Differences between single-wavelength imaging and two-wavelength ratio imaging

Disadvantage of single-wavelength imaging
Overview of two-wavelength ratio imaging
Calcium imaging related products
Other experimental methods

Disadvantage of single-wavelength imaging

In calcium imaging, directly interpreting changes in the fluorescence intensity of simple Ca²⁺ probes as the dynamics of the desired phenomenon or target molecule can lead to inaccurate measurements. The fluorescence intensity detected from Ca²⁺ probes varies due to factors such as fading of the fluorescent probe, variations in localization concentration, and sample movement. These variations are artifacts caused by technical measurement issues.

The following is a schematic explanation of the effects of the state of the sample and fluorescent probe on fluorescence brightness, using as an example the measurement of changes in intracellular calcium concentration using a fluorescent probe with a single wavelength whose fluorescence brightness changes in accordance with calcium ion concentration.

1 wavelength - same intracellular calcium concentration

Figure 1: Single wavelength - when the intracellular calcium ion concentration is the same in three cells (cells A, B, and C)

In Figure 1, we have cells A, B, and C, assuming that their resting intracellular calcium ion concentrations are the same. Additionally, let’s consider that cell B has greater thickness compared to cell A, while cell C has absorbed less pigment than cell A. When each cell is excited under the same conditions, the resulting fluorescence intensity, relative to cell A (which we set as 100 %), is approximately 130 % for cell B due to its greater fluorescent dye content. On the other hand, cell C, with lower fluorescent dye content, exhibits a relative fluorescence intensity of about 70 %.

Keep in mind that fluorescence intensity also depends on the probe’s abundance. Therefore, if we rely solely on fluorescence intensity as an indicator, even when intracellular calcium ion concentrations are identical, cell B may appear to have higher calcium ion concentration, and cell C may appear to have lower calcium ion concentration.

Next, we will use the case where the intracellular calcium ion concentration in cell A increases due to stimulation of only cell A, and the fluorescence brightness increases by 30 % compared to the level before stimulation of cell A.

1 wavelength - high intracellular calcium concentration in cell A

Figure 2: Single wavelength - when the intracellular calcium ion concentration is high in cell A

In the state depicted in Figure 2, despite the different intracellular calcium ion concentrations between cell A and cell B, the fluorescence intensity of both cell A and cell B remains the same. As a result, it becomes difficult to discern the difference in calcium ion concentration between the two cells.

In fluorescence live imaging, measured fluorescence intensity values are influenced by sample and fluorescent probe conditions. Thus, we need to consider various interpretations of what the measurement value means.

Overview of two-wavelength ratio imaging

Ratio imaging is a measurement technique that obtains an image based on the ratio of fluorescence intensities from two different fluorescent wavelengths (referred to as the ‘ratio value’). By calculating the ratio between the two wavelengths, it corrects for fluorescence intensity components unrelated to the actual measurement target, such as variations due to sample or fluorescent probe conditions and environmental changes. This method is widely used to achieve more accurate and quantitative imaging.

Ratio Imaging - Principle

Figure 3: Principle of ratio imaging

In Figure 3, the typical temporal variation of fluorescence intensity obtained using ratio fluorescent probes is schematically shown. Even with sample movement or fading, fluctuations in fluorescence intensity occur with ratio fluorescent probes. In this case, the two fluorescence intensities exhibit the same upward and downward trends. Since the numerator and denominator intensity values change in sync, the ratio value of the two fluorescence intensities remains constant.

On the other hand, ratio fluorescent probes are designed so that the changes in two types of fluorescence intensity related to the phenomenon of interest are mirror images of each other. For example, in the case of measuring calcium ion concentration using a fluorescent probe, one fluorescence increases in brightness as calcium ion concentration rises, while the other fluorescence decreases in brightness as calcium ion concentration increases. As a result, significant changes are observed in the ratio value.

By comparing the ratio value of fluorescence intensity over time, it is possible to cancel out the simple brightness variations due to sample movement or fading and extract only the brightness changes related to the desired phenomenon.

Using ratio imaging not only allows tracking of temporal variations but also facilitates comparisons between cells. Taking the example of measuring intracellular calcium ion concentration using two-wavelength fluorescent probes, we can illustrate how the ratio value provides useful information under various measurement conditions.

2 wavelengths - same intracellular calcium concentration

Figure 4: 2 wavelengths - when the intracellular calcium ion concentrations of the three cells are identical (cells A, B, and C)

In Figure 4, similar to Figure 1, we have cells A, B, and C, assuming that their resting calcium ion concentrations are the same. Cell B has greater thickness compared to cell A, while cell C has lower pigment uptake than cell A. The fluorescence intensity is 100 % for cell A, 130 % for cell B, and 70 % for cell C. Let’s denote the intensity values from the two fluorescence wavelengths separately as Ch1 and Ch2. When calcium ion concentration remains constant, the intensity values depend on the presence of pigment in both Ch1 and Ch2. Therefore, cell B exhibits higher Ch1 intensity and also higher Ch2 intensity, while cell C has lower Ch1 and Ch2 intensities. The ratio values (referred to as the ratio) remain the same for cells A, B, and C. Consequently, using the ratio value, we can distinguish between cells with thick structures (which might lead to higher intensity in single-wavelength measurements) and cells with lower pigment uptake or fading (which might incorrectly suggest lower calcium ion concentration). Comparing the ratio values reveals that the calcium concentrations in these cells are indeed identical.

Next, in Figure 5, let’s consider the case where only cell A is stimulated, and the intracellular calcium ion concentration in cell A increases, resulting in a 30 % increase in Ch1 fluorescence brightness compared to before stimulation of cell A. The following is an explanation.

Two wavelengths - high intracellular calcium concentration in cell A

Figure 5: 2 wavelengths - when the concentration of calcium ions in cell A is high

Due to the properties of the ratiometric fluorescent probe, when the calcium ion concentration increases, the fluorescence intensity of Ch2 decreases inversely and becomes 70 %. When comparing only Ch1, cells A and B have the same brightness value. However, when calculating the ratio value, cell A has a value of 1.86, while cell B has a value of 1. This difference indicates that the intracellular calcium concentration in cell A is higher than in cell B.

As we have seen so far, in fluorescence live imaging, fluorescence intensity varies due to various factors, and obtaining useful information from simple brightness has limitations. Ratiometric imaging allows us to remove variations in brightness caused by the sample or fluorescent probe environment, enabling more accurate observation of the intended phenomenon.

Up to this point, we have schematically demonstrated the factors influencing fluorescence intensity in live imaging and the benefits of ratiometric imaging. On the next page, we will explain how to capture biological phenomena using ratiometric imaging with actual measurement data.

Two-wavelength ratio imaging observed with experimental data