Studies On The Technology Of Cryo-scanning Electron Microscope With Aquatic Animals

With the progress and development of science and technology, cryo-SEM (cryo-scanning electron microscope) has been an important tool in biological research. In scientific research and discovery, if biological samples are treated with glutaraldehyde fixation, a series of dehydration of ethanol or acetone, critical point drying and ion plating, biological tissues always wrinkled, which resulted in deformation and distortion, so that it’s difficult to identify detailed structures. While using cryo-SEM to observe samples of aquatic animals could make up the shortage of SEM, and the results won’t appear the sample contraction deformation and the phenomenon that the structure of the cell surface is not clear. Also, the technology of cryo-SEM overcomes the normal SEM’s defect that in the process of sample preparation, structures with extremely most water is sensitive to the change of temperature. Consequently, cryo-SEM is more advantage than SEM in observation for biological samples with high water content.The technology of cryo-SEM always applied in the observational study of botany, zoology, microbiology, biological technology, biological medicine, agricultural science, medicine and so on, and sometimes it is used in chemistry, physics and materials synthesis. Cryo-SEM’s application in zoology has been widespread.However, the report of scientific research and practice about cryo-SEM technological application on aquatic animals is few, especially not reported in domestic. Therefore, targeting kinds of aquatic animals, such as protozoa, cladocera, rotifer and ostracoda as materials, the present study researched a set of cryo-SEM observational technology suitable for aquatic animals, expecting to provide not only accurate and comprehensive knowledge reserves but also precise and convenient research method for further study on aquatic animals.1. The method research of cryo-SEM technology for aquatic animalsIn order to study cryo-scanning electron microscope technique that is used to observe aquatic organism, in the process of observation by cryo-SEM, the study choosing all kinds of protozoan, Cladocera, Ostracoda, Rotifer as experimental materials, explored the above experimental materials from the following six aspects:(1) centrifugation or not, and the centrifugal rotational speed and time; (2) fixation or not and how to fix, using antifreeze sucrose or not, dehydration or not; (3) the selection of holder; (4) sublimation temperature, time and resublimation or not; (5) plating settings; (6) observation parameter settings. The various parameters are researched in the process of cryo-scanning electron microscopy samples preparation through repeated experiments, and it results that we get a whole cryo-scanning electron microscopy techniques which can be used to observe the majority of aquatic organism. The experimental results show that:in the process of using cryo-SEM technology to treat samples, the effect of observation is the best in this condition that samples is needn’t centrifugation, fixation, dehydration and sucrose(while protozoa need fixation by 2%OSO4); the holder should be cyclo-plate; the deep freezing temperature is generally-196℃, time is 2 min; the sublimation temperature is generally-95℃, time is 2 min (while for protozoa the best sublimation temperature should be-100℃); rotating the holder is needed in the plating process, time is 120sec; the acceleration voltage is generally 1 kV. After fracture, the cross section and the internal structure of Paramecium and Euplotes woodruffi can be observed.2. The study of four drying methods applied in aquatic animals for the observation of SEMThe study chooses typical members of Protozoa, Cladocera, Ostracoda, Rotifer as experimental materials, we use four different methods that are natural drying method, vacuum drying method, critical point drying method and cryo-scanning electron microscopy technology to treat them and observe by scanning electron microscope and cryo-scanning electron microscope. The results showed that:in the maintenance original state of the samples, cryo-scanning electron microscopy is clearly better than the other three methods. The organisms which are treated by cryo-scanning electron microscopy have not signs of shrinkage deformation, and remain completely natural state. Moreover, cryo-scanning electron microscopy technique does not require complex operation steps in the process of samples preparation. The whole experiment process is simple and quick.