Biology Reference
In-Depth Information
rapid and convenient method of obtaining digital images) or collected on high-
resolution EM film, which is subsequently digitized with an appropriate scan-
ning device. Both methods are efficient at electron detection. A typical CCD
camera has 1K
×
1K pixels compared with a possible 10K
×
10K digitization of a
sheet of film 8 cm
8 cm. CCD cameras have, therefore, an inherently smaller
field of view than photographic film at the same resolution. The CCD camera,
however, has the advantage of immediate image assessment. The TVIPS system
described in this chapter provides the opportunity for online processing and feed-
back to the microscope for automated data collection.
×
2.
Fast Fourier transforms (FFT) of the image can be used to assess astigmatism,
calculate defocus values (to permit contrast transfer function correction), and to
perform cross-correlation functions for image alignment and averaging.
3.
Image contrast and signal/noise ratio are low in images of frozen-hydrated bio-
logical specimens collected on conventional TEMs. Energy-filtered electron
microscopy removes the inelastically scattered electrons from the image, thereby
giving an enhancement of both contrast and signal/noise
(10)
. Furthermore,
energy filtered EM allows mass mapping of macromolecular assemblies in vitre-
ous ice
(11)
.
Notes for mica ice-wedge procedures.
4.
This technique can be used to visualize many different macromolecules and mac-
romolecular assemblies in the range of less than 10-100 nm in size.
5.
Where molecules are damaged by washing with water and washing is required,
the sample buffer minus any sample or extra additives may be used both for
washing and the assembly of the wedge. It is normally only large assemblies,
such as collagen fibrils, that can become masked after freeze-etching by buffer
constituents. These might require washing.
6.
Most samples containing smaller molecules are not left masked after etching
and therefore do not require washing. In these cases the mica wedge is
assembled (clamped at one end with the spacer at the other), 10
L of the sample
pipeted into the gap, the sample left for 30-60 s to allow the molecules to adsorb
to the mica and then frozen/fractured/etched, and so on, in the same manner as
washed samples.
µ
7.
If replicas fail to detach and float free, the mica may be placed in an atmosphere of
acetic acid for several hours or days to try and free them. If this fails, it may be
possible to dissolve away the mica with concentrated hydrofluoric acid, but this
may cause disruption of the replicas and make collecting them on grids difficult.
8.
By modifying the stereo pair idea, a series of images may be taken at
n
degree
intervals from +60
°
to -60
°
of tilt and these might be used for 3-D reconstruction
of the collagen fibrils.
9.
For 3-D reconstruction of a tilt series or image analysis, the images need to be
digitized, either by capturing them on the microscope with a CCD camera or by
scanning negatives onto a computer with some form of scanner or digital camera.
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