- Email: [email protected]
Halogens standardization by SIMS microscopy for application in biological samples
264
32• Colloque SFME, Rouen-Mont St Aignan, juillet 1992
HALOGENS S T A N D A R D I Z A T I O N BY SIMS MICROSCOPY FOR APPLICATION IN BIOLOGICAL SAMPLES lEUSSET |osette, HALPERN Sylvain, BRIAN(~ON Colette, GARCIA Fatiha, FRAGU Philippe
Equipe de Microscopie ionique, Inserm U66 - Institut Gustave Roussy - 39 rue Camille Desmoulins - 94805 Villejuif Cedex The absolute q u a n t i f i c a t i o n of an e l e m e n t in biological tissues by SIMS microscopy is limited b y physical p a r a m e t e r s w h i c h are difficult to assess. H o w e v e r a relative q u a n t i t a t i v e a p p r o a c h is possible u s i n g an internal reference (e.g. carbon) p r e s e n t in a large, c o n s t a n t a n d h o m o g e n e o u s a m o u n t in specimen as well as in e m b e d d i n g resin. This p r o c e d u r e allows to normalize quantification with s t a n d a r d curves a n d to express the results in ~ g / m g of tissue. The feasability of this a p p r o a c h has been previously d e m o h s t r a t e d for iodine (127I)*. The p u r p o s e of this work was to extend this m e t h o d to other halogens used if necessary as metabolic markers 09F, 35C], 79Br). S t a n d a r d s w e r e p r e p a r e d a c c o r d i n g to c r i t e r i a of h o m o g e n e i t y a n d stability in m a t r i x similar to resins usually used for e m b e d d i n g biological Ossues. They were obtained first by s y n t h e z i s i n g ~ - b u t y l - m e t h a c r y l a t e w i t h the c h o s e n e l e m e n t i n c o r p o r a t e d in the molecule, then c o p o l y m e r i s e d at different concentrations (10, 5, 1, 0.5, 0.1, 0.05 and 0.01 g g / m g ) with methyl m e t h a c r y l a t e . For each s t a n d a r d , q u a n t i t a t i v e analysis w e r e p e r f o r m e d in a C a m e c a IMS 3F on thin sections (2-3 ~m) d e p o s i t e d on gold holders. D e p t h analysis were p e r f o r m e d on areas of 8 g m a n d 60 g m d i a m e t e r for 30 to 60 m i n u t e s u n d e r cesium b o m b a r d m e n t . Results, o b t a i n e d w i t h a precision better than 0,1% on 100-120 m e a s u r e m e n t s for each s t a n d a r d dilution, s h o w that the relation b e t w e e n the m e a s u r e d signal intensity a n d the e l e m e n t a l c o n c e n t r a t i o n is linear a l o n g the d e c a d e s studied w h i c h c o r r e s p o n d to the physiological concentrations. A similar m e t h o d o l o g y of s t a n d a r d i z a t i o n is n o w in progress in our laboratory for other elements (31p, 32S)" *TELENCZAK P., BESSODES M., RICARD M., HALPERN S. and FRAGU P. (1989) CR Acad Sci Paris, 308, 479-484. CRYOFSEM, A NEW CRYOSYSTEM S U B S T I T U T I O N AND LOW T E M P E R A T U R E P R O C E S S I N G BIOLOGICAL SAMPLES.
QUINTANA
Carmen. I N S E H M V,;l.l..~,,,h,'~,ttMor(Ii.~1~ce)
U 303
OF FREEZEE M B E D D I N G FOR
La Da/se
1~/'3
(~2St~
A new integrated system has been developed in c o l l a b o r a t i o n with the U n i v e r s i t y of B a r c e l o n a (Programme MERCURE) (i). In this CRYOsystem the three procedures (FreezeSubstitution, cryoinfiltration and low temperature EMbedding) can be carried out within the same complex. The main c h a r a c t e r i s t i c s of this C R Y O F S E M system , are: * the cold source is a freezer (minimal temperature 180 K), so no use of cryogenic fluids is required, , total p r o t e c t i o n from toxic vapours coming from Lowicryl resins, * easy manipulation, * efficient shaking of samples in freezes u b s t i t u t i o n and c r y o i n f i l t r a t i o n liquids, * control of temperature in all the different procedures, * highly r e p r o d u c i b l e results. The C R Y O F S E M system has been s u c c e s s f u l l y assayed following f r e e z e - s u b s t i t u t i o n in pure acetone and c r y o e m b e d d i n g in HM23 Lowicryl. We present here the first ultrastructural, microanalytical and immunocytochemical (2) results o b t a i n e d with this system. Within the aim of c o m m e r c i a l i s a t i o n by the French Society RUA INSTRUMENTS, some modifications, leading to a maximal s i m p l i f i c a t i o n in the handling of the samples between the different treatments, have been performed. (I) QUINTANA C. ( 1 9 9 1 ) Colloque FrancoIberique Microscopie Electronique (Barcelona) 58-59. (2) SANTA-CRUZ M.C.,VISA N., FIBLA J., LOPEZ-IGLESIAS C., QUINTANA C. et GONZALEZ-DUARTE R. ( 1 9 9 2 ) EUREM'92.
8a
THE CELLULOSE S Y S T E M O F C/.ADOPHORA D E N S A
V U O N G - R o ~ e r 1, CHANZY H e n r i I a n d SUGIYAMA J u n j l 2
1 Centre de Recherches sur les Macromol~cules VOgOtales (CERMAIO, CNRS, BP 53 X, 38041 Grenoble cedex, France 2 Laboratory o f Wood Physics, Department of Forest Products, Faculty o f Agriculture, The University o f Tokyo, Yayoi. Bunkyo-ku, Tokyo 113, J a p a n S p e c i m e n s f r o m t h e g r e e n "alga Cladophora d e n s a were collected at Chlkura, Chlba Prefecture, Japan. Their cell w a l l s w e r e p u r i f i e d b y b o i l i n g i n w a t e r , e x t r a c t e d w i t h d i l u t e KOH, b l e a c h e d w i t h N a C I O 2 , w a s h e d a n d stored in ethanol. The purified walls which consisted e s s e n t i a l l y of c e l l u l o s e w e r e s e c t i o n e d a n d a n a l y z e d b y diffraction contrast transmission electron microscopy a n d m i c r o d l f f r a c t i o n . In a d d i t i o n , t h e w a l l s w e r e a l s o delaminated and their cellulose microfibrils visualized and analyzed by selected area electron diffraction. T h e c e l l u l o s e of Cladophora w a s h i g h l y c r y s t a l l i n e a n d c o n s i s t e d o f c r i s s c r o s s e d l a y e r s of e n d l e s s m ! c r o fibrils h a v i n g a n a v e r a g e l a t e r a l d i m e n s i o n s of 3 0 0 A. In cross section, these mlcrofibrils had a square-like section d e f i n e d a l o n g t h e 1 0 0 a n d 0 1 0 p l a n e s o f c e l l u l o s e I (I). Within a microflbriUar bundle, the individual microfibrils were statistically packed in an antlparallel fashion. The c e l l u l o s e s y s t e m of Cladophora is t h e r e f o r e v e r y s i m i l a r to t h a t of Microdictyon (2) or t h a t of Valonia (3). (I) T h e s e i n d i c e s r e f e r to t h e t r i c l i n i c u n i t cell definition. (2) SUGIYAMA J . , V U O N G R. a n d CHANZY H. (1991). Macromolecules, 2 4 , 4 1 6 8 . (3) REVOL J . F . a n d GORING D.A.I. (1983) Polymer, 2 4 , 1547.
FROM MICROMETER TO ATOMIC SCALE APPLICATIONS OF STM AND AFM IN BIOLOGY J , ~ , ~ P ~ I X ~ , f . I ~ (1) (2), AUDUC Nathalie (2). RINGENBACH Anne (2). STEVENSON Isabelle (2), TRAN MINH DUC (2)
(1)Science et Surface,78 route de PARIS, 69260 CHARBONNIERES (2)CENATS, Universit~ClaudeBernard, 43 boulevard du 11 novembre1918, 69622 VILLEURBANNE We present the possibilities by using STM (Scanning Tunneling Microscopy) and AFM (Atomic Force Microscopy) to image topographical surfaces of biological samples at a few micron down to atomic scale resolution. The morphology of human hair is investigated by AFM in air and in physiological solution as well at suecesive and significant scales, allowing to image overlapping layers of cuticle with 600 nm high steps, then 50 nm diameter depressions, and ultimately the molecular structure of polypeptides from the epicuticle layer. Morphology of erythrocytes as deposited on a glass substrate is observed by AFM and the surf.ace structure of the membrane is shown to be determined at few nanometer scale in ambient atmosphere. Structures of molecules of biological interest such as hyaluronic acid (HA), behenic acid (BA), poly-13-benzyI-L-aspartste (P[3BA) and poly-L-alanine (PA) are studied at submoleoular scale by these new local probes : - STM allows to observe HA deposited on a highly oriented pyrolitic graphite'. The molecular film appears as a network with a distance of 1.6 run between ordered filaments. - AFM allows to determine the hexagonal close-packed geometry of the alkyl tail groups from one monolayer Langmuir Blodgett (LB) film of behenic acid deposited on a glass substrate. This periodic hexagonal structure displays an intermoleoular distance of 0.45 nm. - The molecular distribution of LB monolayer of P[3BA and PA in periodic a helices increases the order and homogeneity of LB films. The a helix conformation of these polyaminoacids is characterized by interchain distances of 0.85 nm and 0.79 nm respectively. The helix turn is measured at 0.54 rim for P~BA. Artefacts and degradations of sample surfaces due to excessive loading forces of the cantilever are discussed.
32• Colloque SFME, Rouen-Mont St Aignan, juillet 1992
HALOGENS S T A N D A R D I Z A T I O N BY SIMS MICROSCOPY FOR APPLICATION IN BIOLOGICAL SAMPLES lEUSSET |osette, HALPERN Sylvain, BRIAN(~ON Colette, GARCIA Fatiha, FRAGU Philippe
Equipe de Microscopie ionique, Inserm U66 - Institut Gustave Roussy - 39 rue Camille Desmoulins - 94805 Villejuif Cedex The absolute q u a n t i f i c a t i o n of an e l e m e n t in biological tissues by SIMS microscopy is limited b y physical p a r a m e t e r s w h i c h are difficult to assess. H o w e v e r a relative q u a n t i t a t i v e a p p r o a c h is possible u s i n g an internal reference (e.g. carbon) p r e s e n t in a large, c o n s t a n t a n d h o m o g e n e o u s a m o u n t in specimen as well as in e m b e d d i n g resin. This p r o c e d u r e allows to normalize quantification with s t a n d a r d curves a n d to express the results in ~ g / m g of tissue. The feasability of this a p p r o a c h has been previously d e m o h s t r a t e d for iodine (127I)*. The p u r p o s e of this work was to extend this m e t h o d to other halogens used if necessary as metabolic markers 09F, 35C], 79Br). S t a n d a r d s w e r e p r e p a r e d a c c o r d i n g to c r i t e r i a of h o m o g e n e i t y a n d stability in m a t r i x similar to resins usually used for e m b e d d i n g biological Ossues. They were obtained first by s y n t h e z i s i n g ~ - b u t y l - m e t h a c r y l a t e w i t h the c h o s e n e l e m e n t i n c o r p o r a t e d in the molecule, then c o p o l y m e r i s e d at different concentrations (10, 5, 1, 0.5, 0.1, 0.05 and 0.01 g g / m g ) with methyl m e t h a c r y l a t e . For each s t a n d a r d , q u a n t i t a t i v e analysis w e r e p e r f o r m e d in a C a m e c a IMS 3F on thin sections (2-3 ~m) d e p o s i t e d on gold holders. D e p t h analysis were p e r f o r m e d on areas of 8 g m a n d 60 g m d i a m e t e r for 30 to 60 m i n u t e s u n d e r cesium b o m b a r d m e n t . Results, o b t a i n e d w i t h a precision better than 0,1% on 100-120 m e a s u r e m e n t s for each s t a n d a r d dilution, s h o w that the relation b e t w e e n the m e a s u r e d signal intensity a n d the e l e m e n t a l c o n c e n t r a t i o n is linear a l o n g the d e c a d e s studied w h i c h c o r r e s p o n d to the physiological concentrations. A similar m e t h o d o l o g y of s t a n d a r d i z a t i o n is n o w in progress in our laboratory for other elements (31p, 32S)" *TELENCZAK P., BESSODES M., RICARD M., HALPERN S. and FRAGU P. (1989) CR Acad Sci Paris, 308, 479-484. CRYOFSEM, A NEW CRYOSYSTEM S U B S T I T U T I O N AND LOW T E M P E R A T U R E P R O C E S S I N G BIOLOGICAL SAMPLES.
QUINTANA
Carmen. I N S E H M V,;l.l..~,,,h,'~,ttMor(Ii.~1~ce)
U 303
OF FREEZEE M B E D D I N G FOR
La Da/se
1~/'3
(~2St~
A new integrated system has been developed in c o l l a b o r a t i o n with the U n i v e r s i t y of B a r c e l o n a (Programme MERCURE) (i). In this CRYOsystem the three procedures (FreezeSubstitution, cryoinfiltration and low temperature EMbedding) can be carried out within the same complex. The main c h a r a c t e r i s t i c s of this C R Y O F S E M system , are: * the cold source is a freezer (minimal temperature 180 K), so no use of cryogenic fluids is required, , total p r o t e c t i o n from toxic vapours coming from Lowicryl resins, * easy manipulation, * efficient shaking of samples in freezes u b s t i t u t i o n and c r y o i n f i l t r a t i o n liquids, * control of temperature in all the different procedures, * highly r e p r o d u c i b l e results. The C R Y O F S E M system has been s u c c e s s f u l l y assayed following f r e e z e - s u b s t i t u t i o n in pure acetone and c r y o e m b e d d i n g in HM23 Lowicryl. We present here the first ultrastructural, microanalytical and immunocytochemical (2) results o b t a i n e d with this system. Within the aim of c o m m e r c i a l i s a t i o n by the French Society RUA INSTRUMENTS, some modifications, leading to a maximal s i m p l i f i c a t i o n in the handling of the samples between the different treatments, have been performed. (I) QUINTANA C. ( 1 9 9 1 ) Colloque FrancoIberique Microscopie Electronique (Barcelona) 58-59. (2) SANTA-CRUZ M.C.,VISA N., FIBLA J., LOPEZ-IGLESIAS C., QUINTANA C. et GONZALEZ-DUARTE R. ( 1 9 9 2 ) EUREM'92.
8a
THE CELLULOSE S Y S T E M O F C/.ADOPHORA D E N S A
V U O N G - R o ~ e r 1, CHANZY H e n r i I a n d SUGIYAMA J u n j l 2
1 Centre de Recherches sur les Macromol~cules VOgOtales (CERMAIO, CNRS, BP 53 X, 38041 Grenoble cedex, France 2 Laboratory o f Wood Physics, Department of Forest Products, Faculty o f Agriculture, The University o f Tokyo, Yayoi. Bunkyo-ku, Tokyo 113, J a p a n S p e c i m e n s f r o m t h e g r e e n "alga Cladophora d e n s a were collected at Chlkura, Chlba Prefecture, Japan. Their cell w a l l s w e r e p u r i f i e d b y b o i l i n g i n w a t e r , e x t r a c t e d w i t h d i l u t e KOH, b l e a c h e d w i t h N a C I O 2 , w a s h e d a n d stored in ethanol. The purified walls which consisted e s s e n t i a l l y of c e l l u l o s e w e r e s e c t i o n e d a n d a n a l y z e d b y diffraction contrast transmission electron microscopy a n d m i c r o d l f f r a c t i o n . In a d d i t i o n , t h e w a l l s w e r e a l s o delaminated and their cellulose microfibrils visualized and analyzed by selected area electron diffraction. T h e c e l l u l o s e of Cladophora w a s h i g h l y c r y s t a l l i n e a n d c o n s i s t e d o f c r i s s c r o s s e d l a y e r s of e n d l e s s m ! c r o fibrils h a v i n g a n a v e r a g e l a t e r a l d i m e n s i o n s of 3 0 0 A. In cross section, these mlcrofibrils had a square-like section d e f i n e d a l o n g t h e 1 0 0 a n d 0 1 0 p l a n e s o f c e l l u l o s e I (I). Within a microflbriUar bundle, the individual microfibrils were statistically packed in an antlparallel fashion. The c e l l u l o s e s y s t e m of Cladophora is t h e r e f o r e v e r y s i m i l a r to t h a t of Microdictyon (2) or t h a t of Valonia (3). (I) T h e s e i n d i c e s r e f e r to t h e t r i c l i n i c u n i t cell definition. (2) SUGIYAMA J . , V U O N G R. a n d CHANZY H. (1991). Macromolecules, 2 4 , 4 1 6 8 . (3) REVOL J . F . a n d GORING D.A.I. (1983) Polymer, 2 4 , 1547.
FROM MICROMETER TO ATOMIC SCALE APPLICATIONS OF STM AND AFM IN BIOLOGY J , ~ , ~ P ~ I X ~ , f . I ~ (1) (2), AUDUC Nathalie (2). RINGENBACH Anne (2). STEVENSON Isabelle (2), TRAN MINH DUC (2)
(1)Science et Surface,78 route de PARIS, 69260 CHARBONNIERES (2)CENATS, Universit~ClaudeBernard, 43 boulevard du 11 novembre1918, 69622 VILLEURBANNE We present the possibilities by using STM (Scanning Tunneling Microscopy) and AFM (Atomic Force Microscopy) to image topographical surfaces of biological samples at a few micron down to atomic scale resolution. The morphology of human hair is investigated by AFM in air and in physiological solution as well at suecesive and significant scales, allowing to image overlapping layers of cuticle with 600 nm high steps, then 50 nm diameter depressions, and ultimately the molecular structure of polypeptides from the epicuticle layer. Morphology of erythrocytes as deposited on a glass substrate is observed by AFM and the surf.ace structure of the membrane is shown to be determined at few nanometer scale in ambient atmosphere. Structures of molecules of biological interest such as hyaluronic acid (HA), behenic acid (BA), poly-13-benzyI-L-aspartste (P[3BA) and poly-L-alanine (PA) are studied at submoleoular scale by these new local probes : - STM allows to observe HA deposited on a highly oriented pyrolitic graphite'. The molecular film appears as a network with a distance of 1.6 run between ordered filaments. - AFM allows to determine the hexagonal close-packed geometry of the alkyl tail groups from one monolayer Langmuir Blodgett (LB) film of behenic acid deposited on a glass substrate. This periodic hexagonal structure displays an intermoleoular distance of 0.45 nm. - The molecular distribution of LB monolayer of P[3BA and PA in periodic a helices increases the order and homogeneity of LB films. The a helix conformation of these polyaminoacids is characterized by interchain distances of 0.85 nm and 0.79 nm respectively. The helix turn is measured at 0.54 rim for P~BA. Artefacts and degradations of sample surfaces due to excessive loading forces of the cantilever are discussed.