Introduction of Flow Cytometry | Flow Cytometry Color Matching Principles: Lesson 2 - Fluorescence Interference

Today, we will discuss the second rule in the six major rules of multicolor flow cytometry color matching: placing weakly expressed antigens in interference-free channels and strongly expressed antigens in channels that don't interfere with other channels. This rule focuses on avoiding fluorescence interference with weakly expressed antigens and preventing interference from strongly expressed antigens with other channels.

To understand fluorescent interference, it is paramount to elucidate the concept of fluorescent spillover. Fluorescence spillover refers to the overflow of fluorescence signals into other channels, which is caused by the emission properties of fluorescent dyes and the optical settings of the instrument. There are three main reasons for fluorescence spillover:

Fluorochromes often have broad emission spectra, which can lead to spectral overlap between different fluorochromes, resulting in fluorescence interference. For instance, when FITC and PE are excited by a 488 nm laser, FITC's emission spectrum has a long tail that can extend into the PE channel, where it is detected. Consequently, the signal received in the PE channel includes not only the signal from the PE channel itself but also the spillover signal from FITC.

Figure 1. Fluorescence Spectral Overlap

Secondly, tandem fluorochromes consist of two fluorescent molecules connected together, and their luminescence mechanism utilizes the phenomenon of fluorescence resonance energy transfer. The donor fluorochromes, such as PE and APC, are excited, and then transfer energy through resonance to the acceptor fluorochrome, such as Cyanine 7 in PE-Cyanine7 and APC-Cyanine7. In the case of PE-Cyanine7, the flow cytometer's laser first excites PE, which in turn emits light to excite Cyanine7. In this process, part of PE's emission light enters the PE channel, where it is detected, thus interfering with the signal in the PE channel.

Figure 2. Fluorescence Interference from Tandem Fluorochromes

Third, multiple laser excitations can lead to interference. For instance, PE-Cyanine5 and Cyanine5 can be excited by both PE's emission light and APC's excitation light, resulting in signal interference in the APC channel. Therefore, Cyanine5 cannot be used simultaneously with APC.

Figure 3. Fluorescence Interference from Multiple Laser Excitation

In multicolor flow cytometry experiments, compensation adjustments are needed to eliminate the impact of spectral overlap. However, while compensation can remove fluorescence background, it cannot resolve the issue of reduced fluorescence sensitivity in channels affected by interference.

Figure 4. Impact of Fluorescence Interference

To avoid the impact of fluorescence interference on marker detection in multicolor staining, we need to pay attention to the following considerations:

(1) Place weakly expressed antigens in channels free of interference, which means the channels won't be affected by other channels. For example, the FITC channel.

(2) Place strongly expressed antigens in channels that do not interfere with other channels. For instance, when conducting multicolor flow cytometry on peripheral blood and detecting CD45, which is typically expressed at a high level and can easily interfere with the detection of weakly expressed antigens, we can use CD45 in combination with Elab Fluor® Violet 450 (PacBlue channel). This way, it will not interfere with dyes excited by the 488 nm or 633 nm lasers.

The above explains the flow cytometry color matching rule regarding "fluorescence interference." In the next session, we will bring you the third lesson on flow cytometry color matching principles—Mutually Exclusive Antigens. Stay tuned to Elabscience®!