In a bright-field microscope, it is possible to insert a
polarizer between the light source and the specimen and an
analyzer. Polarizer and analyzer are polarizing filters. In this
case, different areas of the preparation deviate differently the
polarization plane. This microscope called “polarizing” will
highlight the fibrous structures, lamellar, granular crystals
and examination is often fresh.
Phase Contrast Microscopy
This is one of the few techniques that can be used to observe
living cells. Phase contrast microscopy is based on the principle that cells differ in refractive index from their surroundings and hence bend some of the light rays that pass through
them. Light passing through a specimen of refractive index
different from that of the surrounding medium is retarded.
This effect is amplified by a special ring in the objective lens
of a phase contrast microscope, leading to the formation of a
dark image on a light background. The phase contrast microscope is widely used in research applications because it can
be used to observe wet-mount (living) preparations. It is thus
widely used in cellular engineering.
Differential Interference Contrast Microscope
Differential interference contrast is a form of light microscope that employs a polarizer to produce polarized light. It
uses either unpolarized light or polarized light for the observation of transparent or reflective objects. The most interesting ones use polarized light and produce images whose relief
can be surprising and astonishing. The study of the light
interference leading to the formation of images in different
interference microscopes is beyond the scope of this chapter.
It is widely used to perform micromanipulation of living
cells but also to observe surfaces. Its use in microbial ecology is still quite limited.
Fluorescence Microscopy
It is widely used in microbial ecology. It exploits the ability of
certain molecules such as fluorophores or fluorochromes to
emit fluorescent light upon absorption of photons, which
means after excitation by a light source. A fluorophore can
be excited only by radiation that it can absorb. The absorption
spectra are band spectra that include therefore the excitation
spectra. Obviously, the specimen to study must be illuminated
by a spectral interval that belongs to the excitation spectrum of
the fluorophore. Generally, a spectral interval centered on the
maximum wavelength of the excitation spectrum of the
fluorophore is used. It is therefore important to ensure consistency between the excitation source whose spectral bands vary
qualitatively and quantitatively from one source to another and
the excitation spectrum of the fluorophore.
Light sources can be lamps, lasers, and more and more
often diodes. Most lasers have a major and narrow band and
must correspond to a spectral region of strong absorption to
excite the fluorophore. Conversely, lamps (i.e., mercury vapor
lamp) emit a spectrum with many bands of varying intensity,
which is reflected on the spectrum with peaks more or less
important. We choose a fluorophore which absorbs strongly in
one of these spectral ranges that are produced by the light
source. The fluorescence intensity is important when working
with very small particles such as viruses or bacteria, and it can
be increased by using light sources with a powerful light flow.
If the fluorophore is a molecule naturally present in the
cell of interest, such as a chlorophyll pigment, the source
will be selected based on the excitation spectrum of the
pigment. Conversely, if the fluorescence is not constitutive
and is based on the staining of a cellular component with a
specific fluorochrome, in this case one may choose the
fluorophore depending on the light source of the cytometer
(microscope or flow cytometer). For a given cellular target,
such as nucleic acids, there is a wide variety of
fluorochromes with very varied peak wavelengths of excitation and emission (see website of Molecular Probes®).
In all cases the wavelength of the fluorescence emitted
upon excitation is higher (lower energy) than the wavelength
used to excite the molecule. This results from a loss of
energy between excitation and emission phases. The selection of spectral bands is done with optical filters whose
characteristics are very different. A band-pass filter is a
device that passes frequencies within a certain range and
rejects (attenuates) frequencies outside that range. These
filters are mainly used as excitation filters. Other filters,
high-pass or low-pass, can pass the wavelengths above or
below a given value and are used for transmission, that is to
say that the fluorescence light will be visible to the eye (for
details, see the many websites that specialize in cytometry
instruments). A dichroic mirror is used to reflect the light
rays in order to direct them to the specimen for the excitation
phase and the eyepiece for the transmission phase. The
excitation and emission filters and the dichroic mirror are
often grouped together in a removable block, and the microscope may be equipped with a set of different blocks in order
to combine several fluorochromes within the same preparation. It is possible to use multiple fluorochromes that are
excited simultaneously by a single light source by exploiting
one or more spectral bands and emit with different
wavelengths. This produces a multicolor staining. For example, it is common to combine a fluorochrome associated with a
physiological function with a second fluorochrome associated
with a taxonomic probe.
The Confocal Scanning Laser Microscopy (CSLM)
CSLM is a computerized microscope that couples a laser light
source to a light microscope. A thin laser beam (argon ion) is
bounced off a mirror that directs the beam through a scanning
device. Then the laser is directed through a pinhole that
774
F. Joux et al.
polarizer between the light source and the specimen and an
analyzer. Polarizer and analyzer are polarizing filters. In this
case, different areas of the preparation deviate differently the
polarization plane. This microscope called “polarizing” will
highlight the fibrous structures, lamellar, granular crystals
and examination is often fresh.
Phase Contrast Microscopy
This is one of the few techniques that can be used to observe
living cells. Phase contrast microscopy is based on the principle that cells differ in refractive index from their surroundings and hence bend some of the light rays that pass through
them. Light passing through a specimen of refractive index
different from that of the surrounding medium is retarded.
This effect is amplified by a special ring in the objective lens
of a phase contrast microscope, leading to the formation of a
dark image on a light background. The phase contrast microscope is widely used in research applications because it can
be used to observe wet-mount (living) preparations. It is thus
widely used in cellular engineering.
Differential Interference Contrast Microscope
Differential interference contrast is a form of light microscope that employs a polarizer to produce polarized light. It
uses either unpolarized light or polarized light for the observation of transparent or reflective objects. The most interesting ones use polarized light and produce images whose relief
can be surprising and astonishing. The study of the light
interference leading to the formation of images in different
interference microscopes is beyond the scope of this chapter.
It is widely used to perform micromanipulation of living
cells but also to observe surfaces. Its use in microbial ecology is still quite limited.
Fluorescence Microscopy
It is widely used in microbial ecology. It exploits the ability of
certain molecules such as fluorophores or fluorochromes to
emit fluorescent light upon absorption of photons, which
means after excitation by a light source. A fluorophore can
be excited only by radiation that it can absorb. The absorption
spectra are band spectra that include therefore the excitation
spectra. Obviously, the specimen to study must be illuminated
by a spectral interval that belongs to the excitation spectrum of
the fluorophore. Generally, a spectral interval centered on the
maximum wavelength of the excitation spectrum of the
fluorophore is used. It is therefore important to ensure consistency between the excitation source whose spectral bands vary
qualitatively and quantitatively from one source to another and
the excitation spectrum of the fluorophore.
Light sources can be lamps, lasers, and more and more
often diodes. Most lasers have a major and narrow band and
must correspond to a spectral region of strong absorption to
excite the fluorophore. Conversely, lamps (i.e., mercury vapor
lamp) emit a spectrum with many bands of varying intensity,
which is reflected on the spectrum with peaks more or less
important. We choose a fluorophore which absorbs strongly in
one of these spectral ranges that are produced by the light
source. The fluorescence intensity is important when working
with very small particles such as viruses or bacteria, and it can
be increased by using light sources with a powerful light flow.
If the fluorophore is a molecule naturally present in the
cell of interest, such as a chlorophyll pigment, the source
will be selected based on the excitation spectrum of the
pigment. Conversely, if the fluorescence is not constitutive
and is based on the staining of a cellular component with a
specific fluorochrome, in this case one may choose the
fluorophore depending on the light source of the cytometer
(microscope or flow cytometer). For a given cellular target,
such as nucleic acids, there is a wide variety of
fluorochromes with very varied peak wavelengths of excitation and emission (see website of Molecular Probes®).
In all cases the wavelength of the fluorescence emitted
upon excitation is higher (lower energy) than the wavelength
used to excite the molecule. This results from a loss of
energy between excitation and emission phases. The selection of spectral bands is done with optical filters whose
characteristics are very different. A band-pass filter is a
device that passes frequencies within a certain range and
rejects (attenuates) frequencies outside that range. These
filters are mainly used as excitation filters. Other filters,
high-pass or low-pass, can pass the wavelengths above or
below a given value and are used for transmission, that is to
say that the fluorescence light will be visible to the eye (for
details, see the many websites that specialize in cytometry
instruments). A dichroic mirror is used to reflect the light
rays in order to direct them to the specimen for the excitation
phase and the eyepiece for the transmission phase. The
excitation and emission filters and the dichroic mirror are
often grouped together in a removable block, and the microscope may be equipped with a set of different blocks in order
to combine several fluorochromes within the same preparation. It is possible to use multiple fluorochromes that are
excited simultaneously by a single light source by exploiting
one or more spectral bands and emit with different
wavelengths. This produces a multicolor staining. For example, it is common to combine a fluorochrome associated with a
physiological function with a second fluorochrome associated
with a taxonomic probe.
The Confocal Scanning Laser Microscopy (CSLM)
CSLM is a computerized microscope that couples a laser light
source to a light microscope. A thin laser beam (argon ion) is
bounced off a mirror that directs the beam through a scanning
device. Then the laser is directed through a pinhole that
774
F. Joux et al.