Two-Photon Absorption: An Open Door to the NIR-II Biological Window?

Abstract

The way in which photons travel through biological tissues and subsequently become scattered or absorbed is a key limitation for traditional optical medical imaging techniques using visible light. In contrast, near-infrared wavelengths, in particular those above 1000 nm, penetrate deeper in tissues and undergo less scattering and cause less photo-damage, which describes the so-called "second biological transparency window". Unfortunately, current dyes and imaging probes have severely limited absorption profiles at such long wavelengths, and molecular engineering of novel NIR-II dyes can be a tedious and unpredictable process, which limits access to this optical window and impedes further developments. Two-photon (2P) absorption not only provides convenient access to this window by doubling the absorption wavelength of dyes, but also increases the possible resolution. This review aims to provide an update on the available 2P instrumentation and 2P luminescent materials available for optical imaging in the NIR-II window.

Keywords: fluorescent imaging; infrared dyes; near-infrared II; pulsed lasers; tissue penetration; two-photon absorption; two-photon microscopy.

FIGURE 1

Absorption and scattering coefficients of endogenous chromophores, tissues, and water over the visible and SWIR wavelengths (200–2000 nm). The downwards arrows represent the tissue penetration of light at these wavelengths, according to the values reported for human skin in ref. (Bashkatov et al., 2005). Dashed, dotted and solid outlines on penetration arrows represent the increase in imaging resolution with increased wavelengths. Adapted from values reported in ref (Jacques, 2015). and references therein.

FIGURE 2

(Left) Simplified Jablonski diagram illustrating the double-photon absorption and single-photon emission involved in 1PA vs. 2PA excitation (Middle) Demonstrates the quadratic excitation that arises in 2PA vs. 1PA, due to the requirement of two photons having to arrive simultaneously at a sample to result in excitation (Right) The resulting tissue is capable of producing fluorescence emission due to the location of the excitation photons in 2PA vs. 1PA, resulting in more focused, high-resolution images in 2PA fluorescence imaging.

FIGURE 3

Overview of fluorophore categories and potential biomedical applications with 2PA NIR-II imaging.

FIGURE 4

(Left) Illustration of AIEgen molecules in solution and aggregated state, and their corresponding simplified Jablonski diagrams showing the effect of motion restriction on the radiative (k_r) and non-radiative (k_nr) relaxation pathways after 1PE or 2PE. (Right) Example of in vivo application of 2P microscopy with NIR-II-responsive AIEDots used to reconstruct images of a mouse brain. The 3D reconstruction of mouse brain vasculature is reprinted with permission from ref. (Qi et al., 2018). Copyright 2018 American Chemical Society.

FIGURE 5

(A) 2PA spectrum of Mn²⁺-doped ZnS QDs in the range from 1050 to 1300 nm reported by Subha et al. (B) Comparison of σ₂ values in Mn²⁺-doped ZnS QDs (curve g) with other standard chromophores (curves a–f) and fluorescent proteins (curves h–m): (a) Rhodamine B, (b) Fluorescein, (c) Coumarin 307, (d) Cascade blue, (e) Dansyl and (f) Lucifer Yellow), and (h) tdTomato, (i) mBanana (j) mRFP (k) mCherry (l) mStrawberry (m) mTangerine). Reprinted with permission from ref. (Subha et al., 2013). Copyright 2013 American Chemical Society.

FIGURE 6

1PA and 2PA spectra of fluorescent proteins of the “fruit series”. Left axis shows the 2PA cross-section σ₂ ^(λ) per mature chromophore, right axis shows corresponding σ₂ ^(λ)Φ_f values. Adapted with permission from ref. (Drobizhev et al., 2009). Copyright 2009 American Chemical Society.

FIGURE 7

The approximate 5-years costs of traditional tunable 2PA lasers (green) and alternative fixed wavelengths lasers (yellow), demonstrating the economical benefits of fixed wavelength fibre lasers. Reproduced from the supplementary material of ref (Mohr et al., 2020) with permission from the authors.

FIGURE 8

2PA images of brain blood vessels in a mouse injected with CP dots. (a,b) 3D reconstructed second-harmonic generation images of mouse skull. (c–h) 2PA images at various vertical depths (0–400 μm) at 1200 nm excitation, 660–750 nm emission. (i–l) 3D reconstructed 2PA images of brain blood vessels. (m) comparison of 2PA images at excitation wavelengths of 800, 1040, and 1200 nm, of brain vasculature in a mouse injected with polymer dots. 2PA line intensity profiles with the different wavelengths across the blood vessels on the right were also acquired for the corresponding depths. Emission at 660–750 nm. Reprinted from ref (Wang et al., 2019b). with permission from John Wiley and Sons.

FIGURE 9

Plot of the 2P brightness and corresponding NIR-II wavelengths of the contrast agents presented in this review (σ₂Φ_f > 24 GM). The 2P contrast agents used in vivo are represented with a framed number. For clarity, a linear scale for brightnesses between 0 and 100 GM, and a logarithmic scale above 100 GM. The typical range of wavelengths covered by common SWIR lasers is shown for reference (power attenuation is represented by the fading colour; note that OPOs and OPAs can extend past 2000 nm).