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Fig. 3. Immune electron micrographs of quantum dot-labeled bacteria. Mixtures of
cultured HeLa cells and
Chlamydia trachomatis
elementary bodies (EB) were pre-
fixed, probed with anti-EB rabbit serum, and labeled with goat anti-rabbit IgG/655 nm
quantum dot conjugates. Thin sections examined by TEM
(A)
and cell layers examined
by SEM
(B)
showed numerous quantum dots on bacterial surfaces, but not on host
cells (unpublished data). Bars, 50 nm.
clear compositional contrast against carbon-rich biological material when
imaged by BEI or scanning transmitted electron detectors. Such contrast is
maximized using relatively low accelerating voltages and carbon sputter coating.
1. Follow
steps 1-5
in
Subheading 3.2.1
and
steps 1-3
in
Subheading 3.2.2
.
2. Lightly sputter coat samples with carbon. The coating thickness should be deter-
mined empirically as the minimum thickness that enables viewing without static
charge artifact in the microscope. A thickness setting of 20 Å was used for the
image in
Fig. 3B
.
3. Choose an initial accelerating voltage of 1-3 kV (
see
Note 8
). Check and adjust
beam alignment as necessary. Examine the sample in backscatter mode at ×25,000
or greater magnification. Quantum dots should be visible as relatively bright
particles on exposed surfaces.
4. Notes
1. Sample processing for EM involves multiple fluid exchanges. Such exchanges
must be sufficiently rapid to prevent samples from drying. For example, fluids
can be removed from and immediately replaced for each sample in an experiment
rather than removing fluids from all samples before replacement.
2. It is the practice in this laboratory to centrifuge suspended cells at 600-800 ×
g
for 1-5 min as necessary. After removing the supernatant, pellets are gently
disaggregated into the residual liquid by tapping the tube prior to adding the
next volume of liquid. Vortexing and rapid passage through pipette tips are not
recommended.
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