Ultrastructural Analysis of Bacteria–Host Cell Interactions - Bacterial Pathogenesis: Methods and Protocols - page 167

1.2. Conventional SEM
For conventional SEM, samples must also be cross-linked and dehydrated
during preparation. Treatments with aldehydes and osmium oxides and solvent
dehydration can often be performed identically for both TEM and SEM. Once
fully dehydrated in 100% organic solvent, the SEM samples are dried and
mounted. To minimize shrinkage during drying, intermediate drying agents
are used. This can be an organic compound, such as hexamethyldisilazane
(4,5) , or more typically the agent is liquid carbon dioxide exchanged within a
critical point dryer. Once dried, the samples are coated with a conductive metal
or carbon before examination or examined without coating. Coating, usually
performed in a magnetron or ion beam sputterer, can provide enhanced conduc-
tivity, some degree of protection against beam damage, and perhaps most
importantly, increased production of secondary electrons for imaging. Although
SEM offers several modes of imaging for biological samples, typically samples
are examined using secondary electrons for cell and microbial surface topog-
raphy and backscattered electrons for compositional contrast of immune probes
and labels.
1.3. Immune Labeling with Quantum Dots
Immune labeling of biological material for electron microscopy is a well-
established and powerful tool for localizing and visualizing macromolecules
and molecular complexes with nanometer scale resolution. In recent years,
considerable efforts have been undertaken to develop and utilize techniques
for correlative microscopy involving assessment of equivalent samples by
fluorescent (including single and multi-photon confocal microscopy) and
electron microscopy (1,2,6,7) . Because of the inherent differences between light
and electron beam imaging, relatively few techniques have been developed
that can utilize the same specific labeling reagents. These include (1) use
of fluorescein-conjugated probes for fluorescence microscopy, which can be
subsequently labeled with anti-fluorescein antibodies and electron-dense metals
(8) , (2) use of the green fluorescent protein family of expressed markers
that can also be labeled with antibodies and electron-dense metals (9) , the
ReASH® system, which utilizes a genetically engineered tetracysteine motif
and a bi-arsenical fluorescent dye that can photoactively deposit diaminoben-
zidine for TEM detection of the expressed protein tag (10) , and (4) the use of
fluorescent CdSe nanocrystals known as “quantum dots,” which are photolumi-
nescent, phosphorescent, and electron dense (11,12,13) . Among these options,
the quantum dots are unique in that they enable fluorescent examination of
labeled live or fixed samples and subsequent preparation of the same samples