A Dash of Photophysics Part 1: 2P vs 1P Fluorophore Excitation

par | Mar 5, 2024

When mapping out a fluorescence imaging experiment, it is important to find the optimal probe(s) for your application. When choosing a probe, a major consideration is matching the excitation and emission spectra to your microscope’s settings. For wide-field and confocal imaging, there are a plethora of online resources that can help guide your choice. By comparison, choosing a probe for two-photon (2P) microscopy requires more time and patience, as the field has only a few reference data sets. (Some examples: Drobizhev et al; Xu and Webb; Bestvater et al). 

In 2P microscopy, the energy of each two exciting photons is roughly half the energy compared to single photon (1P)  excitation. Fig.1 illustrates the 1P excitation and the emission spectra of the fluorescent protein tdTomato. Compare this with Fig.2, which shows the 2P excitation and emission. If you inspect the spectra, you will note that the emission profiles for both 1P and 2P are the same. Yet the 2P excitation spectrum is to the right (longer wavelength) of the excitation spectrum, opposite of the 1P case. 

Comparing the shapes of the 1P and 2P excitation spectra, the 2P spectrum is much more complicated than 1P with a broader range of excitation and multiple peaks. 

Going by the rule of “double the 1P wavelength to get the 2P wavelength” we would expect the 2P excitation peak to be close to 1100 nm. On inspection, however, the excitation spectrum for 2P contains two broad peaks, one closer to 700 nm, with a second peak near 1050 nm.

The direct consequence is you can not rely on simply doubling the 1P wavelength when choosing what excitation to use, as there could be more efficient wavelengths available. When working out the acquisition settings, it is a good idea to “tune” the laser and check the fluorescence emission at different wavelengths, generally in steps of about 10 nm to make sure you are using the optimal wavelength. The additional effort is worth it as some excitation peaks offer less photobleaching, and if you are working with thick or scattering specimens longer-wavelength excitation conditions mean that you can image deeper into your sample compared to standard single photon (1P) techniques.

You can also use the complexity of the 2P excitation spectrum to excite multiple fluorophores with a single wavelength. This requires some planning and an understanding of some of the more unusual features in the excitation spectra, but more on that and some additional quirks of 2P excitation next time!

Figure 1: Single-photon (1P) excitation and emission spectra for the common fluorophore tdTomato. Note how the emission spectrum is to the right (longer wavelength) of the excitation spectrum.  [Improved monomeric red, orange and yellow fluorescent proteins derived from Discosoma sp. red fluorescent protein | Nature Biotechnology]

Figure 2: Two-photon (2P) excitation and emission spectra for tdTomato. Note the emission is to the left (shorter wavelength) of the excitation. [Two-photon absorption properties of fluorescent proteins | Nature Methods]

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