The scientific and research laboratories
are using instruments to study and investigate textile fibres, but none has
been more historically indispensable than the
microscope. Today, the number of different types of microscope of potential
use to the textile technologist is greater than ever before and several
fundamental new types of instrument have appeared in recent years.
the potential range of techniques available is so wide that it seems capital
expenditure is the only restriction when selecting a method of examination
and imaging for any specimen. Some gauge of the complexity of microscopy
today may be gleaned by sampling the plethora of acronyms which describes
the sciences currently in use.
intended to encourage the fibre or textile scientist to explore the
facilities and technologies that are available today and it is hoped to
discover what hidden information may be present in his materials, simply
awaiting release by the correct examination technique.
in Microscopy and Imaging
‘Conventional’ Light Microscope
the external appearance of modern research microscopes differs considerably
from that of their equivalents of some years ago, the basic optical
principles of operation remain unchanged. The main reason for the increase
in size and complexity of microscope stands today is the trend towards
making instruments multi-purpose. It is now possible, for example, to
produce images with polarised light, fluorescence, dark ground, phase
contrast and differential interference contrast, using the same microscope.
the different imaging modes may be used simultaneously to provide additional
information about a specimen. Combining interference contrast with incident
fluorescence excitation, for example, would allow variations in intensity of
emitted fluorescence to be related to differences in refractive index or
thickness of the specimen. The second major development in the ‘Conventional’
compound light microscope is not confined to this type of instrument and is
part of a general advance in image recording.
While it may be
said that image formation in the conventional light microscope has remained
basically unchanged, the later 1980s have seen the commercial introduction
of a fundamentally new type of light microscope: The confocal scanning Light
Microscope has been called the most significant optical advance of the
decade and has received so much attention that it has almost overshadowed
all other forms of light microscopy.
key feature of the confocal microscope is that, instead of the whole field
being illuminated and magnified as a complete area, the specimen is scanned
by a finely focused beam, either singly or as a simultaneous array of beams.
The illuminating apertures are in common focus, any light coming from above
or below the illuminating focal plane is effectively de-focused when it
reaches the imaging focal plane, and thus does not contribute to the
confocal image (other than by a negligible amount). This results in an image
being formed of a single optical section of the specimen which is
effectively free from out-of-focus blur.
are two types of confocal microscope in use today and these are described
Confocal Laser Scanning
basis for the CLSM was first published as long ago as 1977. and, although a
monograph on the subject appeared in 1984, it was no until late 1987 that
the instrument was launched commercially by Bio-Rad Lasersharp of Abingdon,
UK. In principle, this instrument produces a magnified image by scanning the
specimen with a diffraction limited spot of laser light and collecting the
resultant reflected/fluorescent light with a light detector and
Figure 2.2.1 Schematic diagram of
the confocal laser scanning microscope The instantaneous responses
of the light detector at each focus illuminated volume element (voxel) of
the specimen are displayed with equivalent spatial position and relative
intensity on the simultaneously scanned and modulated screen of a monitor.
Thus the image is, in fact, built up point by point and is formed into a
complete image by the TV system. Magnification is achieved by making the
area of the specimen scanned small in relation to the size of the monitor.
The CLSM is thus optically analogous to the SEM.
date most application of CLS have been biological or geological and the
three main imaging modes used in those areas effectively describes the
capabilities of the laser scanning confocal instrument.
resolution sectional imaging:
by making the confocal apertures sufficiently small and by rejecting
out-of-focus blur, both the lateral (x, y) and depth (z) resolutions of the
CLSM can be increased significantly over conventional systems.
using the extensive computational abilities of confocal systems, it is
possible to create a four-dimensional (x,y,z,t) sequence of time lapse
images sowing the same space recorded over a period of minutes. This latter
technique is particularly applicable to live specimens.
Scanning Reflected Light Microscope (TSRLM)
The second type of confocal microscope uses exactly the same principles
of aperture confocality as the CLSM but differs in its illumination source.
The tandem-scanning microscope has theoretical origins even older than those
of the laser systems but it was not until the late 1980s that fully
commercial instruments became available.
the TSRLM the imaging radiation is not a laser but a conventional lamp
either filament or mercury vapor. Scanning and confocality are both achieved
by the same means a spinning disc of pinholes arranged in Archimedian
spirals so that for each of the many thousands of pinholes arranged in
Archimedian spirals so that for each of the many thousands of pinholes
transmitting illuminating incident light there is an equal and exact
opposite accepting reflected imaging light. This disc-the Nipkow disc spins
at a speed of 1,200r/min so that the spirals of confocal pinholes scan the
whole field of view and produce a complete image of the optical section in
focus without the digital image storage necessities of the CLSM.
2.2.2 Schematic diagram of the Tandem Scanning Microscope
advantage claimed for the Nipkow disc system of confocal microscope is that
the specimen is being scanned in parallel rather than is series as with the
laser systems and that as such TSRLM offers the only true real time imaging
confocal microscope system.
The essential components of this type of microscope are shown schematically
in the Fgure.
Uses of the TSRLM
of the applications of the TSRLM have been biological and its real-time
imaging feature has been particularly useful in this respect for the study
of live specimens. There have also, however, seemingly more than with the
CLSM, been many ‘Materials’ applications. These have tended to exploit
the optical sectioning capacity of the instrument more than its real-time
image formation, though the latter is described as particularly useful in
allowing rapid location of the area of specimen to be examined.
Among the reported applications have been the following.
measurement of height variations, to a resolution of 0.02µm over a depth of
This has clear advantages over the conventional light microscope and
also over the SEM as no coating is required for insulating materials and
precise quantitative height data are obtained.
examination of surface topographies, non-destructively:
Images of opaque materials may be built up with successive confocal
scans and a three-dimensional image of the detailed surface topography can
be presented by the image processing computer. Such images are capable of
being colour or intensity coded with contours if required to portray the
specimen’s surface most clearly.
time non-invasive sectioning of translucent materials:
This allows the study of dynamic events and has been particularly useful
in the semiconductor industry as well as in biology.
studies in such areas as thermal fatigue of ceramics, fracture surfaces,
three-dimensional mapping of particle distribution, machining damage in
metals, and the structure of electronic components.
date, there do not appear to have been any reported uses of confocal
microscopy in textile fields. This surely must be only a temporary
situation, given the enormous potential of the technique.
While the various types of spectroscopy
currently in use are far from being new analytical techniques, there has
been in recent year a trend towards integrating spectrometers with
microscopes to allow the analysis of very small samples. This section will
attempt to provide a brief outline of some of the different
micro-spectroscopy methods currently available.
principles and analytical capabilities of infra-red spectroscopy are well
understood. However, it is only within the past few years that the coupling
of Fourier Transform Infra-red spectroscopy and microscopy have allowed
analysis of samples as small as single fibres to be performed. One major
advantages of the infra-red microscope is that the specimen to be analysed
may be viewed conventionally and positioned accurately before analysis is
carried out. This feature has been made use of in the identification of
contaminants on fabric surfaces and in the analysis of unnatural streaks in
fabrics. Conclusive identification was provided by the so-called spectral
subtraction facility of the instrument.
spectroscopy measures molecular vibrations by analysing the minor proportion
of scattered light that undergoes an energy change on encountering a
material. It is a complementary technique to infra-red spectroscopy and many
transitions inaccessible to IR will be detected clearly with Raman
spectroscopy; Infra-red emphasises the polar characteristics of molecules
whereas Raman describes their covalent features.
Applications of Microscopy in fibre Science
The principles of polarised light microscopy are well documented and it
is not the intention to report them here. All micrographs on the
accompanying pages were recorded on Kodak Ekta 50 film, using an Olympus OM4
camera body mounted on the phototube of a Vickers M75 transmitted-light-polarising
microscope. The polarisation colours shown by the fibres in the micrographs
are those produced from clean, lightly-dyed fibres. In practice, of course,
fibres may be heavily dyed or pigmented and may be textured or distorted in
processing, all of which will affect to some extent the polarisation colours
The use of light and electron microanalysis systems has enabled the
fibre scientist to gain access to much information that would have been
difficult or impossible to obtain precisely without microscope-based
analytical methods. These instruments are increasingly employed in providing
structural and analytical information about the specimen to which they are
applied and their use will continue to grow.
Transform Infrared Spectrometry
Fourier Transform Infra-red (FT-IR) spectrometry was developed in order
to overcome the limitations encountered with dispersive instruments. The
main difficulty was the slow scanning process. A method for measuring all of
the infra-red frequencies simultaneously, rather than individually, was
needed. A solution was developed which employed a very simple optical device
called an interferometer.
• It can identify unknown materials.
• It can determine the quality or consistency of a sample.
• It can determine the amount of components in a mixture.
interferometer produces a unique type of signal which has all of the
infra-red frequencies “Encoded” into it. The signal can be measured very
quickly, usually on the order of one second or so. Thus, the time element
per sample is reduced to a matter of a few seconds rather than several
minutes. Most interferometers employ a beam splitter which takes the
incoming infra-red beam and divides it into two optical beams. One beam
reflects off of a flat mirror which is fixed in place. The other beam
reflects off of a flat mirror which is on a mechanism which allows this
mirror to move a very short distance (typically a few millimeters) away from
the beam splitter. The two beams reflect off of their respective mirrors and
are recombined when they meet back at the beam splitter. Because the path
that one beam travels is a fixed length and the other is constantly changing
as its mirror moves, the signal which exits the interferometer is the result
of these two beams “Interfering” with each other.
resulting signal is called an interferogram which has the unique property
that every data point (a function of the moving mirror position) which makes
up the signal has information about every infra-red frequency which comes
from the source. This means that as the interferogram is measured; all
frequencies are being measured simultaneously. Thus, the use of the
interferometer results in extremely fast measurements.
the analyst requires a frequency spectrum (a plot of the intensity at each
individual frequency) in order to make identification, the measured
interferogram signal can not be interpreted directly. A means of “decoding”
the individual frequencies is required. This can be accomplished via a
well-known mathematical technique called the Fourier transformation. This
transformation is performed by the computer which then presents the user
with the desired spectral information for analysis.
Microscopy today offers the fibre
scientist far more than the mere ability to observe magnified images of his
specimens and has become a diverse and complex branch of physical
investigation encompassing many previously separate technologies.
types of microscope are now available commercially-the confocal light
microscope, the scanning tunneling and atomic force microscope amongst the
most important. Each of these offers exciting possibilities for the study
and investigation of textile materials and when combined with the
capabilities of the more established traditional methods the analytical and
experimental power of the microscope has never been greater.
coupling of instrumental analysis techniques to the microscope has added
another dimension to microscopy: Available computing power and the intense
monochromatic coherent photons of laser light have enabled the microscope to
be used to provide microchemical information on fibres. The use of Fourier
Transform Infra-red and Raman spectroscopies is certain to expand and they
have already been used to investigate some of the newer fibres.
the continuing developments in fibrous polymer technology mean that there
will always be a stimulating supply of materials towards which the fibres
scientist may direct his research, whichever type of microscopy or imaging
he may wish to use.
An HPLC/FTIR interface is available commercially from Lab Connections, Inc,
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For detailed version of this article please refer the print version of The
Indian Textile Journal November 2008 issue.
Department of Textile Technology,
KSR College of Technology,
Tamil Nadu 637 215.