The Application of Biexponential and Logarithmic Scales in Flow Cytometry Data Analysis

Today, we will mainly discuss the display methods of coordinate axes, the conversion between logarithmic and biexponential scales, and the adjustment of coordinate axes in flow cytometry results.

The commonly used display methods for three types of flow cytometry plots are Linear, Logarithmic, and Biex scales, as shown in Figure 1.

Figure 1: Mouse spleen sample, three axis results

· Left: FSC and SSC axis displayed with Linear scale

· Middle: SSC axis displayed with Linear scale; CD3 axis displayed with Logarithmic scale

· Right: SSC axis displayed with Linear scale; CD3 axis displayed with Biexponential scale

1. Linear Scale

The Linear scale displays the axis with linearly increasing values. Its advantage is that it can clearly present the differences in signals within the display range when the span of the detected signal is small. Examples of using the Linear scale include presenting cell diameter (FSC), intracellular granularity (SSC), and DNA content in cell cycle experiments.

Its disadvantage is that when analyzing fluorescence signals with a large span, the Linear scale cannot effectively display the fluorescence signal, as shown in the left part of Figure 2.

Figure 2: Peripheral blood sample, CD4 and CD8, comparison of results with linear and logarithmic scales

· Left: Both CD4 and CD8 axes are displayed with the Linear scale.

· Right: Both CD4 and CD8 axes are displayed with the Logarithmic scale.

However, there are also special cases. For example, in the analysis of peripheral blood immune phenotyping, logarithmic scaling can be selected for the SSC axis to enhance the resolution of the granulocyte population, as shown in the right part of Figure 3.

Figure 3: Peripheral blood sample, SSC, comparison of results with linear and logarithmic scales

· Left: The SSC axis is displayed with the Linear scale; CD45 axis is displayed with the Biex scale.

· Right: The SSC axis is displayed with the Logarithmic scale; CD45 axis is displayed with the Biex scale.

2. Logarithmic Scale

The Logarithmic scale displays the axis with exponentially increasing values. Its advantage is that it enhances the clustering of populations and highlights the differences in signals within the display range when the detected signal has a large span. Examples of using the Logarithmic scale include fluorescence signal detection in flow cytometry for antibody staining and Annexin V apoptosis detection experiments.

Its disadvantage is the issue of negative values. In certain detection results (due to excessively strong positive signals or low voltage settings), using the Logarithmic scale may cause some signals to be compressed or not visible (percentage can be observed using gating), as shown in the left part of Figure 4.

Figure 4: Mouse spleen sample, CD8, comparison of results with logarithmic and biexponential scales

· Left: The SSC axis is displayed with the Linear scale; CD8 axis is displayed with the Logarithmic scale.

· Right: The SSC axis is displayed with the Linear scale; CD8 axis is displayed with the biexponential scale.

3. Biex Scale

The biex scale displays the axis with exponentially increasing values. Its advantage is that it exhibits linear characteristics when the values are small, allowing for detailed visualization, while demonstrating logarithmic advantages when the values are large, resulting in population clustering. The biex scale is commonly used to present various flow cytometry and related experimental results that involve negative values (weaker fluorescence signals).

However, its drawback lies in the fact that biex scale is automatically calculated based on the minimum value. If the specimen contains a very small number of cells with extremely low fluorescence intensity, the negative population and weakly positive signals can get compressed, leading to a visual alteration in the distribution of populations. In some cases, it may even result in the appearance of two distinct signal populations in the low fluorescence intensity region, as shown in the right part of Figure 5. Therefore, it is often noticed that many instruments default to using the Logarithmic scale instead of the biex scale for fluorescent channel axes.

Figure 5: Mouse spleen sample, CD8, comparison of results with logarithmic and biexponential scales

· Left: The SSC axis is displayed with the Linear scale; CD8 axis is displayed with the Logarithmic scale.

· Right: The SSC axis is displayed with the Linear scale; CD8 axis is displayed with the biexponential scale.

Summary:

· When the detected signal has a small span, such as FSC, SSC, and DNA content in the cell cycle, the coordinate axis is typically chosen as Linear, except for special requirements.

· When the detected signal has a large span, such as fluorescence signals for antigen markers, the coordinate axis is often selected as Logarithmic.

· If the fluorescence signals to be analyzed are close to the negative value region, the coordinate axis can be chosen as biexponential.

The above discussion summarizes the application of biexponential and logarithmic scales in flow cytometry data analysis. If you have any further questions, please feel free to leave a message, we will respond to you ASAP.