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ACOUSTIC MICROSTRUCTURE OF GREEN COFFEE (SANTOS)
ACOUSTIC MICROSTRUCTURE OF GREEN COFFEE (SANTOS) E.
Kolodziejczyk*, M.R. Fernandez-Graf*, J.M. Saure 1+, V. Fryder*, A.Saied+, J. Attal+ and V. Rey*
*
Nestlé Research Centre Nestec Ltd Vers-Chez-Les-Blanc CH - 1000
+
Lausanne
26
(Switzerland)
LAM, USTL F - 34060
Montpellier-Cedex
(France)
The rapid visualization of the actual microstructure of biologic specimens is of high interest and is now rendered feasible on the intact sample by acoustic microscopy. Nevertheless, practical applications of this method are still limited and relatively little has been done to explore its potential. As an example, we describe here the use of acoustic microscopy as a rapid and efficient method for the study of plant microstructure. INTRODUCTION Acoustic microscopes visualize the microdistribution of viscoelastic properties both at the surface and in the interior planes of specimens (1,2,3). Basically, because ultrasonic waves (US) have no lethal or destructive effects on the the specimen, the direct visualization of the interior planes of living specimens becomes possible. They can thus be explored without the need for mechanical slicing, as is the case for conventional light microscopy, and classical dyes are no longer neces sary to obtain a maximum contrast image. Nevertheless, even though several applications of acoustic microscopy (AM) have been reported (4,5), the technique is still limited to specia lized laboratories and has not yet become a widespread tool for morpho logy. In this paper, we wish to show some advantages of reflection AM to study thick specimens. As an example, we have chosen coffee beans and compared their micro-acoustic patterns to those revealed by scanning electron microscopy (SEM). Technical aspects are also considered.
284
Ultrasonics International 87 Conf. Proc.
METHODS a)
Samples Green coffee beans (Santos) were rehydrated and slices 5 mm thick were manually cut with a razor blade to obtain parallel flat surfaces.
b)
Acoustic microscopy For AM, the slices were attached to stretched mylar and examined unfixed under a reflection acoustic microscope emitting either 200 or 600 Mhz US. The raster scanning AM is based on the system of Quate (1) and was described in a previous report (6).
c)
Scanning electron microscopy For SEM, the slices were first fixed in 2.5 % glutaraldehyde, dehydrated by ethanol, dried by the critical point method and sputtered with gold. They were examined in a Philips 505 SEM.
RESULTS a)
200 Mhz At this frequency, the exterior of the sample was sufficiently flat and a topographic survey of the specimen was feasible at a low magnification (Figures 1 and 3). Cell walls reflected US the most, while, the cell cytoplasm attenuated US strongly. At low magnifications, SEM (Figure 2) added no information compared to AM. However, higher AM magnifications were of relatively low interest especially with regard to the cytoplasmic microstructure (Figure 4 ) .
b)
600 Mhz Here, the surface of the specimen was not sufficiently flat and topographic imaging was not easy (Figure 5 ) . However, the limitation resulting from flatness disappeared at higher magnifications and was compensated by increased resolution. Besides the details of cell walls which became obvious, the intracellular acoustic microstructure was also revealed (Figures 6 and 7 ) . Depending on which plane was observed, intracytoplasmic inclusions about 3 micrometers in size were distributed either peripherally or in the entire cytoplasm. The intimate apposition of the cytoplasm to the cell walls was also noticeable. When compared to AM, SEM (figure 8) showed the following differences: first, cell walls were poorly resolved, second, the cytoplasmic substructure was badly defined (Figure 8: arrow heads). Finally, when still present, the cytoplasm had shrunk as a result of dehydration and drying (figure 8: * ) . This did not occur with AM.
Ultrasonics International 87 Conf. Proc.
285
By focusing the acoustic beam inside the specimen, different images were obtained (Figures 9 and 10) . Figure 10 shows such a micro tomography obtained by focusing the acoustic beam approximately 20 micrometers beneath the surface of the sample. The reference plane is seen in Figure 9, and some cells are identified (Figure 9: A, B, C, D ) . Changes of their outlines (Figure 10: A, B, C, D) as well as of the cytoplasmic microstructure (Figures 9 and 10: arrows) are obvious. The maximum focusing depth was limited to about 30 microns because of the appearance of diffraction patterns. But in micrographs represent micrometers. CONCLUSION These data show that AM is a useful tool for studying thick specimens given that the surface is sufficiently flat. However, it also depends on the frequency of US being used, and flatness can easily be optimised by simple methods. The use of different frequencies as a function of the desired resolution allows different levels of observation depending on the aim of the study. For example, topographic studies can be achieved using low frequency US (200 Mhz). In contrast, when more detailed images are needed, especially in the cytoplasm, higher frequencies are necessary and available (600 Mhz or more). The advantages of AM relative to SEM must be considered in terms of its technical simplicity. First, with AM, the preparation of specimens is very simple, since fixation and in general, dehydration and drying are not necessary. However, it should be kept in mind that the micro-acoustic study of fixed specimens might be helpful, especially in view of acoustic staining, since different fixatives might act differently upon the mechanical (i.e. viscoelastic) properties of the specimen. Thus, as a principal advantage, AM at low magnification is far more efficient than SEM and indeed any other microscopic method because the preparation of the specimens needs little time and the images are obtained very rapidly. Moreover, in the absence of chemical fixation these images reflect the actual structure of the specimen. Further, AM works at such, apart from the prepared for SEM or staining undergo far prepared for AM.
atmospheric pressure while SEM requires a vacuum. As changes elicited by chemical fixation, the specimens any other microscopic method involving slicing and more physical and chemical modifications than those
From these considerations, it should be emphasized that, at least at low magnifications (up to x 2.000), AM of thick specimens is seen to be an excellent alternative to SEM, even though it can never be a total substitute. It is nevertheless likely that in many cases AM will become an excellent tool, complementary or preliminary to other techniques, for example, to help in selecting rapidly and accurately specific regions of interest in large specimens. Aknowledgements: We wish to thank Mrs. M. Weber for photography.
286
Ultrasonics International 87 Conf. Proc.
REFERENCES 1.
Lemons, R.A. and Quate. C.F. Acoustic microscope-scanning Appi. Phys. Lett., Vol. 24 (1974) pp 163-165.
version.
2.
Kessler, L.W., Korpel, A. and Palermo, P.R. Simultaneous acoustic and optical microscopy of biological specimens. Nature (London), Vol. 239 (1974), pp. 111-115.
3.
Hildebrand, J.A. and Rugar, D. Measurements of cellular elastic properties by acoustic microscopy. J: Microscopy, Vol. 134(1984), pp 245-260.
4.
Hildebrand, J.A., Rugar, D. , Johnston, N.R. and Quate, C.F. Acoustic microscopy of living cells. Proc. Nati. Acad. Sci. USA, Vol. 78 (1981), pp 1656-1660.
5.
Israel, H.W., Wilson, R.G., Aist, J.R. and Kunoh,H. Cell wall appositions and plant disease resitance: Acoustic microscopy of papillae that block fungal ingress. Proc. Nati. Acad. Sci (USA), Vol. 77(1980), pp 2046-2049.
6.
Neild, T.O., Attal, J. and Saurei, J.M. Images of arterioles in unfixed tissue obtained by acoustic microscopy. J. Microscopy, Vol. 139 (1985), pp 19-25.
7.
Attal, J. The acoustic microscope: a tool for non destructive testing. In Non detructive evaluation of semi-conductor materials and devices (1979). ed. by Zemel, J.N., pp 631-676. Plenum New-York.
Ultrasonics International 87 Conf. Proc.
287
Fig. 1
Acoustic microscopy (AM, 200 Mhz) of green coffee. SS = silver skin
Fig.
Fig.
AM (200 Mhz). E = epidermal cells : W = cell walls
Fig. 4
288
3
Ultrasonics International 87 Conf. Proc.
2
Scanning electron microscopy (SEM) of green coffee. SS = silver skin
AM (200 Mhz). C = cytoplasm SS = silver skin : W = cell walls
Fig. 5
AM (600 Mhz). C = cytoplasm W = cell walls : SS = silver skin
Fig. 6
AM (600 Mhz). High power micrograph showing details in the silver skin
Fig. 7
AM (600 Mhz). Detailed picture of the cytopla sm (C) and cell walls (W)
Fig. 8
SEM. To be compared to fig. 7. C = cytoplasm : W = cell walls
Ultrasonics International 87 Conf. Proc.
289
Fig. 9
290
AM (600 Mhz). Microtomograph (see fig. 10) : Reference plane
Ultrasonics International 87 Conf. Proc.
Fig. 10
The acoustic beam was focused about 20 ym beneath the surface
Kolodziejczyk*, M.R. Fernandez-Graf*, J.M. Saure 1+, V. Fryder*, A.Saied+, J. Attal+ and V. Rey*
*
Nestlé Research Centre Nestec Ltd Vers-Chez-Les-Blanc CH - 1000
+
Lausanne
26
(Switzerland)
LAM, USTL F - 34060
Montpellier-Cedex
(France)
The rapid visualization of the actual microstructure of biologic specimens is of high interest and is now rendered feasible on the intact sample by acoustic microscopy. Nevertheless, practical applications of this method are still limited and relatively little has been done to explore its potential. As an example, we describe here the use of acoustic microscopy as a rapid and efficient method for the study of plant microstructure. INTRODUCTION Acoustic microscopes visualize the microdistribution of viscoelastic properties both at the surface and in the interior planes of specimens (1,2,3). Basically, because ultrasonic waves (US) have no lethal or destructive effects on the the specimen, the direct visualization of the interior planes of living specimens becomes possible. They can thus be explored without the need for mechanical slicing, as is the case for conventional light microscopy, and classical dyes are no longer neces sary to obtain a maximum contrast image. Nevertheless, even though several applications of acoustic microscopy (AM) have been reported (4,5), the technique is still limited to specia lized laboratories and has not yet become a widespread tool for morpho logy. In this paper, we wish to show some advantages of reflection AM to study thick specimens. As an example, we have chosen coffee beans and compared their micro-acoustic patterns to those revealed by scanning electron microscopy (SEM). Technical aspects are also considered.
284
Ultrasonics International 87 Conf. Proc.
METHODS a)
Samples Green coffee beans (Santos) were rehydrated and slices 5 mm thick were manually cut with a razor blade to obtain parallel flat surfaces.
b)
Acoustic microscopy For AM, the slices were attached to stretched mylar and examined unfixed under a reflection acoustic microscope emitting either 200 or 600 Mhz US. The raster scanning AM is based on the system of Quate (1) and was described in a previous report (6).
c)
Scanning electron microscopy For SEM, the slices were first fixed in 2.5 % glutaraldehyde, dehydrated by ethanol, dried by the critical point method and sputtered with gold. They were examined in a Philips 505 SEM.
RESULTS a)
200 Mhz At this frequency, the exterior of the sample was sufficiently flat and a topographic survey of the specimen was feasible at a low magnification (Figures 1 and 3). Cell walls reflected US the most, while, the cell cytoplasm attenuated US strongly. At low magnifications, SEM (Figure 2) added no information compared to AM. However, higher AM magnifications were of relatively low interest especially with regard to the cytoplasmic microstructure (Figure 4 ) .
b)
600 Mhz Here, the surface of the specimen was not sufficiently flat and topographic imaging was not easy (Figure 5 ) . However, the limitation resulting from flatness disappeared at higher magnifications and was compensated by increased resolution. Besides the details of cell walls which became obvious, the intracellular acoustic microstructure was also revealed (Figures 6 and 7 ) . Depending on which plane was observed, intracytoplasmic inclusions about 3 micrometers in size were distributed either peripherally or in the entire cytoplasm. The intimate apposition of the cytoplasm to the cell walls was also noticeable. When compared to AM, SEM (figure 8) showed the following differences: first, cell walls were poorly resolved, second, the cytoplasmic substructure was badly defined (Figure 8: arrow heads). Finally, when still present, the cytoplasm had shrunk as a result of dehydration and drying (figure 8: * ) . This did not occur with AM.
Ultrasonics International 87 Conf. Proc.
285
By focusing the acoustic beam inside the specimen, different images were obtained (Figures 9 and 10) . Figure 10 shows such a micro tomography obtained by focusing the acoustic beam approximately 20 micrometers beneath the surface of the sample. The reference plane is seen in Figure 9, and some cells are identified (Figure 9: A, B, C, D ) . Changes of their outlines (Figure 10: A, B, C, D) as well as of the cytoplasmic microstructure (Figures 9 and 10: arrows) are obvious. The maximum focusing depth was limited to about 30 microns because of the appearance of diffraction patterns. But in micrographs represent micrometers. CONCLUSION These data show that AM is a useful tool for studying thick specimens given that the surface is sufficiently flat. However, it also depends on the frequency of US being used, and flatness can easily be optimised by simple methods. The use of different frequencies as a function of the desired resolution allows different levels of observation depending on the aim of the study. For example, topographic studies can be achieved using low frequency US (200 Mhz). In contrast, when more detailed images are needed, especially in the cytoplasm, higher frequencies are necessary and available (600 Mhz or more). The advantages of AM relative to SEM must be considered in terms of its technical simplicity. First, with AM, the preparation of specimens is very simple, since fixation and in general, dehydration and drying are not necessary. However, it should be kept in mind that the micro-acoustic study of fixed specimens might be helpful, especially in view of acoustic staining, since different fixatives might act differently upon the mechanical (i.e. viscoelastic) properties of the specimen. Thus, as a principal advantage, AM at low magnification is far more efficient than SEM and indeed any other microscopic method because the preparation of the specimens needs little time and the images are obtained very rapidly. Moreover, in the absence of chemical fixation these images reflect the actual structure of the specimen. Further, AM works at such, apart from the prepared for SEM or staining undergo far prepared for AM.
atmospheric pressure while SEM requires a vacuum. As changes elicited by chemical fixation, the specimens any other microscopic method involving slicing and more physical and chemical modifications than those
From these considerations, it should be emphasized that, at least at low magnifications (up to x 2.000), AM of thick specimens is seen to be an excellent alternative to SEM, even though it can never be a total substitute. It is nevertheless likely that in many cases AM will become an excellent tool, complementary or preliminary to other techniques, for example, to help in selecting rapidly and accurately specific regions of interest in large specimens. Aknowledgements: We wish to thank Mrs. M. Weber for photography.
286
Ultrasonics International 87 Conf. Proc.
REFERENCES 1.
Lemons, R.A. and Quate. C.F. Acoustic microscope-scanning Appi. Phys. Lett., Vol. 24 (1974) pp 163-165.
version.
2.
Kessler, L.W., Korpel, A. and Palermo, P.R. Simultaneous acoustic and optical microscopy of biological specimens. Nature (London), Vol. 239 (1974), pp. 111-115.
3.
Hildebrand, J.A. and Rugar, D. Measurements of cellular elastic properties by acoustic microscopy. J: Microscopy, Vol. 134(1984), pp 245-260.
4.
Hildebrand, J.A., Rugar, D. , Johnston, N.R. and Quate, C.F. Acoustic microscopy of living cells. Proc. Nati. Acad. Sci. USA, Vol. 78 (1981), pp 1656-1660.
5.
Israel, H.W., Wilson, R.G., Aist, J.R. and Kunoh,H. Cell wall appositions and plant disease resitance: Acoustic microscopy of papillae that block fungal ingress. Proc. Nati. Acad. Sci (USA), Vol. 77(1980), pp 2046-2049.
6.
Neild, T.O., Attal, J. and Saurei, J.M. Images of arterioles in unfixed tissue obtained by acoustic microscopy. J. Microscopy, Vol. 139 (1985), pp 19-25.
7.
Attal, J. The acoustic microscope: a tool for non destructive testing. In Non detructive evaluation of semi-conductor materials and devices (1979). ed. by Zemel, J.N., pp 631-676. Plenum New-York.
Ultrasonics International 87 Conf. Proc.
287
Fig. 1
Acoustic microscopy (AM, 200 Mhz) of green coffee. SS = silver skin
Fig.
Fig.
AM (200 Mhz). E = epidermal cells : W = cell walls
Fig. 4
288
3
Ultrasonics International 87 Conf. Proc.
2
Scanning electron microscopy (SEM) of green coffee. SS = silver skin
AM (200 Mhz). C = cytoplasm SS = silver skin : W = cell walls
Fig. 5
AM (600 Mhz). C = cytoplasm W = cell walls : SS = silver skin
Fig. 6
AM (600 Mhz). High power micrograph showing details in the silver skin
Fig. 7
AM (600 Mhz). Detailed picture of the cytopla sm (C) and cell walls (W)
Fig. 8
SEM. To be compared to fig. 7. C = cytoplasm : W = cell walls
Ultrasonics International 87 Conf. Proc.
289
Fig. 9
290
AM (600 Mhz). Microtomograph (see fig. 10) : Reference plane
Ultrasonics International 87 Conf. Proc.
Fig. 10
The acoustic beam was focused about 20 ym beneath the surface