Use of lanthanum as a conductive material in secondary electron imaging

Abstract

The use of colloidal lanthanum (La) as a conductive material in preparatory procedures for scanning electron microscopy of dehydrated non-conductive biological specimens has been explored for the present study. The ability of La to bind certain moities of the plasma membrane has been successfully employed to impart conductivity to a wide variety of biological material. LaNO3 solution (2%) in 0.1M cacodylate buffer (CB) was treated with 0.01 N NaOH with vigorous shaking. At pH 7.5 – 7.8 faint flocculent material appeared which indicated the formation of colloidal La. This solution was used for incubation of fresh and/or fixed biological specimen as described. Specimens were incubated in 1% La solution (final concentration) for 3h and 6h followed by fixation in 2.5% glutaraldehyde (glu) for 15 min on specimens were incubated and fixed simultaneously in 1% La and 2.5 glu (final concentration) in 0.1M CB for 3h. Then washed and post fixed in 1% OsO4 in 0.1M CB for 30 min. Tissues were dehydrated in ascending grades of acetone and dried by the CPD technique. After mounting on metallic stubs, the specimens were examined in JEOL 100 CX electron microscope with scanning attachment in the voltage range 20-40 kV and beam current of 50 μA and also in SEM Hitachi S 520 in the voltage range of 1-30 kV and beam current 10-100 μA. It has been shown that La generates enough secondary electrons to form an optimal image having a good contrast. The resolution achieved in this preparation is as good as that obtained in metal-coated samples (Figs. 1,2). In contrast to metal-coated samples, masking of topographical features is not observed in La incubated samples. The later samples are transparent (Fig. 2). The technique is applicable to a variety of specimens such as lipid vesicles (Fig.3), algae (Fig.4), ciliates, single cell suspensions (Fig.2), various tissues of rat (Fig. 5) and radical of germinating beans (Fig.6). La was also used for the preparation of biological material for back-scattered imaging mode. To study the process of phagocytosis, yeast cells were coated with La and incubated with cultures of macrophage tumor AK-5 cells. The engulfed yeast cells could be detected inside the cell as dark bodies (Figs.7,8).