16
of metal across the entrance pupil of the lens which prevents the intense reflection of laser
light from the cylindrical surface of the jet from reaching the optics. Hence, only some 5 %
of the fluorescence is actually reaching the detection optics in this type of instrument. This
intensity is further reduced before it reaches the detector, primarily by optical filters used to
isolate an appropriate portion of the fluorescence spectrum. Instruments with a closed flow
chamber or a flow chamber of the IOOS type (see Figs. 8 and 9) may employ immersion
optics for light collection. Oil immersion lenses can have NA = 1.3 -1.4, and since there is
no need for an obscuration bar, collection efficiency may be as high as 25 %.
Efficient fluorescence measurement requires: a) that the lens which collects the fluorescence
has the highest possible numerical aperture, and b) that the background light, caused by
scattering by the flow chamber and fluorescence of optical components, is as low as possible.
In order to reduce the level of background light it is important that the fluorescence and light
scattering detectors see as little as possible outside the sample flow itself. This can be
achieved by means of what is called spatial filtering, that is a measuring aperture or slit which
is situated in the image plane of the detection lens (Figs. 2 and 3) and which covers only the
image of the sample flow and thereby eliminates all light from other sources, i.e. from other
parts of the excitation focus. Such a measuring aperture is sometimes termed the pinhole.
Such spatial filtering may also be used to reduce the volume of the excitation focus. Thus, an
excitation slit is situated in the image plane of the excitation lens (Fig. 2). Ideally at least, the
excitation and measuring apertures should be imaged in the excitation focus so as to overlap
perfectly. Efficient spatial filtering depends on flow chambers that permit proper imaging of
the cells and optics which produce images with high definition and contrast. In this regard,
closed flow chambers having flat surfaces and the JOOS flow chamber are better than
cylindrical designs or the jet in air, which act as a cylindrical lens and thereby distort the
image. The detection optics should be a microscope objective or similar lens designed for
imaging purposes. The aspherica1 lenses used as detection optics in some instruments are
inferior in this regard and will provide less efficient spatial filtering and correspondingly
higher levels of optical background, i.e. noise. In most flow cytometers the potential of spatial
filtering is not fully utilized. Typically, the measuring aperture in front of the fluorescence
detector is a circular pinhole which is much larger than the image of the exposed sample flow.
of metal across the entrance pupil of the lens which prevents the intense reflection of laser
light from the cylindrical surface of the jet from reaching the optics. Hence, only some 5 %
of the fluorescence is actually reaching the detection optics in this type of instrument. This
intensity is further reduced before it reaches the detector, primarily by optical filters used to
isolate an appropriate portion of the fluorescence spectrum. Instruments with a closed flow
chamber or a flow chamber of the IOOS type (see Figs. 8 and 9) may employ immersion
optics for light collection. Oil immersion lenses can have NA = 1.3 -1.4, and since there is
no need for an obscuration bar, collection efficiency may be as high as 25 %.
Efficient fluorescence measurement requires: a) that the lens which collects the fluorescence
has the highest possible numerical aperture, and b) that the background light, caused by
scattering by the flow chamber and fluorescence of optical components, is as low as possible.
In order to reduce the level of background light it is important that the fluorescence and light
scattering detectors see as little as possible outside the sample flow itself. This can be
achieved by means of what is called spatial filtering, that is a measuring aperture or slit which
is situated in the image plane of the detection lens (Figs. 2 and 3) and which covers only the
image of the sample flow and thereby eliminates all light from other sources, i.e. from other
parts of the excitation focus. Such a measuring aperture is sometimes termed the pinhole.
Such spatial filtering may also be used to reduce the volume of the excitation focus. Thus, an
excitation slit is situated in the image plane of the excitation lens (Fig. 2). Ideally at least, the
excitation and measuring apertures should be imaged in the excitation focus so as to overlap
perfectly. Efficient spatial filtering depends on flow chambers that permit proper imaging of
the cells and optics which produce images with high definition and contrast. In this regard,
closed flow chambers having flat surfaces and the JOOS flow chamber are better than
cylindrical designs or the jet in air, which act as a cylindrical lens and thereby distort the
image. The detection optics should be a microscope objective or similar lens designed for
imaging purposes. The aspherica1 lenses used as detection optics in some instruments are
inferior in this regard and will provide less efficient spatial filtering and correspondingly
higher levels of optical background, i.e. noise. In most flow cytometers the potential of spatial
filtering is not fully utilized. Typically, the measuring aperture in front of the fluorescence
detector is a circular pinhole which is much larger than the image of the exposed sample flow.