Freeze substitution of high-pressure frozen samples: the visibility of biological membranes is improved when the substitution medium contains water

Biological membranes are often poorly visible with the electron microscope after high‐pressure freezing and freeze‐substitution. The water content of the sample and of the substitution medium is one factor among others that strongly influences membrane visibility. In order to investigate this effect, high‐pressure frozen yeast cells, rat‐pancreas tissue and arthropod tissue were freeze‐substituted with and without adding water to the substitution medium. The visibility of the biological membranes was generally improved if the substitution medium contained 1–5% water. The effect was especially pronounced in yeast cells, where membrane visibility was poor after freeze‐substitution with water‐free medium but good after addition of 5% water to the substitution medium.     

MRT letter: Human bloodstains on antique aboriginal weapons: a guiding low-vacuum SEM study of erythrocytes in experimental samples on ethnographically documented biological raw materials

The aboriginal use of reed and bone as raw materials for knives and daggers, respectively, has been well-documented ethnographically in some geographical areas of Melanesia. Because of the significant role that these weapons played in inter- and intra-ethnic aggression, they can potentially have retained smears from the contact with human blood. To carry out a guiding low-vacuum scanning electron microscopy (SEM) study of specific interest to ethnography, the outsides of a fragment of stalk of giant cane (Arundo donax) and tibial diaphysis of domestic sheep (Ovis aries) were smeared with peripheral human blood. No biological specimen preparation was applied to the samples. After just over 1 month, bloodstain boundaries and their neighboring inner areas were examined via secondary electrons by a variable-pressure SEM (VP-SEM) working in low-vacuum mode. On both substrates, bloodstains exhibited micro-scales. No janocyte (erythrocyte negative replica) was observed in the examined areas. However, erythrocytes were seen crowded together as grain-shaped corpuscles in the smear on reed, and several hecatocytes (moon-like shaped erythrocytes) were evidenced in the smear on bone. The results of this study suggest that a VP-SEM working in low-vacuum mode can be used fruitfully to detect blood remains in medium-sized reed and bone antique aboriginal artifacts. This procedure can prospectively help to ethnographic museum curators and aboriginal-art surveyors as an easy guiding test in the valuation of antique traditional weapons prior to acquisition, when the real use of a piece has been claimed by the supplier.     

SEM examination of human erythrocytes in uncoated bloodstains on stone: use of conventional as environmental-like SEM in a soft biological tissue (and hard inorganic material)

Although nowadays the so-called environmental scanning electron microscopes (ESEMs) allow the observation of the samples without metal or carbon coating, many conventional scanning electron microscopes (SEMs) are still in use. On the other hand, the presence of erythrocytes (red blood cells, RBCs) in a smear is considered a blood confirmation. Such a presence has been previously reported even in Lower Stone Age implements. In previous works, I have reported several studies dealing with cytomorphology of RBCs in bloodstains using scanning electron microscopy with standard specimen preparation procedures, i.e. via coating the samples before SEM analysis. In order to explore the potential of conventional SEM as environmental-like SEM in haemotaphonomical studies, two alkaline (limestone) and two acid (flint) rock fragments were smeared with human blood from a male and a female. The bloodstains obtained in this way were then air dried indoors and stored into a non-hermetic plastic box. Afterwards, the smears and their rock substrates were examined directly without coating, via secondary electrons, using a JEOL JSM-6400 scanning electron microscope. Satisfactory results reveal the capability of a conventional SEM to work in secondary-electron mode as an environmental-like SEM on these kinds of biological and inorganic materials, and probably in many other biological and non-biological samples.