to see an image. First click on the Levels button located on the
upper icon menu tool bar of the left-hand monitor. This will display the Brightness and Contrast values at
the bottom of the right-hand monitor.

Adjust brightness to approximately 50% with the Brightness knob located on the right-hand side of the
keyboard (hard panel). Next adjust the contrast to approximately 20-35% with the Contrast knob also
located on the right-hand side of the keyboard.

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3. Focus on your specimen with the FOCUS knob to determine the current working distance. The working
distance will be displayed on the white data zone strip located at the bottom of the left-hand monitor.

4. For high resolution work and/or for imaging at high magnification, a good starting working distance could
be in the range of 7-8 mm. For lower magnification work or where a greater depth of field is needed (e.g.
rough surface with lots of topography), a working distance of 8-12 mm is generally sufficient.

If you need to change working distance, first turn on chamber camera with the CAMERA button which is
located on the upper portion of the keyboard. This will allow you to see inside the chamber.

5. Using the Z direction lever (small joystick), carefully raise or lower

the stage. Be sure to refocus often (you will need to turn off the Camera
to focus on your image). Repeat until you have reached the desired
working distance. THE WORKING DISTANCE SHOULD NEVER
BE LESS THAN 6MM.

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As a general guide, you can safely raise the stage up so that the top of your sample is level with the top of the
white ‘pillar of light’ that is visible inside the chamber. This ‘pillar of light’ is a reflection of the camera
shining off the back wall of the chamber.

6. It is important to focus on the highest point on your specimen especially on specimens with a dynamic
topography AND/OR on the specimen stub with the tallest sample. Focusing in a “valley” may place a
“peak” a few millimeters closer to the detectors than the 6mm.

AGAIN, THE WORKING DISTANCE SHOULD NEVER BE LESS THAN 6 MM. Shorter working
distances run the risk of hitting the detectors inside the chamber. If the working distance is below 6 mm, use
the Z drive lever (small joystick) to adjust the working distance WD to 6mm or greater.

A proper working distance depends on your desired magnification range, your focus and imaging needs, and
the overall topography of your specimen. For high resolution work and/or for imaging at high magnification,
a good starting working distance could be in the range of 7-8 mm. For lower magnification work or where a
greater depth of field is needed (e.g. rough surface with lots of topography), a working distance of 8-12 mm
might work better.

G. CALIBRATING STAGE NAVIGATION (this will ensure you are viewing the correct sample):

1. Start out on a ‘known’ hole on the SEM stage. This can either be an empty hole on the stage or you can
use position #2 as a reference since it is covered with tape and always blank. Take note of which position
you are actually viewing.

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2. From the Stage Navigation panel (right-hand monitor), see if the stage
navigation graphic (pink and grey image) is correct and corresponds to the
actual image/position you are seeing on the left-hand monitor. If it is
correct and the display matches what you see on the actual SEM image,
then the stage navigation graphic is correct and the stage is calibrated.

If the graphic does not match what you are seeing on the left-hand monitor,
then the graphic needs to be calibrated. Proceed to step 3 to calibrate the
stage navigator.

3. Click on the Settings button on the Stage Navigation panel. A Stage Nav Settings panel will appear.
On the right-hand side of this panel, find the Holder Rotation Offset Calibration slider.

4. Move either the slider or click on the + , arrows or click in the

bar to make adjustments until the graphic on the Stage Navigation
panel corresponds to the actual image position you see on the left-
hand monitor.

5. When finished, close the Stage Nav Settings panel by clicking on the small red x. Be sure to leave the
Stage Navigation panel open.

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H. FINDING and OPTIMIZING AN IMAGE:

1. Once the working distance is properly adjusted, begin imaging your specimen. It is probably best to begin
with the following general settings:

Magnification I Probe Current (pA) Scan Mode (Speed) Focus &

Found under Gun tab Found under Scanning tab Stigmation
less than 1000x Large (~700-900pA) Fast Scan Speed (~1-5) Coarse
~1000-5000x Small (~200-700pA) Slower Scan Speed (~3-6) Coarse
~5000-10,000x Smaller (less than 200pA) Slower Scan Speed (~5-7) Coarse
Over 10,000x Smallest (~25-100pA) Slower Scan Speed (~8+) Fine

Magnification: Adjust magnification with the Magnification knob located on the keyboard. The
magnification will be displayed on the white data zone strip located at the bottom of the left-hand monitor.

I Probe Current: Adjust probe current with slider found under the Gun tab of
the SEM Control panel. To increase or decrease the probe current level, either
move the slider or click on the + , arrows or click in the bar to make
adjustments. Caution: Do not go above 900pA on the probe current. Doing
so can damage or burn out the filament.

Reducing the probe current will have two affects on quality of your image. As
you reduce the probe current you will first notice the image gets dark. Adjust
Contrast knob on keyboard to compensate. Image will now appear somewhat
grainy or ‘snowy’. Slow the scan rate down to compensate for te graininess as
described below.

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Scan Speed: Scan speed can be adjusted in two
ways.
You can adjust scan speeds by first clicking on the
Scanning tab found on the SEM Control panel
and then use the drop down menu to select a scan
speed. The higher the number the slower the scan.
Alternatively, you can use the + (plus) and -
(minus) buttons located on the right-hand side of
the keyboard to adjust scan speed. Depressing the
- (minus) buttons slows the scan rate down.

2. Centering Objective Aperture:

a. Increase magnification to approx. 1200x and Focus on a somewhat round shaped object or particle.

b. Place a Reduced box around the object by clicking on the Reduced button located on the left-hand side
of the keyboard. A reduced box with a Green border will appear on the screen. The tiny boxes around the
perimeter of the reduced box are indications this reduced box is active. The active reduced box may be
resized or moved as needed.

c. From the Apertures tab, check the Focus Wobble box and check the Fast Wobble box. Set the Wobble
Amplitude to at least 75% or higher using the slider. Selecting the Focus Wobble box will cause the
round object in the reduced box to pulsate indicating the objective aperture is aligned or it will cause the
object to shift back and forth in one direction or another indicating the aperture is not aligned.

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d. Increase scanning to a speed of 1 or 2 with the Scan Speed + button or
make this selection in the drop down menu on the Scanning tab of the
SEM Control panel. The faster scan rates make it easier to adjust the
objective aperture.

e. Locate the X and Y centering micrometers found on the front of the SEM
column.

If the object within the reduced box is pulsating but remains stationary, then the
objective aperture is aligned properly and no further adjustments are necessary.

If the object within the reduced box is shifting somewhat left and right,
then turn the Y micrometer either clockwise or counterclockwise very
slowly until this side to side motion stops and the object remains
stationary and is pulsating. The micrometers are very sensitive so use
caution when turning them. Turn them very slowly as you watch the
image so you are able to evaluate the affect of turning the micrometer.
If turning the micrometer one way makes the shifting motion worse,
then stop and turn the micrometer slowly in the opposite direction.
Only a slight amount of adjustment is necessary to align this aperture so
GO SLOW.

If the object within the reduced box is shifting in a somewhat up and

down motion, then turn the X micrometer either clockwise or
counterclockwise very slowly until this up and down motion stops and
the object remains stationary and is pulsating. Again, the micrometers
are very sensitive so use caution when turning them. Turn them very
slowly as you watch the image so you are able to evaluate the affect of
turning the micrometer. If turning the micrometer one way makes the
shifting motion worse, then stop and turn the micrometer slowly in the
opposite direction. Only a slight amount of adjustment is necessary to
align this aperture so GO SLOW.

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If the object within the reduced box is shifting from corner to corner or diagonally, then the aperture is out of
alignment in both the X and Y direction. If this is the case, pick one of the micrometers and turn it very
slowly until the object’s direction of motion switches from diagonal to either side to side or up and down
depending on which micrometer you chose first. Now adjust the second micrometer until the image is
stationary and is pulsating.

f. When finished, uncheck the Focus Wobble box to return to normal

viewing.

g. Use the Focus knob on the keyboard to refocus the image if needed.

3. Hysteresis:

a. Increase magnification to approx. 1200x and Focus on a somewhat round shaped object or particle.

b. Place a Reduced box around the object. To do this, click on the Reduced button located on the left-hand
side of the keyboard. A box with a green border will appear on the screen. The tiny boxes on the perimeter
of the reduced box are indications this reduced box is active. The active reduced box may be resized or
moved if necessary.

c. Once the object or structure is in focus, depress both the Shift button and the F2 button on the
keyboard. This will initiate the Hysteresis step as indicated on the screen. Allow this process to finish.

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d. More than likely, the image will come back out of focus after this
process. Refocus the image by turning the Focus knob clockwise.

e. Once the image is back in focus, perform the Hysteresis step a second
time by depressing both the Shift button and the F2 button on the
keyboard again. Allow this process to finish. Refocus the slightly out of
focus image by turning the Focus knob counterclockwise slightly.

f. Remove reduced box from the image by depressing the Reduced button
once again to complete the Hysteresis process.

I. PHOTOGRAPHY:

1. Adjust scanning speed with Scan Speed +/- buttons or go to the

Scanning tab on the SEM Control panel to make changes. For normal
viewing, any scan speed can be used. Under Noise Reduction select
Frame Avg. and N=1

2. For photography: any speed can be used. For low magnification situations, a
scan speed of 3-6 is generally adequate. Use a slower scan speeds for higher
magnifications, higher resolutions and where an image of higher quality is
desired. Photographs may be taken with Noise Reduction = Frame Avg N = 1
option selected. However, switching the Noise Reduction to Line Avg with an
N value of 4 (four) may result in a higher quality image.

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3. Make your scan speed and noise reduction selection and
allow the image to scan. During a slower scan, a Green
scan line marker will appear on the left-hand edge of the
image. The purpose of the marker was to show the
position of the actual scan line. Following a previous
software update, the Green marker now runs ahead of the
actual scan line by a few centimeters.

4. When the actual scan line reaches the bottom

of the screen, either press the FREEZE button
located on the upper portion of the keyboard or
press the FREEZE button located on the
Scanning tab of the SEM Control panel.

A RED dot (C) will appear in the lower right-hand corner of the
image. This in an indication that the image is “frozen.”

5. On the upper tool bar of the left-hand monitor, click on File and then select Save Image from the drop-
down menu.

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6. An Export TIFF panel will now appear on the left-hand monitor. Click on Change Directory and locate
your folder. If you need to create a new sub-folder, scroll to the top of the directory and select Images and
then click on the New button. On the subdirectory panel, type in the new folder name and click OK.

7. Select a folder and click on the Close button.

8. Type in a filename and click on the Save [file].tif button in the lower left corner of the panel. Make sure
your file now appears in the directory list.

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9. Close the Export TIFF panel.

10. Once your image is saved, a BLUE dot (C) will now appear in
the lower right-hand corner of your image. This means your
image file was saved but the image is still “frozen.”

11. Press either the FREEZE button again on the keyboard or click on the Unfreeze button located on the
Scanning tab of the SEM Control panel to unfreeze your image and resume normal scanning.

J. EXCHANGING SPECIMENS:

1. Turn off the beam by first clicking on All: %which is located in the lower right-hand corner of the right-
hand monitor tool bar and then select Shutdown Gun. The beam will “run down” slowly as shown by an
indicator meter at the bottom of the screen.

2. From the SEM Control panel, select the

Vacuum tab and then select Vent. Select Yes
on the confirmation box.

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3. Wait until the specimen chamber has come to room pressure; this will take
approx. 2-3 minutes.

4. Once the chamber is at room pressure, open the chamber by pulling the
chamber door toward you.

5. Before removing aluminum stub, loosen set screw by

turning it counterclockwise one half a turn. Do not
loosen screw all the way. Head of screw should NOT
be showing.

6. Using forceps (NO HANDS/FINGERS), remove

your specimen(s) from the chamber and then place
another set of specimen stub(s) in to the stage specimen
holder. Gently tighten the corresponding stage set
screw. Caution: Do not over-tighten the screw.

7. Gently close the chamber door.

8. Once you are sure the chamber door is closed, from the SEM Control
panel (right-hand monitor) select the Vacuum tab and click on the Pump
button.

Progress of the pump down process will be shown in the Vac Status
window on the Vacuum tab.

The first message displayed will read “Waiting Turbo Speed” ( turbo pump is coming up to speed)
The second message displayed will read “Waiting Penning” (high vacuum penning gauge is activated).
The third message displayed will read “Pumping”.

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9. Chamber vacuum is displayed in the System Vac window on the Vacuum tab. At a vacuum reading of
approx. 1.0e-004 (i.e. 1.0 x 10-4) mbar a GREEN check mark (%) will appear next to the word Vac at the
bottom of right-hand monitor indicating that the vacuum is sufficient enough to turn on the electron beam.

Special note: Under certain circumstances you might also have a GREEN check mark (%) next to the word GUN and a RED X
next to the word EHT (i.e. Vac:% Gun:% EHT:X) or in some cases you might have two RED Xs next to the words GUN and
EHT (i.e. Vac:% Gun:X EHT:X).

10. Turn on the beam by following the instructions provided in Section C: Turning on the Beam

K. END OF SESSION:

1. Turn off the beam by first clicking on All: %which is located in the lower right-hand corner of the right-
hand monitor tool bar and then select Shutdown Gun. The beam will “run down” slowly as shown by an
indicator meter at the bottom of the screen.

2. From the SEM Control panel, select

the Vacuum tab and then select Vent.
Select Yes on the confirmation box.

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3. Wait until the specimen chamber has come to room pressure; this will take
approx. 2-3 minutes.

4. Once the chamber is at room pressure, open the chamber by pulling the
chamber door toward you.

5. Before removing aluminum stub, loosen set screw by

turning it counterclockwise one half a turn. Do not
loosen screw all the way. Head of screw should NOT
be showing.

6. Using forceps (NO HANDS/FINGERS), remove

your specimen stubs from the chamber.

7. Gently close the chamber door.

8. Once you are sure the chamber door is closed, from the SEM Control
panel (right-hand monitor) select the Vacuum tab and click on the Pump
button.

9. Leave the black SmartSEM screen open on the left-hand monitor

10. Leave the EM Server box open (located on the right-hand monitor).

11. Leave the computer on during the day.

12. Leave the SEM (EVO 50) in the ON position (GREEN button lit).

13. Copy your image files to a USB using the USB hub located on the desk just behind the keyboard.

14. Record your SEM usage time in the SEM logbook. This is your actual usage time and not your
reserved time. Please be accurate with your entry. Do not round up or round down your time.

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L. SHUTTING DOWN FOR NIGHTS AND WEEKENDS

1. Turn off the beam by first clicking on All: %which is located in the lower right-hand corner of the right-
hand monitor tool bar and then select Shutdown Gun. The beam will “run down” slowly as shown by an
indicator meter at the bottom of the screen.

2. From the SEM Control panel, select

the Vacuum tab and then select Vent.
Select Yes on the confirmation box.

3. Wait until the specimen chamber has come to room pressure; this will take
approx. 2-3 minutes.

4. Once the chamber is at room pressure, open the chamber by pulling the
chamber door toward you.

5. Before removing aluminum stub, loosen set screw by

turning it counterclockwise one half a turn. Do not
loosen screw all the way. Head of screw should NOT
be showing.

6. Using forceps (NO HANDS/FINGERS), remove

your specimen stubs from the chamber.

7. Gently close the chamber door.

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8. Once you are sure the chamber door is closed, from the SEM Control
panel (right-hand monitor) select the Vacuum tab and click on the Pump
button.

9. Close the black SmartSEM screen on the left-hand monitor. Select YES when the prompted. This will
close the black imaging screen.

10. Close the EM Server box which is located on the right-hand monitor. Select YES when prompted.
This will close the EM Server box.

11. Copy your image files to a USB using the USB hub located on the desk just behind the keyboard.

12. Now shut down the computer. From the main Windows desktop, click on the START button (bottom of
the left-hand monitor) and click on the Shut Down option.

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13. After a few minutes, both monitors will appear in grey-scale and a Shut Down Windows panel will
appear. Select Shut Down from the drop down menu and click OK.

14. Let Windows close completely. Small blue lights on

monitors will change to orange after shut down.

14. Once the computer is off, place the SEM in the STANDBY position by depressing the YELLOW button
located on the front side of the microscope.

14. Record your SEM usage time in the SEM logbook. This is your actual usage time and not your
reserved time. Please be as accurate as possible with your entries. Do not round your time up or down.
Your start time begins when you first enter the SEM room to begin your SEM session and not when you start
imaging your sample. Your stop time is when you are about to leave the SEM room at the conclusion of your
SEM session. Your stop time includes removing your stubs from the chamber and shutting down the SEM.

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USING BACKSCATTER DETECTION

1. From the SEM Control panel, click on the Detectors tab.

2. On the Detectors tab, select QBSD from the drop-down list found next to Signal A.

3. From the top menu tool bar on the left-hand monitor, select Detection. From the drop-down list that
appears, select BSD Control. A BSD Control panel should appear on the right-hand monitor.

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4. On the BSD Control panel, use the drop-down arrow (middle of this panel) and select High.

5. Adjust the quality of the backscatter image with Brightness and Contrast knobs. You may need to
increase brightness and decrease contrast or decrease brightness and increase contrast to get a good image.
The quality of a backscattered image is also dependent on the speed at which the image is scanned. Slowing
the scan speed will improve the quality of the image.

6. To use both the secondary electron (SE) detector and the

backscatered electron (BSE) detector simultaneously, go to the SEM
Control panel and click on the Detectors tab.

From here, click on the Mixing box. This will give you the option to
select one detector in the Signal A channel and a second detector in the
Signal B channel.

With the Mixing box checked, the Signal = slider is now active.

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When the slider is moved fully to the right (Signal = 1.000), 100% of
the image seen on the screen is coming the detector shown in Signal A.

When the slider is moved fully to the left (Signal = 0.000), 100% of
the image seen on the screen is coming from the detector shown in
Signal B.

Moving the slider back and forth will vary the contribution from each of the two detectors.

7. When finished with BSD, please uncheck the Mixing box and return Signal A to the SE detector.

8. Now close only the BSD Control panel on the right-hand monitor.

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ANNOTATIONS

1. If the Annotation panel is not visible on the screen, depress both Ctrl + A on the keyboard. The
Annotation panel should now be visible on the right-hand monitor. Reposition it as necessary.

2. For measurements, click on the Point to Point Measure icon

located on the Annotation panel.

3. Go over to your image and locate the feature you

want to measure. Click and hold down the left mouse
button on the edge of the feature you wish to measure
and while holding down the left mouse button, drag
the mouse and cursor to the other edge and then
release the left mouse button. A line and measurement
data box will appear on your image. The data box will
display line length and line angle.

To move a line or box, simply click either to activate and move with left mouse button. The small blue
boxes that appear on the line or around the data box are indicators that they are active and can be moved.

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4. To delete a single line or box, first activate that line or box by clicking on the item with the left mouse
button. Small blue boxes will appear indicating that item is now active. Now click on that activated item
with the right mouse button and select Delete.

5. To delete all of the annotations on your image, go to the Annotation panel and click on the red X icon
then confirm with Yes when prompted.

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