14
immersion optics. However, the output of these lasers have a ripple of a few percent which
limits resolution, in terms of lowest cv. What may be more important in marine applications
is that the lasers of such instruments are not tunable, but run at only one wavelength, namely
488 nm. This limits the range of applications. For example, chlorophyll is inefficiently
excited, whereas dyes like Hoechst 33258, DAPI, and Chromomycin cannot be excited at all.
The alternative to lasers is the high pressure arc lamps containing either mercury, xenon or
both. As shown in Fig. 4B, the Hg lamp, which is the most common, exhibits a number of
strong emission lines superimposed on a continuum. Hence, it can be used for any dye which
is excitable in the visible or near-UV. In particular, it has a strong line at 366 nm which is
perfect for UV-excitable dyes like Hoechst 33258 and DAPI, and other strong lines at 436 and
546 nm, which coincides nicely with the excitation maxima of chlorophyll, phycocyanins and
other pigments of marine organisms. The excitation intensity obtained with a 100 W Hg arc
lamp in an instrument with epi-illumination and immersion optics is in the range 2 to 20 mW,
depending on wavelength. The lowest power, about 2 mW, is in the region of excitation of
FITC, i.e. around 490 nm. Nevertheless, the detection limit for FITC of such an instrument
(Argus 100, Skatron AS, Tranby, Norway) is 2 000 - 3 000 FlTC molecules, which is only
twice the value achieved in the best laser-based instruments.
The 75 W Xe arc lamp, which produces a continuum covering all of the UV and visible, has
a somewhat higher intensity around 490 nm than the Hg lamp, but has significantly less light
at other wavelengths commonly used in the flow cytometry. It should be noted that no
increase of excitation intensity is achieved by using lamps of higher power. Although larger
lamps, say 200 or 500 W, emit more light, their larger arc exhibits a lower light density than
that of the 100 W lamp. In addition, according to the laws of optics, a conventional light
source cannot be focused to a higher light density than that of the source itself.
Provided a sufficiently stable power supply is employed, the short term stability of arc lamps
is generally excellent. However, some lamps may exhibit flicker, that is, abrupt intensity
changes of a few percent which is caused by arc movement and which occur with irregular
intervals, typically of many minutes. On the long term, the intensity of Hg arc lamps decays
at a rate of 1 - 2 % per day (8 hours). The effects of intensity fluctuations in arc lamp-based
immersion optics. However, the output of these lasers have a ripple of a few percent which
limits resolution, in terms of lowest cv. What may be more important in marine applications
is that the lasers of such instruments are not tunable, but run at only one wavelength, namely
488 nm. This limits the range of applications. For example, chlorophyll is inefficiently
excited, whereas dyes like Hoechst 33258, DAPI, and Chromomycin cannot be excited at all.
The alternative to lasers is the high pressure arc lamps containing either mercury, xenon or
both. As shown in Fig. 4B, the Hg lamp, which is the most common, exhibits a number of
strong emission lines superimposed on a continuum. Hence, it can be used for any dye which
is excitable in the visible or near-UV. In particular, it has a strong line at 366 nm which is
perfect for UV-excitable dyes like Hoechst 33258 and DAPI, and other strong lines at 436 and
546 nm, which coincides nicely with the excitation maxima of chlorophyll, phycocyanins and
other pigments of marine organisms. The excitation intensity obtained with a 100 W Hg arc
lamp in an instrument with epi-illumination and immersion optics is in the range 2 to 20 mW,
depending on wavelength. The lowest power, about 2 mW, is in the region of excitation of
FITC, i.e. around 490 nm. Nevertheless, the detection limit for FITC of such an instrument
(Argus 100, Skatron AS, Tranby, Norway) is 2 000 - 3 000 FlTC molecules, which is only
twice the value achieved in the best laser-based instruments.
The 75 W Xe arc lamp, which produces a continuum covering all of the UV and visible, has
a somewhat higher intensity around 490 nm than the Hg lamp, but has significantly less light
at other wavelengths commonly used in the flow cytometry. It should be noted that no
increase of excitation intensity is achieved by using lamps of higher power. Although larger
lamps, say 200 or 500 W, emit more light, their larger arc exhibits a lower light density than
that of the 100 W lamp. In addition, according to the laws of optics, a conventional light
source cannot be focused to a higher light density than that of the source itself.
Provided a sufficiently stable power supply is employed, the short term stability of arc lamps
is generally excellent. However, some lamps may exhibit flicker, that is, abrupt intensity
changes of a few percent which is caused by arc movement and which occur with irregular
intervals, typically of many minutes. On the long term, the intensity of Hg arc lamps decays
at a rate of 1 - 2 % per day (8 hours). The effects of intensity fluctuations in arc lamp-based