Carl Zeiss SMT - Nano Technology Systems

®
EVO MA and LS Series
Scanning Electron Microscopes
Operator User Guide
Operator User Guide
 EVO Series SEM Operator User Guide

ZEISS EVO MA and LS Series

Scanning Electron Microscopes
Operator User Guide

Contents
♦ Section 1. General Information .............................................1
. 1. Abstract............................................................................................1
. 2. Important Information ......................................................................2
. 3. Safety Precautions (Warnings and Cautions) ....................................2
. 4. Notes about the Mains Supply ..........................................................4
. 5.Use of Liquid Nitrogen......................................................................4
. 6. Do’s and Don’ts ...............................................................................6

♦ Section 2. Switching on the SEM ...........................................7

. 1. User Interface Description ................................................................7
. 2. Start Up ............................................................................................9
. 3. Starting the SEM Software (Log On) ................................................9
. 4. SEM User Interface Log Off........................................................... 10
. 5. SEM User Interface Close .............................................................. 11
. 6. SEM Shutdown .............................................................................. 12
. 7. Emergency Shutdown ................................................................ 12
. 8. How to use Help............................................................................. 13
. 9. Contents of Help Software.............................................................. 15

♦ Section 3. Operation of the SEM ......................................... 16

. 1. Consider the properties of the specimen.......................................... 16
. 2. Guide to SEM Operating Parameters .............................................. 17
. 3. Sequence Guide to Operation of the SEM...................................... 22

♦ Section 4. Basic Design Principles of the SEM .................... 23

. 1. Introduction.................................................................................... 23
. 2. Resolution ...................................................................................... 24
. 3. Depth of field ................................................................................. 25
. 4. Microanalysis ................................................................................. 26
. 5. Beam specimen interactions ........................................................... 28
. 6. Detectors ........................................................................................ 29
. 7. How it works .................................................................................. 31
. 8. Specimen preparation guidance ...................................................... 35

♦ Section 5. Appendices ......................................................... 39

. 1. An introduction to Macros .............................................................. 39
. 2. Table of Pressure Limiting Apertures ............................................ 49
 EVO Series SEM Operator User Guide

Section 1. General Information

1. Abstract
The Operator User Guide introduces new users to the ZEISS EVO® MA and
LS Series Scanning Electron Microscopes (SEM).
It provides information about important safety precautions, start up/shut down
procedures, the operation of the instrument and how to get help. The basic
design principles are also explained.
It is not intended to be an operator manual as all information about the
operation of the SEM is contained within the comprehensive on-line help
facility.
All reasonable steps have been taken to ensure that this publication is correct
and complete, however should any user be in doubt about any detail,
clarification may be sought from Carl Zeiss SMT Ltd or their accredited
representative. The information in this document is subject to change without
notice and should not be construed as a commitment by Carl Zeiss SMT Ltd.
Carl Zeiss SMT Ltd accepts no responsibility for any errors that may appear in
this document.
Copyright © Carl Zeiss SMT Ltd, Cambridge, England, 2008
All rights reserved. The contents of this publication may not be reproduced in
any form, or communicated to a third party without prior written permission of
Carl Zeiss SMT Ltd.
Microsoft and MS-DOS are registered trademarks and Windows is a
trademark of the Microsoft Corporation

Part Number: 3547060275006

Date: January 2008
Version: 1.0
Printed in England

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EVO Series SEM Operator User Guide

2. Important Information
General
Software License Agreement
The software for the ZEISS EVO® MA and LS Series may be used
only in compliance with the provisions of the licence agreement
supplied with the software. The Carl Zeiss SMT Ltd rights for the use
of the SmartSEM software delivered with the instrument are laid
down in the agreement which is delivered with the software for the
instrument and becomes an integral part of the instrument record.
Maintenance
Only correct care and maintenance of the instrument will assure
optimum performance of the microscope. The maintenance work to be
carried out by the user is described in Care, Maintenance and Trouble
Shooting in the Help section. Carefully read and follow these
instructions, because only then will correct operation of the microscope
be guaranteed.
Only if care and maintenance are completed in compliance with these
instructions, will the Carl Zeiss SMT Ltd guarantee apply. After the
warranty period, ask your local ZEISS agent about a service contract.
Regular servicing by ZEISS qualified engineers will guarantee that the
instrument functions properly and help to obtain reliable and
satisfactory results.

3. Safety Precautions (Warnings and Cautions)

Mains Voltage
When the SEM is shut down, mains AC voltage will still be present
both within the instrument plinth and on some of the external cables.

ALWAYS SWITCH OFF THE SEM MAINS SWITCH BEFORE

STARTING ANY MAINTENANCE WORK.
The mains CIRCUIT BREAKER is located on the rear of the SEM
plinth next to the mains input cable.

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IN AN EMERGENCY, PRESS THE RED ILLUMINATED

STOP BUTTON.
The emergency stop button is located at the top of the plinth front
panel. Pressing it will interrupt the distribution of the mains supply
within the plinth.
Do NOT use this switch as a means of isolating the SEM from the
electricity supply, because some parts of the instrument will still be
live. To isolate the SEM use the circuit breaker and the mains switch.
EHT Voltages
The EHT voltages present in this instrument can be lethal. Safety
interlocks are built into the system to protect the operator and to
safeguard the equipment. Do not override the interlocks.
X-Rays
The SEM is designed to ensure that any radiation of X-rays is within
the permitted limits. This is achieved by the use of appropriate
materials, and the fitting of radiation shields. When fitting to the
chamber any accessory that has not been supplied by Carl Zeiss SMT
Ltd or when installing your own equipment, make sure that X-radiation
levels are checked (contact Carl Zeiss SMT Ltd for more information).
If an X-ray shield has been removed for any reason, always replace it
before switching on the electron beam.
Rotary Pump Exhaust
A small quantity of oil mist will be discharged from the rotary pump.
This can contaminate the environment and create a health hazard in
confined surroundings. Always use an exhaust system, such as an oil
mist filter, or vent the pump discharge outside the building.
Venting system
The SEM can be vented using either
• Dry nitrogen gas (the preferred method)
• Atmospheric Air that has been passed through filters in a specially
designed container filled with a dessicant.
• Atmospheric Air that will probably contain moisture (non-preferred).
Solvents
Careless use of solvents can constitute a health hazard. Follow the
safety procedures or the Hazard Data Sheet relevant to the solvent
being used. Take precautions to avoid spillage, skin and eye contact,
vapour inhalation and use only in small quantities. It must be noted that
some solvents may remove the paint surface from the microscope.

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EVO Series SEM Operator User Guide

Corrosion
Although all the components used in the SEM are protected from
corrosion by painting or plating, Carl Zeiss SMT Ltd cannot take
responsibility for corrosion caused by storing or operating the SEM in
adverse atmospheric conditions. To avoid corrosion of the internal
components in the column, always keep the column and chamber under
vacuum, even when the SEM is not being used to examine specimens.
Panels and Covers
For safety reasons and to comply with CE and EMC regulations, never
operate the SEM with any panel or cover removed.

4. Notes about the Mains Supply

Input Requirements
The SEM operates only within the range of 100 – 230V @ 50-60Hz
single phase with a consumption of 2.5kVA. In countries where the
mains voltage is different, an autotransformer should be used. The
rotary pump is powered seperately. All accessories use the same
voltage as the electricity supply to the SEM.
Mains Switching
With the mains input cable connected to the electricity supply and the
circuit breaker switched on, the lamp in the STOP button should be
illuminated. Pressing the STANDBY button will supply power to the
vacuum system components and the vacuum system will operate
automatically. Power is supplied to the computer and the control
electronics only when the START button is pressed.

5.Use of Liquid Nitrogen

Where the EDS accessory for the ZEISS EVO® MA and LS Series uses
liquid nitrogen the following safety precautions must be strictly
observed.

Caution: Danger of cold burns by liquid nitrogen on the skin!

Because of the low temperature, liquid nitrogen may destroy the skin
severely in a way that is similar to scalding by hot fluids or burns.
Splashes or drops of liquid nitrogen generate an insulating layer of
gaseous nitrogen between the skin and the drop due to the body
temperature (Leidenfrost’s phenomenon). However, a greater amount
of liquid nitrogen which comes into contact with the skin or soaks
clothes is hazardous, because it eliminates the protective Leidenfrost
effect. The drastic thermosteresis of liquid nitrogen on the skin will
cause the aforementioned burn symptoms.

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Caution: Implosion risk when pouring liquid nitrogen into

glass dewar vessels!
Dewar vessels are double-walled, with a vacuum between the walls
(thermos bottle principle). Always wear a face mask when handling
glass vessels or, preferably, use metal Dewar vessels. These are of
higher stability and withstand the temperature shock when pouring in
liquid nitrogen.

Caution: Explosion risk if liquid nitrogen penetrates between

the walls of the Dewar vessel!
Liquid nitrogen which penetrates the insulating space through pores or
fissures of a defective Dewar vessel will evaporate due to heat
absorption on the inner side of the outer wall. The vessel will explode
because the then gaseous nitrogen cannot escape quickly enough.

Caution: Fire risk due to enrichment of liquid nitrogen by

liquid oxygen!
Oxygen liquefies at -183°C, and the oxygen in the air condenses on the
surface of the liquid nitrogen (-196°C) and enriches it gradually by
liquid oxygen. At concentrations of more than 5% liquid oxygen,
flammable material such as dust, paper, wood shavings or foam
material will burn when falling into the Dewar vessel.
Therefore:
Always cover the vessel so that evaporating nitrogen can escape, but
the entrance of air is inhibited and no flammable material can fall in.
Use a vessel with a narrow neck which is also more economic because
of slower nitrogen evaporation.

Caution: Risk of oxygen deficiency due to nitrogen enrichment

of respiratory air!
The amount of evaporating nitrogen in the workroom should not
noticeably reduce the relative amount of oxygen in the respiratory air.
The permissible lower limit of the oxygen content is 20% vol.
(normally 21% vol).
Standard values for maximum amount of liquid nitrogen in surface
rooms:
Naturally aired room: 30: l /cubic meter room volume
Constantly air-conditioned
room: 150:1 /cubic meter room volume

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Liquid nitrogen may be used or stored in subterranean rooms only if,

• controlled mechanical ventilation is available
• evaporated nitrogen (which because of its higher density
accumulates at the ground or in depressions) can drain off
without damage.

6. Do’s and Don’ts

Important Information
The following instructions should be followed to ensure correct
operation of the instrument:
• Ventilation ports and cooling surfaces should be left uncovered
and kept free from obstructions.
• Call the maintenance service in case of incidents or irregularities
and if in doubt switch off the instrument to prevent extended
downtime and expensive repairs.
• All safety precautions mentioned in this chapter and in
connection with the routines described in this instruction manual
must be strictly observed. This applies in particular to the safety
precautions for the use of liquid nitrogen.
Important Prohibitions
The following prohibitions must be observed for safe operation of the
instrument. Failure to comply with such prohibitions may damage your
health or the instrument and also forfeit any warranty claims.
• Do not operate the instrument in case of frequent high-voltage
arcs or strong instabilities.
• Do not operate the vacuum system when there are vacuum leaks.
• Do not apply force to stuck mechanical components.
• Do not unscrew sheathings and protective cover sheets.
• Do not shunt safety circuit breakers.
• Do not remove electrical safety fuses and mechanical safety
devices and earth leads.
• Do not use non-approved vacuum grease and pump oil.
• Do not operate the instrument outside the installation
specifications for an extended time.
• Do not mount or connect attachments that are not approved by
Carl Zeiss SMT Ltd.

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 EVO Series SEM Operator User Guide

Section 2. Switching on the SEM

1. User Interface Description
SEM Software
Format and terminology used throughout this help text, and especially the software help are
explained below in detail.

Font Used for Example Explanation

BOLD CAPITAL Command input VENT Push Vent key
Acknowledgements STORING Data storage
Warnings ERROR Error message
<BOLD CAPITAL> Keyboard input <ENTER> Push Enter key
[boldface] Optional parameters [.TIF] Extension which
can be omitted
Boldface Accentuation Note: General
accentuation in
text

Keyboard Entries
<Key1 + Key2> Special codes <CTRL + Z> Push <CTRL>
and <Z> jointly
<Key1, Key2> Standard input <D,1> Push <D> first, then <1>

Other Designations
DR: Disk drive identification A: Disk drive A (Floppy Disk)
\Path Path input C:\TEST Subdirectory TEST on drive C
.TIF Extension NAME.TIF Image named NAME

Icons

Mark for special attention.

Tip for improved operation.

Caution! Safety precautions; if not observed, danger of

damage to persons or instrument.

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EVO Series SEM Operator User Guide

Additional help available, click!

Reference to drop down menu.

The Mouse:
The mouse is the primary adjustment and selection control
of the microscope. There are carefully defined
conventions governing its use:

The left mouse button:

The normal Windows convention of pointing and
clicking is used for icon selection or menu selection,
dragging is used for moving or sizing objects and for
parameter adjustment.

The middle mouse button:

The middle button is used for parameter adjustment.

The right mouse button:

The right mouse button is used to display a pop-up menu
while over the image. When the cursor is over an icon and
the right button is pressed, the control panel associated
with that icon will pop up.

The Keyboard
In addition to the mouse control of parameters, many
keyboard keys have been assigned to special functions
acting as “accelerators” or “short cut keys“ and giving
direct access to the function. For a list of short cut keys
see Keyboard Keys in Software Help.

The Monitor
The monitor serves as the visible link to the user. The
SEM image together with all other necessary information
for the operation is shown here.
(The resolution of the monitor should be set to 1280 x
1024 pixels).

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2. Start Up
Start-up from Cold
After a power failure or planned power interruption, restart the Zeiss EVO according to
these instructions:
• Turn on mains power to microscope.
• Set the Circuit Breaker on rear panel of the plinth to ON (the red
switch (3) should illuminate). 1 2 3
• On the plinth front panel locate the middle Standby Button (2)
and press it. (This applies power to the vacuum system and is
considered as being the standby condition.)
• The vacuum system will start automatically.
• On the plinth front panel locate the green Start Up Button (1) and press it. (This applies
power to the rest of the system and the computer.)
● Switch on the display monitor.
The Windows software is loaded from the hard disk and the desktop
image with the program icons and the Task Bar is displayed on the
monitor.
The next and most important action is to load the user interface as
described in the section below.

3. Starting the SEM Software (Log On)

Locate the SmartSEM Logo Icon on the desktop if
installed and double click on it
or
Select the Windows START, All programs and navigate
to SmartSEM, and click on SmartSEM Interface .
The SmartSEM program is then started. Wait until the EM Server
program (this controls the hardware) is loaded and then log on to the
SEM. You must insert your name and the password that was supplied
by your systems administrator or installation engineer.

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EVO Series SEM Operator User Guide

Alternatively you may sign in as GUEST with no password.

• Click in the text field for User Name and enter your name.

• Change to Password box with the Tab Key and enter your password. Since the
password is hidden during input, only *** are displayed.
• Click on the OK button.
The software is now loaded, and the SEM user interface is displayed
on the monitor.
When the Vac box in the taskbar displays a tick the SEM is ready to be
used.

4. SEM User Interface Log Off

This function is called from the system menu, it closes the SEM user
interface and puts the SEM hardware into standby mode. Any other
applications using the server such as REMCON and the Administrator
are also closed and you will be logged off the server.

In the Menu Bar click on File and Log Off

or

Type <ALT + F> then <G> to start the shut down

sequence.

<Yes> starts the close down action.

<No> breaks it off and returns to the application.

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This includes the following:

• Setting the EHT to standby conditions.
• Setting the lens currents to standby conditions.
• The EM server application is still running which can be seen on the Task Bar.
If the Zeiss icon is double clicked the log on procedure must be followed to re-establish the
SEM control interface.

5. SEM User Interface Close

This function is called from the system menu and terminates the
operation of the SmartSEM User Interface software only. Any other
applications, such as REMCON and The Administrator that are using
the server, remain active and logged on.

In the Menu Bar click on File and Exit

or

Type <ALT + F> then <X> to start the shut down

sequence.
For safety you are requested to confirm the action and to close the User
Interface.

<Yes> starts the close down action.

<No> breaks it off and returns to the application.
The action is as follows:
• Save operating conditions if required.
• Perform an orderly shutdown of each control module. This includes shutting down the
high voltage set (EHT).
• Save any critical data.

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EVO Series SEM Operator User Guide

• Save in files any user specific data in the User Directory, and record the current user
directory.
• The server is still running and should be closed by clicking the server application on the
System Tray and then clicking the application close button .
• Windows can then be closed in the normal way as described below, once the EM server
has been closed.

6. SEM Shutdown
This function shuts down Windows and removes the power from the
computer, the display monitor and the SEM electronics. The vacuum
system stays ON.

Before you attempt to shut down the instrument, always follow

an orderly exit sequence on the SEM application by following the
procedures given in section SEM User Interface Close. Otherwise loss
of data may occur.

Click on in the Desktop Task Bar and select

<Turn Off Computer>.
On the plinth front panel locate the middle Standby
Button (2) and press it. 1 2 3
The instrument electronics and the computer are
powered down. You may notice a delay until the
system switches OFF.

7. Emergency Shutdown
Use this shutdown mode only in case of an emergency!

All current working data and conditions will be lost!

On the Plinth locate the red Emergency Button (3) and press it.
The software is forced to terminate immediately, the
computer and the instrument electronics are switched off and the
vacuum system is stopped.
The power is removed from the instrument’s electronics.

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 EVO Series SEM Operator User Guide

If you need a complete Power OFF shut the instrument down in

an orderly fashion and then use the Circuit Breaker on the back panel of
the plinth. Then turn OFF the mains power to the microscope.

8. How to use Help

There is extensive help available through the SmartSEM User Interface program. Use the
following techniques to extract the information of interest.
• Context Sensitive Help for the functions called from the Image Window.
• Windows Style Help from the Menu Bar by clicking on Help.

Context Sensitive Help

Key(s):Function:

Displays help for the function that is active.

If the Help window is already open, pressing <F1> displays the “Using
Windows Help“ topic.

+ Changes the cursor to so you can get help on a

specific command, screen region, or key.
You can then choose a command, click the screen region, or press a key
or key combination you want to know more about.
(This feature is not available in all Windows applications.)

Cancels the Context Sensitive Help mode. Brings back the

normal cursor.
Windows Style Help
Select Help from the Menu Bar. A Sub-Menu opens with following
topics:
• Help on Help What you should do to get Help

• Search How to access Help Topics

• SmartSEM Help The overview of SEM specific help

(main page)

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• Keys Help Explains which keys have which functions

Clicking on one of the Sub-Menus takes you into more detailed information.

Searching for Topics

The Contents tab shows the structure of the Help System as books , and pages .
• To open a book click on it.
• Pages show the paragraphs in the book.
• Click on a page to show the help text.
• The Index tab shows all topics in a list.
• Use the Scroll Bar to see the text or start typing the topic in the top window.
• Mark the topic and select Display.
• The Find tab provides full text search. Follow the directions showing up on the tabs as
you go through the program.

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9. Contents of Help Software

SmartSEM Help cover page

Contents contents of Help Software

Glossary explains special terms

Important information similar to the information given

in this manual

Description of Instrument describes the components of the

instrument

SEM Operation describes how to operate the

instrument

Operating Functions describes the principles of

operation of components

Care, Maintenance and describes how to look after the

Troubleshooting instrument and achieve best
results

Operating Principles describes the function of system

components

In some of the graphics in the Software Help you can point and click onto features to
obtain additional information.

This is available whenever the normal cursor

changes to the hand cursor.

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EVO Series SEM Operator User Guide

Section 3. Operation of the SEM

1. Consider the properties of the specimen
Specimens that are studied in the SEM can be divided into two main categories, namely
conductors and non-conductors and these will be discussed in greater detail later.
However there are several factors to consider during all specimen preparation and these
are given below.

1. The size and weight might necessitate some reduction of the specimen to fit holders
and to ease specimen manipulation for observation.
2. Mineral and metallurgical specimens may require polishing. Etching and
electropolishing may also be required.
3. The specimen should be able to withstand the vacuum of the SEM as it might
become damaged or deformed.
4. The specimen should be clean and dry if it is to be used in high vacuum mode, i.e.
free of dust, moisture, oils and grease, as their presence can lead to charging,
contamination and longer pump down times. Otherwise use VP or EP vacuum
mode.
5. Porous samples will also take a long time to pump out in high vacuum mode.
6. Coat non-conductors to prevent charging if allowed or VP mode is not available.
Observation of the specimen at low kV (start at 1 kV) is another good alternative
where “charge balance” can be obtained.
7. The specimen should be firmly attached to the specimen stub or holder either
mechanically or by gluing. Silver dag and carbon dag can be used as conductive
glues for small samples. Carbon tabs/tape are also convenient but may not provide
good mechanical stability.
8. There should be good electrical connection between the surface of the specimen
and specimen stub or holder for non-conductors.
9. Health and Safety procedures regarding the handling of the specimen during its
preparation should be observed.

Conductive specimens are generally observed in what might be described as a conventional

SEM however Non-conductive specimens are being increasingly observed in a specially
modified SEM called the Variable Pressure SEM (VPSEM) where gas can be introduced
into the specimen chamber. The benefit is that the specimen can be viewed without the
necessity to “coat” as the negative charge built up on the surface of the non-conductive
specimen by the high energy primary electrons is neutralised by the positively charged ions
that are produced when the gas molecules are ionised by collisions with secondary,
backscattered and primary electrons. It also slows down the dehydration of moist specimens
that leads to structural collapse.

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However it is now possible to observe moist specimens in conjunction with cooling of the
specimen and the water control option using SEMs with EP vacuum pumping modes.

Specimen chamber pressure range

VP 10 to 400 Pascals

EP 10 to 3000 Pascals

2. Guide to SEM Operating Parameters

The conditions given below are suggestions and the operator may find that different
values will give better information for the types of specimens that are being investigated.

Conductive specimens
1. General Microscopy
EHT =20 kV Iprobe = 200 pA WD = 15 mm
Filament set to the “Long Life Mode” (select the option on the Gun panel)
Filament I set to 1st peak for magnifications < 10 kx (gives longer filament life)
Filament I set to 2nd peak for magnifications > 10 kx (for better resolution)
Detector = SE with collector bias > + 300 V
MC∅ = 30 µm
Cycle time to reduce noise = 20 sec

2. EDS
EHT = 20 kV for metals and minerals
EHT = 7.5 kV for semiconductors and organic materials
Filament I set to 1st peak for Qualitative analysis
Filament I set to 2nd peak for Quantitative analysis
Iprobe = 1000 pA or adjust for 30 % deadtime
WD = 8.5 mm for a 35° take off (elevation) angle

Key:
MC∅ = Mid-Column Aperture (final aperture in HV mode)
PLA = Pressure Limiting Aperture

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Detector = BSD
Final aperture = 30 µm
Cycle time = 20 sec or longer for X-ray mapping
3. High resolution
EHT=30 kV Iprobe = 10 pA WD = 5 mm
Filament I set to 2nd peak and untick the “Lonflife Mode”
MC∅ = 30 µm
Detector = SE with collector bias + 400 V
Cycle time to reduce noise =1.3 mins or longer
Warning – remove the BSD to its parked position

Non-conductive specimens
1. High vacuum mode.
EHT = 1 kV Iprobe = 10 pA WD = 5 mm
Filament I set to 2nd peak
MC∅ = 30 µm
Detector = SE with collector bias + 400 V
Use scan speed 3 with frames to average = 30 to reduce noise.

2. Variable pressure (VP) mode.

EHT = 25 kV Iprobe = 250 pA WD = 8.5 mm
Filament I set to 2nd peak
MC∅ = 750 µm
PLA = 100 µm
Detector = BSD or VPSE
Cycle time = 20 sec or longer
Chamber pressure = 10 Pa for BSD detector, 40 Pa for VPSE detector or
adjust to eliminate charge disturbances.

Key:
MC∅ = Mid-Column Aperture (final aperture in HV mode)
PLA = Pressure Limiting Aperture

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3. Extended Pressure (EP) mode for cooled hydrated samples.

EHT = 30 kV Iprobe = 300 pA WD = 8.5 mm
Filament I set to 2nd peak
MC∅ = 750 µm
PLA = 100 µm
Beam Sleeve = 500 µm
Detector = BSD or VPSE
Cycle time = 20 sec or longer
Chamber pressure = 650 Pa to slow dehydration
Temperature of specimen to retain water at 650 Pa = 1°C.

4. EP mode for hydrated samples.

EHT = 30 kV Iprobe = 300 pA WD = 5 mm
Filament I set to 2nd peak
MC∅ = 750 µm
PLA = 100 µm
Beam Sleeve = 500 µm
Detector = BSD or VPSE
Cycle time = 20 sec or longer
Chamber pressure = 2000 Pa to retain water at 20°C.

Key:
MC∅ = Mid-Column Aperture (final aperture in HV mode)
PLA = Pressure Limiting Aperture

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Imaging wet samples using the Peltier coolstage

To study hydrated specimens it is recommended that a Peltier cooling stage and a water
control system be fitted to the SEM. This will enable fresh specimens to be examined with
little or no loss of water in the SEM environment (the sample is kept fully hydrated during the
pump down).

1. Vent the chamber and remove the lens mounted BSD [if fitted] and place it in the
“park” position.
2. Fit the 100µm EP upper aperture.
3. Fit the 500µm lower aperture/ BeamSleeve®.
4. Remove the round right hand blanking plate from the stage door.
5. Detach the Peltier assembly from its parking plate.
6. Feed the Peltier stage holder through the hole in the stage door and carefully rest it
with its pipe work on the stage.
7. Clamp the Peltier vacuum flange to the stage door.
8. Suitably arrange the pipe work and attach the Peltier stage holder to the SEM stage
with the 3mm clamp screw [Deben Manual].
9. Place the sample onto the Peltier sample stub and tighten the clamp screw but do not
over tighten as this deforms the PTFE end of the screw.
10. Re-fit the BSD if required.
11. On the Aperture Control Panel click on “Select Aperture”.
12. Select the EP aperture, the panel with then expand.
13. Then select the 500µm BeamSleeve®.
14. From the main menu select “Tools”, “Administrator” and type your “User name” and
“Password”.
15. In the Administrator panel select “Configuration”, “Other” and tick the “Peltier fitted”
box.
16. Scroll down and select the USB connection and tick the “Humidity Option” box.
17. Remove the lens mounted BSD [if fitted] from the “park” position and place it in the
“active” position.
18. Ensure that the Z is low enough before closing the stage door.
19. Pump the chamber and wait for vacuum ready.
20. Open the docking panel and from the menu select the “Extended Pressure Control”.
21. On the dialog panel tick the “Peltier” option and click on the “Purge Settings” option.

The aim of purging is: i) to remove air from the water bottle (will take a while to be
completed), and ii) to fill the chamber with just water vapour). Choose between step 22 and
28 depending on the condition of the water bottle.

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22. If the Water Kit has recently been fitted, filled with water, or has not been used for a
while:
23. Set the “Purge cycles” to 5.
24. Set Purge Max to 1000 Pa.
25. Set Purge Min to 20 Pa.
26. On the Purge Control dialog panel select “WET” if the EP Mode states “DRY”.
27. The purging then starts:
i) The pressure in the chamber will vary between the Max and Min levels.
ii) Bubbles can be seen in the water bottle.
iii) All air is removed from the water bottle (will take a while to be completed).
iv) Continue from step 29.

28. If the system has recently been used in the “WET” mode:
29. Set the “Purge cycles” to 1.
30. Set Purge Max to 950 Pa.
31. Set Purge Min to 640 Pa.
32. On the Purge Control dialog panel select “WET” if the EP Mode states “DRY”.
33. Ensure the Peltier control box is switched on.
34. On the “Extended Pressure” panel set the “Peltier Target” to 1°C and suitably adjust
the “Humidity Target”.
35. Adjusting the position of the “Green Cross” in the Phase Diagram will change the
environment of the sample; water vapour, water, or ice.
36. Turn the beam on and start with the imaging.

Suitable SEM operating parameters for imaging the sample

These will depend on the type of sample being examined and the nature of the detail you are
expecting to see [be prepared to experiment]
EHT = 20 KV, I Probe = 200 pA or higher, WD = 8.5 mm or less, Detector = VPSE, BSD,
or EPSE

“Use the Chamberscope and Stage Navigation panel for positioning the specimen”.

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3. Sequence Guide to Operation of the SEM

1. Start up the SEM by pressing green Start Up button.
2. Start the SEM software by double-clicking on the SmartSEM logo icon.
3. Vent the chamber, load samples quickly and pump the chamber.
4. Wait for the vacuum "ready".
5. Switch on the beam by either selecting “File” then "Load state" or the "Gun" in
the bottom right of the display.
6. Set "brightness" to 50 % and adjust "contrast" if the image is too bright/dark.
7. Check filament saturation and gun alignment.
8. Focus the beam on the sample/holder at low magnification and then position the
sample as required using the joystick.
9. Progressively increase the magnification, focussing the beam until the sample is
being viewed at greater than 5000x.
10. Align the final aperture if necessary utilising the Focus Wobble mode.
11. Correct for any observed astigmatism.
12. Select a suitable scanning/noise reduction mode to remove any noise from the
image.
13. Suitably adjust the "brightness" and "contrast" of the image.
14. Freeze the image.
15. Annotate the image if required.
16. For printing or exporting the image, just right-click the mouse in the image area
and select the appropriate option from the "send to" menu.
17. To end the SEM session the operator may LOG OFF or shut-down.

For further assistance on above select Help, then SmartSEM Help and SEM operation and
select the appropriate topic.

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Section 4. Basic Design Principles of the SEM

1. Introduction
The scanning electron microscope (SEM) is used to examine microscopic detail of solid
specimens that may have initially been viewed in a light microscope but subsequently found
to require SEM examination in order to provide information not available from the light
microscope.
The image is produced by scanning an extremely small focused beam of electrons (adjustable
down to a few nm in diameter) across the surface of a specimen in an array of picture points
(pixels) usually 1024 by 768 pixels. High-energy electron bombardment of the specimen
causes signals to be emitted at each pixel. These are collected and their intensities are used to
produce images of the specimen by modulating the brightness of equivalent pixels on a TV
monitor.
Initial viewing of the specimen uses the secondary electron emission signal to provide an
image very similar in shape to that seen in the light microscope. Unlike the light optical
microscope that displays images in true colour, the SEM presents intensity images where zero
signal is displayed as black, intermediate signals as shades of grey and maximum signal as
white.

The SEM image shows a small screw and some broken coils of tungsten wire from a car
headlamp.

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In order to see why one would use an SEM it is useful to compare it with the light microscope
as it has three distinct advantages - better resolution, greater depth of field and the ability to
carry out X-ray microanalysis.

2. Resolution
The resolution limit of any microscope is defined as the minimal perceptible distance between
two points and is partly dependent on the wavelength of the illumination. The wavelength of
electrons is considerably smaller than that of light, hence the improvement in resolution.
The better resolution of the SEM enables surface detail not normally visible in the light
microscope to be viewed at high magnifications.
Resolution of the light microscope > 200 nm (wavelength of green light = 500 nm) with
magnifications up to 3,000 times.
Resolution of the SEM < 5 nm (wavelength of 30 kV electrons = 0.007 nm) with
magnifications up to 1 million times.

This SEM image shows silver dendrites on a copper support mesh.

The image is displayed in dual magnification mode where the area shown within the box on
the left side has been further magnified on the right side. The markers on both sides of the
image indicate the scale.

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3. Depth of field
The SEM has a considerably better depth of field (DOF) than a light microscope i.e. the
ability to maintain sharp focus of detail as the specimen surface height changes. This
facilitates the examination of specimens that have a very irregular topography.
If the depth of field of a light microscope is said to be 1, the depth of field of the SEM is
Electron beam

α
F’

Depth of
field Plane of optimum focus

F’’

Region in focus

typically 300 times better.

This image is of conductive foam where the depth between layers is approximately 0.5 mm.

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4. Microanalysis
The elemental composition of the specimen can be determined from the x-ray spectra
excited by high-energy electron bombardment of the specimen. Each element has its own
unique spectra that can be identified, just like a “finger print”. Particles as small as 1
micrometre can be analysed. Detection and quantification of the spectra is achieved in one
of two ways.
1. EDS
An Energy Dispersive X-ray detector System (EDS or EDX) is the more popular as this
detector can display all the elements present in the specimen as they are being collected
and enables rapid identification to be performed. It enables the composition of the
specimen to be determined to an overall accuracy of about 1% and detection sensitivity
down to 0.1% by weight.
A semiconductor material is used to detect the x-rays together with processing electronics
to analyse the spectrum.

The above diagram shows an EDS spectrum from iron oxide. The vertical axis displays
the number of x-ray counts whilst the horizontal axis displays energy in KeV.
Identification lines for the major emission energies for iron [Fe] are displayed and these
correspond with peaks in the spectrum, thus giving confidence that iron has been correctly
identified. Similarly there is a peak appearing at the marker for oxygen (O).

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2.WDS
Where resolution and sensitivity are of the utmost importance particularly for elements of
low atomic number an additional detector called a Wavelength Dispersive X-ray
Spectrometer (WDX or WDS ) would be required. This detector collects one discrete
element at a time and hence is relatively slow in identifying elements in a specimen of
unknown composition. However its detection sensitivity is in the order of 0.01 % by
weight thus making trace analysis possible. Also, because its resolution is approximately
10 times better than that of an EDS, it can clearly identify elements that are severely
overlapped in the EDS spectrum.
A range of diffracting crystals is used to analyse the spectrum with precision mechanics to
control the positional relationship between the specimen, crystal and a gas proportional
detector for counting the x-rays.
The operation of wavelength spectrometers is based upon the principle of diffraction
according to Bragg’s law that states:
nλ = 2d sin θ
Where n = 1, 2, 3,……. The order of diffraction
λ = wavelength of the x-rays being diffracted
d = lattice spacing of the diffracting crystal
θ = Bragg angle (the angle between the crystal surface
and the incident and diffracted x-rays)

The relationship between the energy of an x-ray (E) and its wavelength (λ) is given by the
equation
E = 12.396 E in kev
λ λ in Angstroms
In both systems the number of x-rays generated by the beam for each element in the
specimen is proportional to its concentration.

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5. Beam specimen interactions

The high energy focused beam of electrons PE (primary electrons) excites a variety of
different signals from the specimen surface. Each of these signals shown below has a
detector in order that its information can be displayed as an image.

1. Secondary electrons SE are excited from the top surface layer of the specimen (0 to
10 nm).They are defined as having an energy range from 0 to 50 eV with the
majority having an energy from 3 to 5 eV. The secondary electrons emitted at the
point of impact of the beam are referred to as SE1 type electrons and it is the amount
of these electrons that are dependent on the shape of the sample.
2. Backscattered electrons BSE emerge from depths well below the top surface of the
specimen.They are defined as those electrons having an energy greater than 50 eV
with the majority having an energy approximating to ¾ of the electron beam (probe)
energy. The number of BSEs that are emitted is highly dependent on the mean
atomic number of the specimen at the point of impact of the beam. So as the atomic
number increases the greater the number of electrons that are backscattered. The
emergence of the backscattered electrons from the specimen surface also excite
secondary electrons that are referred to as SE2 type electrons. BSE give depth
infomation and atomic number contrast within the image.
3. X-rays characteristic of the elements in the specimen are generated in an excitation
volume in the order of a cubic micometre below the surface at the point of impact of
the beam hence “microanalysis”.
4. Cathodoluminescence CL is light generated by some specimens that contain electro
luminescent material. It is generated from a similar volume as that of X-rays.
5. Specimen current SC is the electron flow out of the specimen to earth and if an
amplifier is placed in the return path of electrons to earth its value can be measured
and also used for imaging.

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6. Transmitted electrons TE can be detected provided that the sample is exceptionally

thin.
7. Auger electrons are low energy electrons produced in the ionisation of an atom and
are characteristic of the atom.

The depth of penetration of the electron beam into the specimen will depend on the energy of
the beam and the mean density of the specimen at the point of impact of the beam.

6. Detectors
Everhart – Thornley type Secondary Electron Detector

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Secondary electrons are attracted by the positive voltage (up to + 400V) applied to the
collector. They are then attracted to a phosphor (scintillator) biased at a high voltage (+ 8kV),
the electrons then have sufficient energy to cause the phosphor to emit light. This light is
totally and internally reflected through a glass light pipe into a photomultiplier.
The photomultiplier converts the light into an electrical signal and amplifies the signal so that
it can be used to form an image of the surface of the specimen as if viewing it from above.
The analogy with the light microscope is that the gun is the eyepiece and the detector is the
illumination.
The collector can also be biased to a negative voltage (down to – 250V). This will reject all
SE type electrons from entering the detector. However a small solid angle of BSE electrons
will strike the phosphor and the signal produced can be used to image the specimen as if
illuminating it from the side.
Backscatter Detector
There are two types of detector, either a solid state 4Q-BSD or a Scintillator type. The
detector can be a retractable or a fixed position type and is normally positioned beneath the
pole-plate of the final lens. In this position it will collect the maximum amount of
backscattered electrons provided that the specimen is flat and not tilted.
The 4Q-BSD, when in the pole-piece position, has 4 individual elements (diodes) which are
symmetrically placed around the electron beam and is usually configured so that the signals
from the diodes are added together to maximise the atomic number contrast effect within the
image of the specimen.Thus the image displayed will be dark for low atomic number elements
and bright for high atomic number elements. It can also be easily configured, by altering the
polarity of the diodes, to give a topographical image of the specimen.
The Scintillator type gives an image which combines the atomic number contrast with the
topography of the specimen. It also has a better frequency response than the 4Q-BSD thus
enabling the beam to be scanned faster over the specimen surface.

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7. How it works
The basic operating principle of an SEM is shown in the diagram below followed by a brief
description.

Description
The specimen to be viewed has to be suitably adhered to a stub or clamped into a holder. A
frequently used stub has a diameter of 12.5 mm which is clamped either in a single or multi-
stub holder.
The operator should consult the specimen preparation guidance (next section) and operating
parameters (section 3.2) if unfamiliar with an SEM, as obtaining good images is highly
dependent on both.
To facilitate the fitting of the specimen or specimens, the specimen stage is retractable from
the specimen chamber and before imaging can start it is necessary to pump out the air from
within the chamber and column.

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This will take a few minutes and when a sufficiently good vacuum in the column has been
achieved the electron beam is switched on. The good or high vacuum in the column is
required to prevent any electrical discharges occurring due to the high voltages that are used
to generate the beam and to prevent the electron beam being diffused/absorbed by the gas
molecules.
The stage on which specimens are mounted can be moved in a horizontal plane (X and Y ),
raised or lowered (Z), rotated and tilted so that each one in turn can be imaged from the most
appropriate view.
Above the specimen is the electron optical column which, starting from the top, consists of:
1. An electron gun to generate a source of illumination called the electron beam.
2. A set of deflection coils to direct the electron beam straight down the column called
the gun align coils.
3. Two electromagnetic lenses to focus the electron beam to a very small spot. These
lenses are called condenser lens 1 and 2.
4. A final aperture to assist in defining the size and slenderness of the electron beam
(similar in function to the variable aperture in a camera).
5. A set of stigmation coils to shape the beam so that it is circular in cross section (not
shown in above diagram).
6. A set of scan coils to deflect the beam across the specimen surface in what is usually
referred to as a raster scan, very similar to that of a TV. These coils are driven from a
Scan Generator through a Magnification Control which controls the size of the area
scanned on the specimen surface and hence the “magnification” of the image
displayed on the TV monitor. The scan generator also controls the speed of scanning
the beam, the slower the speed the less “noisy” or “cleaner” the image appears.
7. A third condenser lens, sometimes known as the objective lens, is used to focus the
beam on to the specimen surface.
8. All three condenser lenses are under the control of a software program known as
“Optibeam” that maximises the amount of electron current (probe current) in the
beam as it is focused on the specimen surface. The amount of current is indicated by
the parameter “Iprobe” that controls the “Spot Size”of the beam and greatly influences
the resolution of the image.
9. A secondary electron collector within the specimen chamber is the detector that is
normally used to produce the image of the surface of the specimen. As the beam is
scanned across the specimen it attracts the low energy secondary electrons excited
from its surface. The emission of secondary electrons from each point on the specimen
surface is mainly dependent on its shape. Other signals are also excited from the
specimen; each has its own detector and can be used to display informative images.

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10. The signal from each point on the specimen surface having been collected by the
appropriate detector is placed within the memory of an Image Store. This store resides
within an Image Processor that reduces “noise” in the image in combination with the
selection of beam scanning speed. The image displayed on the TV monitor is the
output of the Store and is constantly being updated at TV frequencies. This enables
changes to be seen very rapidly for example when the specimen is being moved to
look for areas of interest. The image is “frozen” in the Store before it is saved/
exported to a printer or network device.
11. Beneath the specimen chamber is fitted a turbo molecular pump which removes the air
to enable a high vacuum to be achieved in the column and the chamber. A rotary pump
at the rear of the SEM then expels the air to atmosphere.

Gun
Penning Gauge
Measures
Vacuum
System

Chamber

Vent Valve

Turbo
Rotary Dry Air / N2
Pump

12. The specimen chamber pumping system can also be adapted to provide a controllable
poor (low) vacuum within the chamber whilst maintaining the essential high vacuum
within the column. The low vacuum is monitored with a Pirani gauge whilst high
vacuum is monitored with a Penning gauge. This mode enables the viewing of
electrically insulating and volatile specimens.

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Variable Pressure Principle

When the Pressure Limiting Apertures are fitted (see Appendix 2 for sizes and vacuum range)
a controllable amount of gas molecules, usually air, can be introduced into the specimen
chamber whist maintaining a good vacuum in the electron optical column and the electron
gun. Collisions between the high energy electrons of the beam and the gas molecules will
cause the gas to ionise.The positive ions that are produced will neutralise the negative charge
that has formed on the surface of an insulating material and will enable disturbance free
images of the specimen to be obtained.
The secondary electrons (SE) passing through the gas will interact with the molecules and
cause photons of light to be emitted. These photons can be collected by another detector
(VPSE detector) to give an image of the surface of the specimen similar to that produced by
the Everhart Thornley SE detector which cannot work in poor vacuum conditions.
The introduction of water into the chamber slows down the dehydration of specimens
containing water and helps to maintain the shape of the specimen.

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8. Specimen preparation guidance

Specimens which are studied in the SEM can be divided into two main categories, namely
electrical conductors and non-conductors. Initial guidance has already been given in section
3.1 regarding factors for consideration before examination of specimens in the SEM.

Conductors
These fall into two groups:

1. Metallic: these are generally excellent conductors and need no preparation for surface
topography examination. However for microstructural morphology studies and precise
microanalysis it will be necessary to polish the specimen. Etching and electropolishing
may also be a requirement.
2. Semi-conducting (semi-insulating) specimens be examined without special
preparation and can be satisfactorily observed under high vacuum conditions.
Non-Conductors
This group can be further divided into non-volatiles and volatiles.
Non-Volatile
If it is not possible to obtain suitable resolution by using a low accelerating voltage and
leaving the sample uncoated, then there two approaches to follow; either coat the specimen or
use a Variable Pressure in the specimen chamber.

1. Coating the specimen.

For most non-conductors which contain no volatile components, eg water that would
outgas in the vacuum system, it is sufficient to coat the sample with a thin layer of
conducting medium such as Au, Au/Pd, etc. This layer is typically 20-30 nm in thickness
and there are several reasons for coating:

a. Increased conductivity of the sample, thus minimising sample charge up,

which results in deflection of the incident beam and severe degradation of the
final image.
b. Increased mechanical stability of the sample due to increased heat conduction.
c. Increase in primary and secondary electron emission.
d. Decrease in beam penetration, resulting in better spatial resolution.

The two important current techniques of applying a coating are vacuum evaporation and ion
sputtering.

Gold is generally used for the following reasons:

a. High secondary emission co-efficient

b. High conduction of electrons and heat
c. Does not oxidise
d. Good granularity of evaporated or sputtered particles

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Carbon coating by evaporation is generally used if X-ray microanalysis is to be undertaken on

the specimen unless, of course, the element under consideration happens to be carbon.
Aluminium could be used in this case.

More recently Pt/Pd, Au/Pd and Cr have been used since their granularity is smaller.
Aluminium can also be used, but it has low mechanical strength and can oxidise.

2. Variable Pressure
The specimen can be observed at high kV either with the VPSE, EPSE, and/or BSD detector.
It will be necessary to adjust the chamber pressure to reduce beam charging effects. Under
these conditions micoanalysis can be performed.

Volatile
Biological and botanical samples, by their nature, require relatively more complex preparation
procedures. The specimens generally fall into two main categories: hard and soft.

1. Hard samples (eg bone, teeth, wood).

These, if necessary, can be washed to remove extraneous fluids such as blood and mucus,
dried in air and coated in the normal way or left uncoated and observed in the VP mode or
at low kV in high vacuum.

2. Soft samples
Once a living system, be it a plant or animal, is removed from its life support system it
begins to degrade and deform. The aim of the microscopist is to see the specimen in its
most natural state and therefore a more specialised approach is required and this will
depend to a large extent on the type of equipment available. As the specimen will
probably contain large amounts of water as soon as it is placed in a conventional high
vacuum SEM partial collapse and distortion will occur due to the vacuum environment.

For users of a high vacuum SEM

There are several approaches to preparation of the specimen that will initially require a
Chemical Pre-Treatment.
This technique involves chemical fixation of the material to strengthen the tissue. There is a
large range of chemicals used in this process (eg glutaraldehyde and osmium textroxide) and
there are numerous publications discussing the benefits of each. After fixation, it is necessary
to replace the water in the sample by a solvent to aid drying. The method must be such that
the specimen suffers no physical change. The most common drying agent used is a series of
ethanol/water mixtures through to 100% ethanol. Having replaced the water present in the
sample there is a choice of two methods for drying:

Critical Point Drying

The critical point has been defined as “that point at which the liquid, owing to expansion, and
the gas, owing to compression, acquire the same specific gravity, and consequently mix with
each other.”

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The specimen is dehydrated as previously described and the solvent replaced with a liquefied
gas,usually CO2 in a small pressure vessel. The vessel is then heated to above the critical
temperature of the selected gas. Under these conditions the liquid and vapour phases have the

sample physical properties so that on venting the liquid vapourises across cell boundaries and
therefore minimum sample distortion occurs.

Freeze Drying

The sample is quench-frozen and maintained at low temperatures (about -130°C) until the
sublimation process is complete.
The advantage of freeze drying over critical point drying is that it can be done from water. It
is not necessary to treat the sample with organic solvents that might attack membranes or
surface materials soluble in non-polar solvents. There is an advantage in freeze drying when
X-ray analysis of ions such as calcium, potassium, sodium, etc is to be done since they will be
dried in place, hopefully undisturbed by fixation and substitution of cryoprotectant.

Freeze drying involves the following steps: the sample is frozen in liquid nitrogen (N2) or
Freon 22 chilled by N2; some specimens require the use of a cryoprotectant to avoid ice
crystal damage while others (eg a leaf) do not. The frozen sample is transferred onto a cold
stage in a vacuum chamber; drying occurs by sublimation with pressures less than 10-3 torr
and at temperatures less than -70C. The drying process may take several hours (for cells in
suspension) to several days (for leaf samples) depending on the size of the sample.
Most specimens must be treated with a cryoprotectant such as 16% glycerol in water or
saturated chloroform water. This is necessary to avoid ice crystal damage that could poke
holes in the surface of the sample. For the scanning electron microscope, chloroform water is
better than glycerol water since after drying, glycerol is left on the “dry” surface of the
sample, whereas chloroform water evaporates during drying.

Low Temperature Scanning Microscopy

This is the closest approach to the goal of examining fresh, untreated, biological material. It
requires specialised equipment but once up and running it is a very fast technique.
A considerable amount of effort has been devoted to establishing the most suitable way to
perform the freezing operation . If freezing is carried out too slowly, or if frozen material is
kept at temperatures above about -80°C, large ice crystals are formed which cause structural
damage to the specimen and provide artificial structures (artefacts) in the micrographs.
Glycerol and other anti-freezing agents (“cryoprotectants”) are often introduced into
specimens to inhibit the process of crystallisation. Increasing the rate of freezing results in
smaller ice crystals being formed, but the idea is to cool the water so rapidly that it remains in
an amorphous vitreous (glass-like) state. For this to occur it is postulated that specimen
cooling rates must be in the range 105-106 degrees per second. Since biological materials are,
in general, poor conductors of heat it is obvious that the outside of a specimen will cool much
faster than the interior, and a frozen specimen will have only a limited thickness for which the
cooling rate is sufficiently high for vitreous ice to have formed; beyond this the specimen may
be unusable because of crystalline ice formation. The vitreous region is estimated to be less
than 5µm in thickness.

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General Considerations
There is not one major preparative technique for biological/botanical samples. Where
possible, several combinations should be tried for a particular type of sample, giving prime
consideration to the information sought. Once a technique has been established, instrument
parameters and specimen coating methods must be carefully considered.

The methods described above for soft tissue preparation are mainly for secondary electron
imaging. The problems facing the biologist or botanist who wishes to undertake X-ray
microanalysis are different in that the requirements in this case are to maintain the element(s)
of interest in their original position in the sample.

For users of a Variable Pressure SEM with EP capability

To study hydrated specimens it is recommended that a Peltier cooling stage and a water
control system be fitted to the SEM. This will enable fresh specimens to be examined with
little or no loss of water in the SEM environment (the sample is kept fully hydrated during the
pump down).

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Section 5. Appendices

1. An introduction to Macros
A macro is a series of stored sequential instructions written and stored by an operator in order
to assist in performing routine tasks.
It is recommended that the operator become familiar with the basic operation of the macro
manager, including the different types of macro entry and how to insert and edit them. This
information is available in the on-line help and will not be covered in this section.
This section will take the form of a tutorial that will develop macros to emulate normal
microscope operation. Therefore there will be no straightforward progression from basics to
advanced topics, but it should contain some useful real world insights into the process of
macro writing that are used in the operation of the SEM.
Before starting to write a macro it is a good idea to be clear about what you are trying to
achieve. In this case the objective is to create a macro to exercise various subsystems and
components of the SEM, specifically to:

• Cycle the vacuum by venting and pumping

• Drive the EHT set between different voltages and perform filament ramp up and down
operations
• Move the stage in X and Y axes
• Toggle detectors
• Exercise the EO board in terms of lens and coil supplies and mag range relays.
• Record images to prove the success of the above operations

Structure
For simplicity a set of macros rather than just a single macro have been written. There will be
one macro that calls a series of other macros that each perform a simple operation. Macros
will be stored as files rather than in the macro library to make everything more portable.
Component macros will be written first before moving on to the top level ones.

Component Macros

Vacuum Cycle
When starting a macro it is important to remember that the status of the instrument is
unknown, therefore rather than making assumptions about the state the machine is in we need
to find out.

Because the first operation we want to do is vent the chamber, we need to ensure the gun is
off. So for this operation we use a decision structure with an IF
THEN clause followed by a Wait For operation on the same state.

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We can now proceed to vent and this time we will wait for a fixed amount of time.

This would appear to complete this simple macro, however there is one enhancement that we
should consider. If the system is column pumped, then it would be sensible to close the
isolation valve before pumping and open it again at the end.

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Thus the full macro is as follows:

Stage Movement
The objective of the stage macro is to drive the stage in a square from its current position and
leave it at the same point it started. This sounds simple enough but there are some potential
hazards.

1. Stage not initialised

Clearly if this is the case then there is little the macro can do. Therefore the best way to
handle this situation is in the top level macro when the whole test is started. If the stage isn’t
initialised, then the user will be prompted and asked if they wish to proceed. Therefore all that
is required in this macro is a check and an exit if the stage is not initialised.

2. Stage too near limits

The problem here is whether the stage should be driven with absolute or relative moves. If
relative moves are used, then depending on where the stage is on starting, the stage limits may
come into play, which means that the stage won’t travel the intended distance and might not
return to the correct location.

If absolute moves are used, then returning to the original location is a problem.
Oddly enough both these problems can be solved by storing the current stage position on
entry to the macro and then moving to that named position at the end.

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3. Detecting stage idle state

This is one of the most tricky operations to do with macro. The problem is that the stage state
that indicates Stage is Busy doesn’t update immediately a command is issued to the stage. For
this reason simply sending a command to move the stage and then waiting for Stage is Idle
isn’t adequate as the stage might not have gone busy yet. The situation is further complicated
by the fact that for short moves there may not be a transition to a busy state at all.
The preferred solution to this problem is to wait for a fixed time (usually 1 second) before
waiting for Stage is Idle.

Taking these points into account the following stage macro can be generated:

Note that the stage moves are performed using the stage delta X/Y parameter pair. If we only
tried to move using X then whatever was last used in Y would also be applied. This macro
would work equally well if absolute stage positions were used.

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Image Save
The objective of this macro is to save an image to the hard drive in TIF format. This is easily
accomplished using the Photo command, after setting the Output Device to Display / File.
The more difficult aspect is getting an acceptable image in the first place. The sequence of
operations here will be:
1. Ensure gun is run up
2. Focus the image and adjust stigmation
3. Adjust the brightness / contrast of the image
4. Scan a frame at slow scan rate and freeze the image on completion
5. Save to a file
6. Restore normal scanning conditions

The following macro is therefore defined:

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There is a similar issue to detecting the completion of the auto focus function as there is when
detecting stage idle. This macro does make a number of assumptions that we should be aware
of:
• A suitable detector is selected
• The probe current is at a reasonable value
• There are enough features on the sample to permit auto focus and stig to work
• The filament has already been saturated.

The delay following the photo command is required to ensure that the image save operation
has completed before continuing.
It is good practice to unfreeze the image again before leaving the macro, so we will set up
some conditions suitable for navigation.

Note that only the end part of the macro is shown here.

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EHT Change
The macro to exercise the EHT set will make use of the image macro so that it is possible to
change the EHT, save and image and then change the EHT again. This will effectively add a
third level of nesting within the overall macro.

There are a couple of points to note about this macro. Firstly the delay following the setting of
the EHT target is to allow time for the voltage to ramp to its new level. Unfortunately there is
no mechanism to show when EHT ramping is complete, so the only option is to delay for long
enough to let the ramping finish.

The other point of interest is the first example of calling a macro from within a macro. The
SWO_Image macro is called at the end of this sequence. This is just like inserting the whole
macro at this point. Macros can be nested to any depth, but of course recursive macros are not
allowed (recursion occurs when A calls B and B calls A, or A calls A, etc.).

Detector Toggle
The objective of this macro is to toggle between different detectors to check that the relays are
working properly. The difficulty here is that different machines will be configured with
different detectors. Under normal circumstances this doesn’t present a problem as a macro
will be written for a known instrument. However for this application the solution will be to
use detectors that are present on all machines, the SE1 detector and the TV camera.

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Signal A is the correct parameter to use to select the detector. The final delay is to allow time
for the electronics to settle before the next operation takes place (whatever that is).

Top Level Macro

It is now possible to write the macro that calls all these others. Before the other macros can be
called, the SEM must be put into a well defined state. This will be done with the user’s
cooperation.

Initialising the Stage

The first step is to check that the stage is initialised. Unlike the stage macro, this time we will
not halt if the stage is not initialised, instead we will initialise the stage if the user agrees:

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The YesNo Message line is a Display Message entry with a yes no check box selected. This
allows a flag parameter to be selected for the response, 0 = No, 1 = Yes. This is also an
example of nested IF statements, the ELSE clause applies to the most recent IF.

Setting Conditions

Having initialised the stage we can then set up some other parameters:

The command to put Optibeam into Low Probe mode is protected by the IF statement because
if Optibeam is already in that mode, then the command will be disabled and an attempt to
execute it will stop the macro.
An alternative approach to setting up the microscope would be to load a set of saved
conditions. This has not been chosen as this would introduce a dependency on the detector
configuration that is to be avoided in order to keep these macros general.
The message command allows the user to complete the set up of the imaging conditions by
adjusting any necessary parameters. The macro will only continue when the user clicks the
OK button.

The Macro Loop

Finally we can enter the main loop of the macro which will continue until stopped by the user.
This is remarkably simple as all the hard work has been done by the other macros.

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The body of the loop simply consists of calls to the macros we have already defined. The
delays are intended to make the macro emulate normal operation to some extent.
Flag 5 is used for controlling the loop to provide an easy termination mechanism. Simply
changing flag 5 to a different value will stop this macro at the end of the loop. This could be
done by a macro assigned to a function key such as:

Admittedly in this particular instance the macro could take up to 50 minutes to terminate after
pressing this key, but it is a useful technique for stopping a macro. In the case of the
SWO_Control macro, it is best stopped using the macro editor command, as it will be run
from within the editor itself.

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Conclusion
The macros described in this section are a demonstration of various techniques that may be
more or less obvious to control the SEM. However they are intended to encourage SmartSEM
users to produce macros that will assist them in their work.

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MA: Charge compensation

SEM Application Hardware * Min/Max pressure Filament

10-400 Pa W

VP aperture
Charge compensation
MA 10
MA 15 10-273 Pa LaB6
MA 25
VP aperture

Charge compensation +
+ 10-400 Pa W + LaB6
Improved image quality +
EP aperture 500 µm
High accuracy EDS analysis

* Both “air” and “water vapour” can be introduced, as a charge compensating gas, into the chamber.

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Extended Pressure MA: Dynamic processes

SEM Application Hardware * Min/Max Filament

pressure

W
MA 10 Hydrated or
MA 15 Specimen +
10 – 3000 Pa and
MA 25 Imaging
EP aperture 500 µm 500 µm LaB6

* Both “air” and “water vapour” can be introduced, as a charge compensating gas, into the chamber.

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LS: Charge compensation

SEM Application Hardware * Min/Max pressure Filament

10-400 Pa W

VP aperture
Charge compensation
LS 10
LS 15 10-273 Pa LaB6
LS 25
VP aperture

Charge compensation +
+ 10-400 Pa W + LaB6
Improved image quality +
EP aperture 500 µm
High accuracy EDS analysis

* Both “air” and “water vapour” can be introduced, as a charge compensating gas, into the chamber.

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LS: Hydrated specimen imaging

SEM Application Hardware * Min/Max Filament

pressure

W
LS 10 Hydrated or
LS 15 Specimen +
10 – 3000 Pa and
LS 25 Imaging
EP aperture 500 µm 500 µm LaB6

* Both “air” and “water vapour” can be introduced, as a charge compensating gas, into the chamber.

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Enabli ng the Nano– Age Worl d®

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