final test of 三维显微术.doc

Final testTo find evidence of the four questions, I designed an experiment to verify the four processes mainly in two steps. One is using scanning electron microscopy, the other is cryoET.Sample preparationChemical fixation: to preserve and stabilize their structure. Fixation is usually performed by incubation in a solution of a buffered chemical fixative, such as glutaraldehyde.Appropriate sample size: sample must be of appropriate size to fit the specimen chamber and generally mounted rigidly on a specimen holder called a specimen stub.Coating: use conventional SEM sputter coating the major processes as the figure shows.First: The conventional sputter chamber is pumped with a rotary pump until the vacuum level is beyond half scale .Second: Leak argon gas into the chamber to flood the chamber, allow the vacuum to recover.Third: Apply the lowest voltage that will allow a plasma to strike.Note: in case of small particles it will be good to tilt it by about 45.SEM scanningThe first three questions can be concluded from the picture below.Like the picture shows, 1.the surface of the cells and the nanoparticle may look grey with many bumps and pits. But the image can be green if using the light microscopy for nanoparticle.2. The nanoparticle can enter the cell on the condition that the receptors have identified the specific soluble substance. The nanoparticle entering the cell by fusion or by endocytosis or other method is determined by the images taken from SEM, each of them has the possibility.Structure of the nanoparticleBriefly sample preparation: nano-particles are spread into thin films across EM grids plunge-frozen in liquid ethane transferred into an electron cryomicroscope.Note: Plunge-freezing prevents ice crystallization, immobilizing the proteins and other cellular structures in their native states and locations.The theory like the pictures show below Projection reconstruction back-projectionOperation: imaged iteratively while being tilted incrementally. Then recording a series of images at different angles allows a full, three-dimensional (3D) reconstruction of the specimen to be calculated.Making model: after the 3D reconstruction, I can build a model to reflect the structure of the nano-particle. From the model, apparently can get some information about the size and shape of the nano-particle .But