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Cryofixation of Heart Tissue for X-Ray Microanalysis

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Cryofixation of Heart Tissue for X-Ray Microanalysis Scanning Microscopy Volume 3 Number 4 Article 25 12-4-1989 Cryofixation of Heart Tissue for X-Ray Microanalysis Alice Warley St. Thomas's Hospital Campus Follow this and additional works at: https://digitalcommons.usu.edu/microscopy Part of the Biology Commons Recommended Citation Warley, Alice (1989) "Cryofixation of Heart Tissue for X-Ray Microanalysis," Scanning Microscopy: Vol. 3 : No. 4 , Article 25. Available at: https://digitalcommons.usu.edu/microscopy/vol3/iss4/25 This Article is brought to you for free and open access by the Western Dairy Center at DigitalCommons@USU. It has been accepted for inclusion in Scanning Microscopy by an authorized administrator of DigitalCommons@USU. For more information, please contact [email protected]. Scanning Microscopy, Vol. 3, No. 4, 1989 (Pages 1247-1252) 0891-7035/89$3.00+.00 Scanning Microscopy International, Chicago (AMF O'Hare), IL 60666 USA CRYOFIXATION OF HEARTTISSUE FOR X-RAY MICROANALYSIS Alice Warley Division of Biochemistry U.M.D.S. St. Thomas's Hospital Campus Lambeth Palace Road London SEl 7EH, U.K. (Received for publication April 11, 1989, and in revised form December 04, 1989) Abstract Introduction Cryofixation of tissues is necessary to be able to Changes in the concentrations of the inorganic study the concentrations of elements by X-ray micro­ elements Na, Mg, Kand Ca are thought to control the analysis. Simple dissection of heart tissue fragments functions of many different cells. Although much is of the size needed for optimum cryofixation by the con­ known about total elemental concentrations especially ventional methods of plunge or slam freezing leads to of normal tissues, more remains to be learned par­ the development of ischaemia in the tissue fragments ticularly about elemental concentrations in individual and a consequent redistribution of the diffusible cells, subcellular organelles, and whether alterations elements. Heart tissue can be frozen in vivo using occur after the onset of disease. The technique of liquid nitrogen cooled Cu clad pliers, but the morpho­ X-ray microanalysis offers a means of unequivocally logical detail is preserved better if the cooled pliers measuring elements at the cellular and subcellular are exposed to liquefied propane immediately before levels and, for the study of the diffusible elements, freezing, Concentrations of Na are lower and concen­ cryofixation and cryopreparation are necessary, to trations of Kare higher in the tissue which has been arrest physiological processes and to retain elements frozen in vivo compared to tissue frozen after in their in vivo situation. Although there has been an dissection. increasing amount of interest in the cryofixation of biological material and several reviews and books on the subject have been published (see, e.g., Plattner and Bachmann, 1982; Robards and Sleytr, 1985; Steinbrecht and Zierold, 1987; Elder and Bovell, 1988), in the majority of instances emphasis has been placed on the development of cryotechniques to produce specimens suitable for morphological studies. However, if the intention is to study the specimen by X-ray microanalysis, then the aim must be to preserve the distribution of elements within the specimen. It is vital that movement of elements does not occur at any stage during specimen preparation and morphological considerations may have to take second place. During the development of X-ray microanalytical techniques attention has tended to focus on the later stages of specimen preparation, sectioning (discussed in Roomans et al., 1982) and transfer to the electron microscope (see, e.g., Frederik and Busing, 1981; Hagler and Buja, 1986) as points at which movement of elements could KEYWORDS:Cryofixation, heart tissue, X-ray micro­ occur. These possibilities have now been well discussed analysis, rats, sodium, potassium. (Hagler and Buja, 1984; Hagler, 1986; von Zglinicki and Uhrik, 1988; von Zglinicki et al., 1988). However it is Address for correspondance: essential to bear in mind that preparation of the Division of Biochemistry specimen for freezing is the most likely step at which U.M.D.S., St. Thomas's Hospital Campus alteration in the distribution of elements may occur; Lambeth Palace Road this is often overlooked. London SEl 7EH, U.K. The various methods which may used for preparing Phone No.: 01-928-9292 ext 3019 tissues for freezing are: dissection of pieces small 1247 Alice Warley enough to give rapid freezing; tissue preparations excess liquid nitrogen to evaporate. The cool dry (e.g., papillary nruscle) isolated under well defined pliers were then placed into the coolant vessel which experimental conditions; and isolation of single cells contained liquefied propane until needed for freeze from the tissue. Each of these methods has its own ad­ clamping of tissues. Male CSE Wistar rats weighing 300 vantages and drawbacks. For dissected fragments of g were used as control animals. The animals were tissue immersion in a liquid cryogen or impact freezing anaesthetised heavily with sodium pentabarbitone (35 against a cooled metal block are the techniques which mg/kg). The thorax was opened, the vena cava cut, and have been used for cryopreservation. There is no the beating heart rapidly clamped in the jaws of the doubt that excellent morphological detail can be cooled copper clad pliers. The flattened piece of obtained using either method of cryofixation (see e.g. tissue obtained was stored under liquid nitrogen until Sitte et al., 1987; Roos and Barnard, 1985) but the sectioning. In one experiment small pieces of heart effects of preparing the tissue for freezing need to be tissue were dissected placed on silver pins and frozen considered. Dissection of a tissue leads to damage at by plunging into liquefied propane using a Reichert the cut surface and loss of elements from the outermost KF80 freezing device. layer of cells (Somlyo et al., 1977; Hagler et al., The method used for sectioning was similar to 1981; Zierold and Schafer, 1988). In addition if a that described by Hagler et al. (1983), but the wafer tissue remains metabolically active after dissection, of tissue is somewhat thicker (approx. 1 mm), because changes due to anoxia can develop rapidly (von of this the block needs to be tr:llllned to an angle Zglinicki et al., 1986) leading to a loss of the ionic before sections can be cut. Sectioning was carried out gradient across the plasma membrane. Isolated using a Reichert FC4D cryoultramicrotome. The flattened preparations of papillary nruscle (Wendt-Gallitelli and tissue was fractured under liquid nitrogen using a Walburg, 1984; Wendt-Gallitelli and Jacob, 1984; cooled razor blade. A small piece of tissue was placed Wheeler-Clarke and Tormey, 1987) or epithelium (Gupta, in the jaws of a vice type chuck (obtainable from 1989; Rick and Schratt, 1989) have proved useful for Reichert-Jung). The knife was set at as large an angle study by X-ray microanalysis. The physiology of such as possible and the block trimmed. Sections 200 mn isolated preparations has been well studied, the thick were cut from the point of well frozen tissue at preparations are of low bulk, and often suitable a temperature of -125°c to -130°c. The sections were apparatus has been designed so that optinrum pressed between two Pioloform coated grids. The grids cryofixation is obtained with the tissue in a known were separated and placed into a precooled brass block functional state. Similarly isolated cells provide a for transfer to a freeze drier. The sections were specimen of low volume, present in a well defined allowed to freeze dry overnight and were coated with a medium, and it possible to control such preparations thin layer of carbon before analysis. right up to the moment of freezing. However, several Analysis was carried out using a JEOL lOOCXelec­ X-ray microanalytical studies have now shown that tron microscope in STEMmode. The accelerating voltage intracellular elemental concentrations can be altered was 100 keV and the beam current (1.3 nA to 1.8 nA) was in freshly isolated cells (Warley, 1986; Zierold and measured with a Faraday cage. The area of analysis was Schafer, 1988; Warley, 1989) and, unless single cells 0.25 pmx 0.25 pm for myofilaments and 0.125 pmx 0.125 of known viability can be selected (Wendt-Gallitelli pm for mitochondria. For analysis the specimen was and Isenberg, 1989), the effects of isolation need to tilted to 35. Spectra were collected and quantitative be taken into account when interpreting results from data were obtained by use of a Link Analytical 860 these preparations. The use of physiological series 2 EDS detection system and Quantem software. preparations or isolated cells might seem the best This program is based on the continuum normalisation approach for preparing specimens for X-ray procedure of Hall and has full facilities for gain microanalysis. However these methods may not be calibration and background subtraction. The energy appropriate for studying changes in element region used for determining the continuum was 6 keV to concentrations due to disease, as it is possible that 16 keV. For further details of this analysis system see any alterations in vivo could be destroyed during the Spencer et al. 1988. isolation process. This paper reports preliminary results obtained by freeze clamping heart tissue in Results vivo using cooled copper clad pliers which have been exposed to liquefied propane immediately before Morphology. freezing. When liquid nitrogen cooled Cu clad pliers were used to freeze clamp intact heart tissue, the results Materials and Methods were disappointing (Fig. 1), good preservation was not achieved to any great depth in the tissue. Invariably a A pair of Cu clad pliers were made after the gradient of ice crystal damage could be seen even in designs in Hagler and Buja (1984) and Ingram and Ingram the first layer of myocytes. The presence of ice .
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