To
understand how fluorescence microscopy works and
why it has become so important to modern biology,
one must understand what the term fluorescence
means. Fluorescence is the luminescence of
a substance when it is excited by radiation.
In microscopy, fluorescence is used as a means
of preparing specific biological probes. Some
biological substances like chlorophyll and some
oils and waxes have primary fluorescence;
that is, they autofluoresce. But most biological
molecules or structures do not fluorescence on
their own, so they must be linked with fluorescent
molecules (or fluorochromes) in order to
create specific fluorescent probes.
Fluorescence of a
substance is seen when the molecule is exposed
to a specific wavelength of light (excitation
wavelength or spectrum) and the light it emits
( the emission wavelength or spectrum) is always
of a higher wavelength. To view this fluorescence
in the microscope, several light filtering components
are needed. Specific filters are needed to isolate
the excitation and emission wavelengths of a fluorochrome.
A bright light source with proper wavelengths
for excitation is also needed. For normal fluorescence
applications, this is a mercury vapor arc burner.
For fluorescence confocal microscope applications
where up to 95% of the emission light is filtered
out, specific wavelength lasers are used as these
are extremely bright.
Mercury arc burners
are very bright lamps with a limited lifetime
and require some maintenance and care to make
sure that they are producing the brightest possible
light beam for fluorescence excitation. For a
discussion of the care of the mercury arc lamp,
please click
here.
One other component
is required: a dichroic beam splitter or partial
mirror which reflects lower wavelengths of light
and allows higher wavelengths to pass. A beam
splitter is required because the objective acts
as a condenser lens for the excitation wavelength
as well as the objective lens for emission. One
only wishes to see the light emitted from the
fluorochrome and not any of the excitation light,
and the beam splitter isolates the emitted light
from the excitation wavelength. This epi-illumination
type of light path is required to create a dark
background so that the fluorescence can be easily
seen. The wavelength at which a beam splitter
allows the higher wavelengths to pass must be
set between the excitation and emission wavelengths
of any given fluorochrome so that excitation light
is reflected and emission light is allowed to
pass through it.
A typical fluorescence
filter setup is shown in the diagram below (Courtesy
of Carl Zeiss Inc., Germany).
Filter sets must
be made to correspond to the excitation and emission
characteristics of a give fluorochrome. Some sets
are made to allow visualization of two or even
three fluorochromes simultaneously.
Scientific personnel
at UCLA may obtain a two page list of fluorochromes
with excitation and emission wavelengths from
the Facility (see Matt Schibler) or you can link
to
Carl Zeiss, Germany, to look at one.
The filter sets available
on the Zeiss LSM 310 microscope in the Facility
are:
Zeiss Filterset
15: Green
excitation for rhodamine (TRITC). (Propidium iodide
(PI), Texas Red and some other red dyes may also
be used, but the filter set is not optimal for
these dyes).
Zeiss Filterset
17: Blue
excitation for fluorescein isothiocyanate (FITC).
This filter set may be used for green
fluorescent protein (GFP), but it is not optimal.
Here are some websites
which show spectra of some available fluorescence
sets and fluorochrome lists:
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