1. Title: How to study the difference in the cell wall between the wild-type and mok1 mutant fission yeasts by electron microscopy Hong Liu

2. How to identify the difference in the cell wall between the two strains? Two types of yeasts: Wild type mok1 (mutant) (MOK1 encode the synthase that can synthesize the altha-glucan)

3. Two types of electron microscopes can be used to observe these changes: SEM (Scanning Electron Microscope) and TME (Transmission Electron Microscope) Component of cell wall Morphology of cell wall shape of cell

4. SEMTEM Tungsten filament Anode Condenser lens Beam of electrons Scanning coils Electromagnetic objective lens Projectors lens Detector specimen

5. Sample preparation Glutaraldehyde Osmium tetroxide and ruthenium tetroxide Sprayed on to the support Coated with evaporated platinum coated with evaporated carbon Viewed under SEM For SEM Glutaraldehyde Potassium permanganate Embedded in resin Section Ultra-thin sections stained with uranyl acetate and lead citrate Transferred to copper grid Viewed under TEM For TEM

6. Figure 1. UHR-LVSEM images of wild-type (a) and -glucan synthase mutant mok1 (b-d) cells. Various shapes of the mok1 mutant are seen showing abnormal swelling in comparison to wild-type cells. Bar = 1 µm Figure 2. UHR-LVSEM images of the cell surface of the -glucan synthase mutant mok1 (a) and wild-type (b) cells. The naked glucan networks ( ) are seen at the cell surface mixed with amorphous particles ( ) of -galactomannan. Bar = 100 nm

7. Figure 3. TEM image of wild- type (1) and mok1 mutant (2) cells showing the thick cell wall and septum with no fibrillar structure of the outermost layer ( ). (a) whole cell; (b) a highly magnified image of the cell wall; (c) part of the septum. Abbreviations: CM, cell membrane; CW, cell wall; M, mitochondrion; N, nucleus; PS, primary septum; SS, secondary septum; V, vacuole. Bar = 1 µm (a, b), 200 nm (c)

8. Why does there exist this difference? The Mok1 can synthesize the alpha-glucan. The absence of the Mok1 in the mutant cell is the only difference between the wild-type and the mutant. Therefore, it is necessary to observe the ultrastructure of the glucan network. In order to do this, the author utilized the negative staining.

9. Negative staining glutaraldehyde Sprayed on to a filmed grid Stained with uranyl acetate View under TEM Note: the negative staining is very similar to the positive staining. But there is also some difference between them. First, The process of negative staining is carried out quickly Second, it is unnecessary for negative staining to remove the excessive stain after staining.

10. Figure 4. UHR-LVSEM of a coated reverting protoplast of a mok1 mutant cell (a) and negatively stained images of a glucan network of the mok1 mutant (b) and wild-type (c) cells. Interwoven microfibrils ( 5 nm diameter; a-c, ) connected at their sides (16 nm diameter; b, ) and orientated parallel to each other were packed in groups 200 nm wide or more. Many more microfibrils were observed in mok1 than in wild- type cells. The inset is a high- magnification image of (b) Bar = 100 nm

11. Conclusion 1.In order to observe clearly the surface of a cell or shape of a molecule, scanning electron microscopy is a better way. It can also give researchers a three-dimensional image. 2.The transmission electron microscopy can give us some useful information about the detailed inner or surface structures of cells. 3.Different staining methods can give good contrast on different samples. Positive staining can highlight what you’re interested in. If you use a negative staining, the edges of the particles are thus seen in outline. Immuno-electron microscopy can used to observe the localization of enzymes or other protein molecules in cells. (not shown) Other methods:

12. Thank you !