Page Index
What is a Fluorescence
Microscope?
Basic Requirements of Fluorescence Microscope Optics
The Dichroic Mirror
Excitation and Emission Filters
The Filter Cube
Problem: The Need for Quick Wavelength Changes
Solution: C&L's Innovative Filter Wheel Approach
What is a
Fluorescence Microscope?
A fluorescence microscope is basically a conventional
light microscope with added features and components that extend its capabilities.
- A conventional microscope uses light to illuminate the sample and produce a
magnified image of the sample.
- A fluorescence microscope uses a much higher intensity light to illuminate the
sample. This light excites fluorescence species in the sample, which then emit light of a
longer wavelength. A fluorescent microscope also produces a magnified image of the sample,
but the image is based on the second light source -- the light emanating from the
fluorescent species -- rather than from the light originally used to illuminate, and
excite, the sample.
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Basic
Requirements of Fluorescence Microscope Optics
Nearly all fluorescence microscopes use the objective
lens to perform two functions:
- Focus the illumination (excitation) light on the sample.
In order to excite fluorescent species in a sample, the optics of a fluorescent microscope
must focus the illumination (excitation) light on the sample to a greater extent than is
achieved using the simple condenser lens system found in the illumination light path of a
conventional microscope.
- Collect the emitted fluorescence.
This type of excitation-emission configuration, in which both the excitation and emission
light travel through the objective, is called epifluorescence. The key to the optics in an
epifluorescence microscope is the separation of the illumination (excitation) light from
the fluorescence emission emanating from the sample. In order to obtain either an image of
the emission without excessive background illumination, or a measurement of the
fluorescence emission without background "noise", the optical elements used to
separate these two light components must be very efficient.
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The
Dichroic Mirror
In a fluorescence microscope, a dichroic mirror
is used to separate the excitation and emission light paths. Within the objective, the
excitation emission share the same optics.
 Figure 1: Dichroic mirror
separates excitation and emission light paths.
Note: This diagram shows the dichroic mirror's position in an inverted fluorescence
microscope: below the sample. In this type of microscope, the sample is illuminated and
imaged from below the stage.
In a fluorescence microscope, the dichroic mirror separates the light paths.
- The excitation light reflects off the surface of the
dichroic mirror into the objective.
- The fluorescence emission passes through the
dichroic to the eyepiece or detection system.
The dichroic mirror's special reflective properties allow it to separate the two light
paths. Each dichroic mirror has a set wavelength value -- called the transition
wavelength value -- which is the wavelength of 50% transmission. The mirror reflects
wavelengths of light below the transition wavelength value and transmits wavelengths above
this value. This property accounts for the name given to this mirror (dichroic, two
color). Ideally, the wavelength of the dichroic mirror is chosen to be between the
wavelengths used for excitation and emission.
The dichroic mirror is a key element of the fluorescence microscope, but it is not able
to perform all of the required optical functions on its own. Typically, about 90% of the
light at wavelengths below the transition wavelength value are reflected and about 90% of
the light at wavelengths above this value are transmitted by the dichroic mirror. When the
excitation light illuminates the sample, a small amount of excitation light is reflected
off the optical elements within the objective and some excitation light is scattered back
into the objective by the sample. Some of this "excitation" light is transmitted
through the dichroic mirror along with the longer wavelength light emitted by the sample.
This "contaminating" light would otherwise reach the detection system if it were
not for another wavelength selective element in the fluorescence microscope: an emission
filter.
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Excitation
and Emission Filters
Two filters are used along with the dichroic mirror:
- Excitation filter -- In order to select the excitation wavelength, an excitation
filter is placed in the excitation path just prior to the dichroic mirror.
- Emission filter -- In order to more specifically select the emission wavelength
of the light emitted from the sample and to remove traces of excitation light, an emission
filter is placed beneath the dichroic mirror. In this position, the filter functions to
both select the emission wavelength and to eliminate any trace of the wavelengths used for
excitation.
These filters are usually a special type of filter referred to as an interference
filter, because of the way in which it blocks the out of band transmission. Interference
filters exhibit an extremely low transmission outside of their characteristic bandpass.
Thus, they are very efficient in selecting the desired excitation and emission
wavelengths.
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The Filter Cube
The dichroic mirror is mounted on an optical block
commonly referred to as a filter cube. The excitation and emission filters are
usually affixed to the filter cube. This cube provides a convenient means to change the
dichroic mirror without direct handling of either the mirror or filters. Figure 2 shows
the light path through the filter cube in a fluorescent microscope. The narrow red line
emanating from the objective to the filter cube represents the scattered and reflected
emission light that must be removed by these optical elements.
Figure 2: Light
path through the filter cube in a fluorescence microscope.
It is often the case that a specific combination of excitation filter, emission filter and
dichroic mirror are needed to visualize and/or quantitate the fluorescence emission from a
particular fluorescent species. In newer models of fluorescence microscopes, manufacturers
have provided a means to change these optical elements in a convenient manner by arranging
a set of four or more filter cubes in a circular (or linear) turret under the
objective. With a turret arrangement, a specific filter cube can be selected in a manner
similar to that of selecting a specific objective.
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Problem: The
Need for Quick Wavelength Changes
In order to use the newer fluorescent probes, which
exhibit changes in their wavelength properties, it is essential to be able to change
excitation and emission wavelengths quickly, for adequate temporal resolution.
These newer probes, also known as wavelength-shift probes, exhibit changes in
their excitation or emission spectra (or both) depending upon their environment. In order
to use the fluorescent probes to determine the nature of their environment, the changes in
spectra must be monitored. This process involves making several measurements of the same
sample at different excitation and/or emission wavelengths.
In order to determine the extent to which wavelength-shift probes have altered their
wavelength characteristics under specific conditions, it is imperative that measurements
of these wavelength properties occur in a reasonable time frame. Thus, if the desired
quantitation requires a measurement of a ratio of intensities at two wavelengths, the time
resolution of the measurement is dependent on how quickly the system can change excitation
and emission wavelengths.
The conventional turret method of changing filters is very slow and is, thus, not
appropriate in the use of wavelength-shift probes.
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Solution:
C&L's Innovative Filter Wheel Approach
C&L Instruments has developed a fluorometer
designed specifically for high-speed multiwavelength applications. In the C&L Dye
Fluorometer, the excitation and emission filters normally found in a filter cube are
each housed, instead, in a filter wheel. These filter wheels are located outside of the
body of the microscope. The filter wheels are computer controlled and filter changes can
be implemented in milliseconds. Both excitation and emission filters are housed externally
from the microscope, permitting vibration free microscopy.
For details about the components of the C&L Dye Fluorometer, please visit
our Product Description pages.
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