Flow cytometry basics

Filters and mirrors

After excitation by a laser, fluorescent reagents bound to cells or particles emit light, which is detected
according to their specific wavelength range. When using a combination of fluorescent dyes, these reagents should have distinct emission spectra. MACSQuant Instruments utilize various optical filters and dichroic mirrors to direct light of specific wavelengths to the according fluorescence detectors. This arrangement creates the so-called fluorescence channels.
Different types of filters define which wavelengths can enter the fluorescence channels. According to their optical properties they are referred to as long-pass, short-pass, and band-pass filters. MACSQuant Instruments use long-pass and band-pass filters.

Long-pass filters are denoted by the letters LP and a specific number, e.g., LP 750. This means that light with a wavelength of 750 nm and longer can pass through the filter. Light of all other wavelengths will be blocked. Short-pass filters have a similar designation, e.g., SP 500. In that case, light of wavelengths of 500 nm and shorter can pass through, while the remaining light will be blocked. Band-pass filters allow light of a specific wavelength range to pass through. Band-pass filters are referred to as BP followed by two numbers. The first number represents the midpoint of the wavelength range. The second number specifies the wavelength range in nm that can pass. Thus, light that can pass through a BP 525/50 filter ranges from 500 to 550 nm.
Dichroic mirrors have essentially the same function as filters, but are oriented at a 45° angle to the light path, whereas filters are oriented perpendicular to the light path. The light that is not able to pass through a dichroic mirror is deflected at a 90° angle towards another light detector. 

The optical systems of MACSQuant Flow Cytometers combine three lasers and various filters and mirrors, resulting in up to 14 distinct fluorescence channels and two scatter channels.

Photon detectors – Photomultiplier tubes (PMTs)
The light paths of specific wavelengths, i.e., fluorescence channels, are monitored by photomultiplier tubes (PMTs). PMTs amplify the fluorescence signal emitted from the fluorochrome bound to the cell or particle and generate an electrical current.
The magnitude of amplification is dependent on the voltage applied to each PMT. When the voltage is increased, the amplification of the detectable fluorescence is also increased, resulting in a higher mean fluorescence intensity (MFI). Conversely, when the voltage is decreased, the amplification is also decreased, resulting in a lower MFI. The electrical current generated by the PMT is converted into a voltage pulse within the electronics system.

The electronics system of a flow cytometer converts the light signals into proportional electronic signals (voltage pulses), digitizes the signal according to the bit depth of the analog-to-digital converter (ADC), and interfaces with the computer for data transfer.

MACSQuant Instruments are digital flow cytometers, which means that the conversion from analog to
digital signals happens at the point of signal detection and amplification. Advantages of digital flow
cytometers include:

• Voltage pulse height, width, and area can all be a measurable parameter.
  
• Compensation of spectral overlap is applied after digital conversion of signal, meaning compensation can be re-adjusted any time post acquisition.
  
• Detection thresholds or triggers can be set on any of the detection channels.
  
• Fluorescence values detected as zero or negatives will be measured as such and can be displayed in the
  plot choices.

Analog-to-digital conversion
Analog signals are converted into digital signals for display of data according to the ADC's bit depth. MACSQuant Instruments have a 16 bit ADC for all parameters. For the pulse area, an additional calculation allows for 18-bit processing. This means that for the pulse area measurements, the signals are resolved into over 253,000 bins. This enables a five-decade logarithmic scale for data display.