In situ conservation of diffusible elements in liver cells after cryofixation, cryosubstitution, and low temperature embedding at 193 K in HM23 Lowicryl resin

The advantages of 3CRYO-methods (cryofixation by quick-freezing, cryosubstitution in pure acetone and cryoembedding in new Lowicryl resin Kl lM or HM23) for combined ultrastructural, immunocytochemical and microanalytical studies a t subcellular level have been reported earlier (Edelman, 1991; Quintana 1991a, 1991b, ). For microanalytical purposes the method of reference is still based on the analysis of freezedried cryosections of quick-frozen fresh tissues. Therefore, the validity of other alternative approaches, such as the 3CRYO-metods, requires further comparison with the previous ones (Wrobleswki and Wrobleswki,l984). Edelman has compared results on muscle cells cryosubstituted in pure acetone (193 K) and cryoembedded in Kl lM Lowicryl (213 K) , using a commercial instrument (CS-Auto), with those obtained from freeze-dried cryosections, (Edelman, 1991) . In this case a retention of about 70% of intracellular potassium was obtained following the best cryoembedding procedure. We here present the first results obtained after cryosubstitution of liver at 183 K in pure acetone followed by infiltration and cryoembedding in Lowicryl HM23 at 193 K. The cryofixation of liver fragments of about 1 mm3 from anaesthetized r a t , was performed by rapid immersion in liquid propane (Halpern and Quintana, 1989; Quintana, 1991a). For cryosubstitution and cryoembedding we have utilised a new home made cryosytem (Quintana 1991~). The main characteristics of this integrated cryosystem are: (1) the cold source is a freezer (minimal temperature 180 K), so no use of cryogenic fluids is required, (2) efficient shaking of samples in freeze-substitution and cryoinfiltration liquids, (3) easy manipulation of samples between the different modules of work, (4) control of temperature throughout the successive steps, especially during cryopolymerisation. ( 5 ) total protection from toxic vapours coming from Lowicryl resins. Cryosubstitution was performed at 183 K for 3 days in pure acetone. The cryoembedding process was as follows: Infiltration: resin-acetone 1:l (V/V),193 K, 4 h. ; resin-acetone 2:l (v/v), 193 K, 4 h. ; pure resin 193 K, 4 h. ; pure resin 193 K, overnight. Cryopolymerisation was carried out in open flat embedding moulds under UV and an inert atmosphere of N,, for 4 days. The freezer was then switched off and the blocks were allowed to warm at room temperature under UV radiation for about 2 days. Dry -cut sections ( about 1 pm thick) were obtained using glass knives and were spread on Ni grids without ariy solvent . Energy-dispersive X-ray spectra were recorded a t 200 kV in a JEOL 2000FX TEM by nieans of a Link ultrathin window (UTW) Si(Li) detector and a Link AN10000 multichannel analyscr and microanalysis system. Ultrastructure was found to be satisfactory over 2 or 3 cell layers in the surface of cryofixed liver fragments (Fig 1). Ice crystals were not observed in those nuclei located near the cryofixation front . The X-ray analysis of several regions of cytoplasm and nucleus is shown in Fig 2. A semiquantitative method for the analysis of thin films without standard (Cliff-Lorrimer, 1975) was used in order to evaluate the relative concentrations of Na/P, Mg/P S/P, CI/P, K/P, Ca/P and K/Na. The results are shown in table I and are compared with those obtained by Somlyo (1985), von Zglinicki (1987) and Zierold (1988) on freeze-dried cryosections of liver cells. Concerning the conservation of diffusiblModernnts, it is worth noting that results provided by the 3CRYO-methods are similar to those obtained from freeze-drying cryosections. results indicate that the use of lower temperature during cryosubstitution and cryoembedding has a positive effect on the preservation of di! Tusible elements as had been pointed out by Kellenberger (Kellenberger et a1.1986). In comparison with the freezedrying cryosection technique, the 3CRYO-methods offer the advantage of a better ultrastructure which is a necesary condition for microanalysis at subcellular level . In summary, the present