11
Arc lamp-based flow cytometers: The optical configuration of arc lamp-based flow cytometers
is different from that of laser instruments in several respects (Fig. 2). The light from the high
pressure mercury or xenon arc lamp is focused in the sample flow by an oil immersion
microscope objective with high numerical aperture, i.e. NA = 1.3. That implies that the cone
of excitation light is highly convergent. The arc lamp-based instruments employ
epi-illumination, which is to say that the fluorescence is collected by the same lens as that
MICROSCOPE OBJECTIVE
DICHROIC
-OCULAR
DICHROIC
;'
O
\
IL IMMERSION N.A.: 1.3
MIRRORS)
MIRROR\
I
~ " ./".
I LONG PASSFllTER-·' _ --\ A _~u ul) /
V in t~f~ --:~L-~-I~ == = = +
~ ___ J.
\\- OBJECT
FILTERS
...,.T
\
SLIT
'---I""-BANDPASS
\
PLANE
RETRACTABLE
~ EXCITATION
\
MIRROR
/
FILTER
\ I
-):--EXCITATION SLIT
1\
I
\
I
\
I
\
~ j= = = = \: ~-HEAT FILTER
~ __ CONDENSOR LENS
,
/
D 1'2/ 0
ARC LAMP J
/ ' «.
SPHERICA~
MIRROR
Figure 2. Epi-illumination optical configuration for arc lamp-based flow cytometers. The excitation filter isolates
a relatively narrow band of excitation wavelengths from the essentially white light of the light source. The
dichroic mirror reflects the excitation light into the microscope objective, while it transmits the fluorescence
which has higher wavelength than the excitation light. The emission filter eliminates the traces of excitation
light that leak through the dichroic mirror. The excitation slit and the measuring slit are used to eliminate
background light from other parts of the focus than the sample flow itself (spatial filtering) and thereby
increase the signal to noise ratio. Up to three different dyes or color components can be detected by separate
detectors (photomultiplier tubes). Thus, the fluorescence is split by means of dichroic mirrors into the various
wavelength components, which are further purified by band filters. To reduce filter fluorescence as much as
possible the optical filters, especially the filter in the excitation light path, are usually interference filters, i.e.
filters which reflect the light that is not transmitted rather than absorb it. The configuration shown here
employs critical illumination: the light source is imaged in the object plane of the microscope objective, i.e.
in the sample flow. Critical illumination provides somewhat higher intensity than is achieved with Kohler
illumination, which is used in most microscopes. Kohler illumination has the advantage that the excitation
intensity is less susceptible to lamp flicker - the sudden movements of the arc that is seen with some lamps.
Arc lamp-based flow cytometers: The optical configuration of arc lamp-based flow cytometers
is different from that of laser instruments in several respects (Fig. 2). The light from the high
pressure mercury or xenon arc lamp is focused in the sample flow by an oil immersion
microscope objective with high numerical aperture, i.e. NA = 1.3. That implies that the cone
of excitation light is highly convergent. The arc lamp-based instruments employ
epi-illumination, which is to say that the fluorescence is collected by the same lens as that
MICROSCOPE OBJECTIVE
DICHROIC
-OCULAR
DICHROIC
;'
O
\
IL IMMERSION N.A.: 1.3
MIRRORS)
MIRROR\
I
~ " ./".
I LONG PASSFllTER-·' _ --\ A _~u ul) /
V in t~f~ --:~L-~-I~ == = = +
~ ___ J.
\\- OBJECT
FILTERS
...,.T
\
SLIT
'---I""-BANDPASS
\
PLANE
RETRACTABLE
~ EXCITATION
\
MIRROR
/
FILTER
\ I
-):--EXCITATION SLIT
1\
I
\
I
\
I
\
~ j= = = = \: ~-HEAT FILTER
~ __ CONDENSOR LENS
,
/
D 1'2/ 0
ARC LAMP J
/ ' «.
SPHERICA~
MIRROR
Figure 2. Epi-illumination optical configuration for arc lamp-based flow cytometers. The excitation filter isolates
a relatively narrow band of excitation wavelengths from the essentially white light of the light source. The
dichroic mirror reflects the excitation light into the microscope objective, while it transmits the fluorescence
which has higher wavelength than the excitation light. The emission filter eliminates the traces of excitation
light that leak through the dichroic mirror. The excitation slit and the measuring slit are used to eliminate
background light from other parts of the focus than the sample flow itself (spatial filtering) and thereby
increase the signal to noise ratio. Up to three different dyes or color components can be detected by separate
detectors (photomultiplier tubes). Thus, the fluorescence is split by means of dichroic mirrors into the various
wavelength components, which are further purified by band filters. To reduce filter fluorescence as much as
possible the optical filters, especially the filter in the excitation light path, are usually interference filters, i.e.
filters which reflect the light that is not transmitted rather than absorb it. The configuration shown here
employs critical illumination: the light source is imaged in the object plane of the microscope objective, i.e.
in the sample flow. Critical illumination provides somewhat higher intensity than is achieved with Kohler
illumination, which is used in most microscopes. Kohler illumination has the advantage that the excitation
intensity is less susceptible to lamp flicker - the sudden movements of the arc that is seen with some lamps.