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Technical support - Clinical products
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E-Mail: macs@miltenyibiotec.nl
As a global market leader with numerous subsidiaries and distributors, Miltenyi Biotec is committed to providing our customers around the world with the highest quality products. In addition to direct selling in more than 20 countries in North America, Europe and Asia/Pacific, Miltenyi Biotec also provides support for our customers through an extensive distributor network covering dozens of additional countries.
rapidly. As cells or particles travel through the light path, the fluorescent probes are excited, resulting in the
detection of the emitted light and ultimately of the specific cellular properties at a rate of 10,000 events/second.
monitoring patients after treatment. Some clinics also use flow cytometry to characterize donor and recipient
cells prior to transplant to lower the risk of adverse effects following transplantation.
Examples of flow cytometry applications
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To enable the light scatter and fluorescence measurements of cells and particles, MACSQuant® Instruments
utilize a combined system of fluidics, optics, and electronics.
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 “fluorescence channels”.
There are different types of filters that are used to define which wavelengths can enter the fluorescence
channels. According to their optical properties they are designated 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 nanometers (nm) and longer can pass through the filter. Light of all other wavelengths will be absorbed. 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 absorbed. 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 mirror is deflected at a 90° angle towards another light detector.
With this optical setup, eight distinct fluorescence channels and two scatter channels are created by combinations of the light emitted after excitation by the three lasers, as well as various filters and mirrors.
Photon detectors – Photomultiplier tubes (PMTs)
The light paths of specific wavelengths, i.e., fluorescence channels, are monitored by so-called photomultiplier tubes (PMTs). PMTs amplify the fluorescence signal emitted from the fluorochrome bound to the cell or particle.
The magnitude of amplification is dependent on the voltage applied to each PMT. As the voltage is increased, the amplification of the detectable fluorescence is increased, resulting in an increased mean fluorescence intensity (MFI). Conversely, as the voltage is decreased, the amplification is decreased, resulting in a lower MFI.
As each channel’s PMT detects and amplifies the light emission of a specific fluorescent reagent, it is
recommended to assign the correct nomenclature to these channels. The PMT not only amplifies the
detectable light, but converts the optical signal into a voltage pulse that is relayed to the electronics for signal
conversion and display.
Analog-to-digital conversion
Analog signals are converted into digital signals for display of data according to the bit depth of the analog-to-digital converter (ADC) of the flow cytometer. The MACSQuant Instruments have a 16 bit ADC for all parameters. However, 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 allows for a five-decade logarithmic scale for data display.
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 pulses
As a cell is passing through the laser path, the attached fluorochrome will begin to fluoresce. This light is
detected by the PMT and starts the generation of a voltage pulse. As the cell fully enters the laser path, the
fluorescence reaches peak emission, resulting in the highest peak of the voltage pulse. Finally, as the cell
leaves the laser path, this detectable fluorescence decreases, finishing the voltage pulse.
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