INTRODUCTION TO FLOW CYTOMETRY - Flow Cytometry in Drug Discovery and Development - page 5

1.4 OPTICS
Flow cytometers depend on the laws of optics, such as reflection, refraction, and other
principles, which are not new but based on works established centuries ago [56].
Optics are present on both the excitation and the emission side. The excitation optics
encompass the lasers and the lenses that focus the laser beam. The emission optics are
involved in collecting the emission following excitation. These involve lenses to
collect emitted light and mirrors and filters to route specified wavelengths of the
collected light to designated optical detectors. Light coming out of a laser may be
considered a beam but fluorescence must be considered as a photon.
1.4.1 Light Scatter
Due to differences between the refractive indices of cells and the surrounding sheath
fluid, light impinging upon the cells is scattered. The forward light scatter (FSC)
provides empirical information on cell size. Light scattered in an orthogonal direction
or side scatter (SSC), which is collected by a different detector, provides information
about granularity.
1.4.2 Types of Lasers
Laser stands for light amplification by stimulated emission of radiation. Gas lasers
have mirrors at each end of a cylinder or plasma tube filled with an inert gas. The gas is
ionized to a higher energy state by a high-voltage electric current. When these excited
atoms return to the ground state, they give off photons of a characteristic wavelength.
The photons can be reflected by the mirrors and the excitation of the atoms in the
plasma can be amplified but thewavelengths of the emission still are the characteristic
wavelengths for that gas [57]. In the front of the laser there is a small optic that allows
the transmitted light to form a laser beam of desired output wavelengths. The light
from lasers is a stimulated emission and it has uniform characteristics. For current
stream-in-air instrumentation, it is desirable to have at least 50mWof power for each
laser line in use, since the fluorescence signal (and thus sensitivity) increases with
laser power. Cytometers use multiple lasers that are positioned spatially such that
there is a time delay for each laser beam intercept with the cell. Newer solid-state
diode lasers [58-60] are becoming prevalent and these are significantly cheaper than
the older gas ion lasers. Diode lasers are pumped by input of electric current. A partial
list of different lasers is presented in Table 1.1.
The most common lasers for flow cytometers are the argon ion lasers that run at
488 nm. The lasing medium in an ion laser is plasma. A high-voltage pulse is used to
ionize the gas to start the plasma. Ion lasers require a high current to maintain the
plasma discharge. In addition to the 488 nm emission, argon ion lasers also emit at
515 nm (green) and 457 nm (violet-blue). Other emissions can be obtained using
specially coated mirrors. The new low-power, air-cooled argon laser gives out 25 mW
at 488 nm. To obtain other lines of emission, large lasers capable of giving 100mWin
UV must be used.