Transfomation of SEM into TEM without any atachment or remodel! Yoshinori Muranaka & Isao Ohta

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1 Transfomation of SEM into TEM without any atachment or remodel! Development and evaluation of specimen holder of scanning electron microscope for observation of transmitted ultra-thin sections by secondary electron image Yoshinori Muranaka & Isao Ohta Research Equipment Center, Hamamatsu University School of Medicine, Handayama, Hamamatsu Japan

2 Summary We developed and evaluated the special specimen holder of scanning electron microscope (SEM) to observe the transmitted electron images of ultra-thin section for transmitted electron microscope (TEM) by secondary electron of SEM without any attachments or remodels. The specimen holder made of aluminum or carbon is equipped with the grid stage, the plate for generation of secondary electron and the objective aperture. Whole grid areas can be easily observed by the functions of tilting and rotation. By the holder, the quality of secondary images of TEM specimens by the field emission SEM was comparable to that of TEM images. Moreover, developed holder is useful for observation of the backscatter electron image and X-ray element analysis in the section. Introduction There are SEM and TEM for an electron microscope. The former observes the surface of the bulk-shaped sample, and the latter is for transmitted image observation of the film-shape sample such as ultra-thin section. An observational purpose and the shape of the sample differ each. We tried to develop a special specimen holder of SEM that can be used for an observation of transmitted electron images of ultra-thin section for TEM by secondary electron of SEM 1) (fig. 1). The advantages of the scanning transmitted image are easily adjusting contrast and fewness of the sample damages 2). Scanning transmitted image may be able to be observed by an attached scanning transmitted electron detector. However, this device is not common because of a high price. Requirements of developments Requirements for the holder were as follows. (1) SEM images can be observed by the secondary electron detector. (2) Whole area of specimen grids for TEM can be observed as secondary electron images in any SEM. (3) Resolution of the images should keep that of SEM. Resolution of the images by field emission SEM should be comparable to that by conventional TEM, and resolution of the images by small desktop-type SEM should be high enough to use for clinical diagnosis. (4) Any types of specimen for TEM observations, such as ultra-thin sections, negative stained materials and replica membranes, should be applicable. (5) Backscatter electron images and X-ray element analysis can be obtained by SEM equipped with a each detector for backscatter electron or X-ray. Fig.1 The comparison of the principal between general SEM observation and transmission secondary electron image observation. Left: general SEM specifications to observe a bulk sample. Right: specifications to observe a transmission secondary electron image for ultra-thin section. Specifications of developped sample holder The development holder became the following structure according to a developmental requirements (fig. 2). (1) The main body was made by duralumin, and the analysis specifications use carbon for a part. The size of the main body were 10 mm in diameter and 15 mm in height to load it to the sample stage of various SEM. (2) A hole of 3 mm in diameter was drilled on the top surface of main body for fixing a sample grid and locking mechanism. (3) The aspect to convert a transmitted electron beam into a secondary electron was duralumin abrasion side of horizontal angle 60 degrees. (4) A objective aperture disk made of molybdenum was loaded in the holder for highly resolving power and ratio of S/N. The aperture size prepared from 60 µm in diameter for high magnification to 300 µm in diameter for wide view. (5) Objective areas were removed roughly by using the function of incline and revolving system in SEM (fig. 3) and tuned in exactly by original X-Y movement of the specimen stage. Results The development holder wsa tried out and showed the Fig.2 Upper drawing and photograph of the developed sample holder, grid mesh and locking ling. Left drawing is locking structure.

3 following results, the functionality, the applied nature and performance. (1) The specimen grid could be kept horizontally on the upper surface of the specimen holder without specimen drift. Magnetic grids, such as nickel grid could be also kept stably by fastening a holding key (fig. 4). (2) Objective areas could be easily and smoothly observed by using the function of incline and revolving in SEM (fig. 2 and fig. 5). (3) Resolution and image quality under an observation by high resolution SEM such as field emission SEM were comparable to that by TEM when maximum accelerating voltage in SEM and high beam current were used. Transmitted secondary electron images of various cellular organelles from the ultra-thin sections could be seen (fig. 6 and fig. 7). When the TEM images at the same sites were compared with the transmitted secondary electron images in SEM by applying the developed holder, the both images at the same site were almost the same quality in any magnifications (fig. 8). Furthermore, resolution of the scanning transmitted secondary electron image by the small SEM of the desktop type is enough for the use of the clinical diagnosis such as membraneous nephritis from kidney biopsy (fig. 9). (4) Transmitted secondary electron images could be obtained also from other membrane-type specimens, such as replica membrane, negative stain with virus and thinning materials as the ultra-thin sections by using the developed holder (fig. 10). (5) Both transmitted secondary electron image and backscatter electron image could be conveniently observed in the ultra-thin sections labeled with colloidal gold for immunoelectron microscopy. TEM cannot provide this function, so this is one of the unique characteristics (fig. 11). (6) To effectively increase the detection of X-ray from the membrane-type specimens and to avoid production of X-ray surround the specimen, the holder for X-ray element analysis was developed, and some parts of that was made of carbon (fig. 12). If the new holder is used, a X-ray analysis is enabled in Fig.3 A moving the objective area into the objective aperture by using the function of incline and revolving system in SEM. Fig.4 Whole (left) and top surface (right) view of the developed sample holder loaded grid mesh and locking ling Fig.5 Grid images under the search for objective area in ultra-thin section Left: The aperture at center of grid. Middle: Movement of aperture for objective area. Right: Enlargement objective area. both the bulk and the film by a expensive analyzer just installed to SEM. Conclusions With the developed holder, transmitted secondary electron images can be obtained from any thin-membrane type specimen, moreover, the observation of backscatter electron images and X-ray element analysis are available. There are far many numbers of SEM than TEM in universities, institutes, hospitals and high schools. Observation of TEM images would be also to do at Fig.6 Transmitted-secondary electron images of ultra-thin section in rat liver by SEM Left shows whole image of liver cell at x4,000 and right is enlarged image at x200,000

4 Fig.7 Transmitted secondary electron images of ultra-thin section in rat small intestine and medulla oblongata by SEM Left shows the cross section image of microvilli magnified at x300,000, middle hows cross section image of myelin magnified at x200,000, and right is longitudinal section image of rat skeletal muscle magnified at x200,000 Fig.8 The comparison of TEM images of rat liver at the same sites of the same section with the transmitted secondary electron images by SEM using the dveloped sample holder. Upper line is TEM images and lower line is the transmitted secondary electron images. The magnification is x10,000, x50,000 and x100,000 from the left. The differences are not observed in each magnification in a clearness and resolution. Fig.9 Application to the clinical diagnosis by observing ultra-thin section using small SEM. Left image shows CarryScope (JCM-5100; JEOL, Tokyo Japan) which is small desk top SEM. Middle image shows canning transmitted secondary image of the glomerulus from human kidney biopsy specimen by CarryScope with the developed holder. Light is enlarged image of the part which is surrounded by white square in the glomerulus, and immune complex as are located in the basement membrane (arrows).

5 seminars of electron microscopy for engineer, and classes for boys and girls if SEM cloud have a function of TEM (fig. 13). There are a small number of TEM in the developing countries, our developed holder would provide great opportunity to observe TEM images of virus and bacteria in SEM (fig. 14). References [1] E. Oho, T. Sasaki, K. Adachi, Y. Muranaka and K. Kanaya; An inexpensive and highly efficient device for observing a transmitted electron image in SEM; J. Electron Microsc. Technique. 5. (1987) p [2] Y. Muranaka, M. Hakamata, I. Ohta, E. Oho and K. Kanaya. STEM utilization for the better observation of biological specimens; J. Clinical Fig.10 Transmitted secondary electron image of HBs virus on the thin film Electron Microsco. 19, (1986) p stained by the negative stain meythod with uranyl acetate by using the developed holder. Direct magnified is x100,000. Fig.11 The observation of ultra-thin section for immune-electron microscopy with transmitted image and backscatter electron image by SEM. An external secretion cell of the rat pancreas labeled by colloidal gold of 10 nm in diameter. Left image is scanning transmitted secondary electron image and right image is backscatter electron image. Direct magnified were 50,000. Fig.12 Special specifications holder for X-ray analysis and a result of analysis for ultra-thin section. Left drawing and center photograph shows special specifications holder for X-ray analysis. A part of the incident electron irradiation is made of carbon. Right graph is energy spectrum of X-ray element analysis by SEM at the field indicated by arrow in the ultra-thin section.

6 Fig.13 The class of a microscope or nature observation for children where only SEM could be brought in for TEM observation. Left photograph is the class of natural observation for children in National Olympics Memorial Youth Center in 2006 and right is a seminars of electron microscopy for engineer in Fig.14 The request from an anti-infectious disease measure of the developing country Upper line images are bird influenza virus type H5N1 infected with human in Vietnam and cultured in the Vero cell. Left image is many virus budding off from kidney cell of african green monkey kidney origin. Middle is enlarge image of budding virus. Right is more enlarged image of virus by SEM with the developed holder. Direct magnified is x500,000. Lower line images are a kind of the cigarette mosaic virus infected with a fruit and the leavies of the papaya. Left and middle are leavies and fruit. Right is TEM image of a kind of the cigarette mosaic virus stained by negative stain method with uranyl acetate. Author; Yoshinori Muranaka, Ph.D. Laboratory for Ultrastructure Research, Research Equipment Center, Hamamatsu University School of Medicine Handayama, Higashi-ku, Hamamatsu, JAPAN Tel & Fax; , Mobile phone; , ; muranaka@hama-med.ac.jp