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.

Now 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.

This is 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.

Developments in Microscopy and Imaging

The ‘Conventional’ Light Microscope

Although 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.

Sometimes, 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.

Confocal Light Microscopy

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.

The 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.

There are two types of confocal microscope in use today and these are described below.

Confocal Laser Scanning Microscope (CLSM)

The theoretical 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 photomultiplier.

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.

Uses of CLSM

To 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.

i)High 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.

ii)Four dimensional imaging:
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.

Tandem 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.

In 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.

Figure 2.2.2 Schematic diagram of the Tandem Scanning Microscope

The 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

Many 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.

1.The measurement of height variations, to a resolution of 0.02µm over a depth of 50µm:
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.

2.The 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.

3.Real 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.

4.Detailed 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.

To 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.

Microspectroscopy

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.

Infra-red Microscopy

The 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.

Microprobe Raman Spectroscopy

Raman 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.

Practical Applications of Microscopy in fibre Science

Photomicrography:
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 produced.

Analytical Microscopy:
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.

Fourier 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.

FT-IR provides?
• 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.

The 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.

The 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.

Because 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.

Conclusion

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.

New 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.

The 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.

Finally, 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.

References

1. An HPLC/FTIR interface is available commercially from Lab Connections, Inc, Marlborough, MA.
2. A C Scott. Geological Apllications of Laser Scanning Microscopy, Microscopy and analysis,1989,17.
3. C J R Sheppard and a.Choudry, Image formation in the scanning microscope, Optica Acta, 1977, 24,1051.
4. D R Lide, ed, CRC Handbook of Chemistry and Physics, 75th ed (Boca Raton, FL: CRC Press, 1994),9-79.
5. G C Pandey.FTIR Microscopy for the determination of copolymer acrylic fibres, The analyst, 1989,23-26.
6. R M Silverstein, G C Bassler, and T C Morrill, Spectrometric Identification of Organic Compounds, 4th ed. (New York: Wiley, 1981), 166.
7. S P Tear. The use of STM for surface structure determination, Microscopy and analysis, 1990,7.
8. U S Environmental Protection Agency, Methods for Chemical Analysis of Water and Wastes, 3rd ed., Report No. EPA-600/4-79-020 (Springfield, VA: National Technical Information Service, 1983), 413.2-1, 418.1-1.

Note: For detailed version of this article please refer the print version of The Indian Textile Journal November 2008 issue.

O L Shanmugasundaram.
Department of Textile Technology,
KSR College of Technology,
Tiruchengode,
Tamil Nadu 637 215.