INTRODUCTION TO FLOW CYTOMETRY - Flow Cytometry in Drug Discovery and Development - page 6

TABLE 1.1 Partial List of Laser with Their Excitation Wavelength Line and the
Fluorochromes which Can be Detected

Laser Excitation Line (nm) Fluorochrome

UV 355 Hoescht 33342, 33250

He-Cd 325 DAPI, ELF-97, AMCA (AlexaFluor 350),

INDO-1

Mercury lamp
Violet

405 Pacific Blue, CasB

Krypton ion 435 CasY, AlexaFluor 405 (AF405)

Blue (argon) 488 PE-TR, 6FP, FITC, PE, AF488, PE-Cy7, PerCP,

PE-Cy5, SYTO 9, PerCP-Cy5.5

Red (solid state) 640 APC, APC-Cy7, SYTO 59-61

He-Ne 633 AF647, APC-Cy7

Red diode 635 APC-Cy5.5, AF700

Yellow/green 561 PE-Texas Red, PerCP, PE

Krypton lasers can give out strong blue-green lines and UV and violet lines.
Krypton lasers need to be water cooled and optimized and the alignment is very
difficult. Another type of laser is a dye laser and the lasing medium in a dye laser is a
fluorescent dye. The selection of dye depends on the wavelength at which the
operation is desired. Helium-neon (He-Ne) lasers are also small, air cooled, and
stable. The most common lasers emit at 633 nm and have power outputs ranging
from 1 to 50 mW. He-Ne lasers are available at 633, 543, 594, and 611 nm.
Helium-cadmium (He-Cd) lasers emit 5-200mW in blue (441 nm) and 1-50 mW
in UV (325 nm). They plug into the wall and do not require water cooling.

1.4.3 Filters for Emission

All signals that are emitted from fluorochromes that are excited as the cells to which
they are bound are interrogated by the laser beams are routed to detectors via a system
of mirrors and optical filters. In addition, beam splitters direct light of different
wavelengths in different directions. The most commonly used filters are short-pass
filters (which transmit wavelengths of light equal to or shorter than the specified
wavelength), long-pass filters (which transmit wavelengths of light equal to or larger
than the specified wavelength), and band-pass filters (which allow a narrow range of
wavelengths to reach the detector). An example of these types of filters is presented in
Figure 1.3. Because each fluorochrome has an emission spectrum, the choice of filters
optimizes detection of the specific fluorochrome by one detector or photomultiplier
tube (PMT).
Detection of fluorochromes requires selection of appropriate filters that are placed
before each detector or PMT. The type of filter selected must collect as much emitted
light from the primary fluorochrome for high sensitivity, but as little as possible from
other fluorochromes to reduce the compensation required. A partial list of filters is
presented in Table 1.2.