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TV-based analysis of Giemsa-banded chromosomes
Printed in Swedm Copyright 0 1974 by Academic Press, Inc. Ail rights of reproduction in any form reserved
PRELIMINARY
NOTES
TV-based analysis of Giemsa-banded chromosomes
are two-fold. Firstly, photography greatly helps in the detailed scrutiny of each individual chromosome part. Different banding T. CASPERSSON, Institute for Medical Cell Research methods demand photography of different and Genetics, Medical Nobel Institute, Karolinska Znstitutet, S-104 01 Stockholm, Sweden degrees of refinement, the greatest demands applying to the fluorescence procedures. A Summary. In large scale chromosome aberration second reason is that, because of the great studies by Giemsa-based banding techniques the photographic work is the most time-consu&g. A number of chromosomes in most mammalian TV-based procedure which entirely eliminates photometaphase plates, there must be some congraphy is described. This involves as main fiatures image contrast enhancementon arbitrarily selected venient way in which to mark individual gray scale levels, continuously regulable magnification chromosomes as they are analysed in order to and a special device for marking analyzed chromosomes during the progressof the work. monitor the progress of the work. This is easily done with the aid of a photograph. Working time could be greatly reduced by The slowness and also the tediousness of the conventional procedures of karyotype anal- the use of a TV procedure which eliminated ysis with banding techniques are serious most of the photographic work involved in obstacles to their wider application in such fluorescence analysis [l-4]. The negative of important fields as the analysis of frequency the fluorescence photograph was observed by and character of chromosome aberrations a TV camera. The image on the monitor was caused, e.g. by external mutagens or carcino- manipulated by means of an optical zoom gens, in which studies large series of meta- system before the TV camera and an image phases must always be analysed. contrast enhancement system [2] incorporated The main delaying factors in the conven- in the electronic system. The arrangement not tional procedures lie in the photographic only saved working time but also made the work. The reasons for the use of photography whole work considerably less tiring.
Fig. 1. Overall view of the
instrument. Exptl Cell Res 92 (1975)
498
Prrliminury
notpx
~. :
&
These results led to efforts to build an analogous system for Giemsa stained preparations in which photography could be completely eliminated. The applicability of such a procedure to different types of chromosomeanalytical work has been discussed in an earlier publication [5]. Expt~ Cell Res 92 (197Sj
F$. 2. The mode of function, cl text.
Experiences from the development of the instrument for low-level absorption image analysis, described in the preceding paper [6’j, have now also led to the generation of an improved and simplified apparatus for more rapid analysis of preparations stained by Giemsa or Giemsa-like procedures.
Preliminary notes
In this apparatus (fig. 1) a TV camera looks directly into the microscope. No photography is used. A zoom system permits easy and continuous change of magnification within a range 1-3. The optical system is adjusted so that by the lowest magnification the field of the picture covers the area of an ordinary metaphase. For detailed analysis of each individual chromosome, suitable higher magnifications are used. A picture contrast enhancement arrangement is incorporated, in principle the same as that described in reference [q for low level absorption work. The range is extended, however, so that it is possible to obtain great enhancement of small differencesin absorption in faintly stained chromosome regions as well as in intensely stained parts, where fine banding details are often hidden from observation by the overall high absorption. To the naked eye, the effect of this contrast enhanckment of the picture on the monitor is quite striking and the combination of regulable zooming and regulable contrast enhancement facilitates the analysis of chromosome details very much and increases the speed of work. A videotape recorder and a second monitor are incorporated so that individual chromosomes can be marked as work proceeds. The videotape is arranged as a closed loop. When the metaphase has been selected, its picture is recorded on the loop at such a low magnification that all chromosomes are within the field of vision. This picture is later on continually displayed on the second monitor during the whole analysis. One chromosome after the other is then analysed by aid of the first monitor, where the picture can be modified by the zooming and contrast enhancement systems. It is very easy to recognize the individual chromosomes, analysed at high magnification, and to mark them off on a sheet of 33-751808
499
transparent materials arranged on the faceplate df the monitor with the survey picture. The upper part of fig. 2 presents such a survey picture. The four smaller pictures in the lower part of the illustration demonstrate for one small chromosome group (also marked by a ring in the upper picture) at high magnification the principle of analysis, by aid of pictures with different levels of contrast. It should be noted that, whereas in the more elaborate apparatus described in the preceding paper, professional high quality TV equipment is necessary,ordinary standard. TV camera equipment can be used for the metaphase analytic work described here. Thus the cost for a complete TV arrangement including both monitors, the video recorder and the contrast enhancement “black box” is comparable to the cost of the high quality microscope used. Thesestudieswere supported by research grants from the Swedish Natural Science Research Council and the Swedish Cancer Society. References 1. Caspersson, T, Lindsten, J, Lomakka, G, Wallman, H & Zech, L, Exptl cell res 63 (1970) 477. 2. Caspersson, T, Issler, P & Lomakka, G, Exptl cell res 75 (1972) 543. 3. Caspersson, T, Fluorescence techniaues in cell biolbgy (ed A- A Thaer & M Sern&) p. 107. Springer, Berlin, Heidelberg, New York (1973). 4. Caspersson, T, Haglund, U, Lindell, B &Zech, L, Exptl cell res 75 (1972) 541. 5. Caspersson, T, Lomakka, G, Zech, L, Issler, P, Kudynowski, J & Kvarnstriim, K, Exptl cell res 88 (1974) 427. 6. Caspersson, T, Exptl cell res 92 (1975) 333. Received December 19, 1974
Isopynic separation of ram spermatids in colloidal silica gradients M. LOIR and M. LANNEAU, Znstitut National de la Recherche Agronomique, Nouzilly, F-37380 Monnaie, France Summary. By a two-step separation procedure, round and elongated spermatid populations were obtained with purities of 90-97 % and an intermediate spermatid population with a purity of 85 %. Cells of ram Exptl Cell Res 92 (1975)
PRELIMINARY
NOTES
TV-based analysis of Giemsa-banded chromosomes
are two-fold. Firstly, photography greatly helps in the detailed scrutiny of each individual chromosome part. Different banding T. CASPERSSON, Institute for Medical Cell Research methods demand photography of different and Genetics, Medical Nobel Institute, Karolinska Znstitutet, S-104 01 Stockholm, Sweden degrees of refinement, the greatest demands applying to the fluorescence procedures. A Summary. In large scale chromosome aberration second reason is that, because of the great studies by Giemsa-based banding techniques the photographic work is the most time-consu&g. A number of chromosomes in most mammalian TV-based procedure which entirely eliminates photometaphase plates, there must be some congraphy is described. This involves as main fiatures image contrast enhancementon arbitrarily selected venient way in which to mark individual gray scale levels, continuously regulable magnification chromosomes as they are analysed in order to and a special device for marking analyzed chromosomes during the progressof the work. monitor the progress of the work. This is easily done with the aid of a photograph. Working time could be greatly reduced by The slowness and also the tediousness of the conventional procedures of karyotype anal- the use of a TV procedure which eliminated ysis with banding techniques are serious most of the photographic work involved in obstacles to their wider application in such fluorescence analysis [l-4]. The negative of important fields as the analysis of frequency the fluorescence photograph was observed by and character of chromosome aberrations a TV camera. The image on the monitor was caused, e.g. by external mutagens or carcino- manipulated by means of an optical zoom gens, in which studies large series of meta- system before the TV camera and an image phases must always be analysed. contrast enhancement system [2] incorporated The main delaying factors in the conven- in the electronic system. The arrangement not tional procedures lie in the photographic only saved working time but also made the work. The reasons for the use of photography whole work considerably less tiring.
Fig. 1. Overall view of the
instrument. Exptl Cell Res 92 (1975)
498
Prrliminury
notpx
~. :
&
These results led to efforts to build an analogous system for Giemsa stained preparations in which photography could be completely eliminated. The applicability of such a procedure to different types of chromosomeanalytical work has been discussed in an earlier publication [5]. Expt~ Cell Res 92 (197Sj
F$. 2. The mode of function, cl text.
Experiences from the development of the instrument for low-level absorption image analysis, described in the preceding paper [6’j, have now also led to the generation of an improved and simplified apparatus for more rapid analysis of preparations stained by Giemsa or Giemsa-like procedures.
Preliminary notes
In this apparatus (fig. 1) a TV camera looks directly into the microscope. No photography is used. A zoom system permits easy and continuous change of magnification within a range 1-3. The optical system is adjusted so that by the lowest magnification the field of the picture covers the area of an ordinary metaphase. For detailed analysis of each individual chromosome, suitable higher magnifications are used. A picture contrast enhancement arrangement is incorporated, in principle the same as that described in reference [q for low level absorption work. The range is extended, however, so that it is possible to obtain great enhancement of small differencesin absorption in faintly stained chromosome regions as well as in intensely stained parts, where fine banding details are often hidden from observation by the overall high absorption. To the naked eye, the effect of this contrast enhanckment of the picture on the monitor is quite striking and the combination of regulable zooming and regulable contrast enhancement facilitates the analysis of chromosome details very much and increases the speed of work. A videotape recorder and a second monitor are incorporated so that individual chromosomes can be marked as work proceeds. The videotape is arranged as a closed loop. When the metaphase has been selected, its picture is recorded on the loop at such a low magnification that all chromosomes are within the field of vision. This picture is later on continually displayed on the second monitor during the whole analysis. One chromosome after the other is then analysed by aid of the first monitor, where the picture can be modified by the zooming and contrast enhancement systems. It is very easy to recognize the individual chromosomes, analysed at high magnification, and to mark them off on a sheet of 33-751808
499
transparent materials arranged on the faceplate df the monitor with the survey picture. The upper part of fig. 2 presents such a survey picture. The four smaller pictures in the lower part of the illustration demonstrate for one small chromosome group (also marked by a ring in the upper picture) at high magnification the principle of analysis, by aid of pictures with different levels of contrast. It should be noted that, whereas in the more elaborate apparatus described in the preceding paper, professional high quality TV equipment is necessary,ordinary standard. TV camera equipment can be used for the metaphase analytic work described here. Thus the cost for a complete TV arrangement including both monitors, the video recorder and the contrast enhancement “black box” is comparable to the cost of the high quality microscope used. Thesestudieswere supported by research grants from the Swedish Natural Science Research Council and the Swedish Cancer Society. References 1. Caspersson, T, Lindsten, J, Lomakka, G, Wallman, H & Zech, L, Exptl cell res 63 (1970) 477. 2. Caspersson, T, Issler, P & Lomakka, G, Exptl cell res 75 (1972) 543. 3. Caspersson, T, Fluorescence techniaues in cell biolbgy (ed A- A Thaer & M Sern&) p. 107. Springer, Berlin, Heidelberg, New York (1973). 4. Caspersson, T, Haglund, U, Lindell, B &Zech, L, Exptl cell res 75 (1972) 541. 5. Caspersson, T, Lomakka, G, Zech, L, Issler, P, Kudynowski, J & Kvarnstriim, K, Exptl cell res 88 (1974) 427. 6. Caspersson, T, Exptl cell res 92 (1975) 333. Received December 19, 1974
Isopynic separation of ram spermatids in colloidal silica gradients M. LOIR and M. LANNEAU, Znstitut National de la Recherche Agronomique, Nouzilly, F-37380 Monnaie, France Summary. By a two-step separation procedure, round and elongated spermatid populations were obtained with purities of 90-97 % and an intermediate spermatid population with a purity of 85 %. Cells of ram Exptl Cell Res 92 (1975)