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An EFTEM analysis (PEELS and ESI) of bacteria granules located in the tube wall from deep-sea worm
Abstracts Trinoculaire ‘98 des Microscopies, Strasbourg-lllkirch,
France, l-3 July 1998
287
An EFTEM analysis (PEELS and ESI) of bacteria granules located in the tube wall from deep-sea worm
Cryo-scanning electron microscopy applied to preadipocyte cell line 3T3Ll
Jean-Pierre Lechaire, GhislaineFrebourg and Francoise Gail1
SylvieMarull’, AshleyWilson**,MagaliZbinden’, Frkdkrique
Laboratoire de Bioiogie Marine, CNRS UPR 9042 Roscoff/ CIME-Jussieu, UPMC , 7, quai St Bernard Bit. A, 75252 Paris cedex 05, France The tuba wall of Rift&a pachyprila, a tube worm from deep-sea hydrothermal vent, is composed of parallel bundles of chitin microlibrils. Microorganisms arc observed at the external side of the tube wall and in the thickness of the mature portion. In the samples we analyscd. these microorganisms are absent from the internal side of the tube wall and from the freshly sccrctcd material. Their study by WEM (Diffraclion Contrast by Transmission Electron Microscopy) shows \hat they contain one or two electron dense granules. The lirst results (Lechaire J-P. ct al. (1997). &of. Ceff 89. 161) by EFTEM: Energy Filtered Transmission Electron Microscopy (PEELS: parallel electron energy loss spcclrum and ESI: Energy Spectroscopic Imaging) showed that they conlain iron. Biological specimens often challenge the limits of microanalysis because of the low concentrations of elements that they contain (Leapman R.D. and Andrews S.B. (1991). Microsc. Micrmnul. Microstruct. 2,387-394). Our purpose was to test the pcrfonnance of the TEM LEO 912 at 120 kV with the in-column omega speclrometcr lo obtain the best results on the same biological specimen. the bacteria granules, and for the same clcmcnt (Fe). In order to optimize the acquisition of the diffcrcnt elements in these granules we attempted to standardize the. PEELS and ES1 analysis procedures. The main parameters maintained constant are: thickness of the thin section, magnification. irradiation dose and acquisition time for the same clement (Fe). The energy filtered images were recorded using a SS-CCD and a ESlvision softwarc working at 16 bits. WC have studied several criteria in PEELS mode: the cffcct of the irradiation dose as a function of time, the signal intensity as a function of dose and the signal intensity as a function of examined area. Our results show that the characteristic signal in PEELS procedure is dose sensitive and dcpcnds on the radiation damage lolerdtcd by the sample. Thus the obtained signal is stable when acquisition patametcrs are maintained constant, it varies after one acquisition with the ES1 method with a higher irradiation dose. The amplitude of the signal depends on the area occupied by the granule in lhe entrance aperture (4oOFm) of the spectrometer.
Poirier’,
Gaille
Saintigny’,
Dominique
Lutz’
andGilbertFuchs’** ‘Centre de Recherche Yves Rocher. IO1 ,, Quai do Pdt Roosevelt, 92444 lssy /es Moulineaux cedex, France ; “Universrty of York. Department of Biology, PO Box 373, YORK YOI 5wV, United Kingdom ; ‘Elf Atochem, Centre de Recherche RhdneAlpes, BP 63, 69493 Pierre Bknite cedex, France l
l
In order to characterize and quantify the adipocytes conversion process, several microscopic technics were used. For this work, we used primary cultures of the preadipocyte cell line 3T3Ll (Ref,ECACC#86052701) growing in an appropriate medium. Cells were studied 10 days after the induction of differentiation. Cell morphology was examined using oil red 0 and Mayer Hemalun staining medium. Presence or absence of lipids was determined by phase contrast microscopy. Because of its high lipid content, we had to treat the cell culture in a particular way for the transmission electron microscopy in its different preparation process : after a classical chemical fixation, the dehydration and impregnation steps were processed in reduced time and temperature. Even then, many cells suffered, lipid droplets were extracted by the dehydration solvents. To preserve this kind of structure, cryo-methods are most appropriate (Wergin W.P., Erbe E.F. 1991, Scanning Microscopy, 5,927.936). We obtained images of 3T3Ll cells with intact lipid droplets using a highresolution cryo scanning electron microscope coupled to a cryo preparation unit. We used a VG aPolaron>> LT 7400 SEM cryo preparation unit coupled with a Philips XL 40 FEG SEM. Chemical fixation and dehydration procedures were avoided and no cryoprotectant was used. To quantify the adipocytes conversion, fixed and stained cells culture were observed under a photonic microscope equipped with a CCD camera. The cells fields recordings were digitized, and images analysed by Perf& Image software.
Visualization of the expression of the mRNA of growth hormone by in situ RT-PCR
Study of fast water movements in bacteria by cryoelectron microscopy
Sophie Recher, Sylvie Ricard-Blum and GBrard Morel
Jean-Paul Rolland,ChristianDelamarche, lsabellePellerin, Alexandrine Froger,AnnieCavalier,StephaneDeschamps, Jean-Francois Hubert,ValerieLagrBe,JeanGouranton
CNRS UMR 5578,
Universti6Claude Bernard Lyon I, Villeurbanne, France
Growth hormone (GH) which is synthesized by the somatouophs in the anterior pituitary lobe, acts on a wide range of tissues through binding to its specific receptor. Growth hormone gene expression has been demonstrated in immune organs by in virro amplification of GH mRNA using a reverse transcription-polymerase chain reaction (RT-PCR) (Binder er al. 1994 J Endocrinol 140, 137-143). The aim of our study was to investigate the expression of the mRNA of GH by in situ RT-PCR at light microscopic level in rat lymphoid organs (spleen, thymus) and lymphocytes. The presence of GH and its specific receptor in other tissues than pituitary would suggest a paracrine effect of GH in these tissues. Preliminary experiments were performed using mt pituitary as a positive tissue control and liver as a negative tissue control. Liquid RT-PCR was performed on total RNA extracted from pituitary and liver to assess the feasablity of the amplification by PCR (30 cycles). The amplified products were analyzed by electrophoresis and by Southern blotting with a digoxigenin-labelled oligonucleotide probe specific of rat GH. Indirect in situ RT-PCR was performed using an hybridization step with a digoxigenin labelled probe to detect the amplified products. Visualization was done with an anti-digoxigenin conjugated to alkaline phosphatase and NBT/BCIP as chromogenic substrate. Specific primer for GH mRNA was used for the reverse transcriptase reaction. PCR was carried out with a hot start procedure. The finalization of the in siru RT-PCR protocol required optimization of the following parameters: the temperature of the reverse transcriptase reaction and of the annealing step during amplification, MgC12 and primer concentrations, the number of cycles and the presence of additives (DMSO, betaine). A single band was detected in pituitary after in virro amplification by liquid PCR, indicating that the designed primers were functional. The expression of GH mRNA was detected only in anterior pituitary after 25 PCR cycles. The specificity of the reaction was assessed by the fact that posterior pituitary remained constantly negative and by the absence of signal when Taq polymerase, primers or reverse transcriptase were omitted.
and Daniel Thomas UPRES-A CNRS, Biologic Cellulaire et Reproduction, 35042 Rennes cedex, France
Universitk
de Rennes I,
All biological membranes exhibit some water permeability as a result of diffusion through the lipid bilayer, under the driving force of the osmotic gradient. However some cells are able to transfer water at greatly accelerated rates by the way of water-selective channels called aquaporins. In order to study the dynamics of water movements in bacterial cells, we have monitored the response of E. co/i to an osmotic shock by cryoelectron microscopy. On a microscope grid, freshly harvested bacteria were subjected to an hyperosmotic shock by quickly mixing a sucrose solution with the bacterial cell suspension. Then immediately, or after a delay of 15, 30,60 or 90 set, the suspension was blotted and quickly frozen into liquid ethane. E. cdib cells, immediately frozen after sucrose injection look very similar to control bacteria; cells are still in a turgor state with a perisplasmic space reduced. A delay of 15 set after the sucrose injection results in a dramatic decrease of the cytoplasmic volume corresponding to a huge and fast exit of water. No visible effect was observed for longer times, indicating that most of the exit of water outside the cell occurs within the first seconds. To verify if fast water flow exists also inward, the inverted experiment was also carried out. E. cofi cells were maintained in a hi h osmolarity solution for 5 min, then submitted to an hypoosmotic shock Bor 15 set before freezing. Cryoelectron microscopy permits to observe that a fast entry of water occurs into the cell : bacteria with a shrunk cytoplasm can quickly recover their turgor state. Thus, it appears that cryoelectron microscopy could be a simple and pertinent technique to study the physiological implication of bacterial aquaporins in response to a fast changing environment.
France, l-3 July 1998
287
An EFTEM analysis (PEELS and ESI) of bacteria granules located in the tube wall from deep-sea worm
Cryo-scanning electron microscopy applied to preadipocyte cell line 3T3Ll
Jean-Pierre Lechaire, GhislaineFrebourg and Francoise Gail1
SylvieMarull’, AshleyWilson**,MagaliZbinden’, Frkdkrique
Laboratoire de Bioiogie Marine, CNRS UPR 9042 Roscoff/ CIME-Jussieu, UPMC , 7, quai St Bernard Bit. A, 75252 Paris cedex 05, France The tuba wall of Rift&a pachyprila, a tube worm from deep-sea hydrothermal vent, is composed of parallel bundles of chitin microlibrils. Microorganisms arc observed at the external side of the tube wall and in the thickness of the mature portion. In the samples we analyscd. these microorganisms are absent from the internal side of the tube wall and from the freshly sccrctcd material. Their study by WEM (Diffraclion Contrast by Transmission Electron Microscopy) shows \hat they contain one or two electron dense granules. The lirst results (Lechaire J-P. ct al. (1997). &of. Ceff 89. 161) by EFTEM: Energy Filtered Transmission Electron Microscopy (PEELS: parallel electron energy loss spcclrum and ESI: Energy Spectroscopic Imaging) showed that they conlain iron. Biological specimens often challenge the limits of microanalysis because of the low concentrations of elements that they contain (Leapman R.D. and Andrews S.B. (1991). Microsc. Micrmnul. Microstruct. 2,387-394). Our purpose was to test the pcrfonnance of the TEM LEO 912 at 120 kV with the in-column omega speclrometcr lo obtain the best results on the same biological specimen. the bacteria granules, and for the same clcmcnt (Fe). In order to optimize the acquisition of the diffcrcnt elements in these granules we attempted to standardize the. PEELS and ES1 analysis procedures. The main parameters maintained constant are: thickness of the thin section, magnification. irradiation dose and acquisition time for the same clement (Fe). The energy filtered images were recorded using a SS-CCD and a ESlvision softwarc working at 16 bits. WC have studied several criteria in PEELS mode: the cffcct of the irradiation dose as a function of time, the signal intensity as a function of dose and the signal intensity as a function of examined area. Our results show that the characteristic signal in PEELS procedure is dose sensitive and dcpcnds on the radiation damage lolerdtcd by the sample. Thus the obtained signal is stable when acquisition patametcrs are maintained constant, it varies after one acquisition with the ES1 method with a higher irradiation dose. The amplitude of the signal depends on the area occupied by the granule in lhe entrance aperture (4oOFm) of the spectrometer.
Poirier’,
Gaille
Saintigny’,
Dominique
Lutz’
andGilbertFuchs’** ‘Centre de Recherche Yves Rocher. IO1 ,, Quai do Pdt Roosevelt, 92444 lssy /es Moulineaux cedex, France ; “Universrty of York. Department of Biology, PO Box 373, YORK YOI 5wV, United Kingdom ; ‘Elf Atochem, Centre de Recherche RhdneAlpes, BP 63, 69493 Pierre Bknite cedex, France l
l
In order to characterize and quantify the adipocytes conversion process, several microscopic technics were used. For this work, we used primary cultures of the preadipocyte cell line 3T3Ll (Ref,ECACC#86052701) growing in an appropriate medium. Cells were studied 10 days after the induction of differentiation. Cell morphology was examined using oil red 0 and Mayer Hemalun staining medium. Presence or absence of lipids was determined by phase contrast microscopy. Because of its high lipid content, we had to treat the cell culture in a particular way for the transmission electron microscopy in its different preparation process : after a classical chemical fixation, the dehydration and impregnation steps were processed in reduced time and temperature. Even then, many cells suffered, lipid droplets were extracted by the dehydration solvents. To preserve this kind of structure, cryo-methods are most appropriate (Wergin W.P., Erbe E.F. 1991, Scanning Microscopy, 5,927.936). We obtained images of 3T3Ll cells with intact lipid droplets using a highresolution cryo scanning electron microscope coupled to a cryo preparation unit. We used a VG aPolaron>> LT 7400 SEM cryo preparation unit coupled with a Philips XL 40 FEG SEM. Chemical fixation and dehydration procedures were avoided and no cryoprotectant was used. To quantify the adipocytes conversion, fixed and stained cells culture were observed under a photonic microscope equipped with a CCD camera. The cells fields recordings were digitized, and images analysed by Perf& Image software.
Visualization of the expression of the mRNA of growth hormone by in situ RT-PCR
Study of fast water movements in bacteria by cryoelectron microscopy
Sophie Recher, Sylvie Ricard-Blum and GBrard Morel
Jean-Paul Rolland,ChristianDelamarche, lsabellePellerin, Alexandrine Froger,AnnieCavalier,StephaneDeschamps, Jean-Francois Hubert,ValerieLagrBe,JeanGouranton
CNRS UMR 5578,
Universti6Claude Bernard Lyon I, Villeurbanne, France
Growth hormone (GH) which is synthesized by the somatouophs in the anterior pituitary lobe, acts on a wide range of tissues through binding to its specific receptor. Growth hormone gene expression has been demonstrated in immune organs by in virro amplification of GH mRNA using a reverse transcription-polymerase chain reaction (RT-PCR) (Binder er al. 1994 J Endocrinol 140, 137-143). The aim of our study was to investigate the expression of the mRNA of GH by in situ RT-PCR at light microscopic level in rat lymphoid organs (spleen, thymus) and lymphocytes. The presence of GH and its specific receptor in other tissues than pituitary would suggest a paracrine effect of GH in these tissues. Preliminary experiments were performed using mt pituitary as a positive tissue control and liver as a negative tissue control. Liquid RT-PCR was performed on total RNA extracted from pituitary and liver to assess the feasablity of the amplification by PCR (30 cycles). The amplified products were analyzed by electrophoresis and by Southern blotting with a digoxigenin-labelled oligonucleotide probe specific of rat GH. Indirect in situ RT-PCR was performed using an hybridization step with a digoxigenin labelled probe to detect the amplified products. Visualization was done with an anti-digoxigenin conjugated to alkaline phosphatase and NBT/BCIP as chromogenic substrate. Specific primer for GH mRNA was used for the reverse transcriptase reaction. PCR was carried out with a hot start procedure. The finalization of the in siru RT-PCR protocol required optimization of the following parameters: the temperature of the reverse transcriptase reaction and of the annealing step during amplification, MgC12 and primer concentrations, the number of cycles and the presence of additives (DMSO, betaine). A single band was detected in pituitary after in virro amplification by liquid PCR, indicating that the designed primers were functional. The expression of GH mRNA was detected only in anterior pituitary after 25 PCR cycles. The specificity of the reaction was assessed by the fact that posterior pituitary remained constantly negative and by the absence of signal when Taq polymerase, primers or reverse transcriptase were omitted.
and Daniel Thomas UPRES-A CNRS, Biologic Cellulaire et Reproduction, 35042 Rennes cedex, France
Universitk
de Rennes I,
All biological membranes exhibit some water permeability as a result of diffusion through the lipid bilayer, under the driving force of the osmotic gradient. However some cells are able to transfer water at greatly accelerated rates by the way of water-selective channels called aquaporins. In order to study the dynamics of water movements in bacterial cells, we have monitored the response of E. co/i to an osmotic shock by cryoelectron microscopy. On a microscope grid, freshly harvested bacteria were subjected to an hyperosmotic shock by quickly mixing a sucrose solution with the bacterial cell suspension. Then immediately, or after a delay of 15, 30,60 or 90 set, the suspension was blotted and quickly frozen into liquid ethane. E. cdib cells, immediately frozen after sucrose injection look very similar to control bacteria; cells are still in a turgor state with a perisplasmic space reduced. A delay of 15 set after the sucrose injection results in a dramatic decrease of the cytoplasmic volume corresponding to a huge and fast exit of water. No visible effect was observed for longer times, indicating that most of the exit of water outside the cell occurs within the first seconds. To verify if fast water flow exists also inward, the inverted experiment was also carried out. E. cofi cells were maintained in a hi h osmolarity solution for 5 min, then submitted to an hypoosmotic shock Bor 15 set before freezing. Cryoelectron microscopy permits to observe that a fast entry of water occurs into the cell : bacteria with a shrunk cytoplasm can quickly recover their turgor state. Thus, it appears that cryoelectron microscopy could be a simple and pertinent technique to study the physiological implication of bacterial aquaporins in response to a fast changing environment.