Macromol. Mater. Eng.
2001, 286, 663–668
Full Paper: Confocal microscopy is increasingly becom-
ing recognized as a valuable analytical tool in material
science although long established in life sciences. It is
shown that this technique can be employed successfully in
investigating polymers, coatings, emulsions, etc. Using
the scattered light not only for imaging but also for analy-
sis in a Raman spectrometer chemical information from
an exactly defined volume smaller than 1 lm3 within the
sample can be achieved.
Confocal Microscopy: Applications in Materials
Science
Werner Hoheisel,* Wolfgang Jacobsen, Bernhard Lüttge, Wolfgang Weiner
Bayer AG, Central Research, 51368 Leverkusen, Germany
Introduction
Confocal microscopy is an optical imaging technique
based on laser scanning which according to the confocal
principle of the optical arrangement leads to 3-dimen-
sionally resolved images of the specimen under investiga-
tion: When the focus of the optical system is positioned
into the interior of the sample the system produces an
image of exclusively the focal plane. This is due to a pin-
hole positioned in front of the detection system which
rejects all light that originates from the outside of the
detection volume. This volume is defined by the focal
plane and the shallow field of depth being between 0.5
and 1.5 lm depending on the applied objective. This so
called optical sectioning behavior is unique for the confo-
cal light microscope and has so far not been realized with
other imaging systems like scanning electron micro-
scopes.
The contrast mechanisms conventionally used are
based on reflection (ordinary Fresnel reflection as well as
generation of scattered light due to local variations of the
refractive index) and fluorescence. Generating the con-
Macromol. Mater. Eng. 2001, 286, No. 11 i WILEY-VCH Verlag GmbH, D-69451 Weinheim 2001
663
trast by Raman scattering is also a valuable tool, carrying
chemical information about the material under investiga-
tion.[1] Contrast generation by fluorescence lifetime is
possible but is less used due to the required advanced
experimental setup. Absorption does not contribute to a
confocal contrast. Readers interested in further details of
the confocal principle and its instrumentation are referred
to the extensive literature.[2]
The application of confocal microscopy started more
than 15 years ago in the quality control business of the
silicon chip industry but was later on more and more
dominated by biological application. Here it was common
to use fluorescent dyes as selective markers for proteins,
specific parts of cells and tissues. Hence, one could use
confocal microscopy by minor changes of the experimen-
tal techniques but with the additional advantage of an
enormous improvement in image quality and resolution
due to the possibility of the optical sectioning.
Confocal microscopy is also used more and more in
materials science. The underlying principle is well
adapted to the investigation of polymers, ceramics, wood,
1438-7492/2001/1111–0663$17.50+.50/0
664
bones, teeth and any other solid or liquid material show-
ing a moderate intrinsic absorption so that light can pene-
trate the respective material at least some tens of a
micron. Running the microscope in the reflection mode,
the inner morphology of a specimen can be visualized by
moving the focus plane within the detectable sample
volume. Using the fluorescence contrast appeared to be
less feasible for this type of materials since they usually
do not show a significant inherent fluorescence. But in
specific cases even this type of contrast may be useful.
Staining with a fluorescent dye is, on the other side, not
selective regarding the different morphological structure
elements of interest.
Surprisingly only few reports about the application of
confocal microscopy to materials science problems are
found in the literature.[3– 6] The annual international con-
ference on 3-dimensional microscopy (cf., for example,
list of participants in ref.[7]) is still dominated by scientists
investigating biological samples and by only a small
group of materials science participants. This paper gives
three representative examples of our activities in confocal
microscopy applied to material science problems and
focus to instrumental aspects rather than to scientific
background.
Experimental and Instrumental Details
We started using confocal microscopy in our lab in the cen-
tral research division of the BAYER AG in Leverkusen/Ger-
many in 1989 with a Wild-Leitz confocal laser scanning
microscope type commercial microscope equipped with an
Argon ion laser (488/514 nm) and a Helium-Neon-Laser
(632 nm). Since 1997 a Leica Lasertechnik TCS/NT type
microscope is used, again equipped with an argon ion and a
helium-neon laser but now additionally with an UV-argon
ion laser (354/362 nm). This microscope has a significantly
better spatial resolution and many new features. The TCS/
NT has been upgraded by adding a Dilor LabRam type
Raman spectrometer via an optical multimode fiber as a cou-
pling unit. The spectrometer is equipped with a 1 800 lines
grid and a Peltier element cooled CCD camera. The wave-
length resolution is about 0.05 nm and the noise about 3
counts/min. The spatial resolution is better than that of a con-
ventional Fourier transform-Raman microscope.[8] It is
experimentally verified to be in the range of 1 lm in the hor-
izontal direction and only slightly more in the vertical direc-
tion for the objective “63 6 1.32 oil”. The confocal micro-
scope/Raman spectrometer combination is usually run as fol-
lows:
First, the sample is investigated with the microscope with-
out using the spectrometer. Second, a point of interest is set
within the image reproduced on the screen and the laser illu-
mination is started again, but now in the point scan mode
meaning that the laser focus is fixed to the point of interest
marked before. Simultaneously, the Raman spectrometer
detects the signal which is backscattered from the sample.
The time period for detection is typically in the range of 1 to
W. Hoheisel, W. Jacobsen, B. Lüttge, W. Weiner
10 min depending on the chemical composition of the sam-
ple and on the detection volume of interest. The described
equipment offers the possibility to achieve chemical infor-
mation about the samples with a similar spatial resolution as
given in the confocal images. Further instrumental details
will be published elsewhere.
In the following section three typical examples are pointed
out to underline that a confocal microscope is a very useful
tool for industrial materials science research.
Results
Polymers are samples which are well suited for the inves-
tigation with a confocal microscope: They all are translu-
cent in thin layers unless they are filled with strongly
absorbing (e. g. black carbon) or scattering (e. g. TiO2)
particles. According to this, the first example demon-
strates possibilities and limits for the use of the micro-
scope in polymer science. Since already small differences
of the index of reflection generate a significant contrast,
it is possible not just to reproduce polymer blends show-
ing phase separations or semicrystalline polymers show-
ing spherulites but also to visualize inclusions or particles
within a polymer easily. As an example, Figure 1 a pre-
sents an image of acrylonitrile-butadiene-styrene (ABS)
being a 2-component thermoplastic graft polymer con-
taining polybutadiene (rubber) particles in a continuous
phase of styrene-acrylonitrile (SAN) copolymer. A com-
mercially important type of this polymer contains many
inclusions of SAN within the rubber particles. The chemi-
cal and morphological structure of this product is well
known and described elsewhere.[9 –11]
The specific type of the investigated sample contains
relatively large rubber particles with an average diameter
of about 5 lm containing a large number of SAN inclu-
sions with a size of some 400 nm. The transmission elec-
tron microscope image shows clearly the well known
morphology of the ABS polymer. The image taken by the
confocal microscope being used in reflection contrast
with a 63 6 1.32 oil objective indicates small bright
spheres being just beyond the resolution limit of the con-
focal microscope in the reflection contrast image. They
depict the SAN inclusions and are concentrated in clus-
ters with a size which reflects very well the size of the
big rubber particles. This interpretation is verified by
using the Raman spectroscopic facility. Figure 1 b shows
a spectrum of a test volume taken in the region with high
reflection contrast (bright area) as well as in the region
with low reflection contrast (dark area). As a reference,
the Raman spectra of pure SAN and of pure polybuta-
diene were measured with the same equipment (curve (3)
and (4) in Figure 1 b). The interpretation of the spectra is
straight forward (acrylonitrile band at 2 250 cm–1 and the
butadiene band at 1 680 cm–1) and clearly supports the
explanation given above: The SAN matrix can be identi-
fied to appear as the dark areas of the confocal reflection
Confocal Microscopy: Applications in Materials Science 665

Figure 1. (a) Confocal microscopic image of acrylonitrile-

butadiene-styrene (ABS). The polybutadiene phase contains
many inclusions of SAN as is clearly recognizable in the elec-
tron microscopic image which is displayed in the same scale as
the confocal image. (b) Raman spectra taken from the sample
displayed in Figure 1a. Spectrum (1) and (2) is taken from the
dark and bright area, respectively. The spectra (3) and (4) taken
from reference materials demonstrate that the dark area corre-
sponds to pure SAN whereas the bright area represents the
grafted SAN. Figure 2. Confocal microscopic image of (a) a polybutadiene/
polyamide blend reinforced by short glass fibers, and (b) the
same sample after the application of a thermo-mechanical stress
image whereas the bright regions contain both SAN and
treatment.
butadiene. This is consistent with the morphology of the
rubber particles shown in the electron micrograph of Fig-
ure 1 a. It demonstrates the reliability of the instrument material was investigated by applying various types of
which might be used for identifying inner morphologies test methods (including application of thermo-mechanical
and chemical compositions of polymers, polymer blends stress) followed by microscopic analysis including confo-
or coatings in an unbeatable fast and cost effective way. cal microscopy.[12] Using the reflection contrast of the
Not only the interior morphology of a polymeric mate- undamaged material (Figure 2 a), the polyamide itself
rial can conveniently be investigated but also changes of generates some reflection intensity due to its partial crys-
a polymer or a composite due to an applied mechanical talline structure. However, the typical dimension of the
stress. This is demonstrated in the next example. The spe- crystalline and amorphous regions are close to or even
cimen is a polybutadiene/polyamide blend reinforced by below the resolution limit of the microscope generating a
30 wt.-% (ca. 15 vol.-%) of short glass fibers with a mean semi-dark background. The bright spots in the image are
length of about 250 lm and a diameter of about 11 lm formed by the polybutadiene particles. The dark elon-
(BAYER AG, Leverkusen). The fatigue behavior of this gated areas correspond to parts of glass fibers whose
666 W. Hoheisel, W. Jacobsen, B. Lüttge, W. Weiner

orientations are randomly distributed in all dimensions.

Considering that the confocal microscope exclusively
detects objects in the focal plane the measured length of a
dark stripe, unlike the width, usually does not correspond
to the true length of a fiber but the width does. In Figure
2 b a corresponding section of a sample is depicted that
has been exposed to a bending and shear stress for 375
cycles. In the local area around a fiber the main linear
stress induced by this mechanical treatment is probably
oriented almost parallel to the fiber. Therefore the planes
of maximum shear stress in the matrix material are
oriented at an angle of 45 8 to the fiber axis. And precisely
in this orientation a sequence of lines is formed by poly-
butadiene particles with enhanced brightness in the
reflection image. This gives a strong reference to the nat-
ure of the inelastic damage process induced in this sam-
ple, namely an at least partly destructive change of the
rubber particle/thermoplastic matrix interfaces which
enhances the discontinuities of the refractive index in this
layer. Surprisingly, these destructive changes are not
homogeneously distributed over the fiber but concentrate
on cone-shaped planes around the fiber with a mean dis-
tance of about 2 lm. Mechanical stress may also lead to a
detachment of the polymer from the fiber. Consequently,
the refractive index variations at the interface increases,
resulting in a brighter contour of the fibers. This phenom-
ena can be recognized in Figure 2 b as well.
Another well suited object for the confocal microscope
being important for research laboratories in the chemical
industry is paper. Paper mainly consists of cellulose
fibers, pigments (e. g. kaolinite, TiO2), retention aids and
sizing agents (e.g. starch, latices) to control the printabil-
ity of the paper. More valuable “premium papers”
designed for ink jet applications have an additional coat-
ing of porous silicon oxide or pigmented polymer on at
least one side of the paper sheet.[13] Since paper is a
strongly light scattering material the penetration depth of
light is limited. But the top most 15 or 20 lm are accessi-
ble and this is precisely the range where ink jet dyes Figure 3. Confocal microscopic image of a conventional paper
penetrate into the paper during the print processes. Wet- used for copying and printing (Agfa 701) after printing with an
ting processes of the fibers, weathering dynamics of the inkjet magenta ink. Green color represents the reflection contrast
dyes or phase separation processes can be very easily and and red the fluorescence contrast. (a): xz-section, the inset shows
the same section with higher magnification; (b) xy-section 10
quickly studied due to the advantage of the optical sec-
lm below the paper surface.
tioning. Many inks are slightly fluorescing whereas the
ingredients of the paper fluoresce much less or even not shows the corresponding xy-section lying 10 lm below
at all. Consequently, an image of an individual section the surface. The fibers are clearly visible. They are
can be taken in reflection contrast as well as in fluores- obviously just wetted but not penetrated by the ink as can
cence contrast. Both images are overlaid and displayed in be seen by the much stronger fluorescing surfaces com-
false colors. In Figure 3 a a xz-section of a conventional pared to the interior of the fibers. The filler materials
paper used for copying and printing (Agfa 701) is (kaolinite, TiO2) reflect the incoming light better and
depicted which is printed with a magenta ink (Hewlett- appear as green or yellow (overlay of red and green)
Packard, C1823D) from a HP Deskjet 895CXI. The pene- spots.
tration depth of the incoming laser light is about 15 lm Using the same magenta ink which is now printed on a
which is almost the penetration depth of the printed ink premium paper (Hewlett-Packard Premium Inkjet Paper),
which fluorescence is represented in red color. Figure 3 b the differences to the ordinary paper are clearly visible.
Confocal Microscopy: Applications in Materials Science
Figure 4. Confocal microscopic image of a premium paper
used for inkjet applications (Hewlett-Packard) after printing
with an inkjet magenta ink. Green color represents the reflection
contrast and red the fluorescence contrast. (a): xz-section, the
inset shows the same section with higher magnification; (b) xy-
section 10 lm below the paper surface.
This paper is coated with a layer of porous silicon oxide
particles with a size of less than 10 lm. These particles
are covered and filled with ink and, consequently, they
absorb or scatter incoming light of specific wavelengths
very efficiently. The color of a printed premium paper
appears much brighter than for a conventional paper. In
Figure 4 a a xz-section of the partially printed paper is
depicted. The printed parts of the paper can easily be
identified by the corresponding particles which appear
red due to the ink (Objective: 63 6 1.32 oil). On the right
hand side of the image the undyed particles appear pale
667
green revealing that the visible contrast is exclusively due
to reflection. Independent of the position of sectioning
within the dyed particles they appear uniformly red
meaning that the particles became fully saturated with ink
confirming the high porosity of the particles. The fibers
lying below the particles are more difficult to identify for
three reasons: First, the penetration depth of light is lim-
ited in this strongly light scattering system. Second, due
to the bright fluorescence of the upper lying particles the
amplification factors for the light detectors have to be
appropriately reduced in order to prevent an overamplifi-
cation which would occur by adjusting the gain to the
weak fluorescence of the fibers. Third, this fluorescence
light is weakened again by its passage through the parti-
cles to be detected in the microscope. Optical sectioning
in xy-direction does not face this problem since the light
detectors can be more easily adjusted to the rather equally
distributed amount of light coming through the layer
above the focal plane. Figure 4 b shows a xy-section of a
dyed paper area 10 lm below the paper surface. This is
roughly both the penetration depth of the ink and the
interface between the particles and the fibers. Both can be
clearly recognized. In contrast to the particles some fibers
appear green and others red depending whether they are
dyed or not. Again, the fluorescence of the dyed fibers is
more intense on the surface indicating that the ink does
hardly penetrate into the fibers. Similar to Figure 3 the
yellow/green spots display the filler materials and their
distribution, respectively.
In conclusion, the confocal microscope proves to be
well adapted to problems in materials science for indus-
trial as well as for academic research. Many objects of
interest are sufficient translucent and within the size
accessible to the microscope. Using a suited combination
of reflection contrast, fluorescence contrast and Raman
spectroscopy valuable information can be gained very
quickly due to the minimized effort in sample prepara-
tion. Serial investigation of samples of one type may be
easily performed. Further technical developments in the
field of confocal microscopy potentially usable for mate-
rials science problems may for example focus on the
investigation of polarization properties of specific parts
of a sample,[14] two photon microscopy to push the limits
in spatial resolution and light penetration[15] and coherent
anti-Stokes Raman scattering (CARS) microscopy to
include the advantage of using a contrast mechanism
based on vibrational spectroscopic features.[16] This
ensures the ongoing establishment of the confocal micro-
scope in materials science.
Received: March 7, 2001
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