Electron Cryomicroscopy of Fibrillar Collagens - Methods in Molecular Biology: Extracellular Matrix Protocols - page 102

Fig. 9. Stereo pair images of reconstituted type I collagen fibrils prepared by the
mica ice-wedge technique. The images were taken at + and - 6 ° tilt on a JEOL 1200

EX transmission instrument. The repeat period is ~67 nm.

3. Stereo pairs are a useful tool for visualizing the fibrils in 3-D. To achieve them

take, two images of the same structure at + and - n ° (5 to 15 ° is generally accept-

able) of tilt on the goniometer stage of the microscope

4. It is important to keep the structures of interest in the specimen centralized in the

field of view while tilting the stage. To achieve this, adjust the eucentric height of
the stage so the area of interest does not move except on the tilt axis. ( See Notes
8 and 9 .).

5. Record two images at + and - the same degree of tilt (both at the same magnifica-

tion and accelerating voltage) on photographic film and then print them onto
photographic paper.

6. View the stereo pair prints on a stereo imager or use one of the other techniques

for converting two images into one 3-D image ( Fig. 9 ).

3.3. Image Analysis of Vitreous Ice and Replicated Samples
Axial projections were computed from fibril images using Semper 6.4 (2) .
An average D -period was obtained from a set of individual D -periods after
cross-correlation alignment.

4. Notes
Notes for vitreous-ice procedures.

1. For image analysis procedures, the image needs to be in a digital format. Images

can either be captured using a slow scan-cooled CCD camera (which provides a