Acoustogenic Probes: A New Frontier in Photoacoustic Imaging

Abstract

Photoacoustic imaging (PAI) is a powerful imaging modality capable of mapping the absorption of light in biological tissue via the PA effect. When chromophores are optically excited, subsequent energy loss in the form of heat generates local thermoelastic expansion. Repeated excitation from a pulsed laser induces pressure fluctuations that propagate through tissue and can be detected as ultrasound waves. By combining ultrasonic detection with optical excitation, PAI enables high-resolution image acquisition at centimeter depths. PAI is also relatively inexpensive and relies on safe, nonionizing excitation light in the near-infrared window, making it an attractive alternative to other common biomedical imaging modalities. Research in our group is aimed at developing small-molecule activatable probes that can be used for analyte detection in deep tissue via PAI. These probes contain reactive triggers that undergo a selective chemical reaction in the presence of specific stimuli to produce a spectral change that can be observed via PAI. Chemically tuning the absorbance profile of the probe and the reacted product such that they are both within the PA imaging window enables ratiometric imaging when each species is irradiated at a specific wavelength. Ratiometric imaging is an important design feature of these probes as it minimizes error associated with tissue-dependent signal fluctuations and instrumental variation. In this Account, we discuss key properties for designing small-molecule PA probes that can be applied for in vivo studies and the challenges associated with this area of probe development. We also highlight examples from our group including probes capable of detecting metal ions (Cu(II)), reactive nitrogen species (NO), and oxygen tension (hypoxia). Each of these targets can be sensed using a modular design strategy based on influencing the electronic and spectral properties of a NIR-absorbing dye platform. We demonstrate that ideal small-molecule PA probes have high molar absorptivity, low fluorescence quantum yields, and selective triggers that can reliably report on a single analyte in a complex biological setting. Probes must also be highly chemo- and photostable to enable long-term imaging studies. We show that these PA probes react rapidly and selectively and can be utilized for deep-tissue imaging in mouse models of various diseases. Overall, these examples represent a new class of biomedical imaging tools that seek to enable high-resolution molecular imaging capable of improving diagnostic methods and elucidating new biological discoveries. We anticipate that the combination of small-molecule PA probes with new PAI technology will enable noninvasive detection of analytes relevant to disease progression and mapping of tissue microenvironments.

Figure 1.

Schematic illustration of ratiometric imaging with acoustogenic probes. Probe activation results in a spectral change, and PAI is performed at wavelengths corresponding to both the unactivated and activated probe forms. A high ratiometric PA signal corresponds to a large proportion of activated probe, indicating target detection.

Figure 2.

a) Chemical structure of APC probes and the products formed after Cu(II) treatment. b) Normalized absorbance of APC-1 and its hydrolyzed product (1). c) Normalized ratiometric PA fold turn-on of APC-2 upon treatment with Cu(II). d) Normalized ratiometric PA fold turn-on of APC-2 upon treatment with various metal ions. e) PA images of solutions of APC-2 in fluorinated ethylene propylene (FEP) tubes in a tissue-mimicking phantom with or without the addition of Cu(II).

Figure 3.

a) Chemical structures and sensing mechanisms of the APNO and tAPNO series. b) Normalized absorbance spectra and c) PA spectra of APNO-5 and tAPNO-5. d) PA images of APNO-5 and tAPNO-5. e) Ratiometric PA signal and f) representative images of APNO-5 in a murine model of inflammation.

Figure 4.

a) Chemical structure of photoNODs and rNODs. b) Normalized absorbance spectra of photoNOD-1 and rNOD-1 in CHCl₃. c) Time-dependent ratiometric PA signal of photoNOD-1 produced with or without irradiation. d) EPR spectra of photoNOD-1 collected after 0, 5 and 40 minutes of irradiation in the presence of Fe(MGD)₂. e) PA images of photoNOD-2 with or without 5 min of irradiation. f) PA images of photoNOD-2 after subcutaneous injection and a 5 min period with/without irradiation. g) Tumor volumes following repeated systemic administration of photoNOD-1 or vehicle with/without irradiation.

Figure 5.

a) Chemical structure of HyP-1 and red-HyP-1. b) PA image HyP-1 treated with RLMs under normoxic or hypoxic conditions. c) Concentration-dependent PA signal of red-HyP-1 in a tissue mimicking phantom. d) Normalized absorbance (solid) and emission (dashed) spectra of HyP-1 and red-HyP-1. e) PA spectra of HyP-1 and red-HyP-1. f) Normalized ratiometric fluorescence of 4T1 cells following treatment with HyP-1 in hypoxic (red, light grey) or normoxic (dark grey) conditions. g) 4T1 cells stained with HyP-1 and incubated under hypoxic conditions. Cy5 and Cy7 filters used for visualization of HyP-1 and red-HyP-1, respectively. h) Representative images and i) quantification of ratiometric fluorescence of HyP-1 following intratumoral and subcutaneous (control) injection. j) Representative images and k) quantification of PA signal of HyP-1 in a murine tumor model following systemic injection.

Figure 6.

a) Chemical structures of the rHyP series. b) Normalized absorbance and c) PA spectra of rHyP-1 and red-rHyP-1 in CHCl₃. d) PA images of rHyP-1 solutions treated with RLMs in normoxic and hypoxic conditions. e) Ratiometric PA signal in 2 mm slices of a PA reconstruction of a 4T1 tumor following administration of rHyP-1. f) Ratiometric PA fold turn-on of rHyP-1 in tumor-bearing and control flanks. g) Representative PA 3D reconstruction of a 4T1 tumor following administration of rHyP-1.