The scanning electron microscope (SEM) is a powerful tool for acquiring surface information of micro and nanostructures under vacuum conditions. Recently, methods for observing samples under atmospheric pressure in a scanning electron microscope (SEM) have been investigated. Hitachi previously released a novel atmospheric SEM (ASEM) technique for observing samples that are present in ambient air conditions and are separated from the electron gun by a membrane [1]. Environment in the specimen chamber can be kept in ambient air conditions while the electron source remains under vacuum ( Fig. 1(a)). By using this system, observation of wet, liquid, and even bulk samples is possible. While wet materials are clearly observed at an optimized distance between the membrane and sample surface, typical ASEM images taken in atmosphere have more distortion when compared to conventional, high vacuum SEM images. The reason why ASEM images appear "blurred" is due to the electron beam being scattered by air molecules as shown in Fig. 1(b). To solve this problem, methods have been developed to reduce the electron scattering effect [2,3]. Here we present an image enhancement algorithm (electron scattering corrector: ES-Corrector) for ASEM image improvement. Blurred images created by scattered electrons can be improved utilizing the ES-Corrector function as demonstrated in figure 2 for leaf surface of Japanese radish collected under atmospheric pressure. The ES-Corrector restored image shows great improvements in clarity and edge sharpness.The separation membrane may incur water vapor from a wet specimen when the sample is close to the membrane. In these cases, observation of clear images is possible after removal of water by reducing the pressure in chamber via an additional vacuum pump. However, this process poses a risk of changing the shape of a wet sample and therefore to alleviate any potential artifacts, a cooling stage with temperature adjusting controller was utilized. Additionally, the phenomenon of water vapor accumulation on the membrane can also be eliminated by the use of the chilled stage at 1 o C. Figures 3 and 4 demonstrate the observation of a cucumber cross section and a frozen carrot. As shown in Fig. 4 it was possible to observe the temporal change of a thawing carrot and confirm differences in the shape of cells between non-frozen and frozen samples. Therefore coupling this device with ASEM, a wide range of applications including wet / hydrated specimens and observation of dynamic freeze/thaw investigations can be observed. References:[1] Y. Ominami et al., Microscopy, 64, 97-104 (2014).[2] K.
We aimed to evaluate if human milk-based fortifier (HMBF) affects human milk fat globule (MFG) size less than cow milk-based fortifier (CMBF), which may impact overall infant feeding tolerance. Measurements of donated human milk were performed before fortification as well as at 1 hour, 24 hours, and 48 hours after fortification with CMBF or HMBF. MFG size in each sample of fortified milk was measured by laser light scattering. MFG size in the fortified milks increased gradually over time. At 24 and 48 hours after fortification, MFG size in the milk with CMBF was larger than that in the milk with HMBF (4.8 ± 0.5 vs 4.3 ± 0.3 μm, p<0.01, 5.1 ± 0.7 vs 4.5 ± 0.4 μm, p = 0.03, respectively). HMBF is associated with less alteration of MFG size than CMBF. This may have an impact on feeding tolerance of very preterm infants.
The usefulness of the transmission electron microscope (TEM) for pathological diagnosis is apparent. However, high operating costs and other disadvantages have limited the ability to maintain and operate a TEM. In recent years, a general-purpose benchtop low-vacuum scanning electron microscope (LVSEM), which is inexpensive and easy to operate, has been developed and is expected to be applied in electron microscopic pathological diagnosis. To date, we have previously observed TEM ultrathin sections of IgA nephropathy with a benchtop LVSEM using an ultra variable-pressure detector (UVD) and a newly developed holder for observing scanning transmission electron microscope (STEM) images (UVD-STEM holder) and compared the images with those obtained with typical TEM observations. We reported the results in the 53rd Annual Meeting of the Japanese Society for Clinical Molecular Morphology and the 64th Symposium of The Japanese Society of Microscopy, and discussed the validity of the methods in the pathological diagnosis of IgA nephropathy and other renal diseases As a result, we demonstrated the potential for pathological diagnosis using benchtop LVSEM. In this study, we similarly examined typical kidney diseases such as membranous nephropathy, lupus nephritis, and amyloidosis. We could obtain sufficient data for the pathological diagnosis of IgA nephropathy, membranous nephropathy, and lupus nephritis. However, it is difficult to detect amyloid fibres that are characteristic of amyloidosis. The development of this method is expected to expand the possibilities for pathological diagnosis using electron microscopy, including its application to other diseases.
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