Two-wavelength ratio imaging observed with experimental data

The following description of the method of capturing changes in biological phenomena by ratio imaging is based on data obtained by measuring membrane potential changes in iPS cell-derived cardiomyocytes, which have attracted attention as cells to be used for evaluating the cardiotoxicity of new drug candidate compounds, using the membrane potential-sensitive dye di-4-ANEPPS.

The measured cardiomyocytes exhibit fluctuations in fluorescence intensity due to spontaneous contractions, thickness variations, and positional changes. Accurate measurements require effective extraction of fluorescence intensity variations originating from membrane potential. The following is the explanation of how components derived from membrane potential are effectively extracted from beating cardiomyocytes using ratio imaging.

Video 1: Measurement example of membrane potential ratio imaging

In Video 1, the two left images represent fluorescence images obtained from measurements at two different wavelengths. Due to pulsation, the sample undergoes significant movement, leading to substantial fluctuations in the di-4-ANEPPS concentration within the observation area. The fluorescence intensity changes combine information about both membrane potential variations and alterations in dye concentration. On the other hand, the image on the right overlays pseudo-color representations of the ratio values for the two-wavelength fluorescence. These ratio values remain unaffected by sample movement, clearly capturing the membrane potential changes just before cardiac muscle cells contract.

The following graph shows how the effects of sample migration and fading are eliminated by ratio imaging, and how the signals that you want to measure can be extracted. The following waveforms are shown as a graph of the change over time of each of the two wavelengths of fluorescence in a specific observation area.

di-4-ANEPPS-Ch1-Ch2

Figure 1: Temporal changes in fluorescence intensity for Ch1 and Ch2 in fluorescent membrane potential observation

The Figure 1 shows a mixture of waveforms with sharp peaks and waveforms that show gradual changes, making it difficult to derive the timing and magnitude of the membrane potential change from this waveform alone. Furthermore, since the entire graph is a rightward sloping graph, it is difficult to use the absolute value of luminance as an index of activity because of the fading of the fluorescent probes.

Next, the ratio values of Ch1 and Ch2 are calculated and added to the graph as follows.

di-4-ANEPPS-膜電位変化にともなう蛍光輝度の変化

Figure 2: Temporal changes in ratio values and waveform comparison in fluorescent membrane potential observation

In Figure 2, the green ratio graph reveals waveforms with distinct peaks. As indicated in the blue region, the ratio graph exhibits peaks, and Ch1 and Ch2 show brightness changes in opposite directions. It is discernible that these peaks correspond to variations in fluorescence intensity associated with membrane potential changes.

di-4-ANEPPS-弛緩による試料移動

Figure 3: Ratio calculation corrects for sample movement's effects on fluorescence intensity

Additionally, in the red region of Figure 3, the alignment of Ch1 and Ch2 brightness fluctuations likely results from changes in probe concentration within the observation area due to myocardial movement or thickness variations. This portion does not exhibit waveform changes in the ratio graph, indicating suppression of factors other than the intended membrane potential.

In summary, ratio graphs remain unaffected by simple brightness changes like sample movement and instead vary in response to membrane potential fluctuations. In Video 2, the sample movement and membrane potential change are shown in conjunction with the image.

Video 2: Differences in brightness changes due to sample movement and membrane potential changes

Video 2 is a portion extracted from Video 1 to make the sample movement more visible. Between 3 seconds and 3.5 seconds of measurement time, it becomes evident that the sample is moving due to relaxation. When looking at the graph on the right, the fluorescence brightness in the Ch1 and Ch2 observation regions changes with sample movement. However, the ratio value remains unchanged. Next, at 3.57 seconds of measurement time, there is a significant change in the ratio value. This moment corresponds to the membrane potential increasing. Interestingly, compared to the rapid change in the ratio value, there is minimal sample movement visible in the image. This confirms that the brightness changes in Ch1 and Ch2 during this moment are not due to measurement issues such as image misalignment. Such discrepancies often arise in measurements using a filter wheel.

di-4-ANEPPS-退色による蛍光輝度の変化

Figure 4: Changes in fluorescence brightness due to bleaching and the correction effect by ratio calculation

The dashed lines in Figure 4 represent the baselines for each graph. Both Ch1 and Ch2 show a gradual decrease in baseline over time, indicating bleaching occurring within the 6-second interval. However, in the ratio graph, the baseline remains horizontal, resulting in a graph unaffected by bleaching.