Fluorescent dyes WORLD OF MICROBIOLOGY AND IMMUNOLOGY222•
nal. Using such means, it is possible to detect single-copy
sequences on chromosome with probes shorter than 0.8 kb.
Probes can vary in length from a few base pairs for syn-
thetic oligonucleotides to larger than one Mbp. Probes of dif-
ferent types can be used to detect distinct DNA types.
PCR-amplified repeated DNA sequences, oligonucleotides
specific for repeat elements, or cloned repeat elements can be
used to detect clusters of repetitive DNA in heterochromatin
blocks or centromeric regions of individual chromosomes.
These are useful in determining aberrations in the number of
chromosomes present in a cell. In contrast, for detecting sin-
gle locus targets, cDNAs or pieces of cloned genomic DNA,
from 100 bp to 1 Mbp in size, can be used.
To detect specific chromosomes or chromosomal regions,
chromosome-specific DNA libraries can be used as probes to
delineate individual chromosomes from the full chromosomal
complement. Specific probes have been commercially available
for each of the human chromosomes since 1991.
Any given tissue or cell source, such as sections of
frozen tumors, imprinted cells, cultured cells, or embedded
sections, may be hybridized. The DNA probes are hybridized
to chromosomes from dividing (metaphase) or non-dividing
(interphase) cells.
The observation of the hybridized sequences is done
using epifluorescence microscopy. White light from a source
lamp is filtered so that only the relevant wavelengths for excita-
tion of the fluorescent molecules reach the sample. The light
emitted by fluorochromes is generally of larger wavelengths,
which allows the distinction between excitation and emission
light by means of a second optical filter. Therefore, it is possi-
ble to see bright-colored signals on a dark background. It is also
possible to distinguish between several excitation and emission
bands, thus between several fluorochromes, which allows the
observation of many different probes on the same target.
FISH has a large number of applications in molecular
biologyand medical science, including genemapping, diag-
nosis of chromosomal abnormalities, and studies of cellular
structure and function. Chromosomes in three-dimensionally
preserved nuclei can be “painted” using FISH. In clinical
research, FISH can be used for prenatal diagnosis of inherited
chromosomal aberrations, postnatal diagnosis of carriers of
genetic disease, diagnosis of infectious disease, viral and bac-
terial disease, tumor cytogenetic diagnosis, and detection of
aberrant gene expression. In laboratory research, FISH can be
used for mapping chromosomal genes, to study the evolution
of genomes (Zoo FISH), analyzing nuclear organization, visu-
alization of chromosomal territories and chromatin in inter-
phase cells, to analyze dynamic nuclear processes, somatic
hybrid cells, replication, chromosome sorting, and to study
tumor biology. It can also be used in developmental biology to
study the temporal expression of genes during differentiation
and development. Recently, high resolution FISH has become
a popular method for ordering genes or DNA markers within
chromosomal regions of interest.See alsoBiochemical analysis techniques; Biotechnology;
Laboratory techniques in immunology; Laboratory techniques
in microbiology; Molecular biology and molecular geneticsFLUORESCENCE MICROSCOPY• see
MICROSCOPE AND MICROSCOPYFFluorescent dyesLUORESCENT DYES
The use of fluorescent dyes is the most popular tool for meas-
uring ion properties in living cells. Calcium, magnesium,
sodium, and similar species that do not naturally fluoresce can
be measured indirectly by complexing them with fluorescent
molecules. The use of probes, which fluoresce at one wave-
length when unbound, and at a different wavelength when
bound to an ion, allows the quantification of the ion level.
Fluorescence has also become popular as an alternative
to radiolabeling of peptides. Whereas labeling of peptides with
a radioactive compound relies on the introduction of a radio-
labeled amino acid as part of the natural structure of the pep-
tide, fluorescent tags are introduced as an additional group to
the molecule.
The use of fluorescent dyes allows the detection of
minute amounts of the target molecule within a mixture of
many other molecules. In combination with light microscopic
techniques like confocal laser microscopy, the use of fluores-
cent dyes allows three-dimensional image constructs to be
complied, to provide precise spatial information on the target
location. Finally, fluorescence can be used to gain information
about phenomena such as blood flow and organelle movement
in real time.
The basis of fluorescent dyes relies on the absorption of
light at a specific wavelength and, in turn, the excitation of the
electrons in the dye to higher energy levels. As the electrons
fall back to their lower pre-excited energy levels, they re-emit
light at longer wavelengths and so at lower energy levels. The
lower-energy light emissions are called spectral shifts. The
process can be repeated.
Proper use of a fluorescent dye requires 1) that its use
does not alter the shape or function of the target cell, 2) that
the dye localizes at the desired location within or on the cell,
3) that the dye maintains its specificity in the presence of com-
peting molecules, and 4) that they operate at near visible
wavelengths. Although none of the dyes in use today meets all
of these criteria, fluorescent dyes are still useful for staining
and observation to a considerable degree.See alsoBiochemical analysis techniques; Biotechnology;
Electron microscope, transmission and scanning; Electron
microscopic examination of microorganisms; Immunofluores-
cence; Microscope and microscopyFFood preservationOOD PRESERVATION
The term food preservation refers to any one of a number of
techniques used to prevent food from spoiling. It includes
methods such as canning, pickling, drying and freeze-drying,
irradiation, pasteurization, smoking, and the addition of chem-
ical additives. Food preservation has become an increasinglywomi_F 5/6/03 2:15 PM Page 222