PerkinElmer Spotlight 200 User Manual

MOLECULAR SPECTROSCOPY

Spotlight 200

User’s Guide

Release History

Part Number Release Publication Date

L1050036 H October 2015

Any comments about the documentation for this product should be addressed to:

User Assistance
PerkinElmer, Inc.
710 Bridgeport Avenue
Shelton, Connecticut 06484-4794
U.S.A.

Or emailed to: [e-mail]

Notices

The information contained in this document is subject to change without notice.

Except as specifically set forth in its terms and conditions of sale, PerkinElmer makes no
warranty of any kind with regard to this document, including, but not limited to, the
implied warranties of merchantability and fitness for a particular purpose.

PerkinElmer shall not be liable for errors contained herein for incidental consequential damages in
connection with furnishing, performance or use of this material.

Copyright Information

This document contains proprietary information that is protected by copyright.
All rights are reserved. No part of this publication may be reproduced in any form whatsoever or
translated into any language without the prior, written permission of PerkinElmer, Inc.

Copyright © 2015 PerkinElmer, Inc.
Produced in the U.S.A.

Trademarks

Registered names, trademarks, etc. used in this document, even when not specifically marked as such,
are protected by law.

PerkinElmer is a registered trademark of PerkinElmer, Inc.
Fluorolube is a registered trademark of the Hooker Chemical Corporation
Nujol is a registered trademark of the Esso Standard Oil Company
Spotlight, Frontier, Spectrum Two and Spectrum are trademarks of PerkinElmer, Inc.

Contents

Introduction ............................................................................................... 7

About this Manual ............................................................................................. 8

Conventions Used ............................................................................................. 9

Notes, Cautions and Warnings ..................................................................... 9

Definitions .................................................................................................12

Related Documents ....................................................................................12

Requirements for using the Spotlight 200 ....................................................12

Connecting the PC to the Local Area Network ..............................................12

Warnings and Safety Information ........................................................... 13

The Spotlight 200 .............................................................................................14

Summary .........................................................................................................15

General Operating Conditions............................................................................16

Electrical Safety ...............................................................................................17

Location and Ventilation ...................................................................................18

Warning Labels ................................................................................................19

Microscope Safety Labels ...........................................................................19

Stage Controller Safety Labels ....................................................................20

Warning Signs on the Microscope ...............................................................21

Mechanical Safety ............................................................................................22

Lifting the Spotlight 200 ...................................................................................23

EMC Compliance ..............................................................................................24

System Requirements .......................................................................................25

Electrical Requirements ..............................................................................25

Safety Specifications .........................................................................................27

Microscope ................................................................................................27

Stage Controller ........................................................................................27

Overview of the Spotlight 200 ................................................................. 29

A Guided Tour of the Spotlight 200 ...................................................................30

Connections .....................................................................................................31

Operation ........................................................................................................33

The Optical System ..........................................................................................34

Visible Light Optics ....................................................................................34

Infrared Optics ..........................................................................................37

Spotlight System Requirements .........................................................................39

Hardware Requirements .............................................................................39

Software Requirements ..............................................................................39

Getting Ready to Use the Spotlight 200 .................................................. 41

Before Using the Spotlight 200 ..........................................................................42

Cooling the MCT Detector .................................................................................43

Setting up the Spotlight 200 .............................................................................46

Focusing the Microscope ............................................................................46

Fitting an Attenuator to the Spectrometer ...................................................48

Setting Scan Parameters ............................................................................48

Preparing Samples ................................................................................... 51

Preparing Samples ...........................................................................................52

Tools for Sample Preparation ............................................................................53

Tools Provided with the Spotlight 200 .........................................................53

Other Useful Tools .....................................................................................55

Specialized Accessories ..............................................................................55

Items to Have Available .............................................................................56

Common Window Materials ...............................................................................57

Techniques for Preparing Samples for Transmittance Measurements ...................58

Flattening Solids ........................................................................................58

Slicing Samples from Solids ........................................................................59

Polymers ...................................................................................................60

Particles ....................................................................................................62

4 . Spotlight 200 User’s Guide

Fibers ....................................................................................................... 63

Fibrous Solids ........................................................................................... 63

Coatings on Substrates .............................................................................. 64

Liquids ..................................................................................................... 64

Techniques for Collecting Spectra ........................................................... 65

Techniques for Collecting Spectra ..................................................................... 66

Using the Compression Cell ....................................................................... 66

Using a Hot/Cold Stage ............................................................................. 67

Collecting the Spectrum of a Thick Sample ................................................. 67

Collecting a Spectrum in an Inert Atmosphere ............................................ 70

Viewing a Sample with the Visible Polarizer ....................................................... 72

The Theory of Light Polarization ................................................................. 72

Applications .............................................................................................. 73

Equipment ................................................................................................ 74

Collecting IR Spectra Using the Infrared Polarizer Accessory .............................. 76

Using the Polarizer .................................................................................... 77

Collecting Spectra With the ATR Objective ........................................................ 78

ATR Objective Specification .............................................................................. 80

Fitting the ATR Crystal Holder to the Microscope ............................................... 81

Measuring the Maximum Energy in Reflectance Mode ........................................ 83

Adjusting the Height of the Crystal ................................................................... 84

Centering the Crystal ................................................................................. 85

Checking the Infrared Alignment ................................................................ 86

Removing the ATR Crystal Holder from the Microscope ...................................... 87

Fitting a Crystal Assembly to the ATR Objective ................................................. 88

Manual ATR Objective ............................................................................... 88

Automated ATR Objective .......................................................................... 88

Cleaning the ATR Objective Crystal ................................................................... 90

Auto ATR Cleaning Procedure ........................................................................... 91

Materials Needed ...................................................................................... 91

Cleaning Procedure ................................................................................... 91

Changing the Weighbridge Battery ................................................................... 96

Collecting Spectra ............................................................................................ 98

Manual ATR Objective ............................................................................... 98

Automated ATR Objective ........................................................................ 100

Analyzing Samples Using the ATR Imaging Accessory ...................................... 104

Correcting for the Effect of the ATR Crystal .............................................. 107

Reflectance FT-IR Microspectroscopy .............................................................. 109

Diffuse Reflectance ................................................................................. 109

Specular Reflectance ............................................................................... 110

Reflection-Absorption .............................................................................. 111

Maintenance ........................................................................................... 112

Maintenance .................................................................................................. 113

Inspecting the Microscope ....................................................................... 113

Protecting the Microscope ........................................................................ 114

Cleaning ................................................................................................. 114

Cleaning the Cover .................................................................................. 114

Replacing the Microscope Fuse ....................................................................... 115

Testing the Stage Controller ........................................................................... 116

Replacing the Stage Controller Fuse ................................................................ 117

Service .......................................................................................................... 118

Accessories ................................................................................................... 119

Sample Preparation Tools ........................................................................ 119

Electrical Connections .................................................................................... 120

Fitting the Plug ....................................................................................... 120

Connecting the Microscope to the Electrical Supply ................................... 121

Spotlight 200 User’s Guide . 5

Connecting the Microscope to the Spectrometer ........................................ 122

Connections to the Stage Controller .......................................................... 123

Appendices ............................................................................................. 127

Appendix 1: Decontamination and Cleaning ..................................................... 128

Appendix 2: WEEE Instructions for PerkinElmer Products .................................. 129

Index ............................................................................................................ 130

6 . Spotlight 200 User’s Guide

Introduction

8 . Spotlight 200 User’s Guide

About this Manual

This introduction gives you information about this manual, to enable you to use it effectively
when learning to operate the Spotlight 200.

Before you start using your Spotlight 200 we recommend that you:

• Read the

• Read the rest of this

• Read

• Read

Four chapters in this User’s Guide contain information on using and maintaining your
Spotlight 200:

Getting Ready to Use the Spotlight 200

•

Spotlight 200 at the beginning of the day's work, to make sure that it is working
properly.

Preparing Samples

•

samples. It includes descriptions of the sample preparation tools provided with, or
available for, the Spotlight 200.

Techniques for Collecting Spectra

•

techniques to collect spectra.

Maintenance

•

for the Spotlight 200 and about the performance checks that we recommend that you
perform routinely.

Warnings and Safety Information

Introduction

Overview of the Spotlight 200

and become aware of conventions and requirements;

starting on page 29;

Getting Ready to Use the Spotlight 200

describes techniques for preparing many types of microscopic

describes how you can use accessories and different

contains maintenance information. It gives you information on how to care

starting on page 13;

starting on page 41.

gives you information on how to set up your

Conventions Used

Introduction . 9

Normal text is used to provide information and instructions.
commentary. Bold text refers to text that is displayed on the screen.

UPPERCASE text, for example ENTER or ALT, refers to keys on the PC keyboard. “+” is
used to show that you have to press two keys at the same time, for example, ALT+F.

All eight digit numbers are PerkinElmer part numbers unless stated otherwise.

“Spectrometer” refers to the Frontier IR System, Spectrum 100 Series, Spectrum 400 Series
or Spectrum One spectrometer supplied with your Spotlight 200.

Italic

text is used to provide

Notes, Cautions and Warnings

Three terms, in the following standard formats, are also used to highlight special
circumstances and warnings.

NOTE: A note indicates additional, significant information that is provided with some

procedures.

10 . Spotlight 200 User’s Guide

We use the term CAUTION to inform you about situations that could result

or other equipment. Details about

Geräteschaden zu vermeiden.

en beskadigelse af apparatet.

circunstancias.

dans un encadré semblable à celui-ci.

come questo.

om beschadiging van het instrument te voorkomen.

CAUTION

in serious damage to the instrument
these circumstances are in a box like this one.

Caution (Achtung)

Bedeutet, daß die genannte Anleitung genau befolgt werden muß, um einen

Caution (Bemærk)

Dette betyder, at den nævnte vejledning skal overholdes nøje for at undgå

Caution (Advertencia)

Utilizamos el término CAUTION (ADVERTENCIA) para advertir sobre
situaciones que pueden provocar averías graves en este equipo o en otros.
En los recuadros como éste se proporciona información sobre este tipo de

Caution (Attention)

Nous utilisons le terme CAUTION (ATTENTION) pour signaler les
situations susceptibles de provoquer de graves détériorations de

l'instrument ou d'autre matériel. Les détails sur ces circonstances figurent

Caution (Attenzione)

Con il termine CAUTION (ATTENZIONE) vengono segnalate situazioni
che potrebbero arrecare gravi danni allo strumento o ad altra
apparecchiatura. Troverete informazioni su tali circostanze in un riquadro

Caution (Opgelet)

Betekent dat de genoemde handleiding nauwkeurig moet worden opgevolgd,

Caution (Atenção)

Significa que a instrução referida tem de ser respeitada para evitar a

danificação do aparelho.

Verletzung des Benutzers kommen kann.

Betyder, at brugeren kan blive kvæstet, hvis anvisningen ikke overholdes.

este tipo de circunstancias.

un encadré semblable à celui-ci.

su tali circostanze in un riquadro come questo.

genomen, dit kan leiden tot verwondingen van de gebruiker.

WARNING

Introduction . 11

We use the term WARNING to inform you about situations that could
result in personal injury to yourself or other persons. Details about these
circumstances are in a box like this one.

Warning (Warnung)

Bedeutet, daß es bei Nichtbeachten der genannten Anweisung zu einer

Warning (Advarsel)

Warning (Peligro)

Utilizamos el término WARNING (PELIGRO) para informarle sobre
situaciones que pueden provocar daños personales a usted o a otras
personas. En los recuadros como éste se proporciona información sobre

Warning (Danger)

Nous utilisons la formule WARNING (DANGER) pour avertir des
situations pouvant occasionner des dommages corporels à l'utilisateur ou
à d'autres personnes. Les détails sur ces circonstances sont données dans

Warning (Pericolo)

Con il termine WARNING (PERICOLO) vengono segnalate situazioni
che potrebbero provocare incidenti alle persone. Troverete informazioni

Warning (Waarschuwing)

Betekent dat, wanneer de genoemde aanwijzing niet in acht wordt

Warning (Aviso)

Significa que a não observância da instrução referida poderá causar um

ferimento ao usuário.

12 . Spotlight 200 User’s Guide

Definitions

OPERATOR: Person operating the equipment for its intended purpose.

RESPONSIBLE BODY: Individual or group responsible for the use and maintenance of the
equipment and for ensuring that the OPERATORS are adequately trained.

Related Documents

Manuals for Spectrometer

Information on using your spectrometer can be found in the manuals that were supplied with
it. The
Systems, Spectrum 400 Series and Spectrum 100 Series spectrometers. The

Manuals CD

IR & Raman Manuals CD

(L1050242) contains the instrument manual for the Spectrum Two spectrometer.

Spectrum Software

Spectrum software has on-screen Help, which you can access by choosing the Contents
command from the Help menu, by pressing the F1 key, or by clicking the Help button in a
dialog. The Help information assumes that you are familiar with the hardware components of
the Spotlight 200 microscope contained in this guide.

(L1050002) includes instrument manuals for the Frontier IR

Spectrum Two

Requirements for using the Spotlight 200

We assume that the Spotlight 200 has been properly set up and aligned. This installation
must be performed by a PerkinElmer Service Engineer.

Connecting the PC to the Local Area Network

The PC provided is connected point-to-point to the Spotlight 200. If you wish to connect the
PC to your local area network to enable transfer of data, we recommend that you contact
your local PerkinElmer Service Engineer.

Warnings and Safety

Information

14 . Spotlight 200 User’s Guide

The Spotlight 200

The Spotlight 200 consists of a microscope, spectrometer, PC, stage controller and joystick.

Figure 1 Spotlight 200 – microscope and Frontier IR System

Figure 2 Spotlight 200 – microscope and Spectrum Two System

Warnings and Safety Information . 15

Summary

The PerkinElmer Spotlight 200 has been designed to comply with a wide variety of
international standards governing the safety of laboratory equipment. In routine use, the
Spotlight 200 poses virtually no risk to you. If you take some simple, common-sense
precautions, you can make sure that you maintain the continued safe operation of the
Spotlight 200.

• DO make sure that all parts of the Spotlight 200 are properly connected to the electrical
supply; in particular, make sure that the ground (earth) wires are securely connected.

• DO disconnect the electrical power supply before opening the main cover of the
microscope and spectrometer.

• DO keep the microscope dry. Avoid spilling liquid into the microscope and spectrometer.
Clean all external spills immediately. If anything that is spilled enters the main body of
the microscope or spectrometer, switch off the power and call a PerkinElmer Service
Engineer.

• If your Spotlight 200 is fitted with an MCT (mercury cadmium telluride) detector,
DO wear safety glasses and protective gloves when you are filling the detector dewar in
the microscope with liquid nitrogen. Slowly pour the liquid nitrogen into the dewar.
Stand back from the detector during filling because liquid nitrogen may be expelled
from the dewar flask. Use only liquid nitrogen.

• DO NOT use a flammable gas to purge the spectrometer or microscope. The
spectrometer contains a hot lamp, and fire or explosion may result. Use only clean, dry,
oil-free nitrogen or air to purge the instrument.

• If your Spotlight 200 is fitted with an automated ATR objective, DO NOT look directly at
either the stage lighting LEDs or the window at the back of the weighbridge, particularly
if you are using an optical magnifier that could focus the visible or infrared beams from
these sources.

• DO read the more detailed information on safety in the following pages.

16 . Spotlight 200 User’s Guide

General Operating Conditions

For information about the spectrometer, refer to the User’s Guide supplied with the
instrument. The
Frontier IR Systems, Spectrum 400 Series and Spectrum 100 Series spectrometers.

The microscope and stage controller have been designed and tested in accordance with
PerkinElmer specifications and in accordance with the safety requirements of the
International Electrotechnical Commission (IEC). The microscope and stage controller

conform to IEC publication 61010−1 (“Safety requirements for electrical equipment for

measurement, control, and laboratory use”) as it applies to IEC Class 1 (earthed) appliances
and therefore meets the requirements of EC low voltage directive 2006/95/EC.

Only use the microscope and stage controller indoors and under the following conditions:
Temperature 15 °C to 35 °C
Relative humidity 80% maximum (non-condensing)

If possible, avoid any adjustment, maintenance and repair of the opened, operating
instrument. If any adjustment, maintenance and repair of the opened instrument is
necessary, this must be done by a skilled person who is aware of the hazard involved.

IR & Raman Manuals CD

(L1050002) includes instrument manuals for the

Whenever it is likely that the microscope and stage controller are unsafe, make them
inoperative. The microscope and stage controller may be unsafe if they:

• Show visible damage;

• Fail to perform the intended measurement;

• Have been subjected to prolonged storage in unfavorable conditions;

• Have been subjected to severe transport stresses.

The microscope and stage controller have been designed to be safe under the following
conditions:

• Indoor use;

• Altitude up to 2000 m;

• Ambient temperatures of 5 °C to 40 °C;

• A maximum ambient relative humidity of 80% for temperatures up to 31 °C decreasing

linearly to 50% relative humidity at 40 °C;

• Mains fluctuations not exceeding ±10% of the nominal voltage. For example,
230 V ± 10%.

If the equipment is used in a manner not specified herein the protection
provided by the equipment may be impaired.

WARNING

Warnings and Safety Information . 17

Electrical Safety

Connect the microscope and stage controller to a power supply line that includes a switch or
other adequate means of disconnection from the electricity supply.

Only plug the microscope and stage controller into electricity-supply sockets that are
provided with a protective ground (earth) connection. The stage control box and the
microscope must be earthed.

If fuses need replacing, use only those with the required current rating and of the specified
type. Do not use makeshift fuses and do not short-circuit fuse holders.

When the microscope and stage controller are connected to its electricity supply, terminals
may be live. Removing covers other than those which can be removed by hand is likely to
expose live parts.

NOTE: There are no user-serviceable parts in the microscope or the stage controller.

Capacitors inside the microscope and stage controller may still be charged even if the
microscope or stage controller has been disconnected from all voltage sources.

Disconnect the microscope and stage controller from all voltage sources before they are
opened for any adjustment, replacement, maintenance or repair. Any adjustment,
replacement, maintenance or repair must be performed by a PerkinElmer Service Engineer.

The microscope and stage controller must only be connected to equipment meeting the
requirements of IEC 61010-1 (Safety requirements for electrical equipment for measurement,
control and laboratory use – general requirements) or IEC 60950 (Safety of information
technology equipment).

Any interruption of the protective ground (earth) conductor inside or
outside the microscope or stage controller or disconnection of the
protective ground (earth) terminal can make the microscope or stage

WARNING

controller dangerous.

18 . Spotlight 200 User’s Guide

Location and Ventilation

The Spotlight 200 is installed by a PerkinElmer Service Engineer, who will be able to advise
on the positioning of the system. To allow for adequate cooling, the system should not be
sited near to room heating equipment, for example, central-heating radiators. There should
be a minimum gap of at least 15 cm (6 inches) from the top and side surfaces of the
microscope and stage control box to permit adequate cooling.

Make sure that the switches at the electrical supply inlet at the rear of
the microscope and stage controller are not obstructed.

WARNING

Warning Labels

Power

When this label is attached to an instrument it means “Caution, risk of
danger”. Refer to the manual to find out the nature of the potential
hazard and any actions which have to be taken.

Microscope Safety Labels

The following safety labels are fixed to the microscope.

Warnings and Safety Information . 19

Figure 3 Rear of Microscope

100–230V ~50/60Hz
75 VA

20 . Spotlight 200 User’s Guide

MAINS INPUT

FUSE 1.6 A T

Warning

cord before opening

Figure 4 Inside Dewar lid – MCT detector versions only

NOTE: See

Cooling the MCT Detector

on page 43 for information on how to fill the Dewar.

Stage Controller Safety Labels

Figure 5 Rear of Stage Controller

Disconnect supply

100–240 V /AC
60 VA
50/60 Hz

Warnings and Safety Information . 21

Warning Signs on the Microscope

Caution, risk of electric shock.

Caution, risk of danger.

Refer to accompanying documents to find out the nature of the potential
hazard and any actions which have to be taken.

22 . Spotlight 200 User’s Guide

Mechanical Safety

When you are using a motorized stage, do not place your fingers
between the moving and fixed parts of the stage. The motors driving
the stage from side to side, front to back, or up and down are powerful

WARNING

and do not stall easily.

Warnings and Safety Information . 23

Lifting the Spotlight 200

The imager and spectrometer are heavy precision instruments, so two
people are required for safe handling.

WARNING

The microscope weighs approximately 35 kg, including the motorized stage.

• Lift the microscope only by the base.

• Do not attempt to lift it by the stage, cassegrain assembly or other attachments.

The spectrometer weighs approximately 34 kg unpacked and has a lifting recess on either
side.

Consult the local codes of practice issued by safety advisors before
attempting to lift these instruments.

Take care not to injure yourself or others, or to drop the instruments.

Do not move the Spotlight 200 after it has been installed without consulting your local
PerkinElmer service department.

24 . Spotlight 200 User’s Guide

EMC Compliance

EC directive

The Spotlight 200 has been designed and tested to meet the requirements of the EMC
Directive 2004/108/EC.

The Spotlight 200 complies with the EMC standard EN61326, (EMC standard for electrical
equipment for measurement, control and laboratory use) and EN55011 (ISM) class A
(rf emissions).

FCC rules and regulations

This product is classified as a digital device used exclusively as industrial, commercial, or
medical test equipment. It is exempt from the technical standards specified in Part 15 of the
FCC Rules and Regulations, based on Section 15.103(c).

Warnings and Safety Information . 25

System Requirements

Give attention to the following points before installing the Spotlight 200.

Electrical Requirements

The power consumption of the microscope does not exceed 75 VA.

The power consumption of the stage controller does not exceed 60 VA.

The nominal power consumption of the spectrometer is 120 VA (65 VA for the Spectrum
Two).

The line supply must be within 10% of the nominal voltage. For example, 230 V ± 10%.

If possible, do not connect any parts of the Spotlight 200 to circuits that have heavy duty
equipment, such as large motors, connected.

If possible, do not use photocopiers, discharge lamps, radio transmitters, and other
equipment with large or frequent transient loads, on the same supply circuit.

Microscope

The microscope can operate on electricity supplies of 50 or 60 Hz and in a voltage range of
100 to 240 V. The primary fuse (2 A T, 250 V, part number 04970839) for the microscope is
in the drawer on the mains inlet connector.

NOTE: No voltage selection is required.

Stage Controller

The stage controller can operate on electricity supplies of 50 or 60 Hz and in a voltage range
of 100 to 240 V. No voltage selection is required. The primary fuse (1.6 A T, 250 V, part
number 09991641) is located in a drawer on the mains inlet.

NOTE: No voltage selection is required.

26 . Spotlight 200 User’s Guide

Site Requirements

A minimum bench space of 170 x 75 cm (68 x 30 inches) is required to accommodate the
microscope, spectrometer, PC and ancillaries.

To get the best performance from your Spotlight 200:

• Place the Spotlight 200 in an environment that is relatively dust-free.

• Make sure that the bench top is free from vibrations or mechanical shocks, and is flat

and level.

• Do not place the Spotlight 200 near to room heating equipment, for example central
heating radiators.

Leave a gap of at least 15 cm (6 inches) to the sides of the microscope and stage controller
box to permit an adequate flow of cooling air.

NOTE: Do not stand the stage controller box on its side as this will cover some of the cooling

vents.

Warnings and Safety Information . 27

Safety Specifications

Microscope

Power supply 100–230 V ± 10%, 50/60 Hz ± 10%

Primary fuse 2.0 A T (time-lag), 250 V

Weight 32 kg without the motorized stage

Stage Controller

Power supply 100–240 V ± 10% , 50/60 Hz ± 10%

Primary fuse 1.6 A T (time-lag), 250 V

The control box is not designed for use with all
three motors simultaneously over extended
periods

Maximum speed 6 mm/s

Maximum motor voltage 24 V dc

Stepping resolution Maximum 6400 steps/rev, 156 nm/step

Baud rate 9600

Travel 75 mm x 50 mm

or 215 mm x 100 mm

Weight Stage 2.7 kg

Control box 2.8 kg

28 . Spotlight 200 User’s Guide

Overview of the

Spotlight 200

30 . Spotlight 200 User’s Guide

A Guided Tour of the Spotlight 200

The Spotlight 200 enables you to collect IR spectra from extremely small samples. The
PerkinElmer cassegrain collection optics give high-performance infrared microspectroscopy.
The microscope includes a camera and viewing system that magnifies the visible-light image
of the sample so that you can see, position, and isolate a point of interest. The image of your
sample is displayed in the Monitor Visible window on your PC monitor. Spectrum software
enables you to control the operation of your Spotlight 200, and collect IR spectra from the
sample.

The microscope includes the following features:

• A high-performance cassegrain mirror system; this has a wide collection angle (high
numerical aperture) and is highly efficient in collecting infrared radiation for
microspectroscopy.

• Spectra can be collected in either reflectance or transmittance modes.

• Internal coaxial LED illumination, with variable intensity.

• An automatic infrared aperture that closes to the size and rotation selected when you

choose a scan or monitor command.

• A motorized stage, controlled by a joystick and the Spectrum software, enables you to
find points on your sample and focus the microscope. Two sizes of stage are available:

– Sample sizes up to 75 × 50 mm (part number L1860160)

– Sample sizes up to 160 × 69 mm (part number L1860161)

Some versions of the microscope feature LED lighting of the stage area.

• Spectrum software, which controls the operation of the microscope, for example: focus,
illumination, stage position, adjusting the correction, and changing between reflectance
and transmittance sampling modes.

• Auto-correction to optimize the position of the lower cassegrain for maximum energy
throughput.

• A micro-ATR objective for data collection from optically thick and non-reflective
samples. Manual and automatic ATR objectives are available; the automatic ATR option
also includes a weighbridge to measure the pressure applied to the sample during
analysis.

• Automatic image analysis, data collection and post-run processing of collected spectra.

• A single cassegrain to send both infrared radiation and visible light to the remote

aperture. The microscope continuously views and monitors infrared concurrently; there
is no beampath switching between visible and infrared.

• A removeable lower cassegrain to provide space for thick samples to be studied in
reflectance mode.

• An InGaAs (indium gallium arsenide), DTGS (deuterated triglycine sulfate), or an MCT
(mercury cadmium telluride) MIR detector.

Overview of the Spotlight 200 . 31

Connections to

SP1 – connects to spectrometer

Video

Connections

Figure 6 to Figure 9 show the input and output connections on the microscope and stage
controller.

stage controller

Figure 6 Connections to Stage Controller

external left port
Single element detector output:
9-way D type, male

Figure 7 Electrical Connections (top rear of imager)

32 . Spotlight 200 User’s Guide

Connects to PC

Connects to

Stage Z –

Connects to imager

Connects to

Connects to

X-motor (stage)

RS232 connects to

Connects to Joystick
9-way, D-type (pins)

Purge gas connection

Connects PC to

automatic ATR (if
present), USB

RS232, 9-way D type

Figure 8 Electrical Connections (bottom rear of imager)

Connects to Stage
controller Z motor

Stage controller
15-way D type

Drive Signals for
the Z-motor

stage, back
connector
Drive signals for
Y-motor (stage)

stage, front
connector
Drive signals for

Figure 9 Stage Controller Electrical Connections

imager
15-way high density
D type (pins)

Overview of the Spotlight 200 . 33

Operation

Spectrum software enables you to mark points of interest on the sample. You can collect
individual spectra (either at the current aperture position or at positions you have marked)
with or without an ATR objective. You can also collect spectra at regularly spaced intervals
along a line or within a marked area.

For further information, see the Spectrum on-screen help.

The table below lists the modes of operation that should be used for particular samples.

Transmittance Single areas of thin solids, fibers and films. The optimal aperture

range depends upon the detector type, as follows:

MCT detector: Between 10 µm and 100 µm.

DTGS detector: Between 50 µm and 200 µm.

InGaAs detector: Between 10 µm and 200 µm.

Reflectance Single areas of coatings and thick solids. The optimal aperture

range depends upon the detector type, as follows:

MCT detector: Between 10 µm and 100 µm.

DTGS detector: Between 50 µm and 200 µm.

InGaAs detector: Between 10 µm and 200 µm.

With ATR objective Thick, non-reflecting samples.

NOTE: In general, the larger the aperture setting, the better the spectral quality. Sample

sizes may be larger than the maximum aperture sizes given in the table above.

34 . Spotlight 200 User’s Guide

The Optical System

The microscope has optics for infrared microspectroscopy and visible light. A video camera
enables you to view the sample. The two systems intersect at the aperture. When a sample
on the sample stage is in focus, its conjugate image is focused at the remote aperture.

Spotlight 200
section describes what happens within the optical system when the system changes from
visible light to infrared mode in transmittance and reflectance operation.

Dichroic mirrors form part of the optical system. The dichroic mirrors used in the microscope
reflect infrared and transmit visible light.

Visible Light Optics

When you view a sample with the video camera, you are really looking at the conjugate
image of the sample, located at the remote aperture (Figure 10).

The Z-control on the joystick enables you to focus the optical image. This control moves the
sample position up and down until the conjugate image is focused at the remote aperture.

enables you to select between transmittance and reflectance operation. This

The joystick also enables you to move the position of the sample so that the area of interest
is in the center of the field of view. The automatic infrared aperture then enables you to
isolate an area of interest.

Overview of the Spotlight 200 . 35

Detector

Camera

Mirror pair

Visible focus

lens assembly

Dichroic mirror

Variable aperture

Upper cassegrain

Sample position

Lower cassegrain

Lower dichroic
Lower fold mirror

Lower mirror assembly

LED

Visible light optics in transmittance

When the microscope is in viewing mode in transmittance (Figure 10):

• The lower fold mirror, beneath the cassegrains, receives light from the LED source and
directs it up through the lower dichroic mirror onto the lower cassegrain.

• The lower cassegrain condenses the beam to an appropriate size for a microscopic
sample and focuses it at the sample position.

• The upper cassegrain collects light from the sample and sends it upward through the
aperture and the upper dichroic mirror.

mirror

Figure 10 Path of the Visible Beam for Viewing a Sample in Transmittance

NOTE: Figure 10, above, illustrates a Spotlight 200 with an MCT detector. Other detector

types may look different, but the optical arrangement is the same in all cases.

36 . Spotlight 200 User’s Guide

Mirror pair

Visible focus lens

Variable aperture

Reflectance illuminator

Upper cassegrain

Sample position

Lower cassegrain

Lower dichroic

mirror

Detector

Camera

Visible light optics in reflectance

When the microscope is in viewing mode in reflectance (Figure 11):

• The reflectance illuminator assembly directs light from the upper LED down through one
side of the cassegrain onto the sample.

• The upper cassegrain collects the reflected light from the sample and sends it upward
through the aperture.

The lower “transmission” illuminator is also active in reflectance mode, giving simultaneous
illumination from above and below.

assembly

assembly

Figure 11 Path of the Visible Beam for Viewing a Sample in Reflectance

NOTE: Figure 11, above, illustrates a Spotlight 200 with an MCT detector. Other detector

types may look different, but the optical arrangement is the same in all cases.

Overview of the Spotlight 200 . 37

Dichroic mirror

Variable

aperture

Upper

Sample position

Lower

Lower dichroic

Detector cassegrain

Detector

Source toroid

Infrared Optics

The upper cassegrain is used for both visible light and infrared radiation. For this reason,
when you adjust the sample position so that the visible image of the sample is in focus, the
sample is also correctly positioned for collecting infrared spectra. Similarly, when you adjust
the aperture so that the required area of the sample is isolated visually, you have also
isolated the area of the sample from which the IR spectrum is to be collected.

IR optics in transmittance

IR optics in transmittance differs from when viewing a sample in transmittance as follows
(Figure 12):

• Instead of receiving light from the illuminator, light from the spectrometer is reflected
off the toroid onto the lower dichroic mirror which sends it through the lower
cassegrain.

• The upper dichroic mirror reflects the beam onto the detector cassegrain.

• The detector cassegrain focuses the beam on to the detector.

cassegrain

cassegrain

mirror

Figure 12 Path of the Infrared Beam for Collecting an Image in Transmittance

NOTE: Figure 12, above, illustrates a Spotlight 200 with an MCT detector. Other detector

types may look different, but the optical arrangement is the same in all cases.

38 . Spotlight 200 User’s Guide

Dichroic mirror

Variable aperture

Reflectance

Upper

Sample position

Lower cassegrain

Detector cassegrain

Detector

Source toroid

IR optics in reflectance

IR optics in reflectance differs from viewing in reflectance as follows (Figure 13):

• The toroid moves to send the beam to the Reflectance illuminator assembly dichroic
mirror, which sends it through the upper cassegrain.

• The beam is reflected off the sample and back through the other side of the cassegrain,
toward the remote aperture.

• The detector cassegrain focuses the beam on to the detector.

illuminator

assembly

cassegrain

Figure 13 Path of the Infrared Beam for Collecting IR Spectra in Reflectance

NOTE: Figure 13, above, illustrates a Spotlight 200 with an MCT detector. Other detector

types may look different, but the optical arrangement is the same in all cases.

Overview of the Spotlight 200 . 39

Spotlight System Requirements

Spotlight 200 requires additional cards to be installed in the PC. If you encounter a problem
with the PC, contact a PerkinElmer Service Engineer. Do not attempt to remove the cards
from the PC or install the software on another PC.

Hardware Requirements

The PC on which you install the software must meet the following specification:

• Intel® Pentium 4, 2 GHz processor (or greater) – dual-core or hyper-threaded
preferable

• At least 3 GB of Random Access Memory (RAM)

• 40 GB Hard disk with at least 1 GB free space as an NTFS drive

NOTE: We have locked the system into using an NTFS drive because the alternative FAT32

file system doesn’t provide enough protection at a folder and file level to ensure that
users and groups of users cannot delete or amend data files, while at the same time
being able to create new data files.

• A graphics card with an ATI chip-set, with the capability of displaying 32-bit color at a
resolution of 1280 x 1024, with a refresh rate of 75 Hz

• DVD drive

• A keyboard and PS/2®-style mouse

• Serial (RS232) port

• USB port

• 1 free PCI slot

• Network port.

NOTE: If you should need to change the PC we recommend that you contact a PerkinElmer

Service Engineer for information on PC configurations.

Software Requirements

This software requires that either Windows® XP Professional Service Pack 3 (or greater), or
Windows® 7 Professional (32-bit or 64-bit), or Windows® 8.x Pro (32-bit or 64-bit)
operating system is installed on the PC before you install Spectrum.

Microsoft Service Packs and Updates can be downloaded from www.microsoft.com/downloads.

40 . Spotlight 200 User’s Guide

Getting Ready to Use the

Spotlight 200

42 . Spotlight 200 User’s Guide

Before Using the Spotlight 200

Before you use the Spotlight 200 you must:

• Make sure the spectrometer is switched on;

• Switch the stage controller and microscope on;

• Cool the detector (MCT detector versions only);

• Set up the microscope;

• Focus the microscope.

We recommend that you use these procedures at the beginning of the day's work, or any
time the Spotlight 200 has not been in use or has been used by others.

Getting Ready to Use the Spotlight 200 . 43

Oxygen depletion in an enclosed space does not trigger a gasping reflex,

Cooling the MCT Detector

If the microscope is fitted with an MCT (mercury cadmium telluride) detector, the detector
must be cooled to −196 °C before you collect spectra. It is mounted in a dewar that can be
filled with liquid nitrogen. As you fill the dewar, the temperature of the detector drops, and
the preamp supplies power to the detector. Use the following procedure to cool the MCT
detector.

NOTE: If your Spotlight 200 is fitted with a DTGS or InGaAs detector, cooling with liquid

nitrogen is not required.

The extremely low temperature of liquid nitrogen can burn skin and
eyes. Avoid exposure by wearing heavy gloves and safety goggles
whenever you work with it.

WARNING

WARNING

WARNING

WARNING

When liquid nitrogen warms to room temperature, nitrogen gas
vaporizes so rapidly that resulting pressures can send a funnel or
detector cap suddenly and forcefully shooting upward from the top of
the microscope.

Be sure to wait the specified time when filling the funnel and before
replacing the detector cap. This enables the bubbling nitrogen to settle
down and the pressure to dissipate. In addition to wearing safety
goggles at all times, stand back from the microscope after each time
you fill the funnel.

Do not site the instrument in a poorly ventilated area.

and errors of judgment, confusion, or unconsciousness can occur in
seconds and without warning.

44 . Spotlight 200 User’s Guide

1. Open the flap covering the dewar.

2. Remove the dewar cap.

3. Place the small funnel supplied with the microscope in the opening in the detector
dewar (Figure 14).

Figure 14 The Dewar Opening with the Funnel Inserted

Stand where you can see the inside of the funnel as you pour the

CAUTION

nitrogen in, but without positioning your head over the funnel itself.
Pour slowly, so that neither the funnel nor the dewar overflows. If liquid
nitrogen runs down the outside of the dewar, it can damage the optics
of the microscope.

4. Carefully fill the funnel with liquid nitrogen. Stand back and let the funnel empty
completely.

The liquid nitrogen bubbles rapidly as it drains into the dewar. This first amount of
liquid nitrogen vaporizes completely as it cools the dewar.

5. Add another one and a half funnels of liquid nitrogen. Stand back and wait two minutes.
This nitrogen also vaporizes as the dewar continues to cool. The two-minute wait

enables the bubbling to settle down and the pressure of the vaporizing nitrogen to
dissipate.

6. Continue to pour liquid nitrogen into the funnel, adding a little more each time the
funnel empties.

The funnel takes longer to empty as the dewar fills. This happens after two to three
more funnels of liquid nitrogen.

Now the dewar has cooled, the liquid nitrogen does not vaporize, but instead fills the
dewar.

Getting Ready to Use the Spotlight 200 . 45

7. Remove the funnel and wait two minutes.
The liquid nitrogen settles down and bubbling slows.

8. When the nitrogen stops bubbling, refit the detector cap.
The filled dewar cools the MCT to the correct operating temperature for several hours.

After that, the dewar begins to return to room temperature, and the preamp switches
off power to the MCT.

46 . Spotlight 200 User’s Guide

Setting up the Spotlight 200

To set up your Spotlight 200:

1. Make sure the spectrometer is switched on.
It can take the spectrometer up to two hours to equilibrate after being switched off

overnight.

2. Switch on the microscope at the rear switch.
The blue LED light at the front comes on.

3. Switch on the stage controller.
The green LED light at the front comes on.

4. At your PC, click Start and then select Spectrum in the PerkinElmer Applications
group under All Programs.

Spectrum software starts.

5. Enter your User name and, if required, your Password.

6. Select the spectrometer connected to your Spotlight 200.

7. Select Instrument from the Setup menu, and click the Setup Instrument BeamPath
tab.

8. Click the microscope detector
The beampath diagram and accessory toolbar are updated to show that the microscope

is connected.

9. Click the microscope

10. Make sure that the lower cassegrain is fitted, that the ATR objective crystal (if fitted) is
retracted, and that there is no sample on the stage, and then click Start.

The microscope initializes.

NOTE: If you want to avoid initializing the microscope at the start of a session, click Skip to

apply the settings from the previous session. This option is only available if the
microscope has previously been initialized.

button on the accessory toolbar.

to direct the beam to the microscope.

Focusing the Microscope

The focus of the microscope is changed by moving the sample stage up or down. The
Z-control on the joystick, or the Auto-Focus button on the Setup Microscope Basic tab,
enables you to focus the image in the Camera View.

The default position for the lower cassegrain gives optimal illumination for thin samples. For
optimal performance it may be necessary to refocus the lower cassegrain to compensate for
the sample’s refractive index.

Getting Ready to Use the Spotlight 200 . 47

Focusing the image

1. Select Microscope > Stage Move > To Load Position to move the sample stage to a
position where a sample can be loaded easily.

2. Place your sample on the sample stage.

3. Select the Sampling Mode in the Setup Microscope Basic tab.

You can also switch between Transmittance and Reflectance modes using the button on
the accessory toolbar.

4. Select Microscope > Stage Move > To Center Stage.

The sample stage moves so that the center of the sample stage is illuminated.

5. Use the Z-control on the joystick to focus on the sample.
You may need to change the illumination of the sample, or the correction.

OR

Click Auto-Focus in the Setup Microscope Basic tab.

OR

Click the
The image of the sample is focused.

NOTE: If your Spotlight 200 system is supplied with a Frontier or Spectrum 400

6. If you need fine focus, use the Adjust Up or Adjust Down buttons on the
accessory toolbar to move the stage a small distance along the z-axis.

You can select the size of the Z-Axis Adjustment in the Setup Microscope Basic tab.

7. If you are examining the sample in transmittance mode, you may need to adjust the
position of the lower cassegrain so that the field of view is evenly illuminated (this is
especially useful if the sample is in a compression cell or held between windows of high
refractive index):

Adjust the Correction control in the Setup Microscope Advanced tab by clicking the up

or down buttons.

button in the Camera View toolbar.

FT-IR/FT-NIR dual-range spectrometer and this has been setup to work in the
near infrared, you may find that there is a red tint to the image when viewed in
the visible range. To remove this, fit a 1%T attenuator to the external beam
window of the spectrometer. See
page 48 for details.

This attenuator should be removed during data acquisition.

Fitting an Attenuator to the Spectrometer

on

OR

Click Maximize Energy in the Correction section of the Setup Microscope Advanced tab.

OR

Click the

button in the Camera View toolbar.

48 . Spotlight 200 User’s Guide

NOTE: If your Spotlight 200 system is supplied with a Frontier or Spectrum 400

FT-IR/FT-NIR dual-range spectrometer and you are using a large aperture in the
near infrared, the software may report an overload error when monitoring an IR
function or collecting data. To resolve this, fit a 32%T attenuator to the external
beam window of the spectrometer. See

Spectrometer

This attenuator should be kept in place during data acquisition.

8. Adjust the Illumination control in the Setup Microscope Basic tab to give the required
level of illumination.

OR

Click Auto in the Illumination section in the Setup Microscope Basic tab.

OR

, on page 48, for details.

Fitting an Attenuator to the

Click the

button in the Camera View toolbar.

Fitting an Attenuator to the Spectrometer

If your Spotlight 200 system is supplied with a Frontier or Spectrum 400 FT-IR/FT-NIR
dual-range spectrometer, you will be provided with an Attenuator kit (part number
L1160560) containing 1%T, 4%T, 6%T, 14%T and 32%T attenuators. The attenuators
attach to the external beam window of the spectrometer magnetically.

When examining samples in the visible range on systems fitted with an MCT detector, use of
the 1%T attenuator is recommended if the image appears with a red tint. However, the
attenuator should be removed during data acquisition.

Use of the 32%T attenuator is recommended if an overload error message is displayed when
using a large aperture. This attenuator should be kept in place during data acquisition.

Figure 15 KBr Window with 14%T Attenuator

Setting Scan Parameters

1. Select Instrument from the Setup menu.

2. Click the Setup Instrument Basic tab.

3. Click the Resolution setting to display the drop-down list.

For mid-infrared measurements, it is recommended that the spectral resolution is set to

−1

8 cm

; for near infrared, this value should be set to either 8 or 16 cm−1.

Getting Ready to Use the Spotlight 200 . 49

4. Select the Start and End values for the scan.

For mid-infrared analysis, it is normal to set the upper wavenumber limit to 4000 cm
and the lower limit to the low wavenumber specification of the detector, as shown in
the table below.

−1

Detector type Low wavenumber limit (cm

)

MCT mid-band 580

MCT wide-band 450

DTGS 380

The Service engineer can advise you of the lower limit to the low wavenumber, at the
time of installation, or it will be recorded on the Service Installation test spectra.

For near infrared measurements, on a system fitted with an InGaAs detector, the upper
wavenumber value is typically set to between 15800 and 8000 cm
to 4000 cm

−1

.

−1

and the lower limit

NOTE: To work in the near infrared range of the spectrum, the Spotlight 200 must be

attached to a dedicated NIR spectrometer or a dual-range spectrometer which has
been set up to operate in the near infrared range.

−1

The Setup Instrument Basic tab also provides an option for selecting the number of
accumulations. There are no firm rules about these selections since the number of
accumulations needed to generate acceptable spectra will depend on the nature of the
sample, the size of the area being analyzed and the requirements of the application.

For single point analyses where the analysis is relatively quick, it is probably better to “over­scan”; 50 accumulations taken at 8 cm

−1

resolution using an MCT or InGaAs detector

(20 accumulations for a DTGS detector) can be collected reasonably quickly.

For mapping or imaging experiments, the total time for the analysis is normally very
important and most analysts set the number of accumulations to be the very minimum
required to get a sufficiently useable spectrum. In mapping, this may be 1 to
5 accumulations per point and for imaging it may be 15 or 30 accumulations per pixel for the
background image and 2 or 4 accumulations per pixel for the sample image.

Optimizing Transmittance measurements

In most cases, samples for transmittance measurements are placed flat on top of an
IR-transmitting window such as NaCl, KBr, BaF
flattened fibers, the sample is simply secured across an open aperture mounted in the
standard sample holder.

These samples should be prepared such that they are thin, ideally 10 to 25 microns, but
certainly thinner than 50 microns (see section on sample preparation) and also they should
sit as flat on the window as possible.

, ZnSe or diamond. In some cases, such as

2

In transmittance measurements, the infrared beam passes through the window supporting
the sample and this will alter the beam characteristics (focus, etc.). This variation will be
different for different window materials depending on their refractive indices. To optimize the
infrared energy through the window (and sample), the lower cassegrain is moved up or
down using the Correction facility on the Setup Microscope Advanced tab to compensate
for this refractive index variation.

50 . Spotlight 200 User’s Guide

1. Place the thin, flat, sample onto a window (or across an aperture) and use the joystick
to bring the sample into visible focus.

If necessary, use the Auto-Focus option to focus the image.

2. Set the apertures to the appropriate size for the sample area, and mark this position
using the Add Marker option from the Microscope menu.

3. Use the joystick to move the sample away from the center of the viewing screen, and
focus on the top surface of the window.

Use the Monitor function (from the Measurement toolbar) to monitor the energy
throughput. Either manually adjust the lower cassegrain for maximum energy using the

up
facility.

4. Once the maximum energy through the window (or aperture) has been achieved, click
Halt on the Measurement toolbar.

5. Run a background spectrum through this point on the window (or aperture) using the
correct aperture size as selected previously.

6. From the Stage Move menu in the Microscope menu, select To Selected Marker and
record the sample spectrum.

or down buttons of the Correction function, or use the Maximize Energy

This procedure for correction of window materials should be done for single point, mapping
and imaging experiments.

If the same window is used repeatedly for analysis, this height correction of the lower
cassegrain need only be done once.

Preparing Samples

52 . Spotlight 200 User’s Guide

Preparing Samples

The Spotlight 200 enables you to examine the sample in the Camera View window to choose
the area where you want to collect images or spectra. To make sure that you collect good
quality spectra, it is important that you prepare samples properly. Sample preparation is
needed when the sample is too thick for transmission work, or the area of interest is on the
inside (for example in a laminated sample).

• If you are going to collect transmission spectra, the sample should ideally be thin
enough (approximately 5 to 20 µm) to give good detail and undistorted absorption
bands.

• The area of the sample must be large enough to give an adequate signal; otherwise,
the scan time must be increased.

Preparing a sample, therefore, often involves flattening it; this both thins it and increases its
area. The sample can be flattened by rolling, squeezing, or pressing.

This chapter tells you how to prepare samples for spectroscopy with the
Spotlight 200. It includes:

• A list of useful tools;

• A list of window materials commonly used for mounting samples;

• Descriptions of special techniques used to prepare particular types of samples.

Preparing Samples . 53

Tools for Sample Preparation

This section lists the tools you need for preparing samples:

• Tools provided with the Spotlight 200;

• Tools in the microsampling toolkit;

• Materials to have available;

• Specialized accessories you may want to purchase.

Tools Provided with the Spotlight 200

The following items for use in sampling are provided in the Sampling Accessories Kit that is
supplied:

Item Use

Holder for 13 mm disks Supporting 13 mm disks on the sample stage

Slides, glass (box) Supporting samples for sample preparation

Rotating 13 mm disk holder Supporting samples and allows rotation of the sample

Support for large samples Clips on to the sample stage; supporting bulky samples so

that the stage clip does not interfere with them

Gold mirror assembly Reflection measurements

KBr windows (2) Supporting samples

54 . Spotlight 200 User’s Guide

Figure 16 Some of the Tools in the Microsampling Toolkit

The following tools are provided in the Microsampling toolkit (see Figure 16).

Tool Use

Steel tweezers Picking up extremely small objects

Roller knife Cutting (knife end) and flattening (roller end)

Steel probe Pulling samples apart, separating fibers

Forceps, 4½ inch, Cd plated Picking up small objects

Tungsten alloy needle Transferring particles

Pin vise Holds needles for sharpening or for flattening samples

Interchangeable handle for micro
tools

Handle for tungsten needle or steel probe

Preparing Samples . 55

Other Useful Tools

Depending on the type of samples that you usually work with, it may be helpful to have
some of the following tools:

Tool Part Number

Microprobe with right angle bend N9302606

Forceps, round tips N9302607

Forceps, narrow needle points N9302608

Windows: All 13 mm diameter

BaF2 (1 mm thick) N9302611

BaF2 (2 mm thick) N9302612

ZnSe (1 mm thick) N9302613

NaCl (2 mm thick) N9302614

KBr (2 mm thick) N9302615

Wide-tipped forceps, hooked 09908138

Wide-tipped forceps, flat 09908400

1.5 mm microdisk; fits in 13 mm disk holder to support
very small samples

The following items are available from your local PerkinElmer sales office or agent:

01861043

Specialized Accessories

The following accessories are extremely useful in preparing certain types of samples (as
described in

• Miniature Diamond Anvil Cell (part number N9302618);

• Fiber Optic Illuminator (part number N9302602);

• Microtome.

Techniques for Preparing Samples

, starting on page 58):

56 . Spotlight 200 User’s Guide

Items to Have Available

In addition to the items provided with the Spotlight 200, we recommend that you have the
following available:

• Tape with adhesive on both sides (“double-sided tape”) for holding long or large
samples on the sample stage;

• Single-edge razor blades for cutting samples.

Preparing Samples . 57

Common Window Materials

Both liquid and solid samples are often mounted on salt windows. Very thin windows,
1 to 2 mm thick, give the best spectra. The following materials are commonly used in
windows:

• KBr: Potassium bromide is inexpensive, and it transmits infrared radiation to below
400 cm

−1

. The major disadvantage of this material is that it is hygroscopic, so that the

windows fog easily.

• BaF

: Barium fluoride is not hygroscopic. Its transmittance cut-off is 750 cm−1. It can

2

break or crack easily.

−1

• NaCl: Sodium chloride transmits infrared down to 600 cm

. Otherwise, its properties

are similar to KBr.

−1

• ZnSe: Zinc selenide is not hygroscopic. Its transmittance cut-off is 650 cm

. ZnSe is
more durable than the other windows but is yellow, so that the field of the Monitor
visible window appears yellow.

58 . Spotlight 200 User’s Guide

Techniques for Preparing Samples for Transmittance Measurements

This section describes some useful techniques for preparing various types of samples.

Flattening Solids

Flattening samples by pressing or squeezing often enables you to make thick samples thin
enough to give good infrared spectra. As the samples are usually quite small, only moderate
force is necessary.

Rolling with the roller knife

The roller end of the roller knife provided in the microsampling tool is one of the simplest
and most effective devices for flattening samples. It is especially useful for flattening fibers
or particles.

You can treat different types of sample in different ways:

• If the sample is soft, you can roll it on a small salt window.

• If the sample is hard, you can roll it on a hard surface, such as glass or metal. A flat,

black cap from a jar makes a good surface for rolling a light-colored sample.

• If you roll the sample on a small, flat piece of metal you can view it and collect spectra
in reflectance mode. Samples rolled on windows transparent to infrared can be
examined in transmittance.

• If you flatten fibers on a glass microscope slide, they can then be peeled off and
mounted either on a window or over the aperture for the microscope slide.

Squeezing with a pellet press

You can squeeze samples between the polished anvils of a KBr pellet press without KBr. To
collect the spectra use one of the following methods:

• Peel the flattened sample off the anvil with a probe or knife and place it on a sample
mount. Collect the transmittance spectrum.

• Leave the sample on the polished anvil and collect the reflection spectrum. Use a clean
area of the anvil as the reference.

Using a diamond anvil cell

See

Diamond anvil cell

diamond anvil cell.

on page 60 for information on flattening samples with a miniature

Preparing Samples . 59

Compressing between infrared transmitting windows

Pressing two windows together, with the sample between them, compresses the sample.
This also provides optical contact between the windows and the sample, reducing surface
scattering.

Windows made of NaCl or KBr are relatively soft. If your sample is hard, or if it is wet, use

or ZnSe.

BaF

2

Pressing with the heel of a probe

Press on small samples with the flat end of the probe handle. Even moderate pressure
usually produces considerable thinning.

Pressing with a needle

Pressing with the point of a needle or probe applies a high force per unit area, because the
area of contact is small.

Rolling a hard sample with the side of a sewing needle held in a pin vise presses it into a
flake.

Slicing Samples from Solids

Cutting a wedge of sample

Cutting a wedge-shaped piece from its edge enables you to produce a thin sample while
destroying very little of the original. This technique can be used with laminates, plastics,
films, paint chips and paper.

To cut a wedge-shaped piece from a relatively thick sample:

 Hold the sample in tweezers as you slice a thin wedge from it with a razor blade. Taper

the wedge to as thin a slice as possible.

To cut a wedge-shaped piece from a relatively thin sample:

1. Place the sample between two offset glass slides. Allow a triangular portion of the
sample to protrude as shown on the left in Figure 17.

Figure 17 Cutting a Wedge-Shaped Sample

60 . Spotlight 200 User’s Guide

β

2. Run a razor blade or the roller knife along the edge of the upper slide.
The triangular piece of the sample is sliced off, giving a wedge-shaped sample.

3. To mount the sample, rotate it so that it is positioned as shown on the right in
Figure 17.

4. Position it under the microscope so that the infrared beam goes through the thin end of
the wedge (circled in Figure 17).

Microtoming

A microtome enables you to slice a sample into thin cross-sections, 0.5 to 20 µm thick. It is
commonly used to prepare samples for light microscopy; the same range of thicknesses is
also appropriate for infrared microspectroscopy.

If you are trying to identify the individual components of a laminate, microtomed samples
give the best results.

Samples are often embedded in a supporting medium before they are microtomed. If you
must use an embedding material, choose it carefully so that it does not alter the sample by
reacting with it, dissolving it, or contaminating it. Some commonly used materials are:

• paraffin wax: This is the preferred medium for infrared spectroscopy. It produces few
spectral interferences, and it can usually be easily removed from the sample with warm
xylene.

•

-pinene wax: This material is similar to paraffin.

• plastic embedding materials: These can be used depending on the size and porosity
of the sample.

• acrylic and epoxy resins: Although these are commonly used in light microscopy,
they are not recommended for infrared, because they are hard to remove and can
cause spectral interferences.

Polymers

Pressing or squeezing enables you to reduce the pathlength of polymer samples such as
paint chips, thick films, elastomers, or fibers.

Diamond anvil cell

The miniature diamond anvil cell, shown in Figure 18, enables you to press polymers (or
other compressible samples). It enables you to both thin the sample and collect its spectrum
in the same device; this is an advantage when you have limited material available. It is small
enough to be easy to manipulate, and fits in the recessed retainer in the support for large
samples. By collecting a background spectrum of an empty area of the cell, you can
completely compensate for the absorption bands of the diamonds.

Preparing Samples . 61

To thin a sample in the miniature diamond anvil cell:

1. Loosen and remove the three screws that hold the cell together.

2. Lift off the top half of the cell and set it aside.

3. Place the sample on the bottom half of the cell. (The sample must be small.)

4. Put the top half back on the cell, lining up the red lines on the top and bottom halves.
Do not tighten the screws yet; applying uneven shear forces may damage the
diamonds.

One diamond can damage another.

CAUTION

5. Press down on the cell with your thumb to thin the sample.

6. Replace the three screws and tighten them finger tight.

NOTE: If the spectrum collected with the diamond anvil cell shows interference fringes,

place some KBr in the cell and collect a background spectrum through it.

Figure 18 The Miniature Diamond Anvil Cell

Pressing elastomers between windows

If your sample is elastic and you are compressing it between windows, you must apply
pressure continuously. Use the following procedure:

1. Press on the windows with a probe, flattening the sample.

2. While maintaining the pressure, apply small amounts of quick-setting nitrocellulose
cement to the edges of the salt plates.

When the cement is dry, the sample remains compressed.

The compression cell (see
this kind of sample.

Using the Compression Cell

on page 66) enables you to compress

62 . Spotlight 200 User’s Guide

Filled polymers

When a polymer contains a high concentration of fillers, and you want to analyze the
polymer, you have to prepare a sample for analysis that is free of filler.

Often you can obtain a suitable sample by cutting a thin wedge of the material with a sharp
blade. If the filler is not uniformly dispersed, you can find clear regions of polymer for
analysis.

You can use pyrolysis to remove the fillers. As you heat the sample, the polymer volatilizes,
and the fillers are reduced to ash. The sample can be pyrolyzed in the following ways:

Using a disposable pipette:

1. Place the polymer in a disposable pipette and seal the large end.

2. When this end cools, tap the polymer into it, then heat the sample gently.
The pyrolyzed polymer condenses on the walls of the pipette. The filler is left behind as

ash.

3. Score and break the pipette between the ash and the pyrolysate.

4. Add a drop of solvent to the pyrolysate to wash it on to a salt plate.

If the amount of sample is small, use a capillary tube instead of a disposable pipette.

Using a microbrush to pyrolyze micro amounts:

1. Seal the end that is away from the brush fibers and tap the sample particle into this end
then heat it gently with a microtorch.

2. After pyrolysis, break off and discard the end of the tube that contains the ash.

3. While holding the fibers of the brush against the salt plate, add a drop of solvent to the
broken end.

4. Allow the solvent containing the pyrolysate to flow into the fibers.
When it evaporates, the pyrolysate remains on the salt plate for analysis.

Particles

Crushing

Crushing enables you to thin samples such as large particles that cannot be sliced. This can
be done in various ways:

• If the sample is small, crush it with the roller end of the roller knife.

• If the sample is larger, use a pestle and mortar.

Preparing Samples . 63

Separating by aperturing

Powders and other particulate solids may contain several different components. Instead of
separating them, use the infrared aperture to isolate the component you want to sample:

1. Spread the sample out with a probe so that you can visually distinguish the
components.

2. Looking at the Camera View window, find a particle of the component you want to
sample.

3. Center this particle in the field of view.

4. Adjust the infrared aperture (shown by the red dashed lines) until only the particle that
is of interest is visible.

You can easily pick up extremely small particles and transfer them with a very fine-pointed
tungsten needle. Scoring the surface of the salt plate with the needle makes a simple map to
help you positively identify the particles under the microscope.

Transferring with a tungsten needle

When necessary, sharpen the tungsten needle.

Nujol or fluorolube mulls

Suspending fine particles of a solid sample in nujol or fluorolube reduces or eliminates the
surface reflections that can distort absorption measurements. These oils also reduce the
amount of radiation lost to reflection or scattering.

If the film is thin enough, you can correct the spectrum for the presence of the oil by
subtracting a spectrum of the pure liquid. It is difficult, however, to obtain the correct
thickness for a good subtraction.

Fibers

You can roll fibers to flatten them (see
in a diamond anvil cell (see

Polymers

Flattening

on page 60).

Solids on page 58), or they can be pressed

Fibrous Solids

If a fibrous sample, such as paper, is too thick, tear it and examine the torn edges. The
edges contain single fibers and thin clumps of fibers.

64 . Spotlight 200 User’s Guide

Coatings on Substrates

If the sample is coated on a substrate, the method for collecting its spectrum depends on the
nature of the substrate:

• If the substrate is reflective, you can analyze the sample in reflectance.

• If the substrate is opaque, scrape off a sample of the coating; use the roller knife to

scrape a small piece on to a KBr or BaF

• Coatings on reflective or opaque substrates can be measured using the micro-ATR
objective. See

Collecting Spectra With the ATR Objective

disk.

2

on page 78 for details.

Liquids

Solutions of samples

Although liquids are seldom analyzed with the Spotlight 200, sometimes the sample of
interest is in solution.

1. Transfer the solution on to a salt plate.

2. Allow the solvent to evaporate, leaving the sample on the plate.

Micropipettes

You can use a micropipette to apply liquid to the surface of a salt plate, or to the edge of the
junction between two salt plates. In the latter case, the liquid flows between the plates by
capillary action.

Preventing liquids from spreading

If the amount of liquid being transferred to the salt plate is very small, restrict it to a small
area of the plate. There are several ways to do this:

• Use a microbrush to transfer solutions. The bristles of the microbrush hold the liquid in
a small region of the salt plate until the solvent evaporates.

Repeatedly jab a small area of the salt plate with a tungsten probe. Leave the resulting
small salt particles in the well that is produced.

The capillary spaces between the salt particles retain the liquid and minimize spreading.

• Place the salt plate on a small metal washer that is being gently heated.
Because there is more heat at the outside of the salt plate than near the center (over

the hole in the washer), the droplet of liquid is forced toward the center.

Techniques for Collecting

Spectra

66 . Spotlight 200 User’s Guide

Techniques for Collecting Spectra

This chapter describes how accessories and collection techniques enable you to collect
spectra from different types of sample.

Using the Compression Cell

The optional compression cell (part number N1870185, Figure 19) enables you to flatten soft
materials. It also enables you to hold specimens flat and in optical contact with salt windows.
The cell consists of an aluminum block, machined to accept salt windows, with window
retainers and a special wrench to apply pressure across the windows. The sample is held
between the two windows. The compression cell fits into the sample slide holder on the
stage of the microscope. Windows with thicknesses equal to 1 mm and 2 mm, and outer
diameter equal to 13 mm can be used with the cell. Two KBr windows (2 mm thickness) are
included with the system. The cell can apply pressure without rotating the windows, and
therefore avoids scratching them.

Figure 19 The Compression Cell

Although samples can be thinned using the compression cell, it does not replace the
miniature diamond anvil cell as a sample-thinning device. The primary application of the
compression cell is for keeping specimens flat over the entire visual field of view.

Techniques for Collecting Spectra . 67

Using a Hot/Cold Stage

An optional hot stage enables you to study temperature-dependent phenomena in
microsamples. A hot stage consists of a temperature controller and a heating block that
accepts infrared windows. The heating block contains an integral thermocouple, and the
temperature is digitally displayed in degrees Celsius on the controller.

A hot stage can heat samples in 1 degree increments. The maximum temperature that can
be achieved depends upon the type of hot stage being used; see
for details. A target temperature can be selected and maintained.

A hot stage is held in the slide clip on the sample stage of the microscope. The microscope
requires no modifications to accept the hot stage.

A hot stage enables you to study phase transitions and temperature-dependent chemical
reactions. Infrared microscopy can provide detailed molecular structural information for
systems undergoing phase transitions; this information is not available from thermal data
only. Polymers, pharmaceuticals and liquid crystals are examples of materials where
investigations of phase transition are important.

NOTE: Some types of hot stage can be used with a cooler. The cooler uses liquid nitrogen to

reduce the temperature of the stage down to −196 °C. See
for details

Accessories

Accessories

on page 119

on page 119

Collecting the Spectrum of a Thick Sample

Lowering the stage using the Z-control on the joystick enables you to focus on a thick
sample. For very thick samples you may have to remove the lower cassegrain assembly; then
only the reflectance method can be used to view the sample and collect spectra.

To collect the spectrum of a thick sample

1. Click Zero on the Setup Microscope Advanced tab.

The lower cassegrain moves to the position where the infrared beam is focused if no
sample is on the stage.

2. Move the sample stage to its highest possible position.

3. Click Park on the Setup Microscope Advanced tab.

The lower cassegrain bracket moves down to a lower position.

4. Release the locking lever located on the right side on the back of the lower cassegrain
assembly (Figure 20).

68 . Spotlight 200 User’s Guide

Figure 20 The Lower Cassegrain

5. Gently slide the cassegrain assembly forward and out of the dovetail connector.

NOTE: To make withdrawal easier, pull the locking lever gently.

6. Click Lower Park.

The lower cassegrain bracket moves to its lowest position.

7. Lower the stage using the Z-control on the joystick.

Once the lower cassegrain is removed, lowering the stage when at the
limit of its travel backwards will cause it to collide with the dovetail

CAUTION

connector. This will cause permanent damage to the stage motor gear
box. If necessary, remove the dovetail connector to avoid damaging the
instrument.

To remove the dovetail connector:

 Loosen the two fixing screws at the front of the connector using a 3 mm hex key, and

pull the connector away from the microscope (see Figure 21).

Techniques for Collecting Spectra . 69

Figure 21 Location of the dovetail connector behind the stage

To refit the lower cassegrain after use

1. If necessary, refit the dovetail connector using the fixing screws.

2. Raise the stage using the Z-control on the joystick to its highest position.

3. Click Zero on the Setup Microscope Advanced tab.

4. Slide the cassegrain assembly back into the dovetail connector, as far as it goes.

NOTE: To make replacement easier, pull the locking lever gently.

5. Tighten the locking lever.

6. Make sure that the cassegrain is correctly seated.

7. Place the gaiter over the cassegrain if the purge facility is required.

70 . Spotlight 200 User’s Guide

Collecting a Spectrum in an Inert Atmosphere

The optional purge system enables you to purge the spaces around the sample and the
infrared beam (typically with nitrogen or dry air), to provide an inert atmosphere.

Do not use flammable gases for purging.

WARNING

Parts of the purging system

Some parts of the purging system are shown in Figure 22.

Figure 22 Parts of the Purge System

The purge system consists of:

• The gas inlet connector on the metal plate at the rear of the microscope and the
connector at the rear of the spectrometer;

• The purge coupling molding connecting the spectrometer to the microscope;

• The lower purge gaiter under the lower cassegrain.

When all of these parts are in place, the gas entering through the inlet displaces air from the
path of the infrared beam.

Techniques for Collecting Spectra . 71

Purging the system

1. Make sure that all parts of the purge system, as listed above, are in place.

2. Set up the microscope.

Getting Ready to Use the Spotlight 200

See

3. Place the sample in position.

starting on page 41.

4. Purge the microscope and spectrometer for 15 to 20 minutes at a rate of
10 l min

−1

.

5. Collect the background spectrum and the spectrum of the sample.
See the on-screen Help for further information about collecting spectra.

Changing the sample

If you have changed a sample, any air that has entered the system must be flushed out.

72 . Spotlight 200 User’s Guide

Viewing a Sample with the Visible Polarizer

Polarized visible light can enable you to identify areas or structures that differ chemically and
to solve problems commonly found in infrared microspectroscopy applications.

The Theory of Light Polarization

Ordinary light and infrared radiation consists of waves vibrating in all possible planes
perpendicular to the direction of propagation. This is represented in the left side of Figure 23.

Conventionally the plane of the light is taken to be the plane of the continuously varying
electric vector.

If the light passes through a
that have their plane of vibration in one particular direction. The light that emerges is said to

polarized

be

Figure 23 Representation of Unpolarized Light (left) and Polarized Light (right)

Because all components of the wave in the plane of polarization are transmitted, the ideal
polarizer allows 50% of the light through.

If a second polarizer is placed in the path of the polarized light, two things may result:

• If the second polarizer is placed in the same direction as the first (as at the top of
Figure 24), the polarized light can pass straight through.

, and is represented on the right side of Figure 23.

polarizer

, the polarizer allows the passage of only those waves

• However, if the second polarizer is placed at a right angle to the first, a situation which
is referred to as crossed polarizers, the passage of the polarized light is blocked, that is,
extinction occurs (as at the bottom of Figure 24).

Techniques for Collecting Spectra . 73

The second situation occurs because the light transmitted by the first polarizer oscillates in
exactly the plane that is blocked by the second polarizer.

Figure 24 Polarizers Parallel (top) and Polarizers Crossed (bottom)

Some materials are
their orientation. These materials can alter the polarization of light passing through them;
this is dependent on the wavelength of the transmitted light.

When you look at an anisotropic sample using polarized light, the change in polarization
caused by the sample means that some light leaks through the second polarizer. Because the
change in polarization is dependent on the wavelength, the color of the light emerging
changes with the distance traveled through the sample and the amount of birefringence
encountered.

anisotropic

(or

birefringent

), that is, their refractive index depends on

Applications

Differences in the birefringence of an object or area may be an indication of chemical
disparity. This can be useful in visibly separating or identifying an object or area of interest
before collecting an infrared spectrum. Some examples are given below.

Laminates

Many polymer structures consist of different layers of material and adhesives of varying
thicknesses bonded together in order to meet physical requirements. If you view a cross
section of the structure using polarized light, you can identify the individual layers and set
the apertures to collect a separate infrared spectrum from each layer. This is useful for
identifying the materials used to create the structure.

74 . Spotlight 200 User’s Guide

Inclusions in polymer films

Although they may appear identical when using non-polarized light, the film and inclusion
present in a polymer film may exhibit different degrees of birefringence when using polarized
light. If this is so, you can visibly identify the inclusion and collect an infrared spectrum to
determine its composition.

Rocks minerals and crystals

Most crystals are characteristically birefringent and thus are ideally suited to this technique.
Viewing a mixture consisting of crystals, such as an artificial sweetener, pharmaceutical
powder, or an illicit substance, enables you to visibly separate the components by their
relative size, shape and birefringence.

Fibers

Polarization may enable you to identify and separate fibers in a clump or to view a
bicomponent fiber. Most fibers in their natural state are optically thick and their cylindrical
shape can cause lensing effects. For these reasons, fibers are usually flattened in preparation
for infrared microspectroscopy. This flattening affects the birefringence of the structure and
may degrade the usefulness of this technique.

Biological substances

Birefringence can occur in some biological substances. You can collect infrared spectra of
thin sections of these materials. In some cases, polarized light can reveal structures or
chemical disparities in these structures, and infrared spectra can be collected of the different
regions of interest.

Equipment

The equipment for visible polarization studies consists of two parts: the polarizer and the
analyzer (Figure 25).

The polarizer polarizes the incoming beam from the illuminator and the analyzer contains a
polarizing element that you can rotate to any orientation. It is placed in front of the camera.

The polarizers for both transmittance and reflectance are built into the Spotlight 200, and are
automatically switched into the beam when the analyzer is inserted.

Figure 25 The Analyzer

Techniques for Collecting Spectra . 75

Visible Analyzer
Slot

Operating the Analyzer

1. Insert the analyzer into the slot in the right side of the front cover (Figure 26).
Push the analyzer in with the wheel facing towards you. It has two positions:

• The first position allows the full beam to pass through.

• When the analyzer is inserted fully, the polarizing element is in the beam.

Figure 26 Analyzer Position

2. When the polarizing element is in the beam, rotate the wheel while viewing the Camera
View window.

76 . Spotlight 200 User’s Guide

Collecting IR Spectra Using the Infrared Polarizer Accessory

An absorption band in the infrared range occurs when a vibration is accompanied by a
change in dipole moment. The electric vector of the incident radiation must have a
component in the direction of the dipole moment change.

In polarization spectroscopy, the absorption bands of greatest interest are those in which the
direction of dipole moment change is related to a bond direction, for example, the nitrile
stretching vibration. If, in a particular sample, all the bonds of a particular type are aligned in
a specific direction, the strength of the absorption depends on the polarization of the incident
radiation, that is, whether the electric vector is parallel to or perpendicular to the bond
direction.

For example, stretching an acrylic fiber aligns the molecules with the general direction of the
polymer chains parallel to the fiber axis, and the nitrile groups tend to be oriented
perpendicular to the axis. If the spectrum is collected with the infrared radiation polarized
perpendicular to the axis, the nitrile absorption peak is much stronger than if the spectrum is
collected with radiation polarized parallel to the axis. The ratio of the two intensities (called

dichroic ratio

the
the polymer chains.

) is a measure of the extent of alignment of the nitrile groups and thus of

The Spotlight 200 enables you to collect polarization spectra of very small samples. These
include:

• Single filaments (typically 14 x 70 µm);

• Films;

• Single crystals;

• Liquid crystals.

Equipment

The optional polarizer has a rotatable silver bromide element in an aluminum mount
(Figure 27).

Figure 27 The Infrared Polarizer

Techniques for Collecting Spectra . 77

Infrared Polarizer

Using the Polarizer

1. Remove the snap-in cover that masks the aperture for the infrared polarizer (Figure 28).

2. Slide the analyzer into the vertical slot on the sample holder that can be seen through
this aperture.

The flat side of the analyzer must be towards the rear of the microscope, and the wheel
facing outwards.

Figure 28 Infrared Polarizer Position

3. Push the analyzer into the slot until it stops.

4. Turn the wheel to orient the polarizing element.

The polarizer element is extremely fragile. Do not touch it with anything.

CAUTION

It cannot be washed, dusted, or blown upon by air. If damaged, it
cannot be repaired. When it is not in use, protect it in the case supplied.

NOTE: Both the scribed line and the uneven coloration are usual, and do not affect the

performance of the element.

78 . Spotlight 200 User’s Guide

2

/

1

2

21

2

1

)

(sin

2

n

n

d

p

−

=

θ

p

λ

1

n

21

n

Collecting Spectra With the ATR Objective

The ATR (attenuated total reflectance) technique enables the collection of spectra from
materials that are too opaque for transmission measurements, and too strongly absorbing for
good reflectance measurements. Spectra can be collected with little sample preparation.

Spectrum software enables you to automatically map a sample using the ATR objective.
Spectra are collected by touching the ATR objective on the sample and collecting the
spectrum generated from the surface layer of the sample. See the on-screen Help for further
information.

The ATR objective uses a crystal made from a material that transmits infrared radiation, and
has a high refractive index. An infrared beam enters the crystal and is internally reflected
within the crystal, creating an evanescent wave. At each reflection inside the crystal, the
wave continues beyond the crystal surface into a sample that is held in close contact. The
penetration depth depends on the refractive indices of the crystal and the sample. For a
germanium crystal, the penetration depth for most samples is less than 1 µm. The
penetration depth also varies with the wavenumber of the infrared radiation:

Where:

is the refractive index of the ATR crystal (for germanium 4.0)

λ

is the wavelength of the radiation

θ

is the angle of incidence of the beam

is the ratio of the refractive indices of the sample and the ATR crystal.

ATR objective

The ATR objective enables the microscopic examination of samples in order to locate the
exact area of interest. The ATR crystal is moved vertically out of the beam path to enable
you to view the sample, and place the area of interest in the center of the field of view. The
crystal is then placed onto the sample under positive pressure to collect a spectrum.

The crystal has a small contact area formed by the flattened point of a cone; this ensures a
100 µm diameter contact area.

Two versions of the ATR objective are available. The manual ATR objective requires you
to raise and lower the crystal using a toggle bar on the ATR assembly. The automated ATR
objective not only controls the crystal automatically, but also includes a weighbridge to
measure the force applied to the sample by the crystal, which helps you obtain more
repeatable spectra.

NOTE: The weighbridge communicates with the microscope using a small infrared

transmitter located behind a window at the rear of the platform. Take care not to
cover this window with any part of your sample as this will prevent the weighbridge
from transmitting the value of the force applied by the crystal.

Techniques for Collecting Spectra . 79

Sample

ATR Crystal

Infrared In

Infrared
Collection

Optical path

Infrared radiation is directed into the crystal from the front half of the upper cassegrain, and
is focused at the sample position. It is reflected once within the crystal, then the totally
internally reflected beam is collected by the rear portion of the upper cassegrain, which
focuses it on the remote aperture. The radiation is directed onto the detector in the
microscope.

Figure 29 Infrared Radiation Optical Path

80 . Spotlight 200 User’s Guide

ATR Objective Specification

The ATR cassegrain is fitted to the microscope and the ATR crystal holder is supplied
separately.

ATR crystal materials

Germanium (Ge), Silicon (Si), and Diamond Coated Germanium

Range of measurement

Ge: 5500 cm−1 to 600 cm

Ge/Di: 5500 cm−1 to 600 cm−1

−1

Si: 7800 cm

to 800 cm

−1

−1

Area of contact with sample

Nominally 100 µm diameter flat surface. Single internal reflection from surface.

Maintenance

The ATR assembly may be removed (see

Microscope

replaced and aligned by the user (see
page 81).

on page 87), cleaned (see

Removing the ATR Crystal Holder from the

Cleaning the ATR Objective

Crystal on page 90), and

Fitting the ATR Crystal Holder to the Microscope

on

Techniques for Collecting Spectra . 81

ATR cassegrain

ATR crystal holder

Crystal assembly

Knurled

Fitting the ATR Crystal Holder to the Microscope

1. Switch off the microscope at the electricity supply.

2. Stop any laser radiation from entering the microscope by switching the internal beam of
the spectrometer to the internal sample compartment.

For further information on how to do this, see the Spectrum software on-screen Help.

3. Hold the crystal assembly under the upper cassegrain.
Be careful not to change the alignment of the two adjusting levers.

4. Fit the two adjustment thumb-nuts (Figure 30).
The assembly is aligned if the adjusting levers have not been moved.

5. If fitting the automated ATR objective, insert the jack plug into the socket at the rear of
the cassegrain (Figure 31).

Lower the stage if necessary to improve the access to the socket.

6. Direct the infrared beam to your microscope.

7. Switch on the microscope.

Figure 30 The Manual ATR Crystal Holder and Cassegrain

82 . Spotlight 200 User’s Guide

Jack plug socket
(plug not shown)

ATR crystal
assembly

Crystal holder

Motor (lowers and
Knurled thumb-nut

retracts the crystal)

Figure 31 The Automated ATR Crystal Holder and Cassegrain

Techniques for Collecting Spectra . 83

Measuring the Maximum Energy in Reflectance Mode

The following procedure allows you to determine the maximum energy reaching the detector
when the ATR crystal is retracted. The value obtained here can be used to check that the
crystal is aligned correctly; see

1. Make sure that your spectrometer is switched on, and the source has warmed up.

2. Make sure that the microscope, PC, and stage controller are switched on and that
Spectrum software is running on your PC.

3. Ensure that the infrared beam is directed through the microscope and not the
spectrometer by using the Setup Instrument BeamPath tab.

4. Place the slide holding the reference mirror (supplied with the microscope) on the
sample stage.

5. Select the Reflectance sampling mode on the Setup Microscope Basic tab.

Checking the Infrared Alignment

on page 86 for details.

6. Look at the image in the Camera View pane and focus the beam on the surface of the
mirror using the Z-control on the joystick, or the Auto-Focus option.

There are usually dust particles or scratches on the surface that you can use to focus
on.

7. Enter 100 in the Aperture Width and Height text boxes on the Setup Microscope
Advanced tab.

8. Monitor the energy reaching the detector by selecting Monitor from the Measurement
toolbar and choosing the Energy option.

9. Record the energy level measured.

10. Click Halt.

84 . Spotlight 200 User’s Guide

tage

Knurled height-adjustment nut

Adjusting the Height of the Crystal

NOTE: The crystal is mounted on a bayonet assembly similar to that on some light bulbs.

To prevent serious damage to the crystal, do not move the sample s

CAUTION

1. Lower the manual ATR crystal onto the reference mirror by turning the bar clockwise
and slowly lowering the toggle bar.

You must always support both ends of the toggle bar when lowering or raising the
crystal.

OR

while the ATR crystal is in the lower, working position and touching or
near to the sample.

Click the

2. Using the knurled height-adjustment nut, adjust the height of the crystal so that it just
rests on the mirror under positive pressure (Figure 32 and Figure 33).

Figure 32 Adjusting the Crystal Height (Manual ATR Objective)

button to lower the automated ATR crystal onto the reference mirror.

Figure 33 Adjusting the Crystal Height (Automated ATR Objective)

Techniques for Collecting Spectra . 85

Centering the Crystal

1. Retract the manual ATR crystal using the toggle bar, by lifting the bar then twisting
counterclockwise.

OR

Click the

2. Place a piece of black PVC electrical tape on a microscope slide, and put the slide on the
microscope stage.

3. Move the stage so that the tape is directly below the crystal, and focus the beam on the
tape.

4. Lower the crystal onto the tape.
If the crystal height is adjusted correctly, it will just touch the tape and make a shallow

impression in it.

5. Retract the crystal.
If there is no impression on the tape, the crystal height should be lowered slightly by

turning the knurled height-adjustment nut left to right a quarter of a turn.
If there is a deep impression on the tape, which does not disappear within a few

seconds, the crystal height should be raised slightly by turning the knurled height­adjustment nut right to left a quarter of a turn.

6. If appropriate, repeat steps 4 and 5 until the crystal just touches the tape.

7. Once the crystal height has been corrected, look at the impression of the crystal on the
tape. Decide whether the impression is in the center of the field of view.

If the impression is in the center of the field of view, the assembly is aligned.

button to retract the automated ATR crystal.

If the impression is not in the center of the field of view:

1. Loosen the two knurled thumb-nuts under the ATR crystal holder half a turn
(Figure 34).

NOTE: The centering adjustments are the same for both the manual and automated ATR

objectives.

Figure 34 Centering the ATR Crystal

86 . Spotlight 200 User’s Guide

2. Move the ATR crystal holder using the adjusting levers until it is centered.
One lever moves the assembly backwards and forwards and the other from side to side.

3. Tighten the knurled thumb-nuts.

4. Move the microscope slide to a fresh area of tape.

5. Lower the crystal onto the tape.

6. Retract the crystal.

7. Look through the microscope at the impression of the crystal on the tape. Decide
whether the impression is in the center of the field of view.

8. If necessary, repeat steps 1 to 7 until the ATR objective is aligned.

Checking the Infrared Alignment

The following procedure allows you to determine the energy reaching the detector when the
ATR crystal is in place. If the crystal is aligned correctly, the energy level measured should
be at least 20% of the maximum energy measured in reflectance mode; see

Maximum Energy in Reflectance Mode

on page 83 for details.

Measuring the

1. Place the slide holding the reference mirror (supplied with the microscope) on the
sample stage.

2. Look at the image in the Camera View pane and focus the microscope on the surface of
the mirror with the Z-control on the joystick, or the Auto-Focus option.

There are usually dust particles or scratches on the surface that you can use to focus
on.

3. Lower the crystal on to the reference mirror.
You should lower the crystal and then raise the stage slowly with the manual ATR

objective. For the automated ATR objective, click the Contact Sample button on the
Setup Microscope Basic tab once the crystal is lowered.

4. Enter 100 in the Aperture Width and Height text boxes on the Setup Microscope
Advanced tab.

5. To monitor the energy reaching the detector, select Monitor from the Measurement
toolbar and choose the Energy option.

6. Record the level of energy measured and compare it with the value measured when the
crystal is retracted.

If the energy is less than 20% of the maximum energy measured, repeat the alignment
procedure on page 85.

7. Click Halt.

Alternatively, you can measure the energy in reflectance mode before and after lowering the
ATR crystal on to the mirror. The software displays the maximum energy with the crystal
retracted, and then shows the current energy when ithe crystal is lowered.

Techniques for Collecting Spectra . 87

Removing the ATR Crystal Holder from the Microscope

To use the microscope for conventional microspectroscopy, you need only retract the ATR
crystal from its working position. If, however you need a very large working distance, for
example if the sample is recessed, the ATR crystal holder can be removed.

1. Switch off the microscope at the electricity supply.

2. Stop any laser radiation from entering the microscope by switching the internal beam of
the spectrometer to the internal sample compartment.

See the Spectrum on-screen Help for further information.

3. Make sure that the ATR crystal is in the raised position.

4. Screw the plastic protective cover in place, over the crystal.

5. For the automated ATR objective, remove the jack plug from its socket.

6. While holding the crystal assembly, unscrew the two adjustment thumb-nuts
completely. Allow the assembly to drop vertically from the cassegrain. Be careful not to
disturb the two adjusting levers.

Any movement of the adjusting levers will change the alignment of the assembly when
it is refitted.

7. Store the assembly carefully for future use.
When refitted, the assembly will still be aligned, provided that the adjusting levers have

not been moved.

8. Direct the infrared beam back to the microscope.

88 . Spotlight 200 User’s Guide

Fitting a Crystal Assembly to the ATR Objective

Manual ATR Objective

1. Remove the ATR crystal holder, as described on page 87.

2. Place the crystal holder on a bench, with the crystal upwards.

3. While holding the crystal assembly in place, unscrew the knurled height-adjustment nut.
The crystal assembly is under spring pressure. If you do not hold the assembly in place,

the spring may be lost.

4. Remove the crystal assembly.
Leave the spring in the ATR crystal holder.

5. Place the new crystal assembly into the ATR crystal holder and refit the height-
adjustment nut.

Take care not to damage the crystal.

6. Refit the ATR crystal holder to the microscope, as described on page 81.

7. Align and adjust the ATR crystal holder, as described on page 84.

Figure 35 The Manual ATR Crystal Assembly

Automated ATR Objective

1. Remove the ATR crystal holder, as described on page 87.

2. Place the crystal holder on a bench, with the crystal pointing upwards and the motor
pointing away from you.

3. Unscrew the knurled height-adjustment nut.

4. Pull the crystal assembly out of the holder.

5. Hold the new crystal assembly with the thin alignment groove facing you (Figure 36).

Techniques for Collecting Spectra . 89

Alignment groove

Alignment groove

Ball bearing

Figure 36 The Automated ATR Crystal Assembly

6. Slide the crystal assembly into the holder.
You will feel it click into place as it comes into contact with a magnet inside the holder.

7. Rotate the crystal assembly until the alignment groove is in line with the sprung-loaded
ball bearing in the wall of the holder (Figure 37).

You will feel the ball bearing slide into the alignment groove and lock the crystal
assembly in position.

Figure 37 Crystal Assembly aligned in holder

8. Refit the height-adjustment nut.
Take care not to damage the crystal.

9. Refit the ATR crystal holder to the microscope, as described on page 81.

10. Align and adjust the ATR crystal holder, as described on page 84.

90 . Spotlight 200 User’s Guide

Cleaning the ATR Objective Crystal

Because the ATR objective crystal is in contact with the sample under test, it may become
dirty during use. A dirty crystal may give spectra of the contaminant rather than the sample
under test.

Great care must be taken when cleaning the crystal. It should be cleaned using the minimum
of mechanical pressure, with a soft brush or lens tissue. Isopropanol or n-hexane may be
used to clean the crystal.

NOTE: Do not use acetone or xylene to clean the crystal.

1. Pour a small volume of a solvent into a shallow dish.

2. Using the Z-control on the joystick, lower the sample stage, to allow the dish to be
placed on the stage under the crystal.

3. Lower the crystal.

4. Using the Z-control on the joystick, raise the stage, so that the tip of the crystal is just
immersed in the solvent.

NOTE: Do not immerse the whole of the ATR crystal in the solvent.

5. Leave the crystal in the solvent for 5 minutes.
We recommend that you do not leave the crystal in the solvent for longer periods.

6. If any material remains on the crystal, rub the crystal gently with a soft brush or lens
tissue.

Techniques for Collecting Spectra . 91

(Recommend VWR P/N 89022-992)

Auto ATR Cleaning Procedure

If you should see intermittent failures to raise or lower the crystal while running, it is
recommended you clean your Auto ATR Accessory to keep it in good working order.
Regardless of performance we recommend you clean the accessory at least once every
10,000 cycles or per the schedule below:

• Heavy User (25,000 cycles per year) – Every 4-5 months

• Medium User (15,000 cycles per year) – Every 8 months

• Light User (5,000 cycles per year) – Every 12 Months

Materials Needed

Methanol in a squirt bottle

• Kimwipes or similar low lint cleaning wipes

Small Foam Head Swab

Cleaning Procedure

1. If not already done, use Spectrum 10 to raise the crystal into the assembly.

2. Disconnect the motor wire from the left side of the transceiver board.

3. Lower the sample stage, loosen the thumb screws, and carefully pull Auto ATR
accessory from Spotlight microscope to remove it from the sample area.

92 . Spotlight 200 User’s Guide

4. Lay the assembly, crystal up, on a clean work surface. Remove the coned knurled collar
to expose the crystal holder.

5. Carefully grab the crystal holder and remove it from the ATR assembly. Set the crystal
holder aside and be careful not to damage the crystal.

Techniques for Collecting Spectra . 93

6. Look down into the center of the crystal bearing, notice the compression spring and the
plunger retracted and off to one side. Spray methanol into the bore and allow it to drain
from the assembly.

7. Spray methanol in hole above alignment bearing to flush out any free particles.

8. Insert a methanol moistened swab into the larger gap between the bearing and the
retracted plunger and work it all the way around the bore of the bearing.

9. Use a methanol moistened Kimwipe to clean the outside bearing surface, alignment
groove and contact flat opposite the groove on the crystal holder.

94 . Spotlight 200 User’s Guide

It may be necessary to use a non-marring implement to clean any debris on the sliding
surfaces (I use a finger nail). Take care not to damage the crystal. This is a good time
to clean the crystal itself, refer to the User Guide for suggestions.

10. Once all of the methanol has evaporated, it’s time to reassemble the system. Insert the
crystal holder into the bore of the bearing being careful to line up the groove with the
ball in the crystal holder.

11. Gently press down on the crystal holder until it stops. You may need to twist the holder
a small amount from side to side to seat it on the retracted plunger. Do not force it,
you may damage the stop contact.

12. Using another swab, clean any debris from the knurled stop collar. Notice the black
spots in the image below.

Techniques for Collecting Spectra . 95

13. Screw the knurled collar on all the way then back off one and a half (1.5) full
revolutions. This will set the lower limit of travel for the crystal.

14. Reinstall the Auto ATR accessory in your Spotlight system and perform the crystal
alignment and focusing procedure to maximize IR energy.

15. Reset the counter in the Spectrum software on the Setup Microscope Basic tab, click
the Reset button to reset the Auto ATR Crystal Up/Down counter.

96 . Spotlight 200 User’s Guide

Battery cover screws

Changing the Weighbridge Battery

Figure 38 The Weighbridge for the Automated ATR Objective

The weighbridge supplied with the automated ATR objective allows you to apply a known
and repeatable force to the sample when you are collecting spectra. This relies on the
weighbridge communicating with the microscope using a wireless infrared transmitter
powered by a battery. When the battery power starts to decrease, Spectrum displays a
symbol in the Status bar at the bottom of the screen. You should change the battery at this
point. If the symbol changes to
replaced.

, the weighbridge will not function until the battery is

To change the weighbridge battery:

1. Remove the four screws from the corners of the weighbridge and lift it off the
microscope stage.

2. Turn the weighbridge over and place it on a clean, flat surface.

3. Loosen the three captive screws in the cover on the underside (Figure 39).

Figure 39 Weighbridge battery cover

Techniques for Collecting Spectra . 97

4. Remove the battery from inside the cover lid and replace it with a new battery
(Figure 40).

A 3V lithium coin cell (size CR2450) is required (L9004212).

Figure 40 Weighbridge battery

5. Replace the battery cover and tighten the screws firmly.

6. Place the weighbridge on the stage and fix it in place with the four corner screws.
The PerkinElmer logo should be at the front of the weighbridge.

NOTE: The battery status indicator in Spectrum will update the next time you perform an

operation with the weighbridge. If the battery is correctly fitted, a symbol will be
displayed.

98 . Spotlight 200 User’s Guide

tage

Collecting Spectra

Manual ATR Objective

Collecting a Background Spectrum

1. Select the ATR sampling mode on the Setup Microscope Basic tab.

2. Set the infrared aperture to a suitable size and rotation.
For information on how to do this see the on-screen Help.
The contact area of the ATR objective is approximately 100 µm in diameter. We

therefore recommend that you use a maximum aperture size of 100 x 100 µm.

3. Choose the Scan Settings you want on the Setup Instrument Basic tab.

4. Click
The stage will be lowered so that the background spectrum can be collected in air.

5. When prompted, lower the ATR crystal to the working position, and click Retry.

The spectrometer starts to scan and the spectrum is displayed as it is collected.

6. When the scan is completed, retract the ATR crystal.

.

Collecting a Sample Spectrum

Once you have collected a background spectrum, follow the procedure below to collect a
spectrum of your sample.

To prevent serious damage to the crystal, do not move the sample s

CAUTION

while the ATR crystal is in the lower, working position and touching, or
near to, the sample.

1. Make sure that the ATR crystal is retracted.

2. Place the sample on the stage, either on the slide holding the reference mirror or on its
own slide.

3. Move the sample to the center of the field of view.
Even though the microscope may not be focused on the sample, you can see a change

in the light intensity as the sample moves into the center of the field of view.

4. Look at the image in the Camera View and focus on the sample using the Z-control on
the joystick or the Auto-Focus option, and then move the area of interest into the
center of the field of view.

Before you collect the sample spectrum, you should monitor the spectrum to make sure that
there is good contact between the crystal and the sample.

Techniques for Collecting Spectra . 99

5. Lower the ATR crystal to the working position, and slowly raise the stage until the
sample touches the crystal.

To prevent accidental damage to the ATR crystal, always bring the

CAUTION

sample slowly up to the crystal, rather than lowering the crystal directly
on to the sample.

6. Click on the Measurement toolbar.
The Live tab is displayed.

7. Select Sample.

8. Make sure that the spectrum is satisfactory.
If your sample is soft or there is poor contact, you may need to increase the contact of

the crystal with the sample. To increase the contact, raise the stage slightly using the

Adjust Up

lower it using the Adjust Down

9. When the spectrum is satisfactory, click
The spectrometer starts to scan and the spectrum is displayed as it is collected.

button on the accessory toolbar. If the stage position is too high,

button.

.

Always retract the ATR crystal immediately after data collection has

CAUTION

been completed, in order to avoid accidental damage.

Scanning a Line or Map

Spectrum software enables you to collect spectra at marked points using the manual ATR
objective. These points can be individual markers, or groups of markers arranged in lines or
maps across the surface of the sample.

1. Collect a background spectrum.
Refer to

Collecting a Background Spectrum

on page 98.

2. Ensure that the sample is flat and securely fixed to the microscope sample holder or
window.

This can be done by placing adhesive tape around the edge of the sample.

3. Define a sample area that is bigger than the area you intend analyzing using the Stage
View Range menu in the Microscope menu.

4. Look at the image in the Camera View and focus on the sample using the Z-control on
the joystick or the Auto-Focus option, and then move the area of interest into the
center of the field of view.

100 . Spotlight 200 User’s Guide

5. Click on the Stage View toolbar to collect an image survey.

6. Ensure that the ATR sampling mode is selected in the Setup Microscope Basic tab.

7. Use Monitor to find the most suitable stage height to give a staisfactory spectrum of
your sample.

Refer to
not change the stage height.

8. Place markers, lines or maps on to your image survey as required.
For information on how to do this, see the on-screen Help. The markers will all be set at

the stage height (Z-axis position) that you determined previously using Monitor.

Collecting a Sample Spectrum

on page 98. Click Halt when completed, but do

9. Click the Scan Markers

10. When prompted, lower the ATR crystal to the working position and click Retry.

The stage will move to the position of each marker in turn and collect a spectrum. The
results will be displayed in an Image View.

button on the Measurement toolbar.

Always retract the ATR crystal immediately after data collection has

CAUTION

been completed, in order to avoid accidental damage.

Automated ATR Objective

Collecting a Sample Spectrum

1. Select the ATR sampling mode on the Setup Microscope Basic tab.

2. Choose the Scan Settings you want on the Setup Instrument Basic tab.

3. Move the sample to the center of the Camera View.
Even though the microscope may not be focused on the sample, you can see a change

in the light intensity as the sample moves into the center of the field of view.

4. Look at the image in the Camera View pane and focus on the sample using the
Z-control on the joystick, or the Auto-Focus option, and then move the area of interest
into the center of the field of view.

5. Set the infrared aperture to a suitable size and rotation.
For information on how to do this see the on-screen Help.
The contact area of the ATR objective is approximately 100 µm in diameter. We

therefore recommend that you use a maximum aperture size of 100 x 100 µm.

6. Click
The stage moves downwards a small distance and the ATR crystal is lowered to collect

the background spectrum. Once complete, the crystal is raised and the stage returns to
its original position.

to collect a background spectrum.