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TEM examination of four different tissues prepared for SEM either by OTOTO, GOTO or GOTU
438
Abstracts of The Netherlands Society o[ Electron Microscopy
number of small vesicles in the PZ containing gold increased and eFcR-gold was also present in the peripheral and perinuclear multivesicular endosomes. At this time-point no decrease in number of gold particles along the PM was seen. Within 60 min eFcR-gold was also found in electron-dense bodies. After 120 m i n uFcR-gold was still present along the PM and in small vesicles in the PZ, though in smaller amounts. Multivesicular endosomes and an increasing number of electron-dense endosomes still contained gold particles. Organelles involved in uptake of Fc receptors and in pinocytosis were compared. Using BSA-gold as a fluid-phase marker, we found BSA-gold in small vesicles in the PZ and in multivesicular endosomes. These organelles were also labeled in the pulse-chase experiments with sFcRgold. Therefore we suggest that BSA-gold and the Fc receptor share a common pinocytotic pathway.
THE INTESTINE GLYCOCALIX LAYER TREATED ACCORDING TO OTOTO OR URANYLACETATE EXAMINED BY SEM, TEM AND STEM D. Kalicharan and W.L. Jongebloed Centre for Medical E l e c t r o n M i c r o s c o p y , U n i v e r s i t y o f Groningen, O o s t e r s i n g e l 69/2, 9713 EZ Groningen, N e t h e r l a n d s
The non-coating technique, in particular the OTOTO method, gives excellent results with biological specimens, although at high magnifications blurring of the image can occur due to accumulation of osmium. Comparison was made of SEM, TEM and STEM images of the intestine glycocalix of the enterocyte microvilli, treated with either OTOTO or OsO4-Uranylacetate. Results: (SEM 25-30 kV) The glycocalix is only visible as some granular material on the surface of the microvilli, both with OTOTO and Ur.Acet.treated samples. SEM (30-40 kV) In cross view the glycocalix is clearly visible; in top view the microvilli top appears very dark, surrounded by mucopolysaccharides of the glycocalix. STEM (80-100 kV) mh~ ~ , ~} ~ ' • 11~ ~^..L~ _n ~.... mlcrovi brane is poorly visible while the projections of the actine filaments appear very black, both in OTOTO and Ur.Acet. samples. TEM (80-10 kV) The glycocalix appears very prominent with OTOTO, the actine filaments are poorly visible. With Ur. Acet. aggregation of the mucopolysaccharides is observable; the projections of the actine filaments and the double membrane are quite well preserved.
TEM EXAMINATION OF FOUR DIFFERENT TISSUES PREPARED FOR SEM E I T H E R BY OTOTO, GOTO OR GOTU D. Kalicharan and W.L. Jongebloed C e n t r e for M e d i c a l E l e c t r o n M i c r o s c o p y , University of Groningen, Oostersingel 69/2, 9713 EZ G r o n i n g e n , N e t h e r l a n d s
Non-coating techniques like OTOTO and GOTO give excellent results with biological tissues in SEM, particularly at fracture faces. Tissue sections of intestine, kidney, liver and lung, prepared according to OTOTO, GOTO and GOTU, were compared over a wide range of magnifications for their application in a combined TEM/SEM study. At low m a g n i f i c a t i o n good results were obtained at all 3 techniques with intestine, kidney and liver; with lung GOTO and GOTU gave superior results over OTOTO. At higher magnifications only GOTU (and to a lesser extent GOTO) showed a good preservation of the glycocalix of the intestine. W i t h GOTO a well pre[erved kidney m o r p h o l o g y was observed in contrast to OTOTO w h e r e podocyte m e m b r a n e s w e r e damaged. For liver OTOTO was superior to GOTU; GOTO failed, due to contrast reversal which decreased image quality. Cell organelles of lung treated with GOTO or GOTU are well preserved, though for TEM/SEM studies GOTO seems the best.
HIGH MAGNIFICATION SEM AT LOW AND HIGH ACCELERATION VOLTAGES: A NEW TOOL FOR SURFACE ANALYSIS OF BULK SPECIMENS Klaus-Ruediger Peters Yale u n i v e r s i t y ,
New Haven,
CT,
USA
Recent u n d e r s t a n d i n g of signal generation and c o l l e c t i o n (BSE and SE) and improvements of instrumental performance (h~gh brightness electron sources, reduced hydrocarbon contamination and very low accelerating voltages) allow new unique applications of the scanning electron microscope in biology and materials science. Subnanometer electron probe diameters, established at high acceieratina voltages (>3 kV) make it possible to image topographic details of nm dimensions at high magnifications of iOO,OOOx to l,OOO,OOOx, i.e., macromolecular fine structures on biological tissue specimens or Farticulate details on bulk electrical conductors. Using lower voltages (1-3 kV) gooc resolution can be obtained even on instlators of magnifications of iO,O00 -
Abstracts of The Netherlands Society o[ Electron Microscopy
number of small vesicles in the PZ containing gold increased and eFcR-gold was also present in the peripheral and perinuclear multivesicular endosomes. At this time-point no decrease in number of gold particles along the PM was seen. Within 60 min eFcR-gold was also found in electron-dense bodies. After 120 m i n uFcR-gold was still present along the PM and in small vesicles in the PZ, though in smaller amounts. Multivesicular endosomes and an increasing number of electron-dense endosomes still contained gold particles. Organelles involved in uptake of Fc receptors and in pinocytosis were compared. Using BSA-gold as a fluid-phase marker, we found BSA-gold in small vesicles in the PZ and in multivesicular endosomes. These organelles were also labeled in the pulse-chase experiments with sFcRgold. Therefore we suggest that BSA-gold and the Fc receptor share a common pinocytotic pathway.
THE INTESTINE GLYCOCALIX LAYER TREATED ACCORDING TO OTOTO OR URANYLACETATE EXAMINED BY SEM, TEM AND STEM D. Kalicharan and W.L. Jongebloed Centre for Medical E l e c t r o n M i c r o s c o p y , U n i v e r s i t y o f Groningen, O o s t e r s i n g e l 69/2, 9713 EZ Groningen, N e t h e r l a n d s
The non-coating technique, in particular the OTOTO method, gives excellent results with biological specimens, although at high magnifications blurring of the image can occur due to accumulation of osmium. Comparison was made of SEM, TEM and STEM images of the intestine glycocalix of the enterocyte microvilli, treated with either OTOTO or OsO4-Uranylacetate. Results: (SEM 25-30 kV) The glycocalix is only visible as some granular material on the surface of the microvilli, both with OTOTO and Ur.Acet.treated samples. SEM (30-40 kV) In cross view the glycocalix is clearly visible; in top view the microvilli top appears very dark, surrounded by mucopolysaccharides of the glycocalix. STEM (80-100 kV) mh~ ~ , ~} ~ ' • 11~ ~^..L~ _n ~.... mlcrovi brane is poorly visible while the projections of the actine filaments appear very black, both in OTOTO and Ur.Acet. samples. TEM (80-10 kV) The glycocalix appears very prominent with OTOTO, the actine filaments are poorly visible. With Ur. Acet. aggregation of the mucopolysaccharides is observable; the projections of the actine filaments and the double membrane are quite well preserved.
TEM EXAMINATION OF FOUR DIFFERENT TISSUES PREPARED FOR SEM E I T H E R BY OTOTO, GOTO OR GOTU D. Kalicharan and W.L. Jongebloed C e n t r e for M e d i c a l E l e c t r o n M i c r o s c o p y , University of Groningen, Oostersingel 69/2, 9713 EZ G r o n i n g e n , N e t h e r l a n d s
Non-coating techniques like OTOTO and GOTO give excellent results with biological tissues in SEM, particularly at fracture faces. Tissue sections of intestine, kidney, liver and lung, prepared according to OTOTO, GOTO and GOTU, were compared over a wide range of magnifications for their application in a combined TEM/SEM study. At low m a g n i f i c a t i o n good results were obtained at all 3 techniques with intestine, kidney and liver; with lung GOTO and GOTU gave superior results over OTOTO. At higher magnifications only GOTU (and to a lesser extent GOTO) showed a good preservation of the glycocalix of the intestine. W i t h GOTO a well pre[erved kidney m o r p h o l o g y was observed in contrast to OTOTO w h e r e podocyte m e m b r a n e s w e r e damaged. For liver OTOTO was superior to GOTU; GOTO failed, due to contrast reversal which decreased image quality. Cell organelles of lung treated with GOTO or GOTU are well preserved, though for TEM/SEM studies GOTO seems the best.
HIGH MAGNIFICATION SEM AT LOW AND HIGH ACCELERATION VOLTAGES: A NEW TOOL FOR SURFACE ANALYSIS OF BULK SPECIMENS Klaus-Ruediger Peters Yale u n i v e r s i t y ,
New Haven,
CT,
USA
Recent u n d e r s t a n d i n g of signal generation and c o l l e c t i o n (BSE and SE) and improvements of instrumental performance (h~gh brightness electron sources, reduced hydrocarbon contamination and very low accelerating voltages) allow new unique applications of the scanning electron microscope in biology and materials science. Subnanometer electron probe diameters, established at high acceieratina voltages (>3 kV) make it possible to image topographic details of nm dimensions at high magnifications of iOO,OOOx to l,OOO,OOOx, i.e., macromolecular fine structures on biological tissue specimens or Farticulate details on bulk electrical conductors. Using lower voltages (1-3 kV) gooc resolution can be obtained even on instlators of magnifications of iO,O00 -