Flow Cytometry (FACS) Fluorescence Measurement

Colours

Fluorescence measurements taken at different wavelengths can provide quantitative and qualitative data about fluorochrome-labeled cell surface receptors or intracellular molecules such as DNA and cytokines. Flow cytometers use separate fluorescence (FL-) channels to detect light emitted. The number of detectors will vary according to the machine and its manufacturer. Detectors are either silicon photodiodes or photomultiplier tubes (PMTs). Silicon photodiodes are usually used to measure forward scatter when the signal is strong. PMTs are more sensitive instruments and are ideal for scatter and fluorescence readings.

The specificity of detection is controlled by optical filters, which block certain wavelengths while transmitting (passing) others. There are three major filter types. 'Long pass' filters allow through light above a cut-off wavelength, 'short pass' permit light below a cut-off wavelength and 'band pass' transmit light within a specified narrow range of wavelengths (termed a band width). All these filters block light by absorption.

Band-pass filter in a Flow Cytometer

Long‐pass filters transmit wavelengths above a cut‐on wavelength
Band‐pass filters transmit wavelengths in a narrow range around a specified wavelength

Fluorescent labbeled antibody detected in a Flow Cytometer

As the fluorescing cell passes through the laser beam, it creates a peak or pulse of photon emission over time. These are detected by the PMT and converted to a voltage pulse, known as an event. The total pulse height and area is measured by the flow cytometer. The measured voltage pulse area will correlate directly to the intensity of fluorescence for that event.

The pulse area is calculated by adding the height values for each time slice of the pulse, determined by the speed of the analog to digital converter (ADC), which is 10 MHz (i.e. 10 million per second or 10 per microsecond).

Histogram

These events are assigned channels based on pulse intensity (pulse area). This signal can be amplified by turning up the voltage going through the PMT. Each event is given a channel number depending on its measured intensity; the more intense the fluorescence, the higher the channel number the event is assigned. The channels are usually viewed on a log scale on the x axis.

Dot plot