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Trends in biological scanning electron microscopy
0739-6260/90
Micron and Microscopica Acta, Vol. 21, No. 4. PP. 229—232, 1990. Printed in Great Britain.
$3.00 +0.00 Pergamon Press plc
TRENDS IN BIOLOGICAL SCANNING ELECTRON MICROSCOPY W.L.Jongebloed Laboratory of Histology and Cellbiology, University of Groningen Oostersingel 69/1, 9713 EZ Groningen, The Netherlands
If one would formulate certain trends in biological SEM, one could go for the following scheme: A. Microscopical technics: 1. the use of LaB 6 and field-emission guns to improve image resolution; 2. automatic focussing and stigmating programs. 3. the use of low voltage microscopy in particular for the biomedical research; 4. use of digitalized images for storage, processing for better image quality and (almost) real time stereoscopy. 5. Desk-top scanning electron microscopy or fully digitalized scanning electron microscopy in which almost all functions are taken over by the computer. B. Analytical technics: 1. on-line morphometry, to express visual differences in the pictures in numbers; 2. energy- and wavelength-dispersive X-ray microanalyses of the lighter elements p.e. at biodegradation processes. 3. hnmuno-Au-labelling both with secundary and backscatter electrons for cytochemical determination with very small probes (appr. 1 nm). C. Preparation technics: 1. non-coating techniques to replace conventional fixation method (G.A. + 0s04) to improve image quality and resolution. 2. CRYO-SEM, in particular high pressure freezing systems, to avoid possible artifacts due to chemical fixation, dehydration or drying procedures. We will conform ourselfs to the preparation technics and in particular the non-coating techniques. The CRYO-method is a rapid method in comparison with the time consuming non-coating techniques. A disadvantage of the CRYO-SEM often overlooked is the non-versatility of the small frozen specimen, compared to a dried non-coating treated sample which easily can be fractured in different directions. Moreover there is a trend to come to a better integration of images made with LM, SEM and TEM, the more because preparation methods for these techniques are alike up till dehydration with ethanol, as can be seen in the following scheme.
Preparation SEM: 1. Cleaning/washing of the organ/tissue. 2. Pre-fixation of protein compounds: a) chemical fixation: perfusion with glutardialdehyde and/or b) chemical fixation: immersion in glutaraldehyde or c) cryo-fixation >> CRYO-SEMITEM or >> Freeze drying (Kalicharan et al., 1986) 3. Washing with buffer solution. 4. Post-fixation of lipid compounds: a) conventional osmiumtetroxide fixation or b) non-coating technique for SEM. 5. Washing with buffer solution and aquabidest. 6. Dehydration with ethanol, aceton. 229
230
W. L. Jongebloed
7. Drying of the tissue: a) chemical drying with dimethoxypropane (DMP) or b) physical drying: i. drying at air or ii. freeze-drying from t.-butanol (Bartlett and Burstyn, 1975) or iii.critical point drying with liq. CO 2 8. Mounting on specimenholder. a) with double sticked tape or b) with fast hardening epoxy glue. c) with (conducting) glue: colloidal Ag, C or protein. 9. Making specimen conductive: a) metal or carbon sputtering/coating, thickness 5-15 nm. b) Non-coating technique with biological tissues. Because SEM-specimens often have a relative large size and penetration of the fixatives is rather slow an initial perfusion fixation is very important. Nevertheless the internal fixation and conductivity of the samples is rather poor. Methods to overcome these problems to a large extend are the non-coating technics, which are based on a ligand that binds either with osmificated tissue components (TCH) or with collagen-like compounds by means of a galloylglucose compound (TAO). Non-coating technics. Convent
: G.A. + 0s04
O.T.O. : G.A. O.T.O.T.O. : G.A. G.O.T.O. : G.A. G.O.T.U. : G.A. T.A.O. : G.A.
+ Os04 + T~ff(Thiocarbohydrazide) + 0s04 + 0s04 + TCH + 0s04 + I~ff+ OsO4 + 0s04 + Tannic acid + Os04 + 0s04 + Tannic acid + Ur.-Acet. + Tannic acid + Arginine-HCI + 0s04
In practice there two other techniques based on the use of mediated L-lysine with or without tannicacid can be used, but they are inferior at medium or high magnifications. TCH = thiocarbohydrazide is capable to bind with osmificated tissue. TCH can bind more 0504 than conventionally. Tannic acid with its compound galloylglucose reacts with carboxyl-, hydroxyl- and sulfonic groups in the amino acids of the sample proteins. In the TAO method the complex galoylglucose + arginine + guanidine is capable to couple with aminoacids in particular collageneous tissue and to bind extra molecules of 0s04. Obviously the TAO gives a better result than the GOTO or the GOTU, despite the fact that in these cases the sample is being treated with 0504 before the tannic acid. Possibly the Os04 blocks to a certain entend penetration of tannic acid. With images from different fields of interest like adherence of bacteria/cells to certain substrates and degradation processes of biomedical products/cells the usefullness of non-coating techniques and LM/SEM/TEM combination research will be stressed.
ADHERENCE: The adherence of plaque bacteria onto tooth surfaces is a problem that these days is attacked by trying to decrease surface adherence by adding certain agents into p.e. a toothpaste or a mouthwash. In fact the other way around is the problem that certain biomedical products difficult can be covered by p.e. endothelial cells like nylon suturing material in comparison with stainless steel. DEGRADATION: Haptics of intraocular lenses often are made of supramid or polypropylene, while the lens itself is made of PMMA. Both haptic materials are vunerable to attacL of enzymes present in the chamber water. Cataractous lensfibre material shows often a swollen character in comparison with sound fibres. Sometimes the protrusion of the cytoplasm is visible as existing globulus both in SEM and TEM.
Trends in Biological SEM
231
Finally we can come to following conclusions:
1. The SEM is a valuable tool on itself and in combination with LM and TEM research. 2. The use of non-coating techniques as an alternative for conventional 0504 fixation is certainly worthwile to do. 3. Improvement of preparation methods for SEM will contribute very much to the increase of
resolution. 4. An extra effort should be made to develope CRYO-SEM towards a real alternative for the timeconsuming chemical fixation and elaborate drying methods. 5. The use of immunolabeling techniques with very small Au-probes should be stimulated in the analytical SEM. 6. The use of more SEM-images in teaching of biology will lead to a better 3-D knowledge of these tissues. References A.A. Bartlett and HP Burstyn (1975). Scanning electron microscopy. Part I. Scanning electron microscopy symposium. liT Research Institute Chicago. D. Kalicharan and W.L. Jongebloed (1986). TEM of CPD and FD samples of rat liver prepared according to OTOTO, GOTO and GOTU methods, respectively. Ultramicroscopy 19: 98-99.
ADHERENCE Fig. 1 Topview of plaque bacteria onto tooth enamel. x 5370 Fig. 2 Cross-section of plaque (P1) onto tooth enamel (B). x 1340 Fig. 3 Nylon suture in cornea with a few fibroblasts (Pb) and a lot of inflammation cells (IC) and
collagen material (Cm). x 2150 Fig. 4 Stainless steel suture in cornea with macrophages on top of steelsurface, note fibroblasts (Pb) at edge of suture. x 3220
Vt I. i~ti~eIsl~ed
V
7
8
DEGRADATION Fig. 5 Partly degradated surface of polypropylene haptic of l.O.L. after 2 years of residence in the eye. x 600 Fig. 6 Partly degradated surface of supramid haptic after 4 years of residence. x 300 Fig. 7 Swollen cataractous lensfibre. x 3000 Fig. 8 Globular particles as result of protrusions of the cytoplasm of cataractous lensfibres. x 360
Micron and Microscopica Acta, Vol. 21, No. 4. PP. 229—232, 1990. Printed in Great Britain.
$3.00 +0.00 Pergamon Press plc
TRENDS IN BIOLOGICAL SCANNING ELECTRON MICROSCOPY W.L.Jongebloed Laboratory of Histology and Cellbiology, University of Groningen Oostersingel 69/1, 9713 EZ Groningen, The Netherlands
If one would formulate certain trends in biological SEM, one could go for the following scheme: A. Microscopical technics: 1. the use of LaB 6 and field-emission guns to improve image resolution; 2. automatic focussing and stigmating programs. 3. the use of low voltage microscopy in particular for the biomedical research; 4. use of digitalized images for storage, processing for better image quality and (almost) real time stereoscopy. 5. Desk-top scanning electron microscopy or fully digitalized scanning electron microscopy in which almost all functions are taken over by the computer. B. Analytical technics: 1. on-line morphometry, to express visual differences in the pictures in numbers; 2. energy- and wavelength-dispersive X-ray microanalyses of the lighter elements p.e. at biodegradation processes. 3. hnmuno-Au-labelling both with secundary and backscatter electrons for cytochemical determination with very small probes (appr. 1 nm). C. Preparation technics: 1. non-coating techniques to replace conventional fixation method (G.A. + 0s04) to improve image quality and resolution. 2. CRYO-SEM, in particular high pressure freezing systems, to avoid possible artifacts due to chemical fixation, dehydration or drying procedures. We will conform ourselfs to the preparation technics and in particular the non-coating techniques. The CRYO-method is a rapid method in comparison with the time consuming non-coating techniques. A disadvantage of the CRYO-SEM often overlooked is the non-versatility of the small frozen specimen, compared to a dried non-coating treated sample which easily can be fractured in different directions. Moreover there is a trend to come to a better integration of images made with LM, SEM and TEM, the more because preparation methods for these techniques are alike up till dehydration with ethanol, as can be seen in the following scheme.
Preparation SEM: 1. Cleaning/washing of the organ/tissue. 2. Pre-fixation of protein compounds: a) chemical fixation: perfusion with glutardialdehyde and/or b) chemical fixation: immersion in glutaraldehyde or c) cryo-fixation >> CRYO-SEMITEM or >> Freeze drying (Kalicharan et al., 1986) 3. Washing with buffer solution. 4. Post-fixation of lipid compounds: a) conventional osmiumtetroxide fixation or b) non-coating technique for SEM. 5. Washing with buffer solution and aquabidest. 6. Dehydration with ethanol, aceton. 229
230
W. L. Jongebloed
7. Drying of the tissue: a) chemical drying with dimethoxypropane (DMP) or b) physical drying: i. drying at air or ii. freeze-drying from t.-butanol (Bartlett and Burstyn, 1975) or iii.critical point drying with liq. CO 2 8. Mounting on specimenholder. a) with double sticked tape or b) with fast hardening epoxy glue. c) with (conducting) glue: colloidal Ag, C or protein. 9. Making specimen conductive: a) metal or carbon sputtering/coating, thickness 5-15 nm. b) Non-coating technique with biological tissues. Because SEM-specimens often have a relative large size and penetration of the fixatives is rather slow an initial perfusion fixation is very important. Nevertheless the internal fixation and conductivity of the samples is rather poor. Methods to overcome these problems to a large extend are the non-coating technics, which are based on a ligand that binds either with osmificated tissue components (TCH) or with collagen-like compounds by means of a galloylglucose compound (TAO). Non-coating technics. Convent
: G.A. + 0s04
O.T.O. : G.A. O.T.O.T.O. : G.A. G.O.T.O. : G.A. G.O.T.U. : G.A. T.A.O. : G.A.
+ Os04 + T~ff(Thiocarbohydrazide) + 0s04 + 0s04 + TCH + 0s04 + I~ff+ OsO4 + 0s04 + Tannic acid + Os04 + 0s04 + Tannic acid + Ur.-Acet. + Tannic acid + Arginine-HCI + 0s04
In practice there two other techniques based on the use of mediated L-lysine with or without tannicacid can be used, but they are inferior at medium or high magnifications. TCH = thiocarbohydrazide is capable to bind with osmificated tissue. TCH can bind more 0504 than conventionally. Tannic acid with its compound galloylglucose reacts with carboxyl-, hydroxyl- and sulfonic groups in the amino acids of the sample proteins. In the TAO method the complex galoylglucose + arginine + guanidine is capable to couple with aminoacids in particular collageneous tissue and to bind extra molecules of 0s04. Obviously the TAO gives a better result than the GOTO or the GOTU, despite the fact that in these cases the sample is being treated with 0504 before the tannic acid. Possibly the Os04 blocks to a certain entend penetration of tannic acid. With images from different fields of interest like adherence of bacteria/cells to certain substrates and degradation processes of biomedical products/cells the usefullness of non-coating techniques and LM/SEM/TEM combination research will be stressed.
ADHERENCE: The adherence of plaque bacteria onto tooth surfaces is a problem that these days is attacked by trying to decrease surface adherence by adding certain agents into p.e. a toothpaste or a mouthwash. In fact the other way around is the problem that certain biomedical products difficult can be covered by p.e. endothelial cells like nylon suturing material in comparison with stainless steel. DEGRADATION: Haptics of intraocular lenses often are made of supramid or polypropylene, while the lens itself is made of PMMA. Both haptic materials are vunerable to attacL of enzymes present in the chamber water. Cataractous lensfibre material shows often a swollen character in comparison with sound fibres. Sometimes the protrusion of the cytoplasm is visible as existing globulus both in SEM and TEM.
Trends in Biological SEM
231
Finally we can come to following conclusions:
1. The SEM is a valuable tool on itself and in combination with LM and TEM research. 2. The use of non-coating techniques as an alternative for conventional 0504 fixation is certainly worthwile to do. 3. Improvement of preparation methods for SEM will contribute very much to the increase of
resolution. 4. An extra effort should be made to develope CRYO-SEM towards a real alternative for the timeconsuming chemical fixation and elaborate drying methods. 5. The use of immunolabeling techniques with very small Au-probes should be stimulated in the analytical SEM. 6. The use of more SEM-images in teaching of biology will lead to a better 3-D knowledge of these tissues. References A.A. Bartlett and HP Burstyn (1975). Scanning electron microscopy. Part I. Scanning electron microscopy symposium. liT Research Institute Chicago. D. Kalicharan and W.L. Jongebloed (1986). TEM of CPD and FD samples of rat liver prepared according to OTOTO, GOTO and GOTU methods, respectively. Ultramicroscopy 19: 98-99.
ADHERENCE Fig. 1 Topview of plaque bacteria onto tooth enamel. x 5370 Fig. 2 Cross-section of plaque (P1) onto tooth enamel (B). x 1340 Fig. 3 Nylon suture in cornea with a few fibroblasts (Pb) and a lot of inflammation cells (IC) and
collagen material (Cm). x 2150 Fig. 4 Stainless steel suture in cornea with macrophages on top of steelsurface, note fibroblasts (Pb) at edge of suture. x 3220
Vt I. i~ti~eIsl~ed
V
7
8
DEGRADATION Fig. 5 Partly degradated surface of polypropylene haptic of l.O.L. after 2 years of residence in the eye. x 600 Fig. 6 Partly degradated surface of supramid haptic after 4 years of residence. x 300 Fig. 7 Swollen cataractous lensfibre. x 3000 Fig. 8 Globular particles as result of protrusions of the cytoplasm of cataractous lensfibres. x 360