Biology Reference
In-Depth Information
num wire in a jig at a distance of 10 cm from the rotating table. Adjust the wire/
rotating table angle to 4
°
(
see
Note 11
).
9.
Attach mica sheets to the rotating table with double-sided adhesive tape. Carbon-
film grids may be placed towards the centre of the rotating table without using
adhesive tape.
10.
Follow manufacturers instructions to evacuate the coating unit. Rotate the table
to a speed of 60 rpm. Increase the voltage across the tungsten/platinum wire until
the platinum begins
to bubble
(
see
Note 12
).
11.
Carbon coat the shadowed mica sheets (protocol
Subheading 3.3., step 1
, using
carbon braided carbon fiber)
12.
Leave the shadowed and carbon-coated mica over an atmosphere of 10% (v/v)
acetic acid for 24 h.
13.
Float the carbon films onto distilled water and pick up the carbon on 400 mesh
copper or nickel grids.
3.6. Rotary Shadowing Transmission Electron Microscopy
Imaging of rotary shadowed macromolecules may be carried out in any con-
ventional TEM.
3.7. Scanning Transmission Electron Microscopy
The digital STEM system employed in our laboratory is based on a JEOL
1200EX TEM with a JEOL ASID10 scanning unit and the standard bright field
and annular dark-field detectors (
see
Note 13
). A microcomputer interface
(
Fig. 6
) permits digital scan control, signal acquisition, beam blanking, and
digitization of the signal from the ADF detector
(11)
. Digital images are acquired
using a spot size of approximately 3 nm. No correction for beam-induced mass
loss is required where the specimen is exposed to an electron dose of less than
10
3
e nm
-2
. Detector geometries are illustrated in
Fig. 7
.
Standard STEM images consist of 512
×
512 points with an acquisition time
s per point (
see
Note 14
). Magnification calibration can be carried out
with a carbon replica of a diffraction grating ruled at 2160 lines/mm Image
analysis requires an interactive programming or macro-driven environment
(
see
Note 15
). Fibrillar collagens and microfibrils are filamentous structures,
the mass characteristics of such assemblies are usually expressed as mass per
unit length (in kDa/nm).
The following protocol demonstrates the steps in calibrating a STEM
system using tobacco mosaic virus (TMV) particles which have a well-
defined mass per unit length of 131 kDa/nm
(1)
. When calibrated, the system
may be used to determine the absolute mass characteristics of a large range of
ECM macromolecules.
of 200
µ
1.
Capture dark-field STEM images of TMV supported on carbon film (
Fig. 8
, panel
A). Record the signal from the bright-field detector (BF) and the zero probe level
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