A Dash of Photophysics Part 2: Absorbing the nuances of 2P spectra

by | Apr 16, 2024

With the advent of high-powered, pulsed lasers, two-photon (2P) microscopy is now a routine technique for imaging biological samples. As described in earlier posts, 2P microscopy is ideally suited for thick and/or scattering samples, such as organoids, tissue slices, plants, small organisms, or intravital preparations. 

When mapping out a fluorescence imaging experiment, one of the first steps is to choose appropriate fluorescent probes for the application. This requires analysing a probe’s properties, starting with their excitation and emission spectra. Often, 2P excitation spectra of fluorescent proteins and organic probes exhibit more intricate features than the corresponding 1P spectra. By taking time to understand these features, researchers can maximise the effectiveness of their experiments. 

Consider the following scenario: you are characterising a mixed population of cells, with each population expressing either EBFP or tdTomato (but not both). The 2P excitation (dotted lines) and emission spectra (solid lines) of EBFP and Tomato are illustrated in Figure 1A and B. Recall that 2P emission, compared to 1P emission, is shifted to shorter wavelengths compared to the excitation.

Using 1P confocal microscopy, it’s typical to image each channel sequentially, such as by exciting the EBFP with a 405 nm laser and the tdTomato with a 565 nm laser. By contrast, most 2P systems are equipped with one laser that can be tuned over a range of wavelengths. As with 1P imaging, 2P imaging often is carried out using sequential acquisition. As 2P lasers can be tuned to different wavelengths, it is possible to select wavelengths that preferentially excite one fluorophore over another. 

In our scenario, the excitation spectra of EBFP and tdTomato guide us as we select appropriate excitation wavelengths. For example, the 2P laser can be used to excite each fluorophore separately at 740 nm (EBFP) and at 1040 nm (tdTomato). When we overlay the 2P excitations for both EBFP and tdTomato (Figure 2) we see that we can excite EBFP at around 780 nm while minimally exciting tdTomato. Likewise we can excite tdTomato without exciting EBFP at 1040 nm.

Of note, the spectral peaks may change according to the environment, or there may be system factors that affect the quality of imaging at different wavelengths. When working out image acquisition, it is important to check how the image quality varies by wavelength, by recording images over  a range of wavelengths close to the reported peak excitation wavelength. In this way, you can ensure that your imaging settings yield the highest quality data, as defined by your research question. 

As described, the imaging approach requires switching between two two wavelengths during acquisition. Though standard, wavelength switching can be slow compared to switching lasers in a 1P system. A tip is to consider choosing a wavelength that excites both fluorescent probes simultaneously, as it streamlines acquisition and reduces imaging time. Based on the spectra in Figure 2, 720 nm is a promising option for exciting both EBFP and tdTomato efficiently. This wavelength offers the advantage of minimising the need for wavelength switching while still effectively exciting both fluorophores.

Figure 3 depicts the fluorescence emission that would be detected using the different wavelengths, corresponding to exciting each probe individually [A, B] or together [C] .

When carrying multi-colour imaging, don’t neglect single-label controls (in our example, samples containing cells positive for EBFP or tdTomato). Single-label controls are imaged identically to the dual-labelled samples. These controls are vital for assessing whether there is appreciable spectral overlap in emission, and also provides the data needed to correct for the overlap if it is detected. 

Another point is that the complexity of 2P excitation spectra also applies to autofluorescent compounds. Do image an unstained sample as well to gauge whether the 2P fluorescence from endogenous compounds is appreciable compared to the emission from the stained specimen. Without the single-label and autofluorescence controls, you may misinterpret the data and undermine the integrity of your experiment. 

By now, you may be wondering about possible drawbacks when using one wavelength to excite both EBFP and tdTomato. In fact, photobleaching can be a disadvantage of using shorter/higher-energy wavelengths. When choosing to use a shorter wavelength, the  2P emission may bleach more quickly compared to when using longer wavelengths. As in all fluorescence imaging, it is important to take the time to test the different conditions before choosing those that work best for your experiment. Ideally, there would be a perfect solution with no compromise, but this is rarely the case in microscopy. In our experience, general guidelines are a starting point, and every researcher has to work out the optimal conditions for their specific experimental needs.

In our next blog post, we will review the options for lasers used in 2P imaging. 

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