Evaluation of lanthanide tracer methods in the study of mammalian pulmonary parenchyma and cardiac muscle by electron energy-loss spectroscopy

Summary Lanthanum (La) has widely been used as a tracer to study the integrity of plasma membranes. With conventional transmission electron microscopy (cTEM), the absence of electron scattering deposits from the cytoplasm has generally been assumed to reflect an intact cell membrane. However, the application of electron spectroscopic imaging (ESI) and electron energy-loss spectroscopy (EELS) reveals that electron scattering deposits may be present which do not contain La. However, La could be detected in regions of pulmonary parenchyma and cardiac muscle that were devoid of electron scattering deposits. Therefore, to exclude misinterpretations based on cTEM the application of microanalytical techniques is strongly recommended for the study of the integrity of plasma membranes by means of La tracers. In addition, ESI and EELS are shown to distinguish between different tracers in simultaneous applications of La and terbium (Tb) which were used at the different faces of the pulmonary air-blood barrier. The analysis of the distribution of both tracers which form electron scattering deposits, indistinguishable by cTEM, may help us to understand the different functional significances of cellular alterations of both cellular borders of the barrier. As was shown for La, however, strictly controlled conditions are mandatory during the fixation procedure because an increase in the incubation time to more than 1 h in samples of pulmonary parenchyma may result in the occurrence of La deposits within the cytoplasm. In the absence of electron scattering deposits, the presence of La in glycogen granules and ribosome-containing areas of various types of alveolar septal cells even after 15 min incubation indicates that the absence of deposits does not necessarily correspond to the absence of the tracer.