Dynamic ultrastructure of mouse pulmonary alveoli revealed by an in vivo cryotechnique in combination with freeze‐substitution

Abstract

A morphological approach to cell dynamics is usually difficult, since routine preparative techniques for electron microscopy always induce artifacts due to cessation of the blood supply into organs. An in vivo cryotechnique followed by the freeze-substitution method probably reduces such problems. It was applied for examining the pulmonary alveoli of BALB\c mice in vivo. The following ultrastructural features were revealed. (1) A surfactant layer provided a continuous covering to the alveolar epithelium. (2) Pleural epithelial cells, alveolar cells and endothelial cells contained many small vesicles and pits. In the alveolar epithelium, they were often localised near microtubules. (3) Typical lamellar structures in large alveolar epithelial cells were rarely detected. (4) Circulating erythrocytes with various shapes were observed in branching blood capillaries. (5) A close association between erythrocytes and the endothelium was seen at the peripheral alveolar septum. Such ultrastructural arrangements may be appropriate for the physiological functions of the pulmonary alveoli, such as exchanges of gases or materials in vivo.

Dynamic study of intramembranous particles in human fresh erythrocytes using an “in vitro cryotechnique”

“…Flowing erythrocytes in blood vessels can easily change their shape because of the surrounding conditions, such as the types of vessels or flow speed, as well as some processes for preparing electron microscopic specimens (Terada et al, 1998a). Using the ''in vivo cryotechnique,'' we already reported three-dimensional shapes of flowing erythrocytes in sinusoids of living mouse livers (Terada et al, 1998b) and spleens (Xue et al, 2001), large blood vessels, including the aorta and caudal vena cava (Xue et al, 1998), glomerular capillaries of mouse kidneys (Yu et al, 1998), and capillaries of mouse lungs (Takayama et al, 2000). A shear stress from the flow is one of the factors, which affect the erythrocyte shapes (Fisher et al, 1978).…”

Detection of injected fluorescence-conjugated IgG in living mouse organs using “in vivo cryotechnique” with freeze-substitution

“…This technique was performed by rapid freezing of these tissues with liquid isopentane-propane cryogen (À1938C) with or without cutting using a cryoknife (À1968C) cooled down in liquid nitrogen. In previous studies, the dynamic morphological changes in living cells, tissues, and organs using the cryotechnique were observed by electron and light microscopy (Ohno et al, 1996;Takayama et al, 1999Takayama et al, , 2000Terada et al, 1998;Xue et al, 2001;Zea-Aragon et al, 2004b).…”

Advanced Application of the In Vivo Cryotechnique to Immunohistochemistry for Animal Organs

“…The lack of blood supply into some organs can easily modify their ultrastructure and molecular distributions, presumably induced by anoxia and rapid loss of blood volume and pressure [17,18,26,27,[30][31][32]35]. In addition, such loss of blood supply, inevitable in experimental studies involving conventional chemical fixation or common QF of fresh tissue specimens, sometimes modifies the immunoreactivity of various functional molecules, due to the rapid ischemia, which induces diverse responses in living animal organs [8,29].…”

“…Because in vivo cryotechnique can immediately cryofix cells and tissues of living animals, it avoids problematic changes due to stopping of blood circulation and resection of organs, and is useful for demonstrating not only ultrastructures of living animals with blood circulation but also quick and transient morphological changes in vivo without such unwanted effects [17,18,26,27,31]. Tissue samples directly frozen by in vivo cryotechnique can be processed in the same ways as those by other conventional QF methods, including slamming QF, plunging QF and high-pressure freezing, not only for electron microscopy, but also for light microscopy ( Fig.…”