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Subcellular localization of LamB-LacZ hybrid proteins after cryo-ultramicrotomy or freeze-substitution
Abstracts of The Netherlands SocieO of Electron Microscopy
450
particles represent free IgG or antigen receptors on B-lymphocytes. KI-67, A MONOCLONAL ANTIBODY DIRECTED AGAINST A NUCLEOLAR COMPONENT R. Verheijen, H. Kuijpers, H. Beck, F.J.R. Rietveld and F.C.S. Ramaekers D e p a r t m e n t of P a t h o l o g y , U n i v e r s i t y H o s p i t a l Nijmegen, N i j m e g e n , The Netherlands
Ki-67 is a commercially available monoclonal antibody which reacts with a nuclear antigen expressed in all proliferating cells. In cultured cell lines Ki-67 stains an antigen present in the nucleolus of interphase cells and reacts with an interchromatinous network in mitotic cells. Immunoelectron microscopy revealed that the antigen is localized at the periphery of the nucleolus in interphase cells. Despite many efforts, it was impossible to identify the antigen by means of immunoblotting or immunoprecipitation assays. Testing the species specificity of Ki-67 on cultured cell lines by means of indirect immunofluorescence revealed that the antibody reacted only with human cell nuclei and those of rhesis monkey kidney. With the immunoperoxidase staining technique on tissue sections, the cryptic cells of rabbit small intestine were also found to be positive with Ki-67. Flow-cytometry analysis was performed using two human cell lines, HeLa and Molt-4, stained with propidium iodide for DNA and with FITC-labeled second antibodies for Ki-67. Because of the different FITC signals between mitotic cells and G2 phase cells, we were able to separate the G2/M fraction into highly enriched fractions of G2 and M cells, respectively. In situ fractionation experiments on human monolayer cell lines, resulting in nuclear matrix preparations attached to a glass slide, showed that the Ki-67 antigen remained associated with such structures.
INTEGRATION OF I ~ G E - A N ~ L Y S I S IN ELECTRON MICROSCOPES
SYSTEMS
Teun Vet Zeiss
N e d e r ! a n d B.V.
Scanning and transmission electron microscopes were designed and successfully employed to reproduce submicroscopic
structures. The photograph often was, and is, the final result. Owing to rapid developments in the semiconductor industry, one is at pre~ent able to build computer systems which can optimize and analyze the often complex images produced by the electron microscope. But to obtain a reproducible result an intensive interaction between the electron microscope and the imageanalysis system will be required, particularly when one takes an automatic analysis of several fields into account. With the new SEM (CSM-950) and TEM (CEM-902) Zeiss, W e s t Germany, has managed to completely integrate an imageanalyser, i.e., the image is not only transferred from the electron microscope but every possible parameter and setting of the electron microscope can be regulated and adjusted. In this way one can act on the image information, e.g., changing the setting of the EM or referring the automatic motor stage to another position on the specimen. A special feature of the SEM is that other detector systems can be linked up and their signals can be processed in the imageanalysis part. Thus a relatively simple-to-use and high-performance analytical measuring system for qualitative and quantitative microstructure unalysis has now become available.
SUBCELLULAR LOCALIZATION OF LamB-LacZ HYBRID PROTEINS AFTER CRYO-ULTRAMICROTOMY OR FREEZE-SUBSTITUTION W.F. Voorhout, J.J.M. Leunissen-Bijvelt and T. Van Der Krift D e p a r t m e n t o f M o l e c u l a r Cell Biology, U n i v e r s i t y of Utrecht, P a d u a l a a n 8, 3584 CH U t r e c h t , The N e t h e r l a n d s
The subcellular localization of LamB-LacZ hybrid proteins in the Escherichia coli K-12 strains pop3234 and pop ~299 was investigated by two immunoelectron-microscopical techniques. To induce the synthesis of the LamB-LacZ hybrid proteins, cells were grown for 5 hr in 0.4% maltose. Part of the cell culture was fixed in 2% paraformaldehyde/ 0.5% glutaraldehyde and further processed for cryo-ultramicrotomy. The rest of the cells were gently pelleted, rapidly frozen in liquid propane and submitted to freeze substitution. The cells were substituted for 8 hours at -90°C in 1% osmiumtetroxide, 3% glutaraldehyde and 0.5% uranyl acetate in methanol, embedded in Lowicryl HM20 and pol~merized at -45oc.
Abstracts o/The Netherlands Societ)' of Electron Microscopy
L a b e l l i n g of ultrathin cryosections of the cells with a n t i - L a m B or anti-8g a l a c t o s i d a s e protein serum and protein A-gold complexes revealed that the hybrid p r o t e i n s accumulated in the cytoplasm or w e r e associated w i t h membranelike s t r u c t u r e s in the cytoplasm. Freezes u b s t i t u t i o n clearly r e v e a l e d these membrane-like structures and special areas of a c c u m u l a t e d hybrid proteins. After f r e e z e - s u b s t i t u t i o n in the presence of osmiumtetroxide, the h y b r i d proteins could still be detected w i t h immunogold probes. The accumulated form of the hybrid p r o t e i n could only be detected using a n t i - 8 - g a l a c t o s i d a s e serum and not using a n t i - L a m B serum.
T H R E E - D I M E N S I O N A L V I S U A L I Z A T I O N TECHNIQUES FOR CONFOC~L M I C R O S C O P k H.T.M. van der Voort, and N. N a n n i n g a
G.J.
Brakenhoff
Dept. of E l e c t r o n M i c r o s c o p y and M o l e c u l a r C y t o l o g y , U n i v e r s i t y of A m s t e r d a m , P l a n t a g e M u i d e r g r a c h t 14, 1018 TV A m s t e r d a m , The N e t h e r l a n d s
The optical properties of a Confocal Scanning Laser Microscope (CSLM) operated in fluorescence allow for sampling a specific volume in the specimen. The small, d i f f r a c c i o n - i i m i t e d size of this volume permits the m e a s u r e m e n t of microscopic 3D fluorescence distributions. Therefore, confocal m i c r o s c o p y forms an ideal base for the study of the 3D morphology of biological objects. To e x p l o i t the c a p a b i l i t i e s of a CSLM fully, it is n e c e s s a r y to have it coupled d i r e c t l y to a c o m p u t e r system. This s y s t e m can then p e r f o r m tasks like i n s t r u m e n t control and d a t a acquisition, image processing, image analysis and the v i s u a l i z a t i o n of 3D images. This last task is of prime importance in order to assess a newly scanned image quantitatively. A good v i s u a l i z a t i o n method must meet v a r i o u s contradictory conditions: it should provide clear, and as many as possible, visual clues to enable space perception; presentation of the results should be convenient, and the used computer a l g o r i t h m should be fast enough to allow for interactive usage during a microscop±c session. Currently, the following techniques are used in our laboratory: g e n e r a t i o n of s t e r e o s c o p i c pairs, computer graphics (i.e. solid modelling) and a newly developed technique based on computer simulation of a fluorescence process.
4 51
In the paper presented we discuss these methods and their merits, as well as image p r o c e s s i n g techniques required in reprocessing.
DISLOCATION SUDIrRUCTUkES IN A QUARTZITE WITH A T Y P E I CROSS GIRDLE FABRIC L.E.P. W e n m a k e r s and M~R. Drury Instituut voor Aardwetenschappen, Rijksuniversiteit Utrecht, B u d a p e s t l a a n 4, 3584 CD U t r e c h t
Slip s y s t e m geometry and dislocation substructures have been studied in a naturally d e f o r m e d quartz polycrystal. Measurement of axes orientations shows a great circle distribution with a maximum in the XZ piane 30 ° from the lineation (x). O r i e n t a t i o n relationships across grain b o u n d a r i e s were measured to determine the degree of slip system alignment between a d j a c e n t grains. Close alignment of the slip directions occurs for only 7 out of 23 grain pairs measured. Three a l i g n m e n t s were sub-parallel to Y with the rest sub-parallel to the main maximum. Despite the average misalignment of the slip directions, the occurrence of some dislocations apparently crossing grain boundaries suggests that slip transfer may be possible across general grain boundaries in quartz. Measurement of dislocation densities (p) showed no strong dependence on orientation. All grains had p in the range 1.4 - 5.6 x iO12-m -2. There was a tendency for old unrecrystallised grains and new recrystallised grains oriented for rhomb and prism slip to have a higher p = 2.5-5.6 x iO12.m-2 than new grains o r i e n t e d for basal slip p = 2.2 - 1.4 x lO12-m -2. Analysis of dislocation Burgers vectors showed that multiple slip in all three directions occurred in all grains analysed, including those oriented for easy single slip. A few dislocation-free grains were ~ L ~ ~.~ by static recrystallisation~ which involved o r i e n t e d nucleation, with the [c] axis of the ne.~ grain parallel to en axis of the old grain.
450
particles represent free IgG or antigen receptors on B-lymphocytes. KI-67, A MONOCLONAL ANTIBODY DIRECTED AGAINST A NUCLEOLAR COMPONENT R. Verheijen, H. Kuijpers, H. Beck, F.J.R. Rietveld and F.C.S. Ramaekers D e p a r t m e n t of P a t h o l o g y , U n i v e r s i t y H o s p i t a l Nijmegen, N i j m e g e n , The Netherlands
Ki-67 is a commercially available monoclonal antibody which reacts with a nuclear antigen expressed in all proliferating cells. In cultured cell lines Ki-67 stains an antigen present in the nucleolus of interphase cells and reacts with an interchromatinous network in mitotic cells. Immunoelectron microscopy revealed that the antigen is localized at the periphery of the nucleolus in interphase cells. Despite many efforts, it was impossible to identify the antigen by means of immunoblotting or immunoprecipitation assays. Testing the species specificity of Ki-67 on cultured cell lines by means of indirect immunofluorescence revealed that the antibody reacted only with human cell nuclei and those of rhesis monkey kidney. With the immunoperoxidase staining technique on tissue sections, the cryptic cells of rabbit small intestine were also found to be positive with Ki-67. Flow-cytometry analysis was performed using two human cell lines, HeLa and Molt-4, stained with propidium iodide for DNA and with FITC-labeled second antibodies for Ki-67. Because of the different FITC signals between mitotic cells and G2 phase cells, we were able to separate the G2/M fraction into highly enriched fractions of G2 and M cells, respectively. In situ fractionation experiments on human monolayer cell lines, resulting in nuclear matrix preparations attached to a glass slide, showed that the Ki-67 antigen remained associated with such structures.
INTEGRATION OF I ~ G E - A N ~ L Y S I S IN ELECTRON MICROSCOPES
SYSTEMS
Teun Vet Zeiss
N e d e r ! a n d B.V.
Scanning and transmission electron microscopes were designed and successfully employed to reproduce submicroscopic
structures. The photograph often was, and is, the final result. Owing to rapid developments in the semiconductor industry, one is at pre~ent able to build computer systems which can optimize and analyze the often complex images produced by the electron microscope. But to obtain a reproducible result an intensive interaction between the electron microscope and the imageanalysis system will be required, particularly when one takes an automatic analysis of several fields into account. With the new SEM (CSM-950) and TEM (CEM-902) Zeiss, W e s t Germany, has managed to completely integrate an imageanalyser, i.e., the image is not only transferred from the electron microscope but every possible parameter and setting of the electron microscope can be regulated and adjusted. In this way one can act on the image information, e.g., changing the setting of the EM or referring the automatic motor stage to another position on the specimen. A special feature of the SEM is that other detector systems can be linked up and their signals can be processed in the imageanalysis part. Thus a relatively simple-to-use and high-performance analytical measuring system for qualitative and quantitative microstructure unalysis has now become available.
SUBCELLULAR LOCALIZATION OF LamB-LacZ HYBRID PROTEINS AFTER CRYO-ULTRAMICROTOMY OR FREEZE-SUBSTITUTION W.F. Voorhout, J.J.M. Leunissen-Bijvelt and T. Van Der Krift D e p a r t m e n t o f M o l e c u l a r Cell Biology, U n i v e r s i t y of Utrecht, P a d u a l a a n 8, 3584 CH U t r e c h t , The N e t h e r l a n d s
The subcellular localization of LamB-LacZ hybrid proteins in the Escherichia coli K-12 strains pop3234 and pop ~299 was investigated by two immunoelectron-microscopical techniques. To induce the synthesis of the LamB-LacZ hybrid proteins, cells were grown for 5 hr in 0.4% maltose. Part of the cell culture was fixed in 2% paraformaldehyde/ 0.5% glutaraldehyde and further processed for cryo-ultramicrotomy. The rest of the cells were gently pelleted, rapidly frozen in liquid propane and submitted to freeze substitution. The cells were substituted for 8 hours at -90°C in 1% osmiumtetroxide, 3% glutaraldehyde and 0.5% uranyl acetate in methanol, embedded in Lowicryl HM20 and pol~merized at -45oc.
Abstracts o/The Netherlands Societ)' of Electron Microscopy
L a b e l l i n g of ultrathin cryosections of the cells with a n t i - L a m B or anti-8g a l a c t o s i d a s e protein serum and protein A-gold complexes revealed that the hybrid p r o t e i n s accumulated in the cytoplasm or w e r e associated w i t h membranelike s t r u c t u r e s in the cytoplasm. Freezes u b s t i t u t i o n clearly r e v e a l e d these membrane-like structures and special areas of a c c u m u l a t e d hybrid proteins. After f r e e z e - s u b s t i t u t i o n in the presence of osmiumtetroxide, the h y b r i d proteins could still be detected w i t h immunogold probes. The accumulated form of the hybrid p r o t e i n could only be detected using a n t i - 8 - g a l a c t o s i d a s e serum and not using a n t i - L a m B serum.
T H R E E - D I M E N S I O N A L V I S U A L I Z A T I O N TECHNIQUES FOR CONFOC~L M I C R O S C O P k H.T.M. van der Voort, and N. N a n n i n g a
G.J.
Brakenhoff
Dept. of E l e c t r o n M i c r o s c o p y and M o l e c u l a r C y t o l o g y , U n i v e r s i t y of A m s t e r d a m , P l a n t a g e M u i d e r g r a c h t 14, 1018 TV A m s t e r d a m , The N e t h e r l a n d s
The optical properties of a Confocal Scanning Laser Microscope (CSLM) operated in fluorescence allow for sampling a specific volume in the specimen. The small, d i f f r a c c i o n - i i m i t e d size of this volume permits the m e a s u r e m e n t of microscopic 3D fluorescence distributions. Therefore, confocal m i c r o s c o p y forms an ideal base for the study of the 3D morphology of biological objects. To e x p l o i t the c a p a b i l i t i e s of a CSLM fully, it is n e c e s s a r y to have it coupled d i r e c t l y to a c o m p u t e r system. This s y s t e m can then p e r f o r m tasks like i n s t r u m e n t control and d a t a acquisition, image processing, image analysis and the v i s u a l i z a t i o n of 3D images. This last task is of prime importance in order to assess a newly scanned image quantitatively. A good v i s u a l i z a t i o n method must meet v a r i o u s contradictory conditions: it should provide clear, and as many as possible, visual clues to enable space perception; presentation of the results should be convenient, and the used computer a l g o r i t h m should be fast enough to allow for interactive usage during a microscop±c session. Currently, the following techniques are used in our laboratory: g e n e r a t i o n of s t e r e o s c o p i c pairs, computer graphics (i.e. solid modelling) and a newly developed technique based on computer simulation of a fluorescence process.
4 51
In the paper presented we discuss these methods and their merits, as well as image p r o c e s s i n g techniques required in reprocessing.
DISLOCATION SUDIrRUCTUkES IN A QUARTZITE WITH A T Y P E I CROSS GIRDLE FABRIC L.E.P. W e n m a k e r s and M~R. Drury Instituut voor Aardwetenschappen, Rijksuniversiteit Utrecht, B u d a p e s t l a a n 4, 3584 CD U t r e c h t
Slip s y s t e m geometry and dislocation substructures have been studied in a naturally d e f o r m e d quartz polycrystal. Measurement of axes orientations shows a great circle distribution with a maximum in the XZ piane 30 ° from the lineation (x). O r i e n t a t i o n relationships across grain b o u n d a r i e s were measured to determine the degree of slip system alignment between a d j a c e n t grains. Close alignment of the slip directions occurs for only 7 out of 23 grain pairs measured. Three a l i g n m e n t s were sub-parallel to Y with the rest sub-parallel to the main maximum. Despite the average misalignment of the slip directions, the occurrence of some dislocations apparently crossing grain boundaries suggests that slip transfer may be possible across general grain boundaries in quartz. Measurement of dislocation densities (p) showed no strong dependence on orientation. All grains had p in the range 1.4 - 5.6 x iO12-m -2. There was a tendency for old unrecrystallised grains and new recrystallised grains oriented for rhomb and prism slip to have a higher p = 2.5-5.6 x iO12.m-2 than new grains o r i e n t e d for basal slip p = 2.2 - 1.4 x lO12-m -2. Analysis of dislocation Burgers vectors showed that multiple slip in all three directions occurred in all grains analysed, including those oriented for easy single slip. A few dislocation-free grains were ~ L ~ ~.~ by static recrystallisation~ which involved o r i e n t e d nucleation, with the [c] axis of the ne.~ grain parallel to en axis of the old grain.