Lesson 4.3: Cytometer Subsystems – Optical

Optical Subsystem

The optical subsystem of a flow cytometer is somewhat more complex than the fluidic system. The optical system begins at the excitation source. The most excitation sources used in modern flow cytometers are lasers and arc lamps. Choice of an arc lamp or laser will depend upon application as both systems have their pros and cons.






Lasers as Excitation Sources



  • Inefficient (Power In/Power Out)
  • Often Requires Non-standard Utility Requirements
  • Expensive

Arc Lamps as Excitation Sources


  • Inexpensive
  • Wide Wavelegnth Range (Frequency-Agile)


  • Wide Bandwidth
  • Lower Power Density
  • Unfocussed Emission

Directing Light After the excitation light source strikes the stream, fluorescent molecules attached to, or incorporated within, the cells emit higher-wavelength photons. A wide-angle collection optic (often something as simple as a microscope objective) is placed near the flow cell to collect these photons and focus them into the cytometer detector path. Contained within the detector path are a combination of steering optics (prisms, beam splitters and dichroic mirrors) and bandpass filters. The figure below details the optical bench of the BDBiosciences FACSCalibur.

Notice the notation for the bandpass filters in front of the SSC, FL1, FL2 and FL4 detector. The FL1 filter is labeled as 530/30. This indicates that the filter will has a 30nm bandpass centered on 530nm. Therefore, light ranging from 515nm to 545nm will pass through this filter without reflection or absorption. Notice also, that the FL3 detector does not have a bandpass filter, but rather a 670nm long pass filter. As you have probably guessed, this means that the filter will pass any photons that have a wavelength of 670nm or above. Why might the manufacturers have chosen to place a long pass filter here rather than the standard bandpass?

The FACSCalibur uses a spatially-separated laser system. This means that the two laser beams are not brought in a collinear fashion but, instead, are separated in time and space. This separation enables system to accurately measure fluorochromes with very similar emission spectra without the concerns of significant overlap.

A somewhat more complex optical layout is pictured below. This is the FACSVantage SE optical deck. Notice that the system is able to utilize three spatially separated lasers. Be aware that while the system contains eight fluorescent detectors, the FACSVantage SE is only capable of using six of these detectors simultaneously (plus the two scatter detectors) without significant modification of the digital upgrade option. The core facility has three of these sorters available for your use.

The Final Step After steering and selection (via the bandpass filters), the final step in the optical system is detection. This is generally accomplished with photomultiplier tubes and/or photodiodes.

The photomultiplier tube, or PMT, operates via an electron cascade. That is, a PMT contains a series of charged grids. When a photon strikes the outer grid, a number of electrons (e-) are released. These electrons strike the next grid in the series releasing further electrons. This continues throughout the series of charged grids until the last grid at which point the electrons are collected and a current pulse passes from the detector. The amount of electrons released by each grid strike can be controlled by increasing or decreasing the voltage applied to the grids. Therefore, raising the voltage applied to the detector increases the sensitivity of the PMT while decreasing the voltage lowers the sensitivity. Photodiodes work by a different, though quite interesting mechanism, but for our purposes, you can consider them solid state equivalents of the PMT.

Moving On

Once you’re comfortable with this material, please move on to the next subsystem.

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