INFRARED MICROSCOPY

Application eBook

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INFRARED MICROSCOPY APPLICATIONS | TABLE OF CONTENTS

Introduction to Infrared (IR)Microscopy-spectroscopy
Transmission FTIR Microscopy
Reﬂection Absorption (including Grazing Angle) FTIR Microscopy
Attenuated Total Reﬂectance (ATR) FTIR Microscopy

Material Science
Analysis of Foreign Materials ……………………………4
Microscopic Transmission Method

Dynamic Imaging ………………………………………………24
Time Course Imaging

Analysis of Foreign Materials in Silicon Wafers

Analysis of the Curing Process of UV Curable Resins

Analysis of Foreign Materials on Rubber Surfaces

Tips Heating and Cooling Stages for IR and Raman
Microscopy

Microscopic Reﬂection Absorption Method
Analysis of Foreign Materials on Solders
Tips Simple Sample Preparation using a Diamond Cell
Tips KnowItAll
Microscopic ATR Method
Analysis of Foreign Materials on Resin Products
ATR Imaging Measurement of Particles Suspended in
Machine Oil
Tips IQ Mapping
Tips Clear-View ATR Objectives
Combined IR and Raman Analysis
Cross-Section Analysis of a Multilayer Film

Analysis of Paints and Coatings ………………………12
Microscopic ATR Method
ATR Imaging Comparison of Viscous Paint Samples
Tips IR Microscope Imaging Analysis Software Analysis
WizardCombined IR and Raman Analysis
Analysis of Car Paint Chips
Tips Combined Analysis using IQ Frame

Evaluation of Rubber Products …………………………18
Microscopic ATR Method
Analysis of a Rubber Sample with Surface Degradation
Component Distribution Analysis of Rubber with a High
Refractive Index
Tips Wide-Area ATR Objectives

Evaluation of Some Physical Properties
of Polymers…………………………………………………21

Microscopic Transmission Method
Orientation Distribution Analysis of Polypropylene
Evaluation of Polymer Crystallinity by IR Mapping
Tips Advantages of a Grazing Angle Reﬂection Objective
Tips Vacuum Option2

Step-Scan Imaging
Fast Time Course Analysis of Liquid Crystals with Applied
VoltageTips Two-Dimensional Correlation

Life Science
Component Distribution Analysis
of Foods…………………………………………………29

Microscopic Transmission Method
Moisture Distribution Analysis of Foods
Microscopic ATR Method
Wide-Area ATR Measurement of Foods
Tips Protein Secondary Structure Analysis

Applications of IR Microscopy in Pharmaceuticals
and Medicine…………………………………………………32

Microscopic Reﬂection Method
Near-IR Imaging of OTC Drugs (Sedatives)
Microscopic Transmission Method
Measurement Example of Mouse Eyeball Cross-Sections

Environmental Science
Analysis of Microplastics ……………………………………34
Microscopic Transmission Method
Imaging measurement and particle size analysis of plastic
particles
Tips JASCO Particle Analysis
Tips IQ IR NAV Microscopy Image Analysis
Tips Advanced Spectral Search
Combined Microscopic Analysis
IR and Raman Study of Plastic Particles

Introduction to Infrared (IR) Micro-spectroscopy
FTIR micro-spectroscopy is an analytical technique for imaging materials using functional group(s) for sample identiﬁcation, surface
analysis, multilayer ﬁlm characterization, and particles characterization. Requiring minimal sample size and preparation, measurements
can be made without lengthy procedures to manipulate samples or the use of solvents that would create additional laboratory waste. The
use of an IR microscope allows the evaluation of small sample areas to provide useful information for quality control in manufacturing,product development, and failure analysis. The non-destructive nature of IR micro-spectroscopy means that fragile samples, suchas biomedical and medical samples, can be readily analyzed. Samples can also be subjected to further analysis by complementary
spectroscopic and non-spectroscopic measurements including Raman, UV-Visible and ﬂuorescence. Upon collection of a spectrum, anever-growing range of spectral libraries can be used to assist in sample identiﬁcation. Alternatively, through functional group analysis
using characteristic absorption bands in the spectra, structural elucidation can be achieved to identify unknown molecular structures
for newly created materials, foreign materials in a sample (i.e. forensic studies), or a mixture of compounds (which can also be identiﬁed
from spectral library searches). There are several measurement methods that can be employed in IR micro-spectroscopy, includingtransmission, reﬂection, and Attenuated Total Reﬂectance (ATR).

Transmission FTIR Microscopy
Transmission microscopy is one of the most common methods
for analyzing solids, liquids, and gases, and it is the method of
choice for analyzing ﬁlms, laminates, and ﬁbers. For successfultransmission measurements, light is focused through thesample using a size-adjustable aperture and then collimated bya condenser onto a detector. Samples must be relatively thin(less than 50 μm) to allow the light to adequately pass throughthe sample. Transmission measurements provide the highest

again before reaching the detector. As the incident and collection
angles approach the grazing angle, the incident beam strikes thecoating at shallower angles and the path length becomes maximal,enhancing the absorption intensity. Infrared Reﬂection Absorption
Spectroscopy (IRRAS) is often used to study monolayer coatings
on metals and other substrates.

Attenuated Total Reﬂectance (ATR) FTIR Microscopy
An ATR sampling accessory consists of a series of focusing mirrors

sensitivity and best detection of all IR sampling techniques.

that direct the spectrometer’s IR beam into a crystal prism (typicallyReﬂection Absorption (including Grazing Angle) FTIR
Microscopy

diﬀerent for some ATR accessories including ATR microscope

Reﬂection measurements work best for reﬂective materials, but
it is not eﬀective for dark materials or non-reﬂective substrates.
Reﬂection measurements are performed with a single Cassegrain
objective, which uses a primary and secondary mirror to direct
the IR beam onto the sample and return the reﬂected light to thedetector, typically with angles from 25 – 35° depending on theat a 45° angle, but for optimal measurement, the angle can beobjectives). Even though total internal reﬂection occurs within
the prism, an evanescent ﬁeld is propagated from the crystal
surface into the sample that is in intimate contact with the prism;
for solid materials this requires a device to compress the material
against the prism. Following molecular interaction with the light,
the waveform is passed onto the detector. The prisms used in
ATR accessories are selected according to speciﬁc application

magniﬁcation of the Cassegrain objective.

requirements and can be manufactured from IR transmissive

Grazing angle reﬂection is often used to examine the thinnest of

sulﬁde (ZnS), and germanium (Ge). The penetration depth (andsurface coatings by ‘grazing’ the sample at a very shallow incident
angle, which results in longer sample path lengths providing
greater sensitivity. Coatings on ‘shiny’, highly reﬂective substrates
are excellent candidates for infrared reﬂection absorption studies.
As the surface coating layer to be measured becomes thinner,incidence and collection angles can be adjusted from 45 – 75°
until the ‘grazing’ angle is reached, generally considered to be
maximal at 85°. The term reﬂection absorption’ describes theprocess of the incident beam as it passes through the coating,reﬂects from the substrate, and passes back through the coating

crystal materials, including diamond, zinc selenide (ZnSe), zinc
hence eﬀective path length) of light interaction with the sample
is dependent on the refractive index of both the sample andcrystal prism, as well as the wavelength of measurement. In the
mid-IR and ﬁngerprint region, the penetration depth is typically inthe region of several microns but is smaller as the wavelengths
approach the near-IR. Spectra measured using ATR can becompared with transmission data, but often need to be corrected
for slight diﬀerences caused by the change in penetration depth(and apparent path length) across the mid-IR spectrum.
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INFRARED MICROSCOPY APPLICATIONS | MATERIAL SCIENCE

Analysis of Foreign Materials
In the manufacturing process of industrial products, the inclusion of foreign materials can cause defective products. FTIR spectroscopy
is an effective tool for the analysis and identification of causes of failure. If a foreign material consists of a single component, it can be
relatively easy to identify the component using macro-ATR; however, since foreign materials often contain several components, they
are harder to analyze and identify using macro-ATR, which in a spatially wide area cannot discriminate a mixture of components. In suchcases, imaging measurements using IR microscopy is more suitable for analysis of foreign materials. During imaging, the measurement
area is finely divided into a grid pattern and a spectrum at each point is measured.
This facilitates the identification of each spatially separated component. In addition, by creating false-color chemical images, the spatial
distribution of multiple components can be imaged, which cannot be seen directly from the observed images. This section includes
examples of foreign material analysis using IR Microscopy.

Macro measurement (ATR method) or Single point measurement by IR microscope

A
B

A
C

B

C

Mixed spectrum of multiple
components is obtained.

Single point measurement by IR
microscope (aperture size: largest
area inside the sample)

ATR measurement

Imaging (multi-points) measurement by IR microscope
Samples consisting ofmultiple components can beseparated and measured
spatially.

A
B

C

Imaging (multi-points) measurement bysmall aperture size

Microscopic Transmission Method
Analysis of Foreign Materials in Silicon Wafers
Foreign materials in silicon wafers (Fig. 1) were measured using
high-speed FTIR imaging, and comparison with a spectral databasewas performed for each measurement position. As a result, it was
found that the foreign materials that could not be identified byvisual observation were composed of three components: protein,calcium carbonate, and cellulose (Fig. 2). A false-color chemical
image was created based on the peak height of the dominantabsorption band for each of the components, which allowed us tospatially separate and display the complex distribution of foreignmaterial in a silicon wafer consisting of protein, calcium carbonate,
and cellulose and their relative concentrations (Fig. 3).

The distribution state ofcomponents can be analyzed
by creating a color-coded
diagram with the key bands ofthe spectra.

100 µm4

Fig. 1 Observation view

Spectrum #1 (Fig. 4): This result suggests that the spectrum is a
mixture of silicone and protein because there are peaks attributed
to the Si-CH3 stretching (near 1,260 cm-1), to the Si-O stretching(near 1,000 to 1,100 cm-1), to the Si-C stretching (near 800 cm-1) andto the amide I and amide II bands (near 1,650 cm-1 and 1,550 cm-1,
respectively).

Protein

4000

3000

2000
-1
Wavenumber [cm ]Spectrum #2 (Fig. 5): This result suggests that the spectrum is a
protein because there are peaks attributed to the amide I andamide II bands and the N-H stretching (near 3,300 cm-1).

1000

Spectrum #3 (Fig. 6): This result suggests that the spectrum isa zeolite (aluminosilicate, a type of silicate) or talc (magnesiumhydroxide and silicate) from the strong peak around 1,020 cm-1.

Cellulose

3000

2000
-1
Wavenumber [cm ]Silicone

1000

Abs

4000

Spectrum #1

Calcium carbonate(CaCO3)

4000

3000

2000
-1
Wavenumber [cm ]

1000

4000

3000

2000

500

1000

Wavenumber [cm-1]

Fig. 2 Database search results(black: measurement result, red: database search result)Fig. 4 Typical spectrum of mixed foreign material
and database search results (Spectrum #1)

Protein (red): 1,647 cm-1
Calcium carbonate (green): 872 cm-1
Cellulose (blue): 1,057 cm-1

ProteinAbs

1.3 0.4 0.3

Spectrum #2

4000

3000

2000

1000

500

Wavenumber [cm-1]

Fig. 5 Typical spectrum of mixed foreign material
and database search results (Spectrum #2)

100 µm0

00Fig. 3 Imaging result (peak height)
Talc

Microscopic Transmission Method
Analysis of Foreign Materials on Rubber Surfaces

Four unique spectra were found at various sample positions in
the mapping measurement. (Figs. 4 – 7). Each spectrum can beinterpreted as containing the following components:

ZeoliteAbs

White foreign materials that were found adhered to a sample ofrubber packaging were removed and compressed between two
KBr plates for imaging measurement (KBr plate method). After
sample preparation, the imaging measurement was performed
using FTIR microscopy.

Spectrum #3

4000

3000

2000

1000

500

Wavenumber [cm-1]

Fig. 6 Typical spectrum of mixed foreign material
and database search results (Spectrum #3)

5

Spectrum #4 (Fig. 7): This result suggests that the spectrum is an oil
because there is a peak attributed to a C-H stretching (near 2,940
cm-1) and a peak attributed to a C=O stretching (near 1,750 cm-1)
with a close match to the spectral database oil spectrum.

Microscopic Reflection Absorption Spectroscopy(RAS) Method
Analysis of Foreign Materials on Solders
The Reflection Absorption Spectroscopy (RAS) method can be used
when an object to be measured is adhered on a highly reflective
surface, such as a metal plate. In this method, the absorption
spectrum of the sample is acquired and evaluated by reflecting IR
light from the reflective surface.

Oil

Abs

Foreign material

Spectrum #4

4000

IR light

3000

2000

1000

500

-1

Wavenumber [cm ]Metal plate

Fig. 7 Typical spectrum of mixed foreign material
and database search results (Spectrum #4)

Fig. 10 Overview of reflection absorption method

Next, a false-color chemical image was created using the peakheights of the key absorption bands of each component (indicatedby the arrows in the spectra), which was then compared with theoptical image (Figs. 8 and 9). The false-color chemical image
shows that most of the foreign material is oil and protein, with trace
amounts of silicone and zeolite or talc.
Imaging measurements enable the identification of eachcomponent in the mixed foreign material and the spatial distribution
throughout the sample. The identification of the components
in the foreign material is expected to lead to a review of the
manufacturing process to avoid introduction of foreign materials
in the end-product.

Here we present the evaluation of an adhered foreign material on
solder using the RAS method. The results indicated that it was rosin(a natural resin derived from pine resin).
The RAS method has the benefit of being a non-contact, nondestructive measurement technique. For optimal sample
measurement, some sample preparation may be needed to avoid
saturation of the signal or baseline curvature in the spectrum. Thicksamples may need to be sliced to avoid signal saturation. If thesample or underlying surface is rough, the spectral baseline may
exhibit curvature due to light scattering, so sample preparation isadvised to improve the surface condition.

Fig. 11 Observation view
(measurement size: 100 µm × 80 µm)

50 µm

Fig. 8 Observation view
0.098

0

0.01

ROSIN (a natural resin derived from pine resin)

Abs

0.1 0.12 2.1

50 µm
0

0

Fig. 9 False-color chemical image showing the spatial distribution of eachcomponent (red: oil, green: zeolite or talc, blue: protein, yellow: silicone)

6

4000

3000

2000

1500

1000

-1

Wavenumber [cm ]Fig. 12 Microscopic reflection absorption spectrumof foreign material on solder

650

Tips
Simple Sample Preparation using a
Diamond Cell
Before applying pressure

For transmission IR spectroscopy, the sample must
be thin enough to allow the transmission of light.
Common sample preparation methods for overly
thick samples include thinning the sample by applying
pressure between KBr plates or sectioning with amicrotome or slicer, which are both effective methods
to measure transmission IR spectra. To simplify such
sample preparation, JASCO offers the DC-500
diamond cell, which enables the user to perform IR
transmission measurements without using a press ora slicer. The sample is placed between two diamondplates and pressure is applied carefully by turning two
knobs, enabling sample thinning for the transmission
measurement. Another feature of the diamond cell is
the surface smoothness of the diamond plates used,which makes it easy to check for foreign materials
during microscopic observation.

Abs

After applying pressure

Fig. 13 Diamond cell

As an example, a transmission FTIR spectrum of arubber sample was measured using a diamond cell(Fig. 14). Sample freezing may be required when
preparing a rubber sample for sectioning with amicrotome. However, the diamond cell can adjust the
thickness of the rubber sample easily without freezing.
From the observation view, the sample could bethinned by applying pressure to the diamond cell. In
the IR spectrum, the peak position around 2,900 cm-1
is saturated and cannot be resolved before pressure
is applied, but applying pressure and thinning
the sample allows the spectrum to be completely
resolved.

4000

3000

2000

1000

600

-1

Wavenumber [cm ]Fig. 14 IR spectra of rubber samples
measured using a diamond cell
(red: before applying pressure, green: after applying pressure)The diamond cell can be easily adjusted to createa sample thickness suitable for a wide variety of IR
transmission methods. When used with an IQ Frame,
it can also be used for correlated analysis using IR
and Raman spectroscopy.

7

Tips

Microscopic ATR Method
Analysis of Foreign Materials on Resin Products

KnowItAll

Attenuated Total Reflectance (ATR) is an effective surface
measurement technique that can be used when a sample is too
thick for the RAS method, which leads to signal saturation, or
when the substrate is non-reflective. It is an ideal technique when
only information about foreign materials adhering to a substrate's
surface is needed..

Wiley's KnowItAll, which is a highly regarded spectral
library and search software, is used as standard forspectral identification. The search software starts
from one click in the IR Microscope Imaging Analysis
software (Fig. 15).

CrystalIR light

Foreign material
Resin

Fig. 16 Overview of the ATR method
Fig. 15 KnowItAll

KnowItAll includes various functions, such as spectral/peak/structure search, mixture analysis, spectralinterpretation, support, and more. It includes 12,600
Wiley original IR spectra and 625 JASCO original
IR spectra as the standard reference library. Wiley's
339,000 IR spectra library is also available as an
option.
Mixture Analysis (ID Expert)
For unknown samples containing multiple components,
up to five components can be identified in a single
spectrum using the IDExpert.
Spectral Interpretation Support (AnalyzeIt)
AnalyzeIt allows users to identify specific functionalgroups that match the absorption peaks in a sample
spectrum.

As an example, a foreign substance adhered to a resin (Fig. 17) was
evaluated using an ATR fitted with a Zinc Selenide (ZnSe) crystal,
which has a typical penetration depth of approximately 1 – 2 μm.
The results show that the adhered particles were azo dyes withcharacteristic peaks of azo compounds in the 1,300 – 1,500 cm-1
region (Fig. 18). If the thickness of the adherent is quite thin (1 μm orless), and information only about the adherent is required without
interference from the substrate, a Germanium (Ge) crystal, which
has a higher refractive index that results in a smaller penetration
depth, may be a better alternative. If the surface of the sample
matrix obstructs the measurement point, it may interfere with theconventional ATR measurement. In this case, it may be necessary to
use a projection-type ATR accessory, such as the ATR-5000-G45,
which has the benefit of extending the prism of the ATR with anarrower projection.

Foreign material

User Database Builder (MineIt)
MineIt allows users to create their own database,including the sample spectra, structural formula, andphysical properties. It enables the user to manage
and share the measurement data that is specific to
the user’s needs.
Multi-Technique Simultaneous Spectral Searching(SearchIt)
SearchIt is a “simultaneous” multi-technique spectral
search, which allows users to search IR and Raman
spectra simultaneously to find the most relevant
matches in each database linked to each other bychemical structure.

8

Fig. 17 Observation viewResin

Foreign material: Azo dye

Abs

Abs

Suspended particle: PMMA

Resin: Epoxy

4000

3000

Oil

2000

1500

1000

650

-1

Wavenumber [cm ]35001000 850

2000-1

Wavenumber [cm ]Fig. 18 Spectra of the resin (green: epoxy)and the foreign material (red: azo dye)Fig. 20 Spectra of the machine oil (blue) and the suspended particle(red: PMMA)

0.7

ATR Imaging Measurement of Particles Suspended in
Machine Oil
When using the ATR method, it can be difficult to confirm that the
crystal is in intimate contact with the sample, especially for fluidic
samples that may move as the crystal encounters the sample.
This problem also makes FTIR mapping measurements of a fluidic
sample with an auto-stage extremely difficult because the ATR
crystal contact point must be moved for each spectrum to bemeasured. By using IQ Mapping with a ClearView ATR objective,
it is possible to visually confirm that the crystal is in contact withthe correct sample measurement area. Since the crystal does not
move during IQ Mapping measurements, accurate sample contact
is maintained without disturbing the position of the sample.
ClearView ATR objectives allow both observation of the sample
and ATR spectral measurement using a single objective, byswitching the crystal position between Survey mode (up) and ATR
mode (down). The ClearView ATR objective is first used in Survey
mode to observe and identify the desired sample measurement
area. Once the crystal is in contact with the sample, the accurate
selection of the sample measurement area is confirmed. This
single-point contact between the sample and the crystal also
prevents contamination during mapping measurements. To
demonstrate the effectiveness of this ATR imaging technique, amapping measurement of particles suspended in machine oil was
performed, and a false-color chemical image was created using
the 1,720 cm-1 peak height, which is a dominant absorption peakin the polymethyl methacrylate (PMMA) spectrum. (Figs. 19–21).
From the observed image and the false-color chemical image, it
was confirmed that the distribution and chemical composition of
the particles could be well mapped and imaged, and from thespectrum the identity of the suspended particles was confirmed
as PMMA.

50 µm

30 µm
0

Fig. 21 False-color chemical image(peak height: 1,720 cm-1)

30 µm

Fig. 19 Observation view(left: sample before crystal contact, right: sample after crystal contact)

9

Tips

Tips

IQ Mapping

ClearView ATR Objectives

In a typical sample mapping measurement using IR
microscopy, it is necessary to move the sample stage
from one measurement point to the next. By scanning
the IR light across the sample (IQ Mapping), JASCO's
IR microscopes can perform mapping measurements
without the need to move the sample stage, making
it possible to perform a mapping measurement evenwhen using a manual sample stage. This uniquefeature is especially beneficial for ATR measurements,
as the sample area in contact with the ATR crystal canbe mapped without repositioning the objective prism,
which can disturb the sample.

In conventional microscopic ATR measurement,
the sample is observed with a standard reflection
Cassegrain objective to determine the measurement
point(s) in advance, and then the Cassegrain objectiveis switched to an ATR objective for measurement.
However, sample observation cannot be performed
after switching to the ATR objective, and it is notcertain that the target measurement area of thesample is in contact with the center of crystal.
Reviewing the measurement results is the only way
to confirm whether the target sample area has beencorrectly measured, which can be a waste of time,
especially when the sample has been disturbed.

Conventional mapping method (stage scanning)
Normal Cassegrain observation

ATR Contact observation

IQ Mapping method (IR light scanning)

Fig. 23 Optical system of of a ClearView ATR objective

Fig. 22 IQ Mapping allows the sample measurement area tobe moved while using a stationary Cassegrain objective tobuild up the sample image.

JASCO's ClearView ATR objectives can be used toobserve the sample surface while the crystal is in
direct contact with the sample, in addition to the usual
sample observation, allowing the target sample area
to be measured with absolute certainty.
Even if the target object shifts and becomes off center
during contact of the crystal and sample, IQ Mapping
allows the aperture controlling the IR light path to be
moved to the target object for measurement. With IQ
Mapping, the measurement position can be adjusted
to any desired location by moving the aperturearound the entire field of view of the objective.

10

Combined IR and Raman Analysis
Cross-Section Analysis of a Multilayer Film

PET
PP

Cross-sections of a multilayer laminate film were prepared;
measurement areas were identified, and the exact same area wasmapped using both IR and Raman microscopes by utilizing the IQ
Frame (Fig. 24). Principal component spectra were calculated byMultivariate Curve Resolution (MCR) from the spectral results (Figs.
25 and 26), and false-color chemical images were created from theprincipal component spectrum scores to illustrate the distributionof the identified components making up each layer (Figs. 27 and
28).

50 µm

CellulosePE

Fig. 27 False-color chemical image obtained using IR microscopy
50 µm

Fig. 24 Observation view
PET
PP

Cellulose (pink)Abs

PP (blue)

PE (green)

PET (aqua)
4000

2000

3000

1000

50 µm

-1

Wavenumber [cm ]Fig. 25 Principal component spectra calculated by MCR (IR)TiO2
PE
Fig. 28 False-color chemical image obtained using Raman microscopy

TiO2 (red)The distribution of polypropylene (PP), polyethylene (PE), andpolyethylene terephthalate (PET) was visualized using both
spectroscopic techniques. Cellulose was selectively identified
using IR microscopy and titanium dioxide (TiO2) was selectively
identified using Raman microscopy as a very thin layer witha thickness of only a few μm. The fact that cellulose is not a
constituent of the film sample measured in this study suggests that
it is likely to be a contaminant. This example demonstrates that byusing both Raman and FTIR, complementary information can beobtained for a more complete analysis.

Int.

PP (blue)

PE (green)

PET (aqua)
3000

1000

2000

500

-1

Wavenumber [cm ]Fig. 26 Principal component spectra calculated by MCR (Raman)

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INFRARED MICROSCOPY APPLICATIONS | MATERIAL SCIENCE

Analysis of Paints and Coatings
Paints and coatings not only improve appearance and protect underlying substrates, but they can also provide functionality. In orderto manufacture paints and coating films with better appearance, performance, and durability, it is necessary to analyze and understandthe chemistry and functionality of these products at the molecular level. In addition, foreign material and impurity analysis is essential toinvestigate the product uniformity and identify defects to determine their cause and ensure the manufacture of defect-free products on
the production line.
Since the main components of paints and coatings are organic compounds, including polymers, resins, pigments, and additives, FTIR
is an effective analytical technique to evaluate their chemical composition, structure, and behavior. The use of an IR microscope also
allows the evaluation of smaller sample areas, providing useful information for quality control in manufacturing, product development,
and failure analysis.
Here we present an example of paint and coating film analysis using an IR microscope.

Microscopic ATR Method
ATR Imaging Comparison of Viscous Paint Samples
When performing ATR imaging, the measurement technique must
be carefully selected to optimize the analysis. In conventional ATR
imaging measurements, a motorized stage is used to repeatedly
move the sample to measure a new area and to remake contact of
the ATR crystal with the sample. Though this is generally an effective
method for measuring wide areas of samples, when measuring
tacky or viscous samples, the sample may adhere to the crystal
after the first contact. This results in contamination of subsequent
measurement points of contact, which makes it difficult to obtainaccurate data due to the spectrum of the cross-contamination of
the ATR crystal and sample surface.
ATR imaging with IQ Mapping requires only a single point of contact
between the crystal and the sample since the IR light is scanned
across the sample without having to move the motorized stage.
This provides contamination-free measurements and minimizes therisk of the sample moving or becoming damaged during analysis.

12(a) ATR imaging measurement with auto stage

Stage movement

Crystal contact(b) ATR imaging measurement with IQ Mapping
IR light movement

Fig. 1 Overview of ATR imaging measurement techniquesIn this example, we measured a mixture of water-based and oilbased paints. First, an ATR imaging measurement was performed
using an auto-stage (Figs. 2 – 4). Since the ATR crystal and thesample were in contact with each other frequently during the stage
movement, contaminants adhered to the crystal and oil-based
components could not be detected (Fig. 4). In addition, the shapeof
the sample changed when the ATR crystal contacted the sample,
resulting in a completely different shape between the observed
image and the false-color chemical image (Figs. 2 and 3).

Next, we show the results of an ATR imaging measurement usinga Zinc Selenide (ZnSe) ATR crystal and IQ Mapping; changing theaperture measurement position without moving the sample stage.
In this example, a sample comprised of a mixture of water-based
and oil-based paints was prepared for measurement (Fig. 5).
Spectra with different components were detected depending on
the measurement position, and each unique spectrum could be
confirmed (Fig. 7). A false-color chemical image was created using
the peak intensities of the main peaks at 1,666 cm-1 and 1,282 cm-1
(Fig. 6) for the water-based and oil-based paints, respectively.
Selection of a suitable measurement technique allows thedetection of different components at specific locations in a sample
that appears to have only one component in the observed image,and to perform accurate sample identification at the microscopic
level.

50 µm

Fig. 2 Observation view
(yellow: water-based paint, white: oil-based paint,aqua: measurement area)

50 µm

Fig. 5 Observation view
(white: measurement area)
0.7

0.38

0.3

0.26

0.11 0.14

30 µm

20 µm
0

Fig. 6 False-color chemical image(green: water-based paint, 1,666 cm-1 peak,
red: oil-based paint 1,282 cm-1 peak)Fig. 3 False-color chemical image (green: water-based paint, blue:
water-based paint + contamination)

Water-based paint

Abs

Abs

Water-based paint

Water-based paint+ contamination (urea)

4000

0

Oil-based paint

2000

800
-1

Wavenumber [cm ]Fig. 4 Spectra of water-based paint (green) andwater-based paint + contamination (urea) (blue)

4000

2000
Wavenumber [cm-1]

1000

700

Fig. 7 Spectra of water-based paint (green)and oil-based paint (red)

13

Tips
IR Microscope Imaging Analysis Software
Analysis Wizard
PVC + Protein

Abs.

Imaging measurements using IR microscopy arewidely used to visualize component distribution in asample. However, when the area being measured islarge, with a significant number of data points, there isa possibility of missing components that are localized
in the sample. Data analysis skills and time are needed
to analyze the large amounts of data generated fromimaging measurements, which can be overcome byusing model analysis software, such as Multivariate
Curve Resolution (MCR). MCR is a function thatleverages multivariate analysis to extract the principal
component spectra from a collection of measured
spectra to create relative concentration distributions.
Therefore, MCR can be used to very quickly create
false-color images of the each chemical distributed ina sample, together with an indication of their relative
concentrations, without missing any of the individualcomponents. In addition, by performing a databasesearch on the obtained principal component spectra,
the components can be readily identified. JASCO's IR
microscope systems include Analysis Wizard, a groupof software tools that make it easy to create falsecolor chemical images by guiding the user throughthe key parameters for sample analysis.

PVC

Polyester

4000

2000

1000

650

Wavenumber [cm-1]

Obtained spectra
0.14 110 0.9AB
100 µm

100 µm-0.01

Observation
view

0

-0.1

False-color chemical image (red:polyester, green: protein, blue: PVC)

Fig. 9 Imaging analysis of multilayer film by key bands(False-color chemical image based on arrowed key bands)

PVC

Abs.

PolyesterProteinFig. 8 Analysis wizard

Imaging analysis of a multilayer film was performed.
Using the obtained spectra, a false-color chemical
image was created from the intensity distribution of
the key absorption bands (Fig. 9). From the obtained
spectra, it was determined that there are threecomponents in the multilayer film, and the distributionof these components was observed in the falsecolor chemical image. The results show that polyvinyl
chloride (PVC) is distributed in layer A and polyester isdistributed in layer B. In addition, the results show that
the PVC layer contains protein as a foreign material.
Next, using the same spectra, principal component
spectra were modeled using MCR, and a false-color
chemical image was created (Fig. 10). The model
analysis was able to extract components that were notevident in the imaging analysis using key functionalgroup bands and was able to determine the pure
spectrum of the foreign material. It can be concluded
that model analysis, such as MCR, can be used toobtain false-color chemical images without missing
any components and allows the user to perform
spectral database searches with high accuracy.

14

Polyethylene
4000

3000

2000

1000

650

-1

Wavenumber [cm ]Principal component spectra5

9 12 3.5

0

0

A

B
100 µmObservation
view

100 µm00

False-color chemical image (red: polyester,
green: protein, blue: PVC, yellow: polyethylene)

Fig. 10 Imaging analysis of multilayer film by MCR

Combined IR and Raman Analysis
Analysis of Car Paint Chips
Acrylic + Styrene

Melamine + Styrene
Abs.

Analysis combining IR and Raman spectroscopy can provide more
information about a sample and can increase confidence in theanalytical results. When performing a combined analysis, correlated
information from both methods is required, meaning the data mustbe accurately acquired from the same sample area. This can beaccomplished by utilizing an IQ Frame. The IQ Frame enables
accurate sample repositioning so that even when the sample is
moved from one instrument to another, the exact measurement
location(s) can be recorded and identified.

Melamine + Kaolin

Epoxy

IR Microscope
4000

2000

3000

1000

-1

Wavenumber [cm ]Principal component spectra

Acrylic + Styrene

Melamine + Kaolin

IQ FrameRaman Microscope

Melamine + Styrene

Epoxy

False-color chemical image
Fig. 13 Measurement results using IR microscopy
Fig. 11 IQ Frame operation between Raman and IR microscopes

In this application, analysis was performed on a paint chip obtained
from a car body panel. A cross-section of the coating was made andsandwiched between two KBr plates (Fig. 12). First, IR microscopy
imaging measurements were performed on a 200 μm square area.
Then, principal component spectra were calculated by Multivariate
Curve Resolution (MCR) from the measured spectra, and a falsecolor chemical image was created from the principal component
spectra scores (Fig. 13). This imaging analysis revealed the
distribution of four layers of mixed multiple organic components:
acrylic + styrene, melamine + styrene, melamine + kaolin, and epoxy.

20 µm

Fig. 12 Observation view

15

Next, Raman imaging measurements were performed on thesame sample area. Principal component spectra were calculated
by MCR using the acquired Raman spectra, and a false-color
chemical image was created from the principal component spectra
scores (Fig. 14). From the Raman imaging analysis, it was found that
there were several layers containing phthalocyanine as pigment,
carbon black, and titanium dioxide (TiO2), as well as the layers
containing acrylic and styrene, which were previously identified by
IR microscopy. In the layer containing epoxy that was identified by
IR microscopy, the Raman scattering peak could not be seen due
to a significant amount of fluorescence caused by the presence of
pigment components.

Int.

Acrylic + Styrene

Phthalocyanine

Carbon + Titanium dioxide
3000

1000

2000

200

-1

Raman Shift [cm ]Principal component spectra

Acrylic + Styrene

Carbon + Titanium dioxide

Phthalocyanine

Not identified due tofluorescence

False-color chemical image
Fig. 14 Measurement results using Raman microscopy

Int.

520 nm

500

600
Wavelength [nm]Fig. 15 Fluorescence spectrum of epoxy layer (405 nm excitation)

16

However, as a side benefit, fluorescence spectroscopy could
be used to characterize and identify the chemical components
in the pigments. Fluorescence measurement was performed
using a shorter excitation wavelength of 405 nm (Fig. 15). The
resulting emission spectrum indicates the presence of pigmentcomponents with a fluorescence maximum at 520 nm, which
could be used to identify different pigment components. By
combining the two techniques of IR and Raman microscopy (andadditionally photoluminescence), a more detailed material analysis
characterization is possible than by using either technique alone.

Tips
Combined Analysis using IQ Frame
Correlation molecular spectroscopy can provide
more useful and detailed analysis of complex
samples; however, when using microscopy,
it can be difficult to locate the same sample
area when transferring between systems formeasurement. To overcome this barrier to correlated
microspectroscopy, JASCO developed the unique IQ
Frame, a sample measurement system that includes
a calibrated sample holder together with advanced
image matching technology (Fig. 16) that allows
simple and accurate transfer between different
microspectrometers for coordinated imaging andanalysis using any combination of IR, Raman, and UVVisible microscopy.

IR microscope
Identification of organic compounds/polymers
Identification of foreign materialetc.

(1) Sample setting

Set the sample(2) Movement by coordinate information

Moves the stage bycoordinate information
obtained in the previous
measurement(3) Movement by coincidence of observation image

Raman microscope
IQ Frame

UV/Vis-NIR
microscopic
spectrophotometer

Identification of organic/inorganic
compounds
Acquisition of dye information
etc.

Corrects the small
displacement of
measurement point byusing image matching
technique

Color evaluation
Optical constants evaluation in UV/Vis-NIR region
etc.

Fig. 17 Overview of image matching techniqueFig. 16 Combined analysis using IQ Frame

17

INFRARED MICROSCOPY APPLICATIONS | MATERIAL SCIENCE

Evaluation of Rubber Products
The chemical composition, physical structure, and quality of rubber are closely related to its performance and specifications. FTIR,
which can be used to identify organic materials and analyze complex mixtures, is as an effective analytical technique for the researchand development (R&D) and quality control of rubber products because it has the following key benefits:••••

Little or no sample preparation is required
The sample is not destroyed during measurement
Analysis of a wide variety of materials is possible
It can be measured relatively quickly

In addition to FTIR macro measurement, an IR microscope can provide the same information and analysis, but on much smaller sample
areas, making it a useful technique for the identification, distribution and potential source of foreign materials in failure analysis.
Furthermore, since chemical component distribution can be obtained, it can also be useful information for product R&D.
This section shows the evaluation of rubber products using IR microscopy.

Microscopic ATR Method
Analysis of a Rubber Sample with Surface Degradation
An IR microscope with an ATR objective was used to investigate
the compositional changes in rubber upon degradation (Fig. 1).
In order to analyze rubber materials that contain carbon black,a Germanium (Ge) crystal is used since it has a higher refractive
index and smaller penetration depth. ATR measurements of therubber samples before and after degradation were performed(Fig. 2), and then the difference spectrum between normal anddegraded products was calculated (Fig. 3). It was concluded thatzinc stearate was deficient in the degraded product.

Abs

Degraded product

Normal productFig. 1 Observation view(left: normal product, right: degraded product)

18

4000

3000

2000
1500
Wavenumber [cm-1]

1000

700

Fig. 2 Spectra of rubber samples before and after degradation(red: degraded product, green: normal product)Difference spectrum: normal product - degraded product-> Zinc Stearate

Abs

Abs

Butadiene rubber

Silicone (glazing and additives)

4000

2000

3000

1500

1000

700

-1

4000

2000

3000

800

-1

Wavenumber [cm ]Wavenumber [cm ]Fig. 3 Difference spectrum

Fig. 5 Spectra of butadiene rubber (red) andsilicone (glazing and additives) (green)

Microscopic ATR Method
Component Distribution Analysis of Rubber with aHigh Refractive Index
ATR imaging measurement was performed on a prepared crosssection of a tire spur (spike) (Fig. 4). In order to measure rubber
containing carbon black, a Germanium (Ge) crystal is used since
it has a higher refractive index and shallower penetration depth.
In addition, a wide-area ATR objective that allows measurement
across a wide single-contact point (mm scale) was used for thismeasurement. Principal component spectra were calculated byMultivariate Curve Resolution (MCR) from the measurement results(Fig. 5), and a false-color chemical image was created from theprincipal component spectra scores (Fig. 6). High-quality spectraof butadiene rubber (the main component of the tire) and silicone(an additive in the tire) were obtained, and the distribution of thecomponents could be evaluated. The false-color chemical image
also shows the mixture of butadiene rubber and silicone as themixed color (yellow).

0.15

0.06

0

0

200 µmFig. 6 False-color chemical image(red: butadiene rubber, green: silicone)

IQ Mapping for ATR
IQ Mapping, which provides wide-area imaging measurement fora single sample contact point, even for elastic samples, such as
rubber, has enabled accurate analysis of the sample shape andchemical component distribution.

200 µmFig. 4 Observation view of rubber tirefragment

19

Tips
Wide-Area ATR Objectives
ATR imaging measurements using IR microscopy
have a wide range of applications, including theanalysis of foreign materials and multilayer films, andthe evaluation of additives, with the required spatial
resolution and measurement area differing dependingon the purpose of each analysis. The analysis offoreign materials in food and industrial products canvisually be confirmed using ATR imaging; however,
ATR measurement over a wide area is required sincethese foreign materials are often several hundred μm
or larger in size. On the other hand, when evaluating
the distribution of additives found in rubbers or resins,high spatial resolution in the micron order is required.
JASCO offers ATR objectives with different prism
crystal materials, including Zinc Selenide (ZnSe), Zinc
Sulfide (ZnS), diamond, and Germanium (Ge). The
choice of Ge ATR objectives includes three different
options for spatial resolution and sampling area witha single contact: ATR-5000-WG for a wide area atthe mm scale, ATR-5000-MG for several hundredμm, and ATR-5000-G45 for high spatial resolution
measurement to the diffraction limit. The features ofeach ATR objective are described below.

ATR-5000-WG

Blue: Polyethylene
Red: Cellulose
Green: Kaolin + Calcium carbonate
Approx. 1600 µm

ATR Objective

ATR Imaging Pixel Resolution
Area (single (using a linear
contact point) array detector)

ATR-5000-WG 1,600 x 1,600("WG")µm

50 x 50 µm

Applications

Screening analysisof large areas

ATR-5000-MG

Analysis of
ATR-5000-MG
distribution states
400 x 400 µm 12.5 x 12.5 µm("MG")
on the order of tens
to hundreds of µm
ATR-5000-G45
70 x 70 µm("G45")

2.2 x 2.2 µm

Analysis of microobjects and microstructures on the
micron-order

Blue: Polyethylene
Red: Cellulose
Green: Kaolin + Calcium carbonate
Approx. 400 µm

To compare the analysis at different scales, a crosssection of a paper beverage container was prepared,
and ATR imaging measurements using the above ATR
objectives were performed for qualitative analysis
of each layer. The entire area of the multilayer ofapproximately 800 μm could be imaged using the
WG ATR objective, and the components of each layer
were visualized (cellulose, polyethylene, and kaolin +calcium carbonate).
The MG ATR objective was used to measure the areaaround the central layer, and the polyethylene layer
between the cellulose layers was detected, which
could not be identified by the WG ATR objective due
to its lower spatial resolution. The G45 ATR objective
was used to measure the smaller area with highresolution, and was able to discriminate the layer as asingle component (calcium carbonate).

20

ATR-5000-G45

Blue: Polyethylene
Red: Cellulose
Yellow: Calcium carbonate
Approx. 70 µm

Fig. 7 Observation view and false-color chemical image (red:cellulose, blue: polyethylene, green: kaolin + calcium
carbonate, yellow: calcium carbonate)INFRARED MICROSCOPY APPLICATIONS | MATERIAL SCIENCE

Evaluation of Some Physical Properties of Polymers
The physical properties of polymers are largely dependent on the chemical structure and orientation of their constituent molecules.
The molecular orientation of polymers is closely related to the functional properties of the material. For example, in the manufacturing ofpolypropylene (PP) films, different orientation states are used depending on the required application, such as strength, abrasion resistance,
moisture resistance, transparency, etc. Some polymers can have the same molecular structure but different crystal structures, whichthese give rise to differences in physical properties, such as hardness, melting point, transparency, and strength, and can be exploitedfor different applications. Therefore, it is very important to elucidate and understand the orientation and crystal structure of polymericmaterials.
IR spectroscopy is extremely effective for evaluating the properties described above. Polarization measurement, where a polarizer isinserted into the optical path, can be used to evaluate the orientation state of a sample. Characteristic peaks of crystals are found in
the low wavenumber region of an IR spectrum, and these specific peaks can be used to determine the state of the crystal structure.
Furthermore, IR imaging can be used to visualize the distribution of orientation state and crystal structure in a sample.
This section shows an example of using IR microscopy to evaluate the distribution of the orientation and crystal structure of polymers.

Microscopic Transmission Method
Orientation Distribution Analysis of Polypropylene
Polarization imaging measurements are useful in evaluating the
distribution of molecular orientation in a sample. The example
described here demonstrates the use of IR polarization imaging
to evaluate the molecular orientation distribution of a stretched
polypropylene material.

that areas with high and low orientation are distributed periodicallythroughout the polypropylene film.
3

13

A comparison of spectra acquired using different angles ofpolarization (perpendicular and parallel directions) revealed a
difference in peak height at 1,304 cm-1, (CH2 wagging and twisting)(Fig. 1), and at 809 cm-1 (C-C stretching band of the polymer chain
backbone).
Exploiting this variation, a false-color chemical image was createdfrom the peak height ratio of 809 cm-1/1,304 cm-1 (Fig. 2). Thereis a correlation between molecular orientation and peak heightratio, indicating that the polypropylene molectular chain is highlyoriented when the peak height ratio is large. It was confirmed

100 µmPolarizer 0°0100 µmPolarizer 90°

Fig. 1 Polarization false-color chemical
images (Intensity ratio between 809 cm-1
and 1,304 cm-1)

210Microscopic Transmission Method
Evaluation of Polymer Crystallinity by IR Mapping

Tips

Some polymers used in plastics and fibers have the same
molecular structure but different crystal structures. The crystal
structure impacts the chemical, physical, thermal, and mechanicalproperties, such as hardness, transparency, and melting point.
Therefore, it is very important to evaluate the crystal structure ofpolymer materials to assess their use for specific applications.

Advantages of a Grazing Angle Reflection
Objective

Here, we present the results of the investigation into the internal
structure of two different polymeric products (fishing lines). The two
fishing lines have different functions, characteristics, and costs, buteach is manufactured from polyvinylidene fluoride (PVDF), which
is known to possess many crystal structures (polymorphs). A thincross-section of each fishing line was prepared, sandwiched
between two KBr plates, and FTIR imaging measurement was
performed using the microscopic transmission method on an IRT5200 FTIR microscope. Since α- and β-type crystal structures havecharacteristic peaks around 763 cm-1 and 840 cm-1 respectively,
a false-color chemical image was created using the peak heightof each characteristic polymorph peak (Fig. 2). Fishing line #1 wasfound to be composed entirely of the β-polymorph. In contrast, it
was confirmed that fishing line #2 has mostly β-crystal structure
at its center with an external coating of approximately 50 μm,
comprised of a mixture of α- and β-type polymorphs.

Grazing angle reflection objectives are used forhigh sensitivity Reflection Absorption Spectroscopy(RAS) measurements. By using a grazing angle
reflection objective (Fig. 3), very high sensitivity
measurements of thin films on reflective metal
surfaces can be measured, which is useful for the
analysis of Langmuir-Blodgett (LB) films, oxide films
on metal surfaces, and processed films produced byvarious methods. Recently, grazing angle objectives
have been used for the in-situ measurement of thevapor deposition processes onto silicon substrates inchemical vapor deposition (CVD) reaction chambers.
RAS can analyze films as thin as several 10 Å if thesample has a high absorption coefficient. The grazing
angle reflection objective can also perform highsensitivity measurements of small areas.

In conclusion, the cross-sectional FTIR imaging of the fishing lines
demonstrated that fishing line #2 has the greater strength due to its
different outer layer of PVDF, comprised of α+β crystal polymorph
structures.

Fig. 3 Grazing-angle reflection objective

A polymethyl methacrylate (PMMA) film of
approximately 50 nm in thickness was made byevaporating a PMMA solution onto a gold mirror. The
film was measured on the gold mirror using a grazing
angle reflection objective by the conventional RAS
method. While only a small amount of absorption
was observed using the traditional RAS method, agrazing angle reflection objective could perform themeasurement with an order of magnitude greater
sensitivity, as seen in the spectra (Fig. 4) below.

100 µmFishing line #1

Grazing angle reflection objective

Abs = 0.010

Abs

Reflection absorption method

0.0025
Abs = 0.001

100 µmFishing line #2

Fig. 2 False-color chemical images(red: β type (840 cm-1), green: α+β type(840 cm-1 and 763 cm-1))

22

4000

1000

2000
-1

Wavenumber [cm ]Fig. 4 IR spectra of PMMA film on gold mirror

800

Tips
Vacuum Option

Atmosphere

Vacuum
4000

1000

Wavenumber [cm-1]

Atmosphere

Vacuum2000

Wavenumber [cm-1]

1200

Fig. 6 IR spectra of tristearin film on gold mirror(Grazing angle reflection measurement)

Atmosphere

%T

50.0

0.002

Abs

To illustrate the effectiveness of a full-vacuum FTIR,
a spectrum of a silicon wafer was measured using
the transmission method under both atmosphericand full-vacuum conditions (Fig. 5). When measuring
a sample with a high refractive index and a highlyreflective surface, such as a silicon wafer, IR light may
be reflected from the surface (back reflection) andreturned to the interferometer. When this light passes
through the sample chamber for the second timeand enters the detector, the effect of atmosphericnoise may increase due to the time between themeasurement light and the reference light measured
without the sample present. When performing
qualitative analysis of a sample, the sample canbe tilted to prevent the light from returning to theinterferometer, but this is not suitable for quantitative
analysis or optical characterization. By using a fullvacuum system, high-quality IR spectra without
atmospheric noise can be obtained, even without
tilting the sample.

In addition, when measuring small peaks, it may benecessary to eliminate environmental interference
due to the IR absorption of H2O vapor and CO2 thatappear in the spectrum. Measurement under vacuum
conditions is very effective at eliminating atmosphericnoise and can provide a clear spectrum free of peaks
caused by H2O vapor and CO2. In the following
example, a grazing angle reflection measurement of atristearin film formed on a gold mirror was performed
under atmospheric and full-vacuum conditions (Fig.
6). The spectrum collected under vacuum is cleaner,
without interfering peaks from atmospheric noise.

Abs

In general, since an FTIR spectrometer only has a single
beam, it cannot ratio the reference light (background)and measurement light (sample) simultaneously,
which creates a time lag between the backgroundand sample measurements. FTIR spectrometers arevery sensitive to the strong vibrational dipoles ofatmospheric water (H2O) vapor and carbon dioxide(CO2). Even small changes in concentration of H2Ovapor or CO2 (both present in the operator’s breath)in the atmosphere results in noise that can be readily
detected in the IR spectrum, adversely affecting
spectral analysis. In measurements that are likely to
be sensitive to this type of interference, it is common
to purge the optical path with an IR-inert gas, suchas nitrogen (N2) or dry air, or to remove atmosphericnoise through data processing. The most effective
solution to remove atmospheric effects is tocompletely evacuate the IR optical path. Performing
measurements under vacuum enables the collectionof high-quality spectra with high accuracy and speed
without atmospheric influence.

30.0
4000

2000
-1
Wavenumber [cm ]

1000

400

Vacuum

%T

50.0

3000

Full vacuum IR microscope system

The vacuum option is available for the FT/IR-6X
and the FT/IR-8X systems such as seen above.

30.0
4000

3000

2000
Wavenumber [cm-1]

1000

400

Fig. 5 Measurement example of epitaxial film on silicon wafer

23

INFRARED MICROSCOPY APPLICATIONS | MATERIAL SCIENCE

Dynamic Imaging
FTIR spectroscopy has three methods for time course measurement: interval measurement, rapid scan, and step scan with time resolutionsof 1 sec, 50 msec, and 5 msec (option: 10 nsec), respectively.
Here, we will review the basic features and scanning method of the moving mirror in the interferometer for each time course measurement.
“Interval measurement” is performed with regular scanning while rapid scan measurement uses a faster scan rate of the moving mirror. In
step scan measurement, the moving mirror stops at each data-sampling point and the sample is perturbed at each of the points.
One of the main features of a Fourier-Transform Infrared (FTIR) spectrometer is the ability to simultaneously measure multiple wavelengths.
FTIR spectroscopy can acquire an interference pattern of waves generated by light with multiple wavelengths in the time domain, whichis created by scanning a moving mirror in the interferometer along its optical axis to obtain an intensity distribution (spectrum) for eachwavelength. This distribution is then converted from the time domain into the frequency domain (cm-1) using a Fast Fourier Transform(FFT). Since an FTIR spectrometer can acquire an IR spectrum faster than a dispersive IR spectrometer, FTIR spectroscopy is a powerful
analytical technique for time-dependent measurements, which can be used to monitor the structural change in a sample over time.
The IRT-7000, a linear array imaging microscope, has three time course measurement methods: interval measurement, rapid scanmeasurement, and step scan measurement. Each measurement method uses a different scanning method for the moving mirror in the
FTIR interferometer as noted above.

Interval Measurement
2 shows the rapid scan spectra over the course of 50 seconds
of a UV curing reaction where the C=C double bond is seen to
decrease as the polymerization proceeds to form C-C single
bonds (not shown in figure).

Applications: Monitoring of structural changes in a sample orchanges in a gas concentration, etc. Figure 1 shows spectra ofroom air over a period of 12 hours. The temporal changes in theenvironmental CO2 concentration can be monitored by plotting theintensity of the 2,360 cm-1 peak against time.

Rapid scan mode can be used for studying kinetics, monitoring
dynamic processes, such as photo-polymerization reactions,orientation relaxation of polymer films, and depth profiling. Figure
24

0.4
Abs

Rapid Scan Measurement is performed by high-speed scanningof the moving mirror, which can increase the minimum time intervalof the IR microscope to 50 msec (with a spectral resolution of 16cm-1).

0.6

0.2
720
480
Time
240
2400
[min.]
02300
Wavenumber
2200
-1
[cm ]

0
-0.1
2500

0.3
0.25
Abs

Interval measurement is performed using normal moving mirror
scanning speeds, offering a typical minimum time resolution ofaround 1 sec.

0.2
50000

0.15
0.1
1650

1640

1630
0
1620
Wavenumber
1610[cm-1]

25000
Time[msec.]

Fig. 1 CO2 concentration monitoring Fig. 2 Curing process of UV curable resin(Rapid scan measurement data)(Interval measurement data)C-H out-of-plane bending
vibration of the vinyl group
C=O stretchingvibration

Cured portion
Abs

Step Scan Measurement can only be applied to measure thedynamics of samples that have a reversible/repeatable response.
Typical examples include studying the effects of applying stressto samples, changes in applied electric field, or changes intemperature. The minimum time resolution in step scan mode is 5
μsec (optionally 10 nsec).
To make a complete spectral scan, the moving mirror is stopped
and dithered at each frequency sampling point while reversible
perturbations are made to the sample.
Applications: Monitoring the orientation relaxation process of liquid
crystals, ferroelectric transitions of polymers, light-induced protein
reactions, and other systems with reactions in the microsecond
time scale.
The FTIR spectra measured during the relaxation process of theorientation of a liquid crystal in response to an applied electricfield are shown in Fig. 3. The peak intensity transition at 2,925 cm-1
(left of Fig. 3) is attributed to C-H stretching. The peak intensity
fluctuated in response to the applied voltage and decreased in
two phases - rapidly and then moderately. It is known that theliquid crystal in the area near the electrode has a faster orientation
relaxation than the bulk crystal and this difference can be readily
observed using an FTIR spectrometer with step scan time course
measurement.
0.81

0.8
10 msec.

0.79
Voltage

10 V
0V
0

25000
Time [msec.]

50000

Peak intensity transition at 2,925 cm

-1

0.7

0.6
3050
3000

2900
Wavenumber
-1
[cm ]

50000
25000
0
2800

4000

1000

2000
-1
Wavenumber [cm ]750

Fig. 4 IR spectra of cured and uncured portions(blue: cured portion, red: uncured portion)

bending vibration of the vinyl group (near 810 cm-1) decreases
over time, which is consistent with the advancement of thepolymerization reaction as the C=C double bond is converted to asingle bond with the curing of the resin. In contrast, the intensity of
the C=O stretching vibration (around 1,730 cm-1) is almost the same
before and after curing, as this functional group does not undergo
any reaction.
To illustrate the distribution of the curing over time across the
sample, a stacked view was created from the ratio of the two
peak heights (810 cm-1/1,730 cm-1) and using the peak at 1,730
cm-1 as an internal standard (Fig. 5). The z-axis indicates the time,
and the change in color against the time at each location shows
the differences caused by the progress in the chemical reaction
distributed across the sample. The locality of UV light intensity andthe degree of curing per unit area can be evaluated, where thelower intensity in the 810 cm-1 peak indicates a greater extent ofreaction (Figs. 6 and 7).

Time[µsec.]

Fig. 3 Orientation relaxation process monitoring of liquid crystal(Step scan measurement data)

In recent years, there has also been an increasing need for timecourse mapping measurements to monitor the structural change ina micro-area plane over time.
This section shows examples of mapping measurements formonitoring the changes in molecular structure when a sample
is irradiated with a UV light or subject to changes in the applied
electric field.
Dynamic imaging measurements can be performed with an
FT/ IR-8X spectrometer fitted with a step scan interferometer, in
combination with an IRT-7200 linear array imaging microscope.

330
300
240
Z [sec.]

Abs

Abs

0.81

Uncured portion

180
120
60
0

0.33

0

Fig. 5 Stacked false-color chemical images(peak height ratio 810 cm-1/1,730 cm-1)
0.4

0.33

Time Course Imaging
Analysis of the Curing Process of UV Curable Resins
Time course mapping measurements were performed of an UVcured resin applied on a metal plate. The measurement wasstarted as the sample was irradiated with UV light to monitor
the subsequent reaction. The spectra of the cured and uncured
portions were also obtained (Fig. 4).
It can be observed that the peak attributed to the C-H out-of-plane

30 µm
0

Fig. 6 False-color chemical image
before UV irradiation (0 sec.) (peakheight ratio 810 cm-1/1,730 cm-1)

30 µm

0

Fig. 7 False-color chemical image
after UV irradiation (330 sec.)(peakheight ratio 810 cm-1/1,730 cm-1)

25

Tips
Heating and Cooling Stages for IR and Raman
Microscopy
Two models of heating and cooling stages are available
for sample measurements in different temperature
ranges. These are operated with a dedicatedprogram that controls the stage temperature, rampingof temperature, and automatically starts measurement
when the sample reaches the measurement
temperature at the set temperature ramping/fallingrate.

Heating and cooling stage

100 µm

100 µm120°C

122.5°C

100 µm

100 µm

124°C

125°C

Fig. 9 Observation views of benzoic acid at each temperature(square: measurement area)

As an example of measurement, the structural changeof benzoic acid was monitored when heated to 150
degrees Celsius. A 3D spectrum of benzoic acid asa function of temperature was obtained (Fig. 8). An
observation image (Fig. 10) and the intensity of thepeak attributed to the O-H bending vibration at 930
cm-1 (Fig. 9) was monitored at each temperature. From
the results, it could be seen that the structure and stateof benzoic acid changed at approximately 120 – 125
degrees Celsius. Since the melting point of benzoic
acid is 122.4 degrees Celsius, it could be seen that
this system accurately captured the intramolecular
structural and state changes associated with themelting process.

Abs

0.6

0.4

0.2
150
140
130
Temperature [°C]

120
110
100
1050

750

Fig. 8 3D spectrum of benzoic acid26

-1

Wavenumber [cm ]Abs.

By combining an IR microscope with a heating/cooling stage, thermophysical properties, observation
images, and structural changes in molecules can becomprehensively evaluated, including crystallization,
melting, and other phase transitions.

100

120

140

150

Temperature [°C]Fig. 10 Peak intensity change at 930 cm-1

Compared to conventional macro FTIR spectroscopy,
since the measurement area of an IR microscope isrelatively small, it allows for accurate uniform heating/cooling) and yields high-precision analysis of thesample. This system can be used in a wide range ofapplications that include structural changes due toheating, such as the curing process of thermosetting
resins, crystallization processes of drugs andpolymers, protein denaturation, and polymorphic
phase transitions.

Step Scan Imaging
Fast Time Course Analysis of Liquid Crystals with
Applied Voltage
Step scan time course measurements were used to monitor theorientation relaxation process of liquid crystals when subjected
toan electric field1). It is well known that the orientation of moleculesin liquid crystals changes when a voltage is applied. Step scan
FTIR measurement was used with an IR polarizer placed incident
to the sample and parallel to the orientation direction of the liquid
crystal to evaluate the changes in orientation. A typical nematic
liquid crystal, 5CB (4-pentyl-4'-cyanobiphenyl), was used for themeasurements (Fig. 11).

C5H11
Before applying the voltage

C N
After applying the voltage

Dynamic imaging measurement of the orientation relaxation
process of liquid crystals was performed using an IR microscope(Fig. 13). The time resolution interval of this measurement was 1.0
msec, and the total measurement time was 120 msec. A voltageof 10 V was applied to the liquid crystal for 11 msec, from 19 msec
after the start of the measurement. The images before and after
the voltage was applied (19 msec and 21 msec, respectively) wereentirely one color, indicating that the orientation of the liquid crystal
molecules was uniform. After the applied voltage was stopped (32msec, 40 msec, 60 msec, and 100 msec), the orientation of theliquid crystal molecules was heterogeneous. This indicates the
localization of the reorientation process of liquid crystal molecules
depends on its distribution through the structure; with fasterreorientation near the point where the electric charge was applied,and slower reorientation in the bulk material.
19 msec.

40 msec.

50 µm

Fig. 11 Overview of sample (5CB) (yellow: sample)

The time resolution interval of the initial measurement was 250
μsec, with a total measurement time of 25 msec. A voltage of 5 V
was applied to the liquid crystal for 10 msec, from 1 msec after thestart of the measurement. Focusing on the intensity change in thepeak attributed to the C-H anti-symmetric stretching vibration of
the methylene group (at 2,925 cm-1), the peak intensity changed insynchronization with the ON and OFF voltage, and the orientationof the molecules in the liquid crystal could be observed in the
spectrum (Fig. 12).

21 msec.

50 µm
60 msec.

50 µm
32 msec.

50 µm
100 msec.

50 µm

50 µm

Abs

Fig. 13 Dynamic imaging results of the orientation relaxation processof liquid crystal (5CB)

25
20

Time
[msec.]As described above, step scan time course measurement can beused to monitor the orientation relaxation process of a liquid crystal
when subjected to an applied electric field. Combining step scan
FTIR measurement with an IR microscope enablesobservation of
the localized changes in the in-plane distribution of the orientation
relaxation process.

10

3050
Wavenumber [cm-1]

0
2800

0

5 [V]

1) Sugiyama, H., Koshoubu, J., Kashiwabara, S., Nagoshi, T., Larsen
R. A., Akao, K., Appl. Spectrosc., 62(1), 17-23 (2008).

Fig. 12 Orientation relaxation process of liquid crystal (5CB)

27

Tips
Two-Dimensional (2D) Correlation
1400

Wavenumber [cm-1]

The two-dimensional (2D) correlation program(optional Spectra Manager software) can be used
to calculate and display the correlation of peaks
observed in the measured spectra acquired in three
dimensions (3D), which can include time interval andtemperature-dependent measurements. This enables
correlation analysis of different functional groupmeasurements to understand how the relationshipbetween functional groups and the aggregation
state of molecules change depending on time ortemperature.

1400

Wavenumber [cm-1]

1000
1000

Fig. 16 Synchronous correlation spectra(red: positive value, blue: negative value)*Synchronous correlation: This indicates the degree
to which the behavior of the changes in peak intensity
are similar. Positive values are indicated when theintensities of each peak are changing in the same
direction while negative values are indicated when
the intensities of each peak are changing in opposite
directions.

Fig. 14 Two-dimensional (2D) correlation program

1400

Wavenumber [cm-1]

In order to understand 2D correlation, simulated
spectra (Fig. 15, Table 1) with varying peak intensities
under the following conditions were synthesized
and analyzed using the 2D correlation program. The
correlation spectra of the composite waveforms aswell as their interpretations are shown (Figs. 16 and
17).
1400

Wavenumber [cm-1]

1000
1000

Fig. 17 Asynchronous correlation spectra(red: positive value, blue: negative value)

Abs

1

0.5

20
Time [sec.]

0
1400

1300

1200
-1

Wavenumber [cm ]

1100

0
1000

Fig. 15 Three-dimensional (3D) spectrum of composite
waveform
Table 1 Summary of Simulation Spectra
Peak Position
Characteristics
of peak

28

1300 cm-1
Increase

1200 cm-1

Around1100 cm-1-1

Slowly1090 cm : Decrease

decrease1110 cm : Increase-1*Asynchronous correlation: This indicates the degree
to which the behavior of the change in peak intensity
is diﬀerent. When the synchronous correlation ispositive, negative values are indicated if the peakintensity change on the X-axis is slower than thepeak intensity change on the Y-axis while positive
values are indicated if the peak intensity change on
the X-axis is faster than the peak intensity change on
the Y-axis.
2D correlation analysis can be applied to data fromdifferent molecular spectroscopy measurements;
these 2D correlation results combining spectral
analysis techniques (including near-IR, Raman, UVVisible, or Circular Dichroism) with IR, and can provide
additional information about peak assignment,
lattice vibrations, and the relationship between
intramolecular vibrations, color, or chiral information.

INFRARED MICROSCOPY APPLICATIONS | LIFE SCIENCE

Component Distribution Analysis of Foods
IR microscopy is one of the most important analytical techniques in the field of food science; one example is the analysis of foreignmaterials to discover the nature and origins of contamination. Complaints about foreign materials discovered by consumers in foodproducts are widespread, and product recalls are not only common but well publicized. Many such incidents are reported to include
rubber or resin contamination caused by deterioration of components used in the production line equipment, and contamination causedby human error, etc. IR microscopy can be used to determine the identity of the foreign material contamination to help in pinpointing theirsource.
IR microscopy can also be used to investigate food quality. In food research and development (R&D), there is an interest in evaluating therelationship between the distribution of food components and its effect on flavor and taste. It is expected that IR microscopy, which canvisualize the distribution of components, such as water and fats, will provide important information in food R&D.
This section shows examples of food component distribution analysis using an IR microscope.

Microscopic Transmission Method
Moisture Distribution Analysis of Foods
FTIR spectroscopy is useful for obtaining information about thenature of trace amounts of moisture in food products. Furthermore,
FTIR mapping measurement of a cross-section of a food productcan be used to visualize the moisture distribution in a food product.

CookingDifferences in moisture distribution were analyzed in noodle crosssections of two types of fried noodles, cooked by amateurs andprofessionals, respectively.
In order to compare moisture distribution in the two different noodle
preparations, the noodles were cut to a few mms thickness andsandwiched between CaF2 windows in order to prevent drying,and fast imaging measurements were performed (Fig. 1).

Cut out a section
Window

Cross sectionof noodle

Measurement light

Hold the sample
with windows

Fig. 1 Diagram of the measurement

29

The water content distribution in both noodle samples cooked
by a professional and an amateur, respectively, was comparedby calculating the peak area ratio of the water (1,882 – 2,321 cm-1)
and the starch (3,872 – 4,165 cm-1), and the moisture distribution
was analyzed (Fig. 2). The results show that the amateur-cooked
noodles had the highest moisture content in the center, while theprofessionally cooked noodles had the highest moisture content
in the middle layer (between the outermost shell and the center),rather than in the center. In parallel with the measurement, a sensory
test was conducted with a group of 20 people, and almost all ofthem agreed that the professionally cooked noodles were more
flavorful. It was confirmed that there is a relationship between the
difference in "taste" and the moisture distribution in fried noodles.

Microscopic ATR Method
Wide-Area ATR Measurement of Foods
FTIR imaging measurements can visualize the distribution ofcomponents on a sample surface. In order to evaluate the
distribution of components on the surface of a cheese sample (Fig.
3), an ATR-5000-WG, which is capable of wide-area ATR imaging,
was used.

Amateur

100 µmFig. 3 Observation view
1 mmObservation view

Moisture distribution

Professional

Principal component spectra were calculated by Multivariate Curve
Resolution (MCR) analysis (Fig. 4), and a false-color chemical image
showing the distribution of principal components was createdusing the peak areas of the key absorption bands (Fig. 5). As aresult of the analysis, fats and proteins were detected as the main
components, and their distribution in the surface was determined
and visualized.

Abs.

Main component: fats

Main component: proteins

1 mmObservation view

Moisture distribution

Fig. 2 Moisture distribution of fried noodles by different cooks
4000

3000

2000

800

-1

Wavenumber [cm ]Fig. 4 Principal component spectra(red: fats, green: proteins)

100 µmFig. 5 False-color chemical image(red: fats, green: proteins)

30

Tips
Protein Secondary Structure Analysis
Many protein structures have been confirmed using
x-ray crystallography, especially since the humangenome has been sequenced and elucidated.
Nuclear Magnetic Resonance (NMR) spectroscopy
also provides excellent structural information aboutproteins and Circular Dichroism (CD) spectroscopy
is an extremely useful technique for aqueous andsolid phase samples. However, it is very difficult toanalyze the secondary structure of a protein in a
multi-component system with a structure like that ofa biological tissue using the above methods while
maintaining the structural integrity. In contrast, IR
spectroscopy can be easily used even for noncrystalline samples, and thus the secondary structureof proteins in biological tissues can be analyzed in amore in-vivo-like state without purification.

IR microscopy can also be used for in-situ surface
analysis of samples with non-uniform structural
distribution. With the Secondary Structure Imaging(SSI) program, spatial analysis can be performed nondestructively on biological samples. In the example
described here, a hair sample was prepared using
the KBr plate method, and an FTIR transmission
measurement was performed (Fig. 7). The FTIR image
confirmed that the percentage of -CH was higher
in the center (medulla) and on the surface (cuticle)of the hair. Focusing on the secondary structure of
the protein, it was confirmed that the percentage of
α-helix was higher in the center (medulla) of the hair.
0.7

JASCO's protein Secondary Structure Estimation(SSE) program can quickly perform protein secondary
structure analysis using the amide I peak in an IR
spectrum.
30 µm

30 µm
0.2

Observation view

IR imaging (CH2)
20

40

30 µm

30 µm
0

SSE imaging (α-Helix)

Fig. 7 Cross-sectional analysis of hairFig. 6 SSE calibration modeling program

In conventional secondary structure analysis ofproteins using IR spectroscopy, a heavy water solution(D2O, deuterium) is often used to minimize the effect of
the water (H2O) peak, which coincides with the amide
I peak. However, D2O is more expensive and moredifficult to handle than ordinary H2O. To compensate
for the effects of water in the amide I band, the SSE
program in Spectra Manager automatically subtracts
the H2O peak from the spectrum so that protein
secondary structure analysis can be performed using
ordinary aqueous solutions.
Conventional secondary structure analysis has
been performed from the amide I peak using band
decomposition, which requires a certain amount ofexperience and can be user subjective. In the SpectraManager SSE program, the protein secondary
structure is estimated by Principal Component
Regression (PCR) or Partial Least Squares (PLS) usinga reference protein dataset with known secondary
structure (confirmed by x-ray crystallography). The
use of multivariate analysis minimizes the need forexperience in data processing and removes the
subjectivity involved with the band decomposition
method initially described.

25

SSE imaging (β-Sheet)This technology is now being used to analyze
pathological tissue sections (e.g., cancer andatherosclerosis)1) and food products (e.g., rice andwheat). Secondary structure analysis using FTIR
microscopy is an effective, nondestructive analyticaltechnique that can be applied to a variety of fields.
FTIR microscopy with the IRT- 5000 Series FTIR
microscope together with software tools, such as
SSE and SSI, supports in-depth secondary structure
analysis and distribution imaging.

1) Yamada, T., Miyoshi, N., Ogawa, T., Akao, K., Fukuda
M., Ogasawara, T., Kitagawa Y., Sano K., ClinicalCancer Research., 8, 2010-2014 (2002).
.

31

INFRARED MICROSCOPY APPLICATIONS | LIFE SCIENCE

Applications of IR Microscopy in Pharmaceuticals
and Medicine
Recently, IR microscopy has been attracting attention in the medical field for being able to discriminate between healthy and diseased
tissues, for blood analysis, drug development, analysis, and identification of contaminants in medicines and medical devices.
In the field of pharmaceuticals, FTIR microscopy is widely used as an analytical method for drug development and quality control because
it allows both qualitative and quantitative evaluation of active pharmaceutical ingredients (API) and additives in tablets. Furthermore,
imaging measurements enable visualization of the distribution of API, fillers, and encapsulation, which is important information for tablet
dissolution, drug uptake in patients, and quality assurance.
FTIR microscopy is also effective for measuring biological samples because it requires minimal sample preparation and can be performed
with little or no damage to the sample. FTIR microscopy is used routinely in the medical field to obtain detailed information about thechemical composition of biological samples (proteins, lipids, carbohydrates, DNA, etc.), which is useful in research. Furthermore, imaging
measurements can visualize the distribution of chemical components in biological samples to provide new insights into developing
technologies.
In the following application, we present examples using IR microscopy for drug evaluation and the analysis of diseased tissue.

Microscopic Reflection Analysis
Near-IR Imaging of OTC Drugs (Sedatives)
Near-Infrared (NIR) spectroscopy is widely used for the nondestructive evaluation of foods and pharmaceuticals because thisregion of the spectrum readily transmits through glass containers
and many background absorption peaks in the NIR region are
weaker than those in mid-IR region, which is especially beneficial
for reducing the interference of water. Recently, NIR has been
introduced into the regulatory framework for Process Analytical
Technology (PAT) proposed by the FDA and is mainly used toevaluate the mixing uniformity of samples in sealed vials and using
in-line measurement to evaluate moisture content. NIR microscopy
can also be used to determine and visualize the chemical content
uniformity in pharmaceutical tablets and capsules.
There are a wide variety of measurement methods that can beused in NIR spectroscopy with instruments that include dispersive,

32

filter, acousto-optic tunable filters (AOTF), and Fourier-transform(FT). FT-NIR is typically faster and has superior wavenumber
range, wavenumber accuracy, and throughput compared to theother methods mentioned. Furthermore, NIR imaging provides
nondestructive visualization of components on the surface of tablets
and other materials, making it useful in drug design and quality
control. NIR is also used to help solve problems in manufacturing
processes and to better characterize final products.
This section describes the use of NIR imaging to evaluate the
distribution of components in a tablet. An IR microscope with an
FT-NIR spectrometer was used in reflection measurement mode
for the imaging of a cross-section and the surface area of an OTC
drug (sedative).

From the NIR mapping measurement of the tablet cross-section(Fig. 1), a false-color chemical image was created using peak heights
specific to the unique functional groups for each component (Fig.
2). As a result, it was confirmed that the tablets consist of four
layers of three components. Next, the tablet surface was analyzed,
and the distribution of the components on the surface coating
layer was expressed as a peak height ratio (4,380 cm-1/4,680 cm-1);
typical of acetylsalicylic acid (Fig. 3). As a result, it was confirmedthat the components in the surface coating layer were unevenly
distributed.

Microscopic Transmission Method
Measurement Example of Mouse Eyeball
Cross-Sections
As an example of a biological sample, IR imaging measurements
were performed on cross-sections of a mouse eyeball. The
sections were mounted on BaF2 slides (10 mm diameter, 1 mm thick)and measured in transmission mode (Fig. 4).

Abs.

Surface coat

Antacid
Acetylsalicylic acid
7500

7000

6000
5000
-1
Wavenumber [cm ]4000

Fig. 1 Average spectra of each component(green: surface coat, blue: antacid, red:acetylsalicylic acid)
0.1

Fig. 4 Observation image (bottom) and H&E-stained image (top)

0.15

0.4

1 mm
0

0

0

Fig. 2 NIR false-color chemical image of tablet cross-section(green: surface coat, blue: antacid, red: acetylsalicylic acid)

A false-color chemical image was created using the area ratio of
the amide I peak (~1,640 cm-1) and the C-H stretching vibration
peak (~2,900 cm-1), and false-color secondary structure distribution
images of α-helix and β-sheets in the protein were also created(Fig. 5). As a result, the correspondence between the Hematoxylin
and Eosin (H&E) stain image in each component tissue wasconfirmed. The two layers corresponding to the retina and optic
disc have an abundance of α-helix structure, which indicatespotential for a proliferative diseased state.
By further analysis of diseased samples, it is hoped that a causal
relationship between disease states and protein structure can berevealed.
We would like to express our sincere gratitude to Dr. Miyoshi of Department
of Tumor Pathology in University of Fukui, Japan, for providing the samples
in this evaluation.

1.5

Vitreouschamber

RetinaChoroid
Sclera

1 mm

Optic
disc
300 µm0

Fig. 3 NIR false-color chemical image of table surface coating
layer (peak ratio 4,380 cm-1/4,680 cm-1)

Fig. 5 False-color chemical images(left: peak area ratio, center: α-helix, right: β-sheet)33

INFRARED MICROSCOPY APPLICATIONS | ENVIRONMENTAL SCIENCE

Analysis of Microplastics

Microscopic Transmission Method
Imaging and Particle Size Analysis of Microplastic
Particles
IR microscopy is one of the best methods to investigate andcharacterize types of environmental plastics. Microplastics can bemeasured without complicated sample preparation and chemical
identification can be made with reference to spectral databasesof known compounds. In addition, imaging measurements enable
not only qualitative analysis, but also a more quantitative approach
to chemical composition, as well as particle size and distribution
analysis.
To confirm the validity of the evaluation of microplastics using an
FTIR microscope, a suspension containing five different plastic
polymers (PE: polyethylene, PP: polypropylene, PS: polystyrene,
PET: polyethylene terephthalate, and PVC: polyvinyl chloride) was
measured. The samples were collected on a polytetrafluoroethylene(PTFE) filter, and then mapping imaging measurements were madeof the entire filter (Fig. 2).

Absorption saturation by PTFE
PS
PE
Abs

Here we present examples of microplastic evaluation using IR
spectroscopy.

Using the average spectrum of each component obtained fromthe imaging measurements (Fig. 1), a false-color chemical image
was created using the peak heights indicated by the arrow ineach spectrum (Fig. 3). By creating a false-color chemical image,it is possible to visualize the distribution of each of the five plastic
polymers.

PP
PVC
PET

4000

3000
2000
Wavenumber [cm-1]

1000

PS
PE
Abs

There is a growing and alarming concern about the effectsof microscopic plastics (microplastics) on the environment.
Microplastics were first identified as contamination in the marine
environment and food chain, but then developed to land-based
ecosystems and the entire food chain, which has led to a long
lasting and potentially devastating impact on the health of all
animal life on the planet, including human health. The source andnature of microplastics is starting to be well understood, but asignificant amount of research is still being done into the natureof microplastics, their distribution in the environment, and how todeal with them.

PP
PVC
PET
1500

Wavenumber [cm-1]

1000

Fig. 1 Average spectrum of each component

34

650

Tips
JASCO Particle Analysis
The JASCO Particle Analysis (JPA) program is an addin to the Spectra Manager Micro Imaging Analysis
program, which allows the user to perform particle
size analysis and statistical processing of sample
data. The program automatically binarizes observedor false-color chemical images and performs particle
size analysis using parameters such as sample size,
area, perimeter, horizontal and vertical Feret diameter,
aspect ratio, circularity, and color. In addition,
statistical processing functions can be used to display
histograms (stacked histograms for each component
are also available), frequency distribution, correlation
distribution, and component presence ratios.

100 µmFig. 2 Observation view

139

100

100

Y [µm]

Y [µm]

139

50

50

100 µm

0

0

50

100

0

134

0

50

Fig. 3 False-color chemical image(pink: PS, aqua: PE, blue: PP, yellow: PVC, red: PET)

100

134

X [µm]

X [µm]

False-color chemical
Image

Observation view

Furthermore, as an example of particle size analysis, a stacked
histogram of the relevant particle size dimensions and frequency
for each component was created (Fig. 4). The JASCO Particle
Analysis (JPA) program allows the analysis of particle size using
both observation images and false-color chemical images.
20

8
2
2
0
1

10

5

0

2
2
0
0
2

3
2
0
0
0

0
1
1
1
2

0
1
1
0
0

Binarization by component
0
1
0
0
1

0
0
0
0
1

0
1
0
0
0

0
0
1
0
0

20

15
0

10

20

30

40

50

60

70

80

90

100

110

120

Size [µm]Fig. 4 Stacked histograms by component (X-axis: particle size)(pink: PS, aqua: PE, blue: PP, yellow: PVC, red: PET)

Count

Count

15

10

5

IR microscopy allows the identification of the type of plastic present,
and by creating a histogram from the results can illustrate trends
in sample data.

0

0

4

8

12

16

20

Size [µm]

Stacked histogram view
We would like to express our sincere gratitude to TOSOH Analysis andResearch Center Co.,Ltd., Japan, for providing this data.

Fig. 5 Overview of JASCO Particle Analysis Program

35

Tips
IQ IR NAV Microscopy Imaging Analysis
JASCO's IQ IR NAV* makes it easy to analyze
microscopic foreign materials and particulates. These
functions are accessed from the Spectra Manager
software through the Microscope Measurement
program.
*IQ IR NAV can only be used with single-element
detectors.

The following additional functions can be usedin combination with the optional add-in software
Advanced Search NAV (ASN).
Reﬁning of the Measurement Target The target
measurement positions can be detected and filteredusing parameters such as sample size, shape,
circularity, contrast, and color.

Observation Optimization IQ IR NAV consists of thefollowing two functions:
Focus NAV**
correction.

executes

auto-focus

and

auto-

**Focus NAV is only available when using the auto
XYZ stage.
View NAV performs automatic brightness adjustment
according to preset conditions with a single click.

Fig. 8 Refining of the measurement target

Fig. 6 Observation optimization

Search NAV Using Search NAV, measurement points
can be automatically detected in real-time using animage recognition algorithm; the locations can thenbe registered and reviewed in a list of measurement
points prior to acquiring data.

Real-Time Analysis Result Display Using Advanced
Spectra Search (ADSS), library search or spectralclassification*** of the target measurement points
can be performed automatically after sample
measurement, and the results can be displayed as acompound name overlaid on top of the measurement
position in the observation image.
*** See page 37 for more details.

Fig. 9 Display of analysis results
Fig. 7 Real-time sample recognition
and registration of measurement points

36

Tips
Advanced Spectra Search (ADSS)
When performing qualitative analysis of an unknown
sample from its FTIR spectrum, identification can bemade using a library search against a database of
known reference spectra. However, a library search
will often output multiple spectra with similar features,and the final selection has to be made by the analyst.
In order to assist the user in making the correct
judgment, knowledge of spectral interpretation
is beneficial, which can be a major obstacle forinexperienced analysts.
JASCO's Advanced Spectra Search (ADSS) program,which incorporates the “interpretation of a skilled
analyst” into the software using machine learning,
can overcome the above complications of spectralinterpretation. The ADSS program has two functions:"Classification" and "Search".

"Classiﬁcation" Function Based on the machinelearning results of approximately 10,000 spectra, theclassification function categorizes a spectrum of an
unknown sample into one of 35 different types ofchemical compounds (Table 1). Since this classification
function has been trained mainly on spectra of foreignmaterials and polymers, it is particularly powerful
for these analyses. Furthermore, since the training
includes characteristics that depend on the difference
in measurement methods and sample preparation,
the acquired spectra can be used without any
additional data processing. In addition to the factthat the type of substance can be estimated from theclassified categories, key absorption bands unique toeach compound and their potential functional groups
are displayed in the spectrum, making it possible tovisually confirm the validity of the results.
Table 1 Classification categories
AcetoneAcrylic resins

Alcohol

Carbonates

Carboxylic acids

Carboxylic acid esters

Carboxylic acid esters

Carboxylic acid salts

Cellulose and sugar(oil)Fig. 10 Advanced Spectra Search
programEpoxy resins

Fluorides

Hydrocarbons

Hydrocarbons

Hydrocarbons

Nitriles(polyethylenes)(polypropylenes)

Phenolic resins

Phosphates

Polyamides

Polycarbonates

Polyethers

Polyethers (polyacetal)

Polyesters

Polyimides

Polyvinyl acetates

Polyvinyl alcohol

Polyvinyl chlorides

Proteins

SilicaSilica (talc)

Silica (kaolin)

Silicone

StyreneSulfates

Urethanes

Water"Search" Function This function performs a library
search. A correlation of similarity is made (scored)
using a selected search algorithm, and the search
results are reported in descending order of the score.
A database library of more than 600 registeredspectra, mainly of foreign materials and polymers, isavailable as standard, but it is also possible to build
custom user-libraries.
In addition, using the dedicated microplastics library,"IR Plastics Library", with 153 spectra of 39 registeredspecies, qualitative analysis specific to plastics andpolymers can be performed.

37

Combined Microscopic Analysis
IR and Raman Study of Plastic Particles
A mixed sample of plastic particles was sprayed onto a substrate
and the distribution of components (Fig. 12) was measured using
both IR and Raman microscopy. Analysis using two complementary
methods, in this case, IR and Raman spectroscopy, can provide
more information about a sample with increased confidence in the
results, compared to one method in isolation. When combining
IR and Raman microscopy, information from the two methods canbe obtained more accurately by utilizing the IQ Frame (Fig. 11,
see page 16), which provides accurate and precise measurement
positioning as the sample is transferred between the FTIR and
Raman microscopes.

Measurement by IR Microscopy
Principal component spectra were calculated using multivariate
analysis (MCR model analysis), and a false-color chemical image
was created using the principal component spectrum scores (Fig.
13). FTIR analysis revealed the presence of proteins, in addition
to four types of plastics: polyethylene terephthalate (PET),
polyethylene (PE), polypropylene (PP), and polystyrene (PS).
ProteinPPPS

IR microscope

30 µm
PET

False-color chemical image (Score of Principal Component Spectra)PE

IQ Frame
PET (green)

Raman microscopeAbs.

PE (red)PP (blue)

PS (aqua)Fig. 11 IQ Frame operation between Raman and IR microscopes

The distribution of components in a sample of sprayed plastic
particles (Fig. 12) was measured using both IR and Raman
microscopy.

Protein (yellow)

4000

3000

2000
Wavenumber [cm-1]

Principal component spectra
Fig. 13 Measurement results from IR microscopy

30 µm

Fig. 12 Observation view

38

700

Measurement by Raman Microscopy
Raman imaging measurements were performed at exactly thesame locations as were measured with the FTIR microscope. Eachprincipal component spectrum was calculated using MCR model
analysis, and a false-color chemical image was created using thepeak heights of the key absorption bands (Fig. 14). The analysis
revealed the presence of carbon, in addition to the same fourtypes of plastics: PET, PE, PP, and PS.
CarbonPS

PP

Using the false-color chemical images derived from the IR and
Raman measurements, it was found that combining the results
revealed the presence of a total of six components, while each of
the methods alone could only detect five components.
In order to visualize the results of this analysis, the falsecolor chemical images from the Raman measurement weresuperimposed on the false-color chemical images from the FTIR
measurement (Fig. 15). The combined false-color chemical image,included carbon, which was detected by Raman spectroscopy, in
addition to the five components (PET, PE, PP, PS, and protein) thatwere detected by IR spectroscopy, demonstrates the advantage ofcombining the different attributes of IR and Raman spectroscopy,
coupled with the benefits of IQ Mapping to be able to synchronize
these measurements.
Thus, the use of IR and Raman spectroscopy together enables
complementary analysis and increases the amount of information
obtained from a single sample, thereby improving the scope,
accuracy, and confidence in the analysis.

Protein

Carbon
PP

PS

30 µm
PET

PE

False-color chemical image

PET (green)

PE (red)

30 µm
PET

Int.

PP (blue)PE

Fig. 15 Superimposition of false-color chemical images obtained
from IR and Raman microscopes

PS (aqua)

Carbon (white)

3500

2000

300

Raman shift [cm-1]

Principal component spectra
Fig. 14 Measurement results from Raman microscopy

In addition to the four plastics, IR microscopy was able to detect
protein, whereas Raman microscopy could not identify the protein
but was able to measure carbon.

39

JASCO CORPORATION

2967-5, Ishikawa-machi, Hachioji-shi, Tokyo 192-8537 Japan
Tel: +81-42-649-5177 Fax:+81-42-646-4515 Web: www.jasco.co.jp
Japan

JASCO INTERNATIONAL CO., LTD.

11-10, Myojin-cho 1-chome, Hachioji-shi, Tokyo 192-0046, Japan
Tel: +81-42-649-3247 Fax: +81-42-649-3518 Web: www.jascoint.co.jp/english/
Australia, Hong Kong, India, Indonesia, Korea, Malaysia, New Zealand, Pakistan,Philippines, Russia and CIS countries, Singapore, Taiwan, Thailand, Vietnam

JASCO INCORPORATED

28600 Mary’ s Court, Easton, Maryland 21601, U.S.A.
Tel: +1-410-822-1220 Fax: +1-410-822-7526 Web: www.jascoinc.com
Argentina, Bolivia, Brazil, Canada, Chile, Colombia, Costa Rica, Guatemala, Mexico,
Paraguay, Peru, Puerto Rico, United States of America, Uruguay, Venezuela

JASCO EUROPE S.R.L.

Via Luigi Cadorna 1, 23894 Cremella (LC), Italy
Tel: +39-039-9215811 Fax: +39-039-9215835 Web: www.jascoeurope.com
JASCO Deutschland www.jasco.de | JASCO UK www.jasco.co.uk | JASCO France www.jascofrance.fr
JASCO Benelux www.jasco.nl | JASCO Spain www.jasco-spain.com
Algeria, Austria, Belgium, Cyprus, Denmark, Egypt, Finland, France, Germany, Greece, Hungary,
Israel, Italy, Jordan, Kuwait, Luxembourg, Morocco, Netherlands, Norway, Poland, Portugal, Romania,
Saudi Arabia, South Africa, Spain, Sweden Switzerland, Tunisia, Turkey,
United Arab Emirates, United Kingdom, Yemen
ISO-9 0 0 1

JASCO CHINA (SHANGHAI) CO., LTD.

Room No.D, 10F, World Plaza, 855 Pudong South Road, Pudong New Area, Shanghai, China
Tel: +86-21-6888-7871 Fax: +86-21-6888-7879 Web: www.jasco-global.com
China

ISO-14001

CERTIFIED

Products described herein are
designed and manufactured by
ISO-9001 and ISO-14001 certified
JASCO Corporation
e6708-2404ENG

The contents of this brochure are for reference and illustrative purposes only. Information, descriptions, and specifications in thispublication are subject to change without notice. JASCO assumes no responsibility and will not be liable for any errors or omissions
contained herein or for incidental, consequential damages or losses in connection with the furnishing, performance or use of this material.