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Nuclear configuration of bladder cancer associated to bilharziosis: Preliminary study by confocal microscopy
C o n g r ~ s A.C.F. - C.F.M.Q., M O N S 1991 3-D IMAGING OF NUCLEAR STRUCTURES BY CONFOCAL MICROSCOPY ROBERT-NICOUD Michel Equipe de Reconnaissance des Forints er de Micro.rcopie Quantaatave. Laboratoire TIMJ. Uniuersit~ Joseph Fourier. lip 53X. F-JS041 Grenoble Cedex. France
Confocal laser scanning microscopy (CLSM) offers several advantages over conventional fluorescence microscopy for the in ~im hx:alization of flL~orescently labeled targets and probes in cells and tissues : (i) rejection of fluorescence signals from out-of-focus planes and from neighboring suructures. Oi) improvement of both lateral and axial resolutions. (iii) electronic adjustement of contrast brightness and magnification, (iv) sinmltaneous visualization of the specimen by using optical phenomena based on incident, scattered. emitted and transmitted light, (v) simultaneous use of different fluorescent probes and types of detectors, (vi) acquisition of serial optical sections for generation of 3-D ~constrnctions without time consuming deconvolution procedures. Qualitative analyses of the can be effectively performed by visual {nspection of reconstructed images using stereo projectioo. For quantitative evaluation of 3-D dismbudons, contour extraction, surface and volume reconstructions can be undertaken. CLSM has proved to be a powerful technique for muhiparameter and tridintensional in .~itu analyses on intact cells, tissues and whole embryos. Several levels of the structural and functional organization of the chromatin in the cell nucleus were investigated : (i) the conformational state of the DNA, (it) the distribution of eu- and heterochromatic regions, (iii) the localization of DNA replication sites, (iv) the redistribution of nuclear and nucleolar proteins during the cell cycle. (v) the condensation aod %patti arrangement of the chromosomes. (vii the localization of parucular DNA SalUencesand of individual genes. Probes used in these studies include : (i) DNA fluorescent dyes, (it) chemical reagents revealed by immunofluorescence, (iii) antibodies recognizing nuclear proteins. DNA conformation, and modified DNA bases. (iv) markers of the cell cycle and proliferation. (v) DNA probes for non-isotopic in sita hybridization. These probes were used either individually or in various combinations with each other, in order to establish possible correlations between the structural state of the chmmatin and its funcuonal state in cycling and in differentiated cells.
NUCLEAR CONFIGURATION OF BLADDER CANCER ASSOCIATED TO BILHARZIOSIS : PRELIMINARY STUDY BY CONFOCAL MICROSCOPY. NASR Safaa*, MOUKHTAR Nadia**, MANSOUR Abbas**. OMRI DE LANGEN FrtM~rique***, KAHN Edmond***. * Nemrock EM Center of EEC - NCRRT - Facully o f Medicine - Cairo - Egypt. ** National Cancer institute - Cairo - Egypt. *** I N S ~ R M ;.166- institut Gustave-Rma~.- 94805 ~7LLEJ&'a'F Cedex - France.
By means of a confocal laser scanning microscope (Sarastro Phoibos I000), ten biopsies of the normal and malignant bladder mucosis were examined. Eight cases of Egyptian origin were subject to bilharziosis, while two other cases, of French origin, were not subject to bilharziosis. Preserved cases in a dilution of formol (I0%) in phosphate buffer at pH 7.8, were cut by means of an Oxford vibratome, in slices of 50 to | r , ..nicrons. Slices were washed in the PBS during I0 to 30 minutes at 4"C, then plunged into a dilutior~ of •ethidinm bromide at 0.2 mg/mi in the PBS during 15 to 60 minutes at 4°C. Two washes in the PBS at pH 7.8 were followed by mounting between printed glass slide and coverslip to reach observation of the DNA by confocal'microscopy. Observation consisted in selection of different optical slices at x I0 and x 40. We show these slice sets and the corresponding three dimensional (3D) reconstructions. Measurements of the diameter of nuclei provided confirmation that malignant samples had nucleus sizes between 60 and 95 microns. while normal samples had nucleus sizes between 12 and 18 microns. Observation of optical s;ice sets shows that chromadn distribution around the central axis of the nucleus is much variable in the medi. gnant cases, while it remains constant in the normal cases. These results could not be Obtained by conventional optical microscopy or electron microscopy on semi and ultra thin sections. 3D reconstructions show that confignradons of benign cases are homogeneous. Malignant cases show nucleolar lobules of variable shape inside the cytoplasm. The two malignant cases of French origin seem to be more uniform than the malignant cases of Egypdan origin. Work will continue on a larger set to correlate malignancy grades and histological types of tumors to 3D configuration of DNA in the nucleus, and therefore to provide precisions on the expression mode of the DNA. Aclmowlcdgemen~:Thisweekwaspartiallys6ppmugby the EEC.
CONTRIBUTION OF TIME-LAPSE MICROCINEMATOGRAPHY TO THE QUANTITATIVE STUDY OF CELLULAR DYNAMICS
"REAL TIMEm IMAGE ANALYSIS OF THE CHANGES OF THE SHAPE AND/OR SIZE OF LIVING CELLS AS A METHOD FOR STUDYING
GU]EULWI'I~ John : Universe. Catkolique de 1.,oueoia Unit~ de Radiobiologie et de Radioprotection (Pro/./.. Wambersie ) Ciiniques Universitm're$ St-Luc, 1200 Bru~iles.
CELL PHYSIOLOGY TRAVO Pierre, LEES Delphine, BODIN Philippe, PUGNEILE Denis, CASTEL Amaud, and PAPIN Serge Centre de Recherche de Biochimie Macromol~:ulaire, UPR 8402 CNRS, U 249 INSERM, Route de Mende, BP $051 34 0:)3 Montpellier Cedex, France
it is well Imown dun cellular kinetics is one of the main problem of the optimization of treatment protocols in ngliodanpy. In this frmmwork we studied mitotic delay after radiation using time-lapse mlcrocincmatography. This technique makes it possible to detamine pedigree of individual cells and to study Iho influence of cycle phase direcdy in exponendal growing cell culnu~ The latter is p.micul~d.y advantagu)us when radiations m combined with cymmxic mugs mat m likely to react with the syncluonis~on chemical ~genta used in the classical methods. Additional infonmdon o0nceming cell mob~ty, moth~ I dmsahta or daugh~ I daughter corre¼dons can be pmyi'dad. which all would aocount for abe Ima/icdon of tomotg response. In respect out knows the need of "pmJicdve assays" in consideredon of the new Ireatmem medalides (hypeffractionadon, high LET radiations, radiation I drug associations) seeking to adjust the treatments to individual cases. Peetimln,w ] results indicate that the avenge mitotic delay of W9 cells increases linearly with dose at the rate of 1.3 and 2.5 horn I Oy ~ter gamma and neutron inmJiadons respectively. The corresponding ncumon RBE (1.9) is significandy smaller than the one for cell lethality (2.8 for 2 Oy), which suggesu that the cell eedisMbodon in the cycle is different after neutron than after gamma inadiations. Analysis of pedigrees also shows that "potentially killed cells" arc able to undergo 3 - 4 normal divisions after gamma while they only divide 1 - 2 times after neumms. Similar experiments are presendy perfomgd for drag alone or in combination with radiations.
From a technical point of view, the instantaneous experimental conditions (hours of shots, temperatme etc.) ate printed on the/'tlm at the very moment of the shots by means of an original "optical incrustation" device. The film, g ~ transferred on video tapes and analysed using a monitor I magnetoscope set. The data are scored in a genealogical nee like diagram from which pcdigXees, cycle dmos and mitotic delays will be computed.
We developed a non invasive non destructive method for studying the individual behaviour (contraction, proliferation, motility) of cultivated vascular smooth muscle cells. This method is based on the quantitative analysis of the images of living cells obtained with an optical microscope. It was first implemented by a human operator using microphotographs and digitizing table (1). We developed a abet capable of obser,ing, recognizing and measuring the spontaneous or modulated (inhibited or stimulated) behaviour (displacements, changes of shape and I or size, division) of 10 to 50 single vascular smooth muscle cells in the same culture dish at chosen intervals (from 30 s to 2 to 3 hours) over periods of time of up to 4 to $ days in a fully automated way (2). In the long run, it appeared that our method could be of general use: it was named morphophysiology.However, the robot needed to be adapted and its use made simpler. Consistently. its capacities were enlarged. At the present time, it is capable of using any type of CCD camera and microscope (with Nomarsky optics), any type of cultureware (provided it fits to the stage), and after specific learning sessions, to study any type of cell (and cell behaviour) which can be grown or simply main~ned in culture (3). (1) TI~AVOPet al. Lab Invest,56, 3:!5- :142, 1987 (2) BODINPh et al, Lab Invest,59, i:J7 - 14), 1988 (:1)TP~VO Pet al, EuropeanMicroscopyand Analysis,12, Sept 1991
31a
Confocal laser scanning microscopy (CLSM) offers several advantages over conventional fluorescence microscopy for the in ~im hx:alization of flL~orescently labeled targets and probes in cells and tissues : (i) rejection of fluorescence signals from out-of-focus planes and from neighboring suructures. Oi) improvement of both lateral and axial resolutions. (iii) electronic adjustement of contrast brightness and magnification, (iv) sinmltaneous visualization of the specimen by using optical phenomena based on incident, scattered. emitted and transmitted light, (v) simultaneous use of different fluorescent probes and types of detectors, (vi) acquisition of serial optical sections for generation of 3-D ~constrnctions without time consuming deconvolution procedures. Qualitative analyses of the can be effectively performed by visual {nspection of reconstructed images using stereo projectioo. For quantitative evaluation of 3-D dismbudons, contour extraction, surface and volume reconstructions can be undertaken. CLSM has proved to be a powerful technique for muhiparameter and tridintensional in .~itu analyses on intact cells, tissues and whole embryos. Several levels of the structural and functional organization of the chromatin in the cell nucleus were investigated : (i) the conformational state of the DNA, (it) the distribution of eu- and heterochromatic regions, (iii) the localization of DNA replication sites, (iv) the redistribution of nuclear and nucleolar proteins during the cell cycle. (v) the condensation aod %patti arrangement of the chromosomes. (vii the localization of parucular DNA SalUencesand of individual genes. Probes used in these studies include : (i) DNA fluorescent dyes, (it) chemical reagents revealed by immunofluorescence, (iii) antibodies recognizing nuclear proteins. DNA conformation, and modified DNA bases. (iv) markers of the cell cycle and proliferation. (v) DNA probes for non-isotopic in sita hybridization. These probes were used either individually or in various combinations with each other, in order to establish possible correlations between the structural state of the chmmatin and its funcuonal state in cycling and in differentiated cells.
NUCLEAR CONFIGURATION OF BLADDER CANCER ASSOCIATED TO BILHARZIOSIS : PRELIMINARY STUDY BY CONFOCAL MICROSCOPY. NASR Safaa*, MOUKHTAR Nadia**, MANSOUR Abbas**. OMRI DE LANGEN FrtM~rique***, KAHN Edmond***. * Nemrock EM Center of EEC - NCRRT - Facully o f Medicine - Cairo - Egypt. ** National Cancer institute - Cairo - Egypt. *** I N S ~ R M ;.166- institut Gustave-Rma~.- 94805 ~7LLEJ&'a'F Cedex - France.
By means of a confocal laser scanning microscope (Sarastro Phoibos I000), ten biopsies of the normal and malignant bladder mucosis were examined. Eight cases of Egyptian origin were subject to bilharziosis, while two other cases, of French origin, were not subject to bilharziosis. Preserved cases in a dilution of formol (I0%) in phosphate buffer at pH 7.8, were cut by means of an Oxford vibratome, in slices of 50 to | r , ..nicrons. Slices were washed in the PBS during I0 to 30 minutes at 4"C, then plunged into a dilutior~ of •ethidinm bromide at 0.2 mg/mi in the PBS during 15 to 60 minutes at 4°C. Two washes in the PBS at pH 7.8 were followed by mounting between printed glass slide and coverslip to reach observation of the DNA by confocal'microscopy. Observation consisted in selection of different optical slices at x I0 and x 40. We show these slice sets and the corresponding three dimensional (3D) reconstructions. Measurements of the diameter of nuclei provided confirmation that malignant samples had nucleus sizes between 60 and 95 microns. while normal samples had nucleus sizes between 12 and 18 microns. Observation of optical s;ice sets shows that chromadn distribution around the central axis of the nucleus is much variable in the medi. gnant cases, while it remains constant in the normal cases. These results could not be Obtained by conventional optical microscopy or electron microscopy on semi and ultra thin sections. 3D reconstructions show that confignradons of benign cases are homogeneous. Malignant cases show nucleolar lobules of variable shape inside the cytoplasm. The two malignant cases of French origin seem to be more uniform than the malignant cases of Egypdan origin. Work will continue on a larger set to correlate malignancy grades and histological types of tumors to 3D configuration of DNA in the nucleus, and therefore to provide precisions on the expression mode of the DNA. Aclmowlcdgemen~:Thisweekwaspartiallys6ppmugby the EEC.
CONTRIBUTION OF TIME-LAPSE MICROCINEMATOGRAPHY TO THE QUANTITATIVE STUDY OF CELLULAR DYNAMICS
"REAL TIMEm IMAGE ANALYSIS OF THE CHANGES OF THE SHAPE AND/OR SIZE OF LIVING CELLS AS A METHOD FOR STUDYING
GU]EULWI'I~ John : Universe. Catkolique de 1.,oueoia Unit~ de Radiobiologie et de Radioprotection (Pro/./.. Wambersie ) Ciiniques Universitm're$ St-Luc, 1200 Bru~iles.
CELL PHYSIOLOGY TRAVO Pierre, LEES Delphine, BODIN Philippe, PUGNEILE Denis, CASTEL Amaud, and PAPIN Serge Centre de Recherche de Biochimie Macromol~:ulaire, UPR 8402 CNRS, U 249 INSERM, Route de Mende, BP $051 34 0:)3 Montpellier Cedex, France
it is well Imown dun cellular kinetics is one of the main problem of the optimization of treatment protocols in ngliodanpy. In this frmmwork we studied mitotic delay after radiation using time-lapse mlcrocincmatography. This technique makes it possible to detamine pedigree of individual cells and to study Iho influence of cycle phase direcdy in exponendal growing cell culnu~ The latter is p.micul~d.y advantagu)us when radiations m combined with cymmxic mugs mat m likely to react with the syncluonis~on chemical ~genta used in the classical methods. Additional infonmdon o0nceming cell mob~ty, moth~ I dmsahta or daugh~ I daughter corre¼dons can be pmyi'dad. which all would aocount for abe Ima/icdon of tomotg response. In respect out knows the need of "pmJicdve assays" in consideredon of the new Ireatmem medalides (hypeffractionadon, high LET radiations, radiation I drug associations) seeking to adjust the treatments to individual cases. Peetimln,w ] results indicate that the avenge mitotic delay of W9 cells increases linearly with dose at the rate of 1.3 and 2.5 horn I Oy ~ter gamma and neutron inmJiadons respectively. The corresponding ncumon RBE (1.9) is significandy smaller than the one for cell lethality (2.8 for 2 Oy), which suggesu that the cell eedisMbodon in the cycle is different after neutron than after gamma inadiations. Analysis of pedigrees also shows that "potentially killed cells" arc able to undergo 3 - 4 normal divisions after gamma while they only divide 1 - 2 times after neumms. Similar experiments are presendy perfomgd for drag alone or in combination with radiations.
From a technical point of view, the instantaneous experimental conditions (hours of shots, temperatme etc.) ate printed on the/'tlm at the very moment of the shots by means of an original "optical incrustation" device. The film, g ~ transferred on video tapes and analysed using a monitor I magnetoscope set. The data are scored in a genealogical nee like diagram from which pcdigXees, cycle dmos and mitotic delays will be computed.
We developed a non invasive non destructive method for studying the individual behaviour (contraction, proliferation, motility) of cultivated vascular smooth muscle cells. This method is based on the quantitative analysis of the images of living cells obtained with an optical microscope. It was first implemented by a human operator using microphotographs and digitizing table (1). We developed a abet capable of obser,ing, recognizing and measuring the spontaneous or modulated (inhibited or stimulated) behaviour (displacements, changes of shape and I or size, division) of 10 to 50 single vascular smooth muscle cells in the same culture dish at chosen intervals (from 30 s to 2 to 3 hours) over periods of time of up to 4 to $ days in a fully automated way (2). In the long run, it appeared that our method could be of general use: it was named morphophysiology.However, the robot needed to be adapted and its use made simpler. Consistently. its capacities were enlarged. At the present time, it is capable of using any type of CCD camera and microscope (with Nomarsky optics), any type of cultureware (provided it fits to the stage), and after specific learning sessions, to study any type of cell (and cell behaviour) which can be grown or simply main~ned in culture (3). (1) TI~AVOPet al. Lab Invest,56, 3:!5- :142, 1987 (2) BODINPh et al, Lab Invest,59, i:J7 - 14), 1988 (:1)TP~VO Pet al, EuropeanMicroscopyand Analysis,12, Sept 1991
31a