Page 1
MOLECULAR SPECTROSCOPY
Spotlight 150
User’s Guide
Page 2
Release History
Part Number Release Publication Date
L1050099 D 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: info@perkinelmer.com
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, and Spectrum are trademarks of PerkinElmer, Inc.
Page 3
Contents
Introduction ............................................................................................... 5
About this Manual ............................................................................................. 6
Conventions Used in this Manual ........................................................................ 7
Notes, Cautions and Warnings ..................................................................... 7
Definitions .................................................................................................10
Related Documents ....................................................................................10
Requirements for using the Spotlight 150 ....................................................10
Connecting the PC to the Local Area Network ..............................................10
Warnings and Safety Information ........................................................... 11
The Spotlight 150 .............................................................................................12
Summary .........................................................................................................13
General Operating Conditions............................................................................14
Electrical Safety ...............................................................................................15
Location and Ventilation ...................................................................................16
Warning Labels ................................................................................................17
Microscope Safety Labels ...........................................................................17
Warning Signs on the Microscope ...............................................................18
Lifting the Spotlight 150 ...................................................................................19
EMC Compliance ..............................................................................................20
System Requirements .......................................................................................21
Electrical Requirements ..............................................................................21
Safety Specifications .........................................................................................22
Microscope ................................................................................................22
Overview of the Spotlight 150 ................................................................. 23
A Guided Tour of the Spotlight 150 ...................................................................24
Connections .....................................................................................................25
Operation ........................................................................................................26
The Optical System ..........................................................................................27
Visible Light Optics ....................................................................................27
Infrared Optics ..........................................................................................30
Getting Ready to Use the Spotlight 150 .................................................. 33
Before Using the Spotlight 150 ..........................................................................34
Cooling the MCT Detector .................................................................................35
Setting up the Spotlight 150 .............................................................................38
Focusing the Microscope ............................................................................39
Fitting an Attenuator to the Spectrometer ...................................................41
Setting Scan Parameters ............................................................................42
Preparing Samples ................................................................................... 45
Preparing Samples ...........................................................................................46
Tools for Sample Preparation ............................................................................47
Tools Provided with the Spotlight 150 .........................................................47
Other Useful Tools .....................................................................................48
Specialized Accessories ..............................................................................49
Items to Have Available .............................................................................49
Common Window Materials ...............................................................................50
Techniques for Preparing Samples for Transmission Measurements .....................51
Flattening Solids ........................................................................................51
Slicing Samples from Solids ........................................................................52
Polymers ...................................................................................................53
Particles ....................................................................................................55
Fibers .......................................................................................................56
Fibrous Solids ............................................................................................56
Coatings on Substrates ..............................................................................57
Liquids ......................................................................................................57
Techniques for Collecting Spectra ........................................................... 59
Techniques for Collecting Spectra ......................................................................60
Page 4
Using the Compression Cell ....................................................................... 60
Collecting a Spectrum from a Thick Sample ................................................ 61
Collecting a Spectrum in an Inert Atmosphere ............................................ 62
Viewing a Sample with the Visible Polarizer Accessory ........................................ 64
The Theory of Light Polarization ................................................................. 64
Applications .............................................................................................. 65
Equipment ................................................................................................ 66
Collecting IR Spectra Using the Infrared Polarizer Accessory .............................. 68
Using the Polarizer .................................................................................... 69
Collecting Spectra With the ATR Objective ........................................................ 70
ATR Objective Specification .............................................................................. 72
Fitting the ATR Crystal Holder to the Microscope ...................................................... 73
Measuring the Energy in Reflectance Mode ....................................................... 75
Adjusting the Height of the Crystal ................................................................... 76
Centering the Crystal ................................................................................. 77
Checking the Infrared Alignment ................................................................ 78
Removing the ATR Crystal Holder ..................................................................... 79
Fitting a Crystal Assembly to the ATR Objective ................................................. 80
Manual ATR Objective ............................................................................... 80
Automated ATR Objective .......................................................................... 80
Cleaning the ATR Objective Crystal ................................................................... 82
Auto ATR Cleaning Procedure ........................................................................... 83
Materials Needed ...................................................................................... 83
Cleaning Procedure ................................................................................... 83
Collecting Spectra with the ATR Crystal ............................................................. 88
Manual ATR Objective ............................................................................... 88
Automated ATR Objective .......................................................................... 89
Reflectance FT-IR Microspectroscopy ................................................................ 91
Diffuse Reflectance ................................................................................... 91
Specular Reflectance ................................................................................. 92
Reflection-Absorption ................................................................................ 93
Maintenance ............................................................................................. 94
Maintenance .................................................................................................... 95
Inspecting the Microscope ......................................................................... 95
Protecting the Microscope .......................................................................... 96
Cleaning ................................................................................................... 96
Cleaning the Cover .................................................................................... 96
Replacing the Microscope Fuse ......................................................................... 97
Service ............................................................................................................ 98
Accessories ..................................................................................................... 99
Sample Preparation Tools .......................................................................... 99
Electrical Connections .................................................................................... 100
Fitting the Plug ....................................................................................... 100
Connecting the Microscope to the Electrical Supply ................................... 101
Connecting the Microscope to the Spectrometer........................................ 101
Appendices ............................................................................................. 102
Appendix 1: Decontamination and Cleaning ..................................................... 103
Appendix 2: WEEE Instructions for PerkinElmer Products ................................. 104
Index ............................................................................................................ 105
Page 5
Introduction
Page 6
6 . Spotlight 150 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 150.
Before you start using your Spotlight 150 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 150:
Getting Ready to Use the Spotlight 150
•
Spotlight 150 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 150.
Techniques for Collecting Spectra
•
techniques to collect spectra.
Maintenance
•
for the Spotlight 150 and about the performance checks that we recommend that you
perform routinely.
Warnings and Safety Information
Introduction
Overview of the Spotlight 150
and become aware of conventions and requirements;
starting on page 23;
Getting Ready to Use the Spotlight 150
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 11;
starting on page 33.
gives you information on how to set up your
Page 7
Conventions Used in this Manual
Introduction . 7
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 Systems, Spectrum 400 Series, Spectrum 100 Series,
Spectrum 65 or Spectrum One spectrometer supplied with your Spotlight 150.
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.
Page 8
8 . Spotlight 150 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.
Page 9
WARNING
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.
Introduction . 9
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.
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10 . Spotlight 150 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 an on-screen Help system, which you can access by selecting
Contents from the Help menu, by using 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 150 microscope contained in this guide.
(L1050002) includes instrument manuals for the Frontier IR
Spectrum Two
Requirements for using the Spotlight 150
We assume that the Spotlight 150 has been properly set up and aligned. This installation
must be performed by a PerkinElmer Service Representative.
Ensure that your PC meets the minimum requirements for hardware and software set out in
PC Requirements
the
section of the
Spectrum Administrator’s Guide
(L1050095).
Connecting the PC to the Local Area Network
The PC provided is connected point-to-point to the Spotlight 150. 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 Representative.
Page 11
Warnings and Safety
Information
Page 12
12 . Spotlight 150 User's Guide
The Spotlight 150
The Spotlight 150 consists of a microscope, FT-IR spectrometer and a PC.
Figure 1 Spotlight 150 – Microscope and Frontier IR System
Figure 2 Spotlight 150 – Microscope and Spectrum Two System
Page 13
Warnings and Safety Information . 13
Summary
The PerkinElmer Spotlight 150 has been designed to comply with a wide variety of
international standards governing the safety of laboratory equipment. In routine use, the
Spotlight 150 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 150.
• DO make sure that all parts of the Spotlight 150 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
Representative.
• If your Spotlight 150 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.
• DO read the more detailed information on safety in the following pages.
Removal of covers and fixtures not specified in this manual may result in
hazardous radiation exposure.
WARNING
Use of controls or adjustments or performance of procedures other than
those specified herein may result in hazardous radiation exposure.
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14 . Spotlight 150 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 has been designed and tested in accordance with PerkinElmer specifications
and in accordance with the safety requirements of the International Electrotechnical
Commission (IEC). The microscope conforms 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 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 is unsafe, make it inoperative. The microscope may
be unsafe if it:
• Shows visible damage;
• Fails to perform the intended measurement;
• Has been subjected to prolonged storage in unfavorable conditions;
• Has been subjected to severe transport stresses.
The microscope has 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
Page 15
Warnings and Safety Information . 15
or disconnection of the protective ground (earth)
Electrical Safety
Connect the microscope to a power supply line that includes a switch or other adequate
means of disconnection from the electricity supply.
Plug the microscope into electricity-supply sockets that are provided with a protective ground
(earth) connection only. 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 is 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.
Capacitors inside the microscope may still be charged even if the microscope has been
disconnected from all voltage sources.
Disconnect the microscope from all voltage sources before opening for any adjustment,
replacement, maintenance or repair. Any adjustment, replacement, maintenance or repair
must be performed by a PerkinElmer Service Representative.
The microscope 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
terminal can make the microscope dangerous.
WARNING
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16 . Spotlight 150 User's Guide
Location and Ventilation
The Spotlight 150 is installed by a PerkinElmer Service Representative, 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 to permit adequate cooling.
Make sure that the switch at the electrical supply inlet at the rear of the
microscope is not obstructed.
WARNING
Page 17
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 . 17
Figure 3 Rear of Microscope
100–230 V ~50/60 Hz
75 VA
Figure 4 Inside Dewar lid – MCT detector versions only
NOTE: See
Cooling the MCT Detector
on page 35 for information on how to fill the Dewar.
Page 18
18 . Spotlight 150 User's Guide
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.
Page 19
Warnings and Safety Information . 19
Lifting the Spotlight 150
The microscope and spectrometer are heavy precision instruments, so
two people are required for safe handling.
WARNING
The microscope weighs approximately 32 kg.
• 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 150 after it has been installed without consulting your local
PerkinElmer Service Representative.
Page 20
20 . Spotlight 150 User's Guide
EMC Compliance
EC directive
The Spotlight 150 has been designed and tested to meet the requirements of the EMC
Directive 2004/108/EC.
The Spotlight 150 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).
Page 21
Warnings and Safety Information . 21
System Requirements
Give attention to the following points before installing the Spotlight 150.
Electrical Requirements
The power consumption of the microscope does not exceed 75 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 150 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.
Site Requirements
A minimum bench space of 170 × 75 cm (68 × 30 inches) is required to accommodate the
microscope, spectrometer, PC and ancillaries.
To get the best performance from your Spotlight 150:
• Place the Spotlight 150 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 150 near to room heating equipment, for example central
heating radiators.
Leave a gap of at least 15 cm (6 inches) between any vertical obstacle and the sides of the
microscope to permit an adequate flow of cooling air.
Page 22
22 . Spotlight 150 User's Guide
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 sample stage)
Page 23
Overview of the
Spotlight 150
Page 24
24 . Spotlight 150 User's Guide
A Guided Tour of the Spotlight 150
The Spotlight 150 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 and position a point of interest. The image of your sample
is displayed on the Setup Microscope tab in Spectrum software. Spectrum enables you to
control the operation of your Spotlight 150, 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 sampling modes.
• Internal coaxial LED illumination, with variable intensity.
• An automatic infrared aperture that closes to the size and rotation selected when you
select a scan or monitor command.
• Spectrum software controls the operation of the microscope: for example, the
illumination of the sample, adjusting the correction, and changing to reflectance or
transmittance. Some versions of the microscope also feature LED lighting of the stage
area which is controlled from the software.
• An optional micro-ATR objective for data collection from optically thick and nonreflective samples. Manual and automatic ATR objectives are available.
• Data collection (including saving the visible image) and post-run processing for IR
spectra.
• The same cassegrain is used to send both infrared radiation and visible light to the
remote aperture. The microscope continuously views and monitors infrared
concurrently; there is no beam path switching between visible and infrared. Spectrum
software allows you to view the visible image and the IR spectrum simultaneously.
• The lower cassegrain can be removed. This provides space for thick samples to be
studied in reflectance mode.
• An InGaAs (indium gallium arsenide), a DTGS (deuterated triglycine sulfate), or an MCT
(mercury cadmium telluride) MIR detector is fitted.
Page 25
Overview of the Spotlight 150 . 25
SP1 – connects to spectrometer
Video
Connects to PC
Connections
Figure 5 and Figure 6 show the connections on the microscope.
external left port
Single element detector output:
9-way D type, male
Figure 5 Connections (top rear of microscope)
RS232, 9-way D type
Figure 6 Connections (bottom rear of microscope)
Page 26
26 . Spotlight 150 User's Guide
Operation
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.
Page 27
Overview of the Spotlight 150 . 27
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 150
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 7).
enables you to select between transmittance and reflectance operation. This
Page 28
28 . Spotlight 150 User's Guide
Detector
Camera
Mirror pair
Visible focus
lens assembly
Dichroic mirror
Variable aperture
Upper cassegrain
Sample position
Lower cassegrain
Lower dichroic
mirror
Lower fold mirror
Lower mirror assembly
LED
Visible light optics in transmittance
When the microscope is in viewing mode in transmittance (Figure 7):
• 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.
Figure 7 Path of the visible beam for viewing a sample in transmittance
NOTE: Figure 7, above, illustrates a Spotlight 150 with an MCT detector. Other detector
types may look different, but the optical arrangement is the same in all cases.
Page 29
Overview of the Spotlight 150 . 29
Mirror pair
Visible focus lens
Variable aperture
Reflectance
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 8):
• 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
illuminator assembly
Figure 8 Path of the visible beam for viewing a sample in reflectance
NOTE: Figure 8, above, illustrates a Spotlight 150 with an MCT detector. Other detector
types may look different, but the optical arrangement is the same in all cases.
Page 30
30 . Spotlight 150 User's Guide
Dichroic mirror
Variable
aperture
Upper
Sample position
Lower
Lower dichroic
Detector cassegrain
Detecto
r
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 9):
• 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 9 Path of the infrared beam for collecting an image in transmittance
NOTE: Figure 9, above, illustrates a Spotlight 150 with an MCT detector. Other detector
types may look different, but the optical arrangement is the same in all cases.
Page 31
Overview of the Spotlight 150 . 31
Dichroic mirror
Variable aperture
Reflectance
Upper cassegrain
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 10):
• 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
Figure 10 Path of the infrared beam for collecting IR spectra in reflectance
NOTE: Figure 10, above, illustrates a Spotlight 150 with an MCT detector. Other detector
types may look different, but the optical arrangement is the same in all cases.
Page 32
32 . Spotlight 150 User's Guide
Page 33
Getting Ready to Use the
Spotlight 150
Page 34
34 . Spotlight 150 User's Guide
Before Using the Spotlight 150
Before you use the Spotlight 150 you must:
• Make sure the spectrometer is switched on;
• Switch the 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 150 has not been in use or has been used by others.
Page 35
Getting Ready to Use the Spotlight 150 . 35
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 150 is fitted with a DTGS or InGaAs detector, cooling 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
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.
WARNING
WARNING
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.
Page 36
36 . Spotlight 150 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 11).
Figure 11 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.
Page 37
Getting Ready to Use the Spotlight 150 . 37
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.
NOTE: If the level of coolant is low, a message prompting you to refill the dewar will be
displayed in the Status bar at the bottom of the Spectrum workspace.
Page 38
38 . Spotlight 150 User's Guide
Setting up the Spotlight 150
To set up your Spotlight 150:
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. At your PC, from the Start menu select Programs; the PerkinElmer Applications
group; the Spectrum sub-group and then the Spectrum application.
The Spectrum start-up splash-screen is displayed, followed by a dialog that may require
your login details:
4. If required, enter your User name and Password, and then click OK.
An instrument selection dialog is displayed.
5. Select the Instrument you want to use.
OR
If you want to work with data that has been collected previously, without connecting to
an instrument, select to work offline.
NOTE: Working offline can free a networked instrument for use by another user.
6. Click OK.
Spectrum starts.
7. If you are connected to an FT-IR instrument, select Instrument from the Setup menu.
The Setup Instrument tabs are displayed.
8. Select the Setup Instrument BeamPath tab.
Page 39
Getting Ready to Use the Spotlight 150 . 39
9. Select the Beam Location that directs the beam path to the Spotlight 150.
Figure 12 Beam path directed to the Spotlight 150
10. Select the Setup Microscope tab.
You are prompted to start the microscope initialization and aperture calibration
procedure.
11. Make sure that the lower cassegrain is fitted, that if the ATR objective is fitted it is
retracted, that the stage is centered and that there is no sample on the stage, and then
click OK.
The microscope initializes and runs the aperture calibration procedure.
NOTE: If you do not want to initialize your instrument at this time, you can click Cancel.
You will not be able to use any of the microscope controls on the Setup Microscope
tab until you have initialized the microscope. To initialize the microscope later, click
Initialize on the Setup Microscope tab.
Focusing the Microscope
Microscope sample positioning and focus controls
The focus of the microscope is changed by moving the sample stage up or down. While
focusing, you can view the image on the Setup Microscope tab.
Turn the coarse or fine focus controls (Figure 13) clockwise to move the stage up and
counter-clockwise to move the stage down.
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.
Page 40
40 . Spotlight 150 User's Guide
Coarse (outer) and fine
(inner) focus controls
X-axis stage control
(left to right)
Y-axis stage control
(towards/away)
Figure 13 Focus and stage position controls on the Spotlight 150 Microscope
Focusing the image
1. Place your sample on the sample stage.
2. Select the Setup Microscope tab.
Figure 14 Setup Microscope tab showing the Sampling Mode group expanded
3. Position your sample using the stage controls on the microscope so that you can see
4. Focus on the sample using the microscope focus controls.
the live image of the object of interest.
You may also need to change the illumination of the sample, or the correction, when
focussing the image.
Page 41
Getting Ready to Use the Spotlight 150 . 41
NOTE: If your Spotlight 150 is supplied with a Frontier or Spectrum 400 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 video image. To remove this, fit a 1%T
attenuator to the external beam window of the spectrometer. See
Attenuator to the Spectrometer
This attenuator should be removed during data acquisition.
5. If you want to examine the sample in reflectance mode, select Reflectance in the
Sampling Mode section.
Transmittance is selected by default.
6. If you are examining the sample in transmittance mode, then you may need to adjust
the position of the lower cassegrain so that the field of view is evenly illuminated:
Adjust the Correction by clicking on the up ( ) or down ( )
buttons.
This is especially useful if the sample is in a compression cell or held between windows
of high refractive index.
7. Adjust the Illumination to give good illumination of your sample.
on page 41 for details.
Fitting an
Fitting an Attenuator to the Spectrometer
If your Spotlight 150 is supplied with a Frontier or Spectrum 400 FT-IR/FT-NIR dual-range
spectrometer or if your Spotlight 150 has an InGaAs detector, 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.
The use of an attenuator is recommended if a detector overload error message is displayed
in the Status Bar when monitoring or collecting data. This attenuator should be kept in place
during data acquisition. For example, a detector overload error message may be displayed
when using your Spotlight 150 with a dual-range spectrometer in the near infrared with a
large aperture size. To resolve this, fit a 32%T attenuator to the external beam window of
the spectrometer.
Figure 15 KBr window with 14%T attenuator
Page 42
42 . Spotlight 150 User's Guide
Setting Scan Parameters
The Scan toolbars at the top of the workspace include the tools you need to collect a
spectrum from a sample.
Figure 16 Scan toolbars
By default, sensible values for the scan and instrument parameters are entered in the
Instrument Settings toolbar. The values applied depend on your instrument and accessory.
To amend any value, select the parameter and then enter your new value, or select a value
from the drop-down list.
8. Set the Start and End points of the scan range (by default in wavenumbers, but the
abscissa units can also be set to nanometers or microns on the Setup Instrument Basic
tab).
−1
For mid-IR analysis it is normal to set the upper wavenumber limit to 4000 cm
the lower limit to the low wavenumber specification of the detector, as shown in
Table 1.
Table 1 Low wavenumber specifications of Spotlight 150 detectors
and
Detector type Low wavenumber limit (cm
−1
)
MCT mid-band 580
MCT wide-band 450
DTGS 400
Your PerkinElmer Service Representative 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-IR 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-IR range of the spectrum the Spotlight 150 must be attached to
a dedicated near-IR spectrometer or a dual-range spectrometer which has been set
up to operate in the near-IR range.
9. Set the Accumulations required, either as a number of scans, or as a length of time.
There are no firm rules about these selections since the number of scans needed to
generate acceptable spectra will depend on the nature of the sample and the
requirements of the application.
−1
It is probably better to “over-scan”; 50 scans taken at 8 cm
resolution using an MCT
or InGaAs detector (20 scans for a DTGS detector) is relatively quick.
10. Enter a unique Sample ID and Description for the sample.
11. If a background scan is required, the Scan button includes a small background flag.
Clear the instrument beam path, or insert a suitable background material, and then click
to collect a background spectrum.
Page 43
Getting Ready to Use the Spotlight 150 . 43
The background spectrum is displayed briefly, and then the Viewing Area is prepared
for data collection from your sample.
A new background scan will be required if you change the aperture dimensions or the
sampling mode. However, you can modify the correction and illumination without
requiring a new background. We recommended that you run a background scan before
every sample.
If you are sampling in transmittance, you may simply chose to move the stage to an
area away from the sample and focus on the surface of the substrate using the
microscope focus controls. If you are sampling in reflectance, you may want to use an
appropriate reference, such as the gold mirror supplied with your Spotlight 150.
See the Spectrum on-screen help for more information.
12. Click to begin scanning your sample.
By default, during scanning the sample data is displayed on the Live tab in the Viewing
Area.
The completed spectrum is displayed on the Graph tab, and added to the current
Samples View in the Data Explorer.
NOTE: If a detector overload error is reported in the Status Bar when monitoring or
collecting data, then fit an attenuator to the external beam window of the
spectrometer. See
Fitting an Attenuator to the Spectrometer
, on page 41, for details.
This attenuator should be kept in place during data acquisition.
13. If, for any reason, you want to stop scanning your sample, click .
Optimizing Transmission measurements
In most cases samples for transmission measurements are placed flat on top of an IRtransmitting 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 as possible on the window.
In transmission measurements, the IR 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
IR energy throughput through the window (and sample) the lower cassegrain is moved up or
down using the Correction facility on the Setup Microscope tab to compensate for this
refractive index variation.
1. Place the thin, flat, sample onto a window (or across an aperture) and focus on the
sample using the microscope controls.
2. Set the apertures to the appropriate size for sample area.
, ZnSe or diamond. In some cases, such as
2
3. Move the sample away from the centre of the viewing screen, and focus on the top
surface of the window, ensuring that the widow is clean.
Page 44
44 . Spotlight 150 User's Guide
4. Select Monitor function from the Measurement menu or click to monitor the
energy throughput.
5. Adjust the lower cassegrain for maximum energy using the up and down arrows of the
Correction function.
Clicking Zero will move the cassegrain to the optimum position for an open beam
(suitable for very thin samples).
Figure 17 Correction control
6. Once the maximum energy through the window (or aperture) has been achieved, press
Halt on the Measurement toolbar.
7. Collect a background spectrum through this point on the window (or aperture) using the
correct aperture size as selected previously.
8. Return the stage to the selected sample position, adjust the correction, and then record
the sample spectrum.
NOTE: If a detector overload error is reported in the Status Bar when monitoring or
collecting data, then fit an attenuator to the external beam window of the
spectrometer. See
This attenuator should be kept in place during data acquisition.
If the same window is used repeatedly for analysis, height correction of the lower cassegrain
need only be done once.
Fitting an Attenuator to the Spectrometer
, on page 41, for details.
Page 45
Preparing Samples
Page 46
46 . Spotlight 150 User's Guide
Preparing Samples
The Spotlight 150 enables you to examine live image of the sample on the Setup Microscope
tab and 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 150.
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.
Page 47
Preparing Samples . 47
Tools for Sample Preparation
This section lists the tools you need for preparing samples:
• Tools provided with the Spotlight 150;
• Tools in the microsampling toolkit;
• Materials to have available;
• Specialized accessories you may want to purchase.
Tools Provided with the Spotlight 150
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
The following tools are provided in the Microsampling toolkit.
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
Page 48
48 . Spotlight 150 User's Guide
Figure 18 Some of the Tools in the Microsampling Toolkit
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
01861043
Page 49
Preparing Samples . 49
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 51):
Items to Have Available
In addition to the items provided with the Spotlight 150, 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.
Page 50
50 . Spotlight 150 User's Guide
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 live image on the Setup
Instrument Microscope tab appears yellow.
Page 51
Preparing Samples . 51
Techniques for Preparing Samples for Transmission 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 53 for information on flattening samples with a miniature
Page 52
52 . Spotlight 150 User's Guide
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 19.
Figure 19 Cutting a wedge-shaped sample
Page 53
Preparing Samples . 53
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 19.
4. Position it under the microscope so that the infrared beam goes through the thin end of
the wedge (circled in Figure 19).
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.
• β-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 20, 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.
Page 54
54 . Spotlight 150 User's Guide
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 20 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 60) enables you to compress
Page 55
Preparing Samples . 55
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.
Page 56
56 . Spotlight 150 User's Guide
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 Monitor Visible 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 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 Solids
on page 53).
on page 51), 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.
Page 57
Preparing Samples . 57
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 70 for details.
Liquids
Solutions of samples
Although liquids are seldom analyzed with the Spotlight 150, 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.
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58 . Spotlight 150 User's Guide
Page 59
Techniques for Collecting
Spectra
Page 60
60 . Spotlight 150 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 21) 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 21 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.
Page 61
Techniques for Collecting Spectra . 61
Collecting a Spectrum from a Thick Sample
If your sample is very thick, you will have to lower the stage to focus the 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 remove the lower cassegrain and lower the stage
1. Remove any sample from the stage.
2. Click Zero in the Correction section of the Setup Microscope tab.
The lower cassegrain moves to the position where the infrared beam is focused if no
sample is on the stage.
3. Move the sample stage to its highest possible position.
4. Release the locking lever located on the right side on the back of the lower cassegrain
assembly (Figure 22).
Figure 22 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 Park in the Correction section of the Setup Microscope tab.
The lower cassegrain bracket moves down. Park is the lowest position to which the
lower cassegrain bracket can move when the lower cassegrain is fitted.
The button text now updates to Lower Park.
7. Click Lower Park again.
The lower cassegrain bracket moves to its lowest position.
8. Lower the stage.
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62 . Spotlight 150 User's Guide
To refit the lower cassegrain after use
1. Raise the stage to its highest position and ensure that the stage is centered.
Before moving the stage up from the Park or Lower park positions you must ensure that
the stage is centered. The calibrated rules marked on the stage can help with this. The
X-axis should be positioned at about 115 mm and the Y-axis should be positioned at
about 10 mm.
2. Choose Zero in the Correction section of the Setup Microscope tab.
The lower cassegrain moves to the position where the infrared beam is focused if no
sample is on the stage.
3. Make sure that the cassegrain is correctly seated.
4. Place the gaiter over the cassegrain if the purge facility is required.
5. Slide the cassegrain assembly back into the dovetail connector, as far as it goes.
NOTE: To make replacement easier, pull the locking lever gently.
6. Tighten the locking lever.
Collecting a Spectrum in an Inert Atmosphere
The 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 23.
Figure 23 Parts of the purge system
Page 63
Techniques for Collecting Spectra . 63
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.
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 150
See
3. Place the sample in position.
4. Purge the microscope and spectrometer for 15 to 20 minutes at a rate of 10 l min
starting on page 33.
−1
.
5. Collect the background spectrum and the spectrum of the sample.
Changing the sample
If you have changed a sample, any air that has entered the system must be flushed out.
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64 . Spotlight 150 User's Guide
Viewing a Sample with the Visible Polarizer Accessory
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 24.
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 24 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 25), the polarized light can pass straight through.
, and is represented on the right side of Figure 24.
polarizer
, the polarizer allows the passage of only those waves
• However, if the second 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 25).
Page 65
Techniques for Collecting Spectra . 65
The second occurs because the light transmitted by the first polarizer oscillates in exactly the
plane that is blocked by the second polarizer.
Figure 25 Polarizers parallel (top) and polarizers crossed (bottom)
Some materials are
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
): their refractive index depends on their
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 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.
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66 . Spotlight 150 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 26).
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 150, and are
automatically switched into the beam when the analyzer is inserted.
Figure 26 The analyzer
Page 67
Techniques for Collecting Spectra . 67
Visible Analyzer
Slot
Operating the Analyzer
1. Insert the analyzer into the slot in the right side of the front cover (Figure 27).
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 27 Analyzer position
2. When the polarizing element is in the beam, rotate the wheel while viewing the sample
image on the Setup microscope tab.
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68 . Spotlight 150 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 150 enables you to collect polarization spectra of very small samples. These
include:
• Single filaments (typically 14 × 70 µm);
• Films;
• Single crystals;
• Liquid crystals.
Equipment
The optional polarizer has a rotatable silver bromide element in an aluminum mount
(Figure 28).
Figure 28 The infrared polarizer
Page 69
Techniques for Collecting Spectra . 69
Infrared polarizer
Using the Polarizer
1. Remove the snap-in cover that masks the aperture for the infrared polarizer (Figure 29).
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 29 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.
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70 . Spotlight 150 User's Guide
2/12
21
2
1
)(sin2 nn
d
p
−
=
θp
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.
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 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 an
approximately 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 controls the crystal automatically.
Page 71
Techniques for Collecting Spectra . 71
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 30 Infrared radiation optical path
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72 . Spotlight 150 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
cleaned (see
the user (see
Cleaning the ATR Objective
Fitting the ATR Crystal Holder to the Microscope
Removing the ATR Crystal Holder
Crystal on page 82), and replaced and aligned by
on page 73).
on page 79),
Page 73
Techniques for Collecting Spectra . 73
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 (see Figure 31).
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 32).
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 31 The ATR crystal holder and ATR cassegrain
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74 . Spotlight 150 User's Guide
Jack plug socket
(plug not shown)
ATR crystal
assembly
Crystal holder
Motor (lowers and
Knurled thumb-nut
retracts the crystal)
Figure 32 The Automated ATR Crystal Holder and Cassegrain
Page 75
Techniques for Collecting Spectra . 75
Measuring the 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 and PC are switched on and that Spectrum is running.
3. Ensure that the IR beam is directed through the microscope on the Setup Instrument
BeamPath tab.
4. Place the slide holding the reference mirror (L1860409, supplied with the microscope)
on the sample stage.
5. Make sure that the microscope is in reflectance mode by selecting Reflectance in the
Sampling Mode section of the Setup Microscope tab.
6. Look at the live image and focus the beam on the surface of the mirror.
There are often dust particles or scratches on the surface that you can use to focus on.
Checking the Infrared Alignment
on page 78 for details.
7. Enter 100 in the Width and Height text boxes in the Apertures section of the Setup
Microscope tab.
The aperture size is set.
Figure 33 Aperture controls
8. Click on the Measurement bar and select to monitor the energy reaching the
detector.
The Live tab displays the energy reaching the detector.
9. Record the energy measured.
10. Click .
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76 . Spotlight 150 User's Guide
Knurled height-adjustment nut
Adjusting the Height of the Crystal
To prevent serious damage to the crystal, do not move the Sample
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
Click the button to lower the automated ATR crystal onto the reference mirror.
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 34).
Stage while the ATR crystal is in the lower, working position and
touching or near to the sample.
Figure 34 Adjusting the Crystal Height (Manual ATR Objective)
Figure 35 Adjusting the Crystal Height (Automated ATR Objective)
Page 77
Techniques for Collecting Spectra . 77
Centering the Crystal
1. Retract the manual ATR crystal using the toggle bar, by lifting the bar then twisting
counterclockwise.
OR
Click the button to retract the automated ATR crystal.
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 carefully 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 clockwise 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 clockwise 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.
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.
NOTE: The centering adjustments are the same for both the manual and automated ATR
objectives.
Figure 36 Centering the ATR crystal
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.
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78 . Spotlight 150 User's Guide
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
Energy in Reflectance Mode
1. Place the slide holding the reference mirror (supplied with the microscope) on the
Sample Stage.
on page 75 for details.
Measuring the
2. Look at the video image on the Setup Microscope tab and focus the microscope on the
surface of the mirror.
There are usually dust particles or scratches on the surface that you can use to focus
on.
3. Lower the crystal onto the reference mirror.
4. Enter 100 in the Width and Height text boxes in the Apertures section of the Setup
Microscope tab.
5. Click on the Measurement bar and monitor the energy reaching the detector.
The energy level measured should be at least 20 of that measured in reflectance mode;
Measuring the Energy in Reflectance Mode
see
6. Click .
on page 75 for details.
Page 79
Techniques for Collecting Spectra . 79
Removing the ATR Crystal Holder
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.
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80 . Spotlight 150 User's Guide
Fitting a Crystal Assembly to the ATR Objective
Manual ATR Objective
1. Remove the ATR crystal holder, as described on page 79.
2. Place the crystal holder on a bench, with the crystal upwards.
3. While holding the crystal assembly in place, unscrew the knurled height-adjusting 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-adjusting
nut.
Take care not to damage the crystal.
6. Refit the ATR crystal holder to the microscope as described on page 73.
7. Align and adjust the ATR crystal holder, as described on page 77.
Figure 37 The Manual ATR Crystal Assembly
Automated ATR Objective
1. Remove the ATR crystal holder, as described on page 79.
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 38).
Page 81
Techniques for Collecting Spectra . 81
Alignment groove
Alignment groove
Ball bearing
Figure 38 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 39).
You will feel the ball bearing slide into the alignment groove and lock the crystal
assembly in position.
Figure 39 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 73.
10. Align and adjust the ATR crystal holder, as described on page 77.
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82 . Spotlight 150 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. Lower the sample stage, to allow the dish to be placed on the stage under the crystal.
3. 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.
4. Leave the crystal in the solvent for 5 minutes.
We recommend that you do not leave the crystal in the solvent for longer periods.
5. If any material remains on the crystal, rub the crystal gently with a soft brush or lens
tissue.
Page 83
Techniques for Collecting Spectra . 83
(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.
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84 . Spotlight 150 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.
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.
Page 85
Techniques for Collecting Spectra . 85
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.
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.
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86 . Spotlight 150 User's Guide
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.
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.
Page 87
Techniques for Collecting Spectra . 87
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.
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88 . Spotlight 150 User's Guide
tage
Collecting Spectra with the ATR Crystal
Manual ATR Objective
Collecting a Background Spectrum
To prevent serious damage to the crystal, do not move the sample s
CAUTION
To collect a background spectrum in air:
1. Select ATR in the Sampling Mode section of the Setup Microscope tab.
2. Select the Scan Settings you want in the Setup Instrument Basic tab.
3. Set the infrared aperture to a suitable size and rotation on the Setup Microscope tab.
For information on how to do this see the Spectrum 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 × 100 µm.
while the ATR crystal is in the lower, working position and touching, or
near to the sample.
4. Select Background from the Measurement menu.
The software will prompt you to lower the stage to prevent a collision.
5. Lower the stage and click OK.
The software will prompt you to lower the ATR crystal.
6. Use the toggle bar to lower the crystal when prompted and click OK.
The spectrometer starts to scan and the spectrum is displayed as it is collected.
7. Retract the ATR crystal when the scan is completed.
Collecting a Sample Spectrum
Once you have collected a background spectrum, follow the procedure below to collect a
spectrum of your sample.
1. Make sure that the ATR crystal is retracted.
2. Place the sample slide on the stage.
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 on the Setup Microscope tab and focus on the sample using the
microscope focus controls, then move the area of interest into the center of the field of
view.
Page 89
Techniques for Collecting Spectra . 89
5. Select Monitor from the Measurement menu.
The Live tab is displayed.
NOTE: 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.
6. Use the toggle bar to lower the ATR crystal to its working position.
7. Raise the stage carefully 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.
8. Select Sample.
The spectrum at the current aperture position is displayed.
9. Make sure that the spectrum monitored 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, by raising the stage slightly.
10. When the spectrum is satisfactory, click Scan.
The software will again prompt you to lower the crystal and raise the stage but these
messages can be cleared by clicking OK. The spectrometer starts to scan and the
spectrum is displayed as it is collected.
Always retract the ATR crystal immediately after data collection has
CAUTION
been completed, in order to avoid accidental damage.
Automated ATR Objective
Collecting a Background Spectrum
To collect a background spectrum in air:
1. Select ATR in the Sampling Mode section of the Setup Microscope tab.
2. Select the Scan Settings you want in the Setup Instrument Basic tab.
3. Set the infrared aperture to a suitable size and rotation on the Setup Microscope tab.
For information on how to do this see the Spectrum 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 × 100 µm.
4. Select Background from the Measurement menu.
The software will prompt you to lower the stage to prevent a collision when the ATR
crystal is lowered.
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90 . Spotlight 150 User's Guide
5. Lower the stage and click OK.
The automated ATR crystal will be lowered automatically. The spectrometer starts to
scan and the background spectrum is displayed as it is collected.
Collecting a Sample Spectrum
Once you have collected a background spectrum, follow the procedure below to collect a
spectrum of your sample.
1. Make sure that the ATR crystal is retracted.
2. Place the sample slide on the stage.
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 on the Setup Microscope tab and focus on the sample using the
microscope focus controls, then move the area of interest into the center of the field of
view.
5. Select Monitor from the Measurement menu.
The Live tab is displayed. The software will prompt you to lower the stage to prevent a
collision when the ATR crystal is lowered.
6. Lower the stage and click OK.
The automated ATR crystal will be lowered automatically.
NOTE: 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.
7. Raise the stage carefully 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.
8. Select Sample.
The spectrum at the current aperture position is displayed.
9. Make sure that the spectrum monitored 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, by raising the stage slightly.
10. When the spectrum is satisfactory, click Scan.
The software may prompt you to raise the stage to make contact with the sample. Click
OK to proceed. The spectrometer starts to scan and the spectrum is displayed as it is
collected.
Page 91
Techniques for Collecting Spectra . 91
Reflectance FT-IR Microspectroscopy
The Spotlight 150 enables you to collect images and spectra from samples that display any of
the three types of reflectance: diffuse reflectance, specular reflectance, or reflectionabsorption.
Figure 40 Three types of reflectance
Figure 40 shows how the incident radiation (I
not uncommon for two or more of these processes to occur simultaneously, depending on
the structure of the sample.
) is reflected in each type of reflectance. It is
0
Diffuse Reflectance
In diffuse reflectance, the incident radiation is reflected in all directions from the surface of
the sample. This type of reflectance is seen in samples with matt surfaces, such as paper and
powders. The broad collection angle of the microscope enables it to capture a large
proportion of the diffusely reflected radiation and send it to the detector.
A problem often encountered when using the diffuse reflectance technique is that there is a
large specular component in the reflected radiation. Figure 41 shows three diffuse
reflectance spectra of polymethyl methacrylate (PMMA) shavings. In the top spectrum, the
shavings were neat. In the next two spectra, the PMMA was diluted with successively larger
amounts of KBr.
In the neat sample, the presence of interfering specular reflectance introduces
non linearities to the spectral data. For example, the relative intensities of the strong C=O
and C-O stretching absorptions are not as expected (arrows on the top spectrum).
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92 . Spotlight 150 User's Guide
As the sample is diluted in a non-absorbing matrix (KBr), specular reflectance is minimized.
The bottom spectrum more closely matches an absorption spectrum.
NOTE: Sample cups for obtaining diffuse reflectance spectra of powders are available from
PerkinElmer: regular cup (part number 01862760), micro cup (part number
01862761).
Figure 41 Diffuse reflectance spectra of PMMA
Specular Reflectance
Specular reflectance is reflection in one direction (Figure 40). This is the type of reflection
that occurs from a smooth, polished surface.
Absorption information cannot be obtained directly from a specular reflectance spectrum of a
dielectric material, because the reflectance spectrum is governed by dispersion in the
refractive index. However, you can use the Kramers–Kronig integration to calculate the
absorbance spectrum from the specular reflectance spectrum. This integration is performed
by the Kramers–Kronig process in Spectrum.
Figure 42 Specular reflectance spectrum of a PMMA plate and the absorption
spectrum calculated from it with the Kramers–Kronig process in Spectrum
Page 93
Techniques for Collecting Spectra . 93
The greatest limitation to this method is the presence of an interfering diffuse reflection
signal. This occurs when the sample surface is not perfectly smooth. The Kramers–Kronig
integration is not appropriate for spectra from such samples.
Reflection-Absorption
Reflection-absorption occurs when the incident radiation passes through a thin, absorbent film
that is on a reflective surface (typically a metal) and is then reflected back through the film
(see Figure 40). The absorbance spectrum of the film can be collected directly. Figure 43
shows the spectrum of residual oil on an electronic contact. The sample diameter was 100 µm.
Figure 43 Reflection-absorption spectrum of residual oil on an electronic contact
Page 94
Maintenance
Page 95
Maintenance
Switch off the mains voltage and remove the mains cord before
cleaning.
Maintenance . 95
WARNING
A Responsible Body must perform routine maintenance on your microscope to make sure
that it is safe and performing well. This involves:
• Inspecting the microscope;
• Protecting the microscope;
• Cleaning the optics;
• Cleaning the microscope cover;
• Renewing the fuse;
• Service.
This chapter contains a list of the available spare parts and optional equipment for the
microscope.
If you need to replace a part, use only PerkinElmer approved spare parts.
The covers of the microscope must only be removed by a PerkinElmer
CAUTION
Service Representative.
Inspecting the Microscope
At least once a year, or whenever the microscope has been subjected to adverse
environmental conditions, visually inspect the housing to make sure that no covers are loose
or distorted.
At the same time, make sure that all required labels are firmly in place; see
on page 17.
Warning Labels
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96 . Spotlight 150 User's Guide
Protecting the Microscope
The most important rule in caring for the microscope is to keep it as free from dust and dirt
as possible. Dust, fingerprints, and smears on the optics reduce the quality of the images it
produces.
Whenever the microscope is not in use, cover it with a plastic cover.
Ensure the power is switched off when the microscope is covered.
CAUTION
Cleaning
The microscope was aligned, cleaned, and sealed at the factory. Do not attempt to take it
apart.
Avoid the excessive use of solvents. Flowing solvents dissolve the
CAUTION
cement on cemented optics; dissolved cement can damage mirror
surfaces.
Cleaning the Cover
Ensure the power is switched off and the supply lead is disconnected
CAUTION
You can clean the outside of the microscope using a damp cloth. Mild detergent may be
used, if necessary. Always perform a patch test on an inconspicuous area, before you clean
the entire instrument.
Avoid spilling liquid into the microscope. Clean all external spills immediately. If anything that
is spilled enters the main body of any part of the system, switch off the power and call a
PerkinElmer Service Representative.
before cleaning the cover.
Page 97
Maintenance . 97
Replacing the Microscope Fuse
The microscope is not voltage sensitive and will operate at voltages between 100–240 V and
at 50–60 Hz.
1. Switch off the microscope and disconnect it from the power supply.
2. Insert a screwdriver into the slot at the side of the fuse drawer; pull out and flip to one
side over the mains inlet.
The fuse may now be removed.
3. Fit the replacement fuse into the fuse drawer.
You require a 2.0 A time-lag, 250 V fuse (part number 04970839).
4. Refit the fuse drawer.
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98 . Spotlight 150 User's Guide
Service
All optical and mechanical equipment requires periodic servicing to keep it performing
properly and to compensate for wear. We recommend that the Spotlight 150 is cleaned,
examined, and adjusted periodically by a PerkinElmer Service Representative.
NOTE: If you experience unexpected problems with the microscope, contact your
PerkinElmer office or representative immediately.
Page 99
Maintenance . 99
Accessories
Accessories can be ordered directly from PerkinElmer at https://shop.perkinelmer.com/default.aspx
Description Part Number
Spare fuse for microscope (2 A time-lag, 250 V) 04970839
Visible Polarizer Kit L1860294
Sampling Accessory Kit L1860250
IR Polarizer Assembly L1860408
Manual ATR Accessory L1860334
Auto ATR Accessory L1862046
Holder for 13 mm Sampling Disks L1860409
Compression Cell N1870185
Rotatable Sample Disk Holder N1873039
Holder for Reflection Transmission Measurements N1873124
BaF2 Window (1 mm thick) N9302611
BaF2 Window (2 mm thick) N9302612
ZnSe Window N9302613
NaCl Window N9302614
KBr Window N9302615
Miniature Diamond Anvil Cell N9302618
Spare Germanium Crystal (Manual) L1860268
Spare Silicon Crystal (Manual) L1860269
Spare Diamond Coated Germanium Crystal (Manual) L1862054
Spare Germanium Crystal (Auto) L1862044
Spare Silicon Crystal (Auto) L1862051
Spare Diamond Coated Germanium Crystal (Auto) L1862047
Sample Preparation Tools
These are described in
Tools for Sample Preparation
on page 47.
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100 . Spotlight 150 User's Guide
Electrical Connections
Fitting the Plug
The power cable for the electrical supply plugs into the back of the microscope. It has a
molded connector at one end. If it is necessary to fit a plug on the power cable, use the wire
color code below:
Plug Pin Wire Color
(100/110/120 V)
Ground (Earth) Green or Green/Yellow Green/Yellow
Line Black Brown
Neutral White Blue
Wire Color
(220/230/240 V)
To ensure safe and satisfactory operation of the instrument, it is
essential that the green or green/yellow ground (earth) wire of the
power cord is connected to a ground that complies with the regulations
WARNING
of the local electricity supply authority (or equivalent body);
circuit continuity is essential for safe operation of the equipment.
ground
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