2P imaging of carbon nanotubes

Imaging deep inside biological tissue is limited by the scattering and absorption of light, both of which decrease at longer wavelengths. The short-wave infrared window around 1700 nm offers one of the longest attenuation lengths in tissue, making it particularly attractive for deep imaging (Horton et al., 2013). At these wavelengths, contrast can be generated by two-photon (2P) or three-photon (3P) excitation: 3P excitation provides better optical sectioning and a higher signal-to-background ratio deep in tissue — at the cost of a weaker signal — while 2P is more efficient but less confined. Choosing the excitation order and the spectral window is therefore a trade-off between penetration depth, contrast and signal.

Attenuation length in tissue versus wavelength: longer wavelengths (e.g. the 1700 nm window) penetrate deeper. The diagrams show the 2P / 3P excitation schemes accessible at each window.

Single-walled carbon nanotubes (SWCNTs) are rolled-up sheets of graphene whose electronic — and therefore optical — properties are set by their “chirality” (the (n,m) indices). They emit in the near-infrared, are remarkably photostable and do not blink or bleach, which makes them excellent luminescent probes for deep, long-term imaging (Godin et al., 2017). Each chirality has a specific excitation/emission fingerprint, and the nanotubes can be excited through multiphoton processes — both of which we characterise by spectroscopy.

Spectroscopy of carbon nanotubes: the excitation–emission map (left) resolves the individual chiralities (n,m), each with its own near-infrared signature; the two-photon excitation spectra (right) characterise their multiphoton response.

Building a dedicated infrared multiphoton microscope, we then demonstrate the two-photon imaging of carbon nanotubes, exploiting their near-infrared luminescence together with the depth and contrast advantages of multiphoton excitation. In parallel, we run numerical simulations to compare standard two-photon excitation with two-photon excitation in the short-wave infrared (2P-IR), in order to identify the most favourable imaging regime.

Demonstration of two-photon imaging of carbon nanotubes recorded with the infrared multiphoton microscope.