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Quantification assay by SIMS microscopy of non organified thyroid iodine after cryopreparation
32e Colloque SFME, Rouen-Mont St Aignan, juillet 1992
THREE-DIMENSIONAL AND
Ag-NOR
MICROSCOPY.
ORGANIZATION
PROTEINS
Jun~ra
OF
STUDIED
H.
R.,
rDNA BY
GENES
CONFOCAL
Robert-Fortel
G#raud
G.
and
Hernandez-Verdun
Monod,
2
Place
Jussieu,75251
D.Institut Paris
Cedex
I., J. 05
France. We u s e d c o n f o c a l laser s c a n n i n g m i c r o s c o p y (CLSM) to i n v e s t i g a t e in i n t e r p h a s e and m e t a p h a s e PtK 1 cells, the 3-D o r g a n i z a t i o n of the r i b o s o m a l genes (rDNA) and of a set of p r o t e i n s s p e c i f i c of the nucleolar organizer regions (NORs) . T h e rDNA l a b e l l e d by n o n i s o t o p i c in situ h y b r i d i z a t i o n was detected by epifluorescence and the proteins labelled by the s i l v e r (Ag-NOR proteins) were d e t e c t e d by r e f l e x i o n light mode. The r D N A was m a p p e d using b i o t i n y l a t e d DNA p r o b e for 45S rDNA sequences. The probe consisted of a 6.6 kb fragment, i n c l u d i n g the d o w n s t r e a m half of the 18S and 90% of the 28S r D N A c o d i n g sequences. In metaphase chromosomes, in situ hybridization d e m o n s t r a t e d the p r e s e n c e of rDNA in the s e c o n d a r y c o n s t r i c t i o n of the X c h r o m o s o m e s with an a x i a l d i s t r i b u t i o n and a l s o l a t e r a l e x p a n s i o n s . The 3-D reconstruction of the Ag-NOR protein signals e v i d e n c e d the p r e s e n c e of these p r o t e i n s in the secondary constriction where they formed a crescent-shaped structure around the axial chromatin pedicule. During interphase, in situ hybridization in intact cell monolayers and i s o l a t e d nuclei s h o w e d the rDNA g e n e s d i s t r i b u t e d as intense fluorescent spots linked by weak s i g n a l s in the inner r e g i o n s of the n u c l e o l i . We conclude that the rDNA is n o t homogeneously distributed in the internal regions of the nucleoli. In the same n u c l e o l a r regions, the AgNOR p r o t e i n s were r e v e a l e d as g r a n u l e s l i n k e d by thin filaments. These images i n d i c a t e s i m i l a r 3-D d i s t r i b u t i o n s for rDNA probes and A g - N O R p r o t e i n s .
SECONDARY ION MASS SPECTROMETRY (SIMS) AND ASSOCIATED IMAGE PROCESSING: AN ENVIRONMENTAL SURVEYPROCEDURE OF PLANT RADIOACTIVE CONTAMINATION CHASSARD-BOUCHAUD Colette~ESCAIG Fran~ois~,BOUMATI Pierre~ Bi31]RL-AT--Yves--6~r~-6-d-DUCOUSSO Roger~'" • Cep~Cre de Microano~y~e app~iqu~e a la Biologie(INSERM),Lab. Biophysique,Facult~ de M~decine, 6 rue du Gl.Sarrail, Cr~teil, • . S ~ R , B . P. 208,9 ; 311 Montlhery Cedex, France. ""SMCB, B.P.208, 913;1 Montlh~y Cedex, France. 239 Pu, one of the most abundant of plutonium isotopes,has been distributed worldwide from nuclear weapons testing inje cted into the atmosphere and then deposited to the earth's surface. As plutonium tends to bind t i g h t l y in soils, the problem of plant uptake had to be investigated. Root uptake and direct deposition on f o l i a r surfaces are the two pathways by which f a l l o u t has contaminated plants. Cazua~na equ~e~foLi.a, also called "FX~zw" or "Ax~to", from the intertropical zone has been investigated. The d i f ferent tissues were fixed chemically, embeddedin paraffin, sectioned ( 5 Nm ) and deposited on a highly pure gold specimen holder. SIMS was performed, using the ion microscope CAMECA IMS 300, associated with the PERICOLOR 2000 Image processing system ( I ). Results obtained from the ion images demonstrated a higher uptake via the leaves than via the roots. These results a l lowed a precise localization of Pu deposits within the cells and no adsorption phenomenawere observed in both tissues. Data obtained, using SIMS microanalysis were in complete agreement with the ones, obtained from bulk specimens, using radiochemical techniques. The advantages of SIMS imaging, in particular i t s very high s e n s i t i v i t y , for the detection of radioactive isotopes, has been discussed and compared to cla ssical autoradiography techniques. ( i ) Chassard-Bouchaud C.,Escaig F.,Boumati P.,Galle C.,1992, Microanalysis and image processing of stable and radioactive elements in ecotoxicology.Current developments using SIMS mi coscope and electron microprobe. Biol.Cell, 74: 59-74. This wo,'d'z u~us fZnane.i.aZZy suppo.,t;ted by .t.he French Idi_nXA.6,uj of Defence and the S~ruiee ~ e de C o n t ~ l e Biologiqae ( c o r t t ~ c t 59 / SMCB ).
259
CILIARY MOTILITY IN CULTURED NASAL E P I T H E L I U M OF NORMAL SUBJECT AND KARTAGENER PATIENT ZAHM Jean-Marie. PIERROT Denis, GILAIN Laurent, PUCHELLE Edith INSERM U314, Universit# de Reims and Centre Hospitalier Intercommunal,Crdteil, Fra.ce. Mucociliary clearance is an important defence mechanism of the respiratory tract. Its effectiveness relies in part on the integrity of ciliary movements. In conditions where ciliary function is impaired, as with primary ciliary dyskinesia, frequent infections are common and may also be accompanied by acquired ciliary changes which are also frequently found in chronic pulmonary disease. Culture of respiratory epithelial cells may be an aherna,ve to objectively determine if ciliary alterations are primary or acquired. The aim of the present work was to study the functional activity of cultured nasal ciliated cells of a Kartagener patient with primary ciliary dyskinesia and to compare the values to that obtained in normal subjects. Explants from a nasal polyp of a normal subject and of a Kartagener patient were cultured on a collagen I matrix in RPMI supplemented culture medium. After 5 days of culture, the culture dishes were placed on the heating stage of a phase-contrast inverted microscope and video recordings of the outgrowths were made. By using a photodetector placed on the videoscreen and a fast Fourier transform analysis, ciliated cell mean beating frequency and the associated standard deviation were calculated. The native polyp tissue from the Kanagener patient exhibited a mean ciliary beating frequency of 6.5 + 1.4 Hz (range: 4.4 to 9.9 Hz). The mean beating frequency of ciliated cells from the Kartagener cultured explants (7.9 + 2. I Hz0 range: 3.4 to 12.5 Hz) was significantly lower (p< 0.001) than the ciliary beating frequency of cells from normal cultured explants (12.1 + 0.8 Hz, range: 10.7 to 12.9 Hz). The ciliary beating variability, reflected by the standard deviation of the mean beating frequency was twice as high in the Kartagener patient compared to the normal subject. The percentage of ciliated cells with normal beating (>8 Hz) was 98% in the normal subject and only 47.5% in the Kartagener patient. These results indicate that different beating patterns may be obtained in cultured ciliated cells from patients suffering from primary ciliary dyskinesia. One can observe either quite normal or decreased and inhomogeneous ciliary beating patterns, whereas the beating pattern of the native tissue seemed abnormal. The culture of ciliated cells from patients with primary ciliary dyskinesia could therefore be useful to precisely determine the extent of the ciliary defect, independently from any acquired defect.
Q U A N T I F I C A T I O N ASSAY BY SIMS MICROSCOPY OF N O N ORGANIFIED THYROID IODINE AFTER CRYOPREPARATION B R I A N ~ O N Colette, JEUSSET Josette, HALPERN Sylvain, a n d FRAGU Philippe Equipe de Microscopie ionique, htserm U66 - Institut Gustave Roussy, 39 rue Camille Desmoulins - 94805 Villejuif Cedex The mean advantage of anhydrous cryopreparation method for microanalysis is to preserve both diffusible and organified elements because it avoids rapid lost of diffusible fraction in aqueous chemical fixative and embedding resin. Our purpose in this study was to evidence non organified iodine (iodide) in thyroid by secondary ion mass spectrometry (SIMS) microscopy. This iodide is reduced to 1-2% in normal thyroid in which almost all of iodine is linked by covalent bounds in thyroglobulin stored in follicular lumens. Iodide is strongly increased in glands submitted to propylthiouracil (PTU). drug prescribed in human treatment of hyperthyroidism which blocks iodine organification by inhibition of thyroid peroxidase. After PTU treatment, organified iodine concentration is greatly decreased (factor 17) as it can be shown by SIMS after chemical fixation. Swiss mice, fed a normal iodine diet, were treated either with PTU 1% (10 days) or kept as controls. Thyroids were cryofixed by ultrarapid immersion (5000K.sec-i, 2m.sec-0 in liquid technic propan (77K), then cryosubstituted in aceton (183K), embedded in Lowicryl K11M (213K) and serially cut. Sections intended to microanalysis (31.tm) were laid on gold holders either on pure water (PW) in which iodide can diffuse, or on ethylene glycol (EG), organic solvent used for minimizing extraction and redistribution of diffusible elements. Sections of 1.51.tm served as histological controls b., photonic microscopy. Measurement by SIMS microscopy of local follicular concentration of 127I evidenced significant differences (p<0.01) not detectable by mapping alone. The total iodine concentration, evaluated o n "EG sections", varied only by a factor 6 between PTU treated (1.14 + 0.23 I.tg/mg, mean + SE) and control mice (6.03 + 0.51 gg/mg). Furthermore, iodine evaluated on "PW sections", which is essentially organified iodine, varies by about a factor 10 between PTU tr~ted (0.70 + 0.21 gg/mg) and control mice (5.58 + 0.32 I.tg/mg). Finally, the diffusible fraction of thyroid iodine, corresponding at least to the difference between measurements on "EG sections" and on "PW sections", is extremely low in controls (less than 1%) while it reaches 44% in PTU treated mice. Our results give proof of the cryopreparation efficiency for maintenance, even if partial, of diffusible iodide, and of its methodological interest for microanalysis of any diffusible element in any tissue.
3a
THREE-DIMENSIONAL AND
Ag-NOR
MICROSCOPY.
ORGANIZATION
PROTEINS
Jun~ra
OF
STUDIED
H.
R.,
rDNA BY
GENES
CONFOCAL
Robert-Fortel
G#raud
G.
and
Hernandez-Verdun
Monod,
2
Place
Jussieu,75251
D.Institut Paris
Cedex
I., J. 05
France. We u s e d c o n f o c a l laser s c a n n i n g m i c r o s c o p y (CLSM) to i n v e s t i g a t e in i n t e r p h a s e and m e t a p h a s e PtK 1 cells, the 3-D o r g a n i z a t i o n of the r i b o s o m a l genes (rDNA) and of a set of p r o t e i n s s p e c i f i c of the nucleolar organizer regions (NORs) . T h e rDNA l a b e l l e d by n o n i s o t o p i c in situ h y b r i d i z a t i o n was detected by epifluorescence and the proteins labelled by the s i l v e r (Ag-NOR proteins) were d e t e c t e d by r e f l e x i o n light mode. The r D N A was m a p p e d using b i o t i n y l a t e d DNA p r o b e for 45S rDNA sequences. The probe consisted of a 6.6 kb fragment, i n c l u d i n g the d o w n s t r e a m half of the 18S and 90% of the 28S r D N A c o d i n g sequences. In metaphase chromosomes, in situ hybridization d e m o n s t r a t e d the p r e s e n c e of rDNA in the s e c o n d a r y c o n s t r i c t i o n of the X c h r o m o s o m e s with an a x i a l d i s t r i b u t i o n and a l s o l a t e r a l e x p a n s i o n s . The 3-D reconstruction of the Ag-NOR protein signals e v i d e n c e d the p r e s e n c e of these p r o t e i n s in the secondary constriction where they formed a crescent-shaped structure around the axial chromatin pedicule. During interphase, in situ hybridization in intact cell monolayers and i s o l a t e d nuclei s h o w e d the rDNA g e n e s d i s t r i b u t e d as intense fluorescent spots linked by weak s i g n a l s in the inner r e g i o n s of the n u c l e o l i . We conclude that the rDNA is n o t homogeneously distributed in the internal regions of the nucleoli. In the same n u c l e o l a r regions, the AgNOR p r o t e i n s were r e v e a l e d as g r a n u l e s l i n k e d by thin filaments. These images i n d i c a t e s i m i l a r 3-D d i s t r i b u t i o n s for rDNA probes and A g - N O R p r o t e i n s .
SECONDARY ION MASS SPECTROMETRY (SIMS) AND ASSOCIATED IMAGE PROCESSING: AN ENVIRONMENTAL SURVEYPROCEDURE OF PLANT RADIOACTIVE CONTAMINATION CHASSARD-BOUCHAUD Colette~ESCAIG Fran~ois~,BOUMATI Pierre~ Bi31]RL-AT--Yves--6~r~-6-d-DUCOUSSO Roger~'" • Cep~Cre de Microano~y~e app~iqu~e a la Biologie(INSERM),Lab. Biophysique,Facult~ de M~decine, 6 rue du Gl.Sarrail, Cr~teil, • . S ~ R , B . P. 208,9 ; 311 Montlhery Cedex, France. ""SMCB, B.P.208, 913;1 Montlh~y Cedex, France. 239 Pu, one of the most abundant of plutonium isotopes,has been distributed worldwide from nuclear weapons testing inje cted into the atmosphere and then deposited to the earth's surface. As plutonium tends to bind t i g h t l y in soils, the problem of plant uptake had to be investigated. Root uptake and direct deposition on f o l i a r surfaces are the two pathways by which f a l l o u t has contaminated plants. Cazua~na equ~e~foLi.a, also called "FX~zw" or "Ax~to", from the intertropical zone has been investigated. The d i f ferent tissues were fixed chemically, embeddedin paraffin, sectioned ( 5 Nm ) and deposited on a highly pure gold specimen holder. SIMS was performed, using the ion microscope CAMECA IMS 300, associated with the PERICOLOR 2000 Image processing system ( I ). Results obtained from the ion images demonstrated a higher uptake via the leaves than via the roots. These results a l lowed a precise localization of Pu deposits within the cells and no adsorption phenomenawere observed in both tissues. Data obtained, using SIMS microanalysis were in complete agreement with the ones, obtained from bulk specimens, using radiochemical techniques. The advantages of SIMS imaging, in particular i t s very high s e n s i t i v i t y , for the detection of radioactive isotopes, has been discussed and compared to cla ssical autoradiography techniques. ( i ) Chassard-Bouchaud C.,Escaig F.,Boumati P.,Galle C.,1992, Microanalysis and image processing of stable and radioactive elements in ecotoxicology.Current developments using SIMS mi coscope and electron microprobe. Biol.Cell, 74: 59-74. This wo,'d'z u~us fZnane.i.aZZy suppo.,t;ted by .t.he French Idi_nXA.6,uj of Defence and the S~ruiee ~ e de C o n t ~ l e Biologiqae ( c o r t t ~ c t 59 / SMCB ).
259
CILIARY MOTILITY IN CULTURED NASAL E P I T H E L I U M OF NORMAL SUBJECT AND KARTAGENER PATIENT ZAHM Jean-Marie. PIERROT Denis, GILAIN Laurent, PUCHELLE Edith INSERM U314, Universit# de Reims and Centre Hospitalier Intercommunal,Crdteil, Fra.ce. Mucociliary clearance is an important defence mechanism of the respiratory tract. Its effectiveness relies in part on the integrity of ciliary movements. In conditions where ciliary function is impaired, as with primary ciliary dyskinesia, frequent infections are common and may also be accompanied by acquired ciliary changes which are also frequently found in chronic pulmonary disease. Culture of respiratory epithelial cells may be an aherna,ve to objectively determine if ciliary alterations are primary or acquired. The aim of the present work was to study the functional activity of cultured nasal ciliated cells of a Kartagener patient with primary ciliary dyskinesia and to compare the values to that obtained in normal subjects. Explants from a nasal polyp of a normal subject and of a Kartagener patient were cultured on a collagen I matrix in RPMI supplemented culture medium. After 5 days of culture, the culture dishes were placed on the heating stage of a phase-contrast inverted microscope and video recordings of the outgrowths were made. By using a photodetector placed on the videoscreen and a fast Fourier transform analysis, ciliated cell mean beating frequency and the associated standard deviation were calculated. The native polyp tissue from the Kanagener patient exhibited a mean ciliary beating frequency of 6.5 + 1.4 Hz (range: 4.4 to 9.9 Hz). The mean beating frequency of ciliated cells from the Kartagener cultured explants (7.9 + 2. I Hz0 range: 3.4 to 12.5 Hz) was significantly lower (p< 0.001) than the ciliary beating frequency of cells from normal cultured explants (12.1 + 0.8 Hz, range: 10.7 to 12.9 Hz). The ciliary beating variability, reflected by the standard deviation of the mean beating frequency was twice as high in the Kartagener patient compared to the normal subject. The percentage of ciliated cells with normal beating (>8 Hz) was 98% in the normal subject and only 47.5% in the Kartagener patient. These results indicate that different beating patterns may be obtained in cultured ciliated cells from patients suffering from primary ciliary dyskinesia. One can observe either quite normal or decreased and inhomogeneous ciliary beating patterns, whereas the beating pattern of the native tissue seemed abnormal. The culture of ciliated cells from patients with primary ciliary dyskinesia could therefore be useful to precisely determine the extent of the ciliary defect, independently from any acquired defect.
Q U A N T I F I C A T I O N ASSAY BY SIMS MICROSCOPY OF N O N ORGANIFIED THYROID IODINE AFTER CRYOPREPARATION B R I A N ~ O N Colette, JEUSSET Josette, HALPERN Sylvain, a n d FRAGU Philippe Equipe de Microscopie ionique, htserm U66 - Institut Gustave Roussy, 39 rue Camille Desmoulins - 94805 Villejuif Cedex The mean advantage of anhydrous cryopreparation method for microanalysis is to preserve both diffusible and organified elements because it avoids rapid lost of diffusible fraction in aqueous chemical fixative and embedding resin. Our purpose in this study was to evidence non organified iodine (iodide) in thyroid by secondary ion mass spectrometry (SIMS) microscopy. This iodide is reduced to 1-2% in normal thyroid in which almost all of iodine is linked by covalent bounds in thyroglobulin stored in follicular lumens. Iodide is strongly increased in glands submitted to propylthiouracil (PTU). drug prescribed in human treatment of hyperthyroidism which blocks iodine organification by inhibition of thyroid peroxidase. After PTU treatment, organified iodine concentration is greatly decreased (factor 17) as it can be shown by SIMS after chemical fixation. Swiss mice, fed a normal iodine diet, were treated either with PTU 1% (10 days) or kept as controls. Thyroids were cryofixed by ultrarapid immersion (5000K.sec-i, 2m.sec-0 in liquid technic propan (77K), then cryosubstituted in aceton (183K), embedded in Lowicryl K11M (213K) and serially cut. Sections intended to microanalysis (31.tm) were laid on gold holders either on pure water (PW) in which iodide can diffuse, or on ethylene glycol (EG), organic solvent used for minimizing extraction and redistribution of diffusible elements. Sections of 1.51.tm served as histological controls b., photonic microscopy. Measurement by SIMS microscopy of local follicular concentration of 127I evidenced significant differences (p<0.01) not detectable by mapping alone. The total iodine concentration, evaluated o n "EG sections", varied only by a factor 6 between PTU treated (1.14 + 0.23 I.tg/mg, mean + SE) and control mice (6.03 + 0.51 gg/mg). Furthermore, iodine evaluated on "PW sections", which is essentially organified iodine, varies by about a factor 10 between PTU tr~ted (0.70 + 0.21 gg/mg) and control mice (5.58 + 0.32 I.tg/mg). Finally, the diffusible fraction of thyroid iodine, corresponding at least to the difference between measurements on "EG sections" and on "PW sections", is extremely low in controls (less than 1%) while it reaches 44% in PTU treated mice. Our results give proof of the cryopreparation efficiency for maintenance, even if partial, of diffusible iodide, and of its methodological interest for microanalysis of any diffusible element in any tissue.
3a