Xanthene-Based Dyes for Photoacoustic Imaging and Their Use as Analyte-Responsive Probes

Abstract

Developing imaging tools that can report on the presence of disease-relevant analytes in multicellular organisms can provide insight into fundamental disease mechanisms as well as provide diagnostic tools for the clinic. Photoacoustic imaging (PAI) is a light-in, sound-out imaging technique that allows for high resolution, deep-tissue imaging with applications in pre-clinical and point-of-care settings. The continued development of near-infrared (NIR) absorbing small-molecule dyes promises to improve the capabilities of this emerging imaging modality. For example, new dye scaffolds bearing chemoselective functionalities are enabling the detection and quantification of disease-relevant analytes through activity-based sensing (ABS) approaches. Recently described strategies to engineer NIR absorbing xanthenes have enabled development of analyte-responsive PAI probes using this classic dye scaffold. Herein, we present current strategies for red-shifting the spectral properties of xanthenes via bridging heteroatom or auxochrome modifications. Additionally, we explore how these strategies, coupled with chemoselective spiroring-opening approaches, have been employed to create ABS probes for in vivo detection of hypochlorous acid, nitric oxide, copper (II), human NAD(P)H: quinone oxidoreductase isozyme 1, and carbon monoxide. Given the versatility of the xanthene scaffold, we anticipate continued growth and development of analyte-responsive PAI imaging probes based on this dye class.

Graphical Abstract

Developing near-infrared absorbing dyes tailored for photoacoustic imaging (PAI) applications will advance this emerging non-invasive deep-tissue imaging approach. Herein, we present strategies for tuning the classical xanthene scaffold for uses in PAI, and how activity-based sensing strategies can be applied to design xanthene-based, analyte-responsive probes to illuminate changes in disease biochemistry.

1. Introduction

Photoacoustic Imaging (PAI), also referred to as optoacoustic imaging, holds great promise for deep-tissue biomedical imaging applications. PAI functions as a light-in, sound-out technique, generating contrast through the optical excitation of chromophores, whether endogenous or exogenous.^{[1, 2]} The resulting detectable ultrasound signal, a manifestation of the photoacoustic effect first described by Alexander Graham Bell in 1880,^[3] is generated by pulsed laser irradiation to induce cycles of excitation and nonradiative relaxation (Figure 1). Ultrasound transducers detect the pressure waves arising from thermoelastic expansion caused by localized, nonradiative relaxation-induced heating. This output affords imaging depths in the centimeter range, a substantial improvement compared to the limited depths of approximately 1 millimeter in purely optical imaging techniques.^{[1, 4]} This enhanced depth capability is attributed to the markedly reduced scattering of sound (approximately 1000-fold less) in biological tissues compared to light. PAI effectively combines the high contrast advantages of optical techniques like fluorescence imaging with the deep-tissue penetration capabilities of ultrasound, serving as a bridge between macroscopic and microscopic imaging approaches.

Figure 1.

A Jablonski diagram showing the electronic state transitions leading to fluorescence, phosphorescence, and photoacoustic emission.

Label-free imaging utilizing endogenous contrast agents such as hemoglobin, deoxyhemoglobin, and melanin has been at the frontier of clinical applications of PAI. Due to the high concentration of these endogenous chromophores, imaging of the vasculature is feasible.^[5] Using the differences in absorbance profiles of oxygenated and deoxygenated hemoglobin, it is possible to glean information regarding tumor size and location.^{[6, 7]} PAI applications extend beyond cancer detection, encompassing fields such as neuroscience,^{[8, 9]} cardiovascular imaging,^{[5, 10, 11]} and functional imaging of organs,^{[12, 13]} further highlighting its emerging role in biomedical research and clinical diagnostics.

While label-free imaging using endogenous chromophores is convenient and minimally invasive, the application of exogenous contrast agents offers additional benefits such as the ability to design analyte-responsive probes for molecular imaging applications.^[14] Although polymeric-organic^[15] and inorganic-based nanoparticles^{[16, 17]} have found utility in PAI, small molecule contrast agents are attractive as PA agents due to their high chemical tunability, excellent biocompatibility, and distinct absorbance profiles that facilitate spectral unmixing.^{[14, 18–20]} These properties enable the design of analyte-responsive agents using activity-based sensing (ABS) principles.^[21–23] In short, ABS relies on the uncaging of a dye through a chemoselective reaction under biologically relevant conditions.^[22] The resulting analyte-responsive probes provide unique insights into the biochemistry of cells and tissues.

To achieve deep-tissue penetration in multicellular organisms, commercially available PA instrumentation typically utilizes wavelengths ranging from 660–1300 nm. Thus, due to the need for near-infrared (NIR) absorbance only a limited array of dye scaffolds such as cyanines,^[24] squarines,^[25] BODIPYs,^{[14, 26–28]} and hemicyanines^[29–32] have been used for PAI. The tail end of the absorbance peak of known azo dyes such as methylene blue and Evans blue can also be visualized using PAI, although these dyes are suboptimal since their absorbance maxima is blue-shifted relative to the window of commercial PAI instrumentation.^[33] BODIPY and hemicyanine dyes bear readily modifiable chemical handles (-OH, -NH₂) which can be decorated with functional groups for ABS. For example, these dyes scaffolds have been utilized to develop ABS probes for glutathione (GSH),^[31] nitric oxide (NO),^{[14, 24, 27, 34–38]} alkaline phosphatase (ALP),^[29] monoacylglycerol lipase (MGL),^[32] and fatty acid amide hydrolase (FAAH).^[32] While these probes enable molecular imaging, the lack of dye scaffold diversity has limited the ability to expand the repertoire of analyte-responsive PA probes. In this Concept, we highlight recent advances in the development of NIR xanthenes and their use as analyte-responsive probes in PAI. These new xanthene-based scaffolds broaden the range of analytes that can be detected using PAI, further expanding the utility of this modality for fundamental research and clinical applications.

2. Tuning Xanthene dyes for PAI

Xanthene dyes, namely fluorescein, rhodol, and rhodamine are among the earliest examples of fluorescent dyes dating back to the 1870s and have found numerous applications in imaging.^{[39, 40]} Canonically, xanthene dyes absorb and emit in the visible range with fluorescein being more blue-shifted (Tokyo Green, λ_abs = 491 nm, λ_em = 510 nm) and rhodamines being more red-shifted (Tetramethyl rhodamine, λ_abs = 549 nm, λ_em = 565 nm, Figure 2a, 2b).^[40] A key structural feature of xanthene dyes is the ability to engineer an absorbance off/on transition by placing a nucleophilic group at the C2 position of the pendant ring that can attack the C’9 position of the dye. The resulting equilibrium between an open (absorbance on) and closed (absorbance off) form leads to blinking properties which can be leveraged for super resolution microscopy (Figure 2c).^{[23, 41–50]} Additionally, leveraging chemoselective functional groups at the C2 position affords analyte-responsive rhodamines that can provide cellular readouts of reactive oxygen species (ROS),^{[51, 52]} reactive nitrogen species (RNS),^[52] and metal ions.^[53] While clearly powerful, the use of xanthenes as imaging probes has largely been limited to fluorescence applications involving shallow imaging depths (e.g. cell culture) due to the lack of NIR absorbance in this dye class.

Figure 2.

a) General structures of the classical xanthene dyes such as fluorescein, rhodol, and rhodamine. b) Table summarizing the photophysical properties for a group of heteroatom-bridged xanthene dyes. c) A schematic highlighting the transition between a ring closed and ring open state for rhodamine.

2.1. Heteroatom Substitution – Beyond the Visible

More recently, several NIR xanthene derivatives have been described. Heteroatom substitution at the bridging (C’10-position, Figure 2a) has emerged as a powerful approach to achieve absorbance in the NIR within this dye class. The approach leverages observations from the Dewar-Knott color rule^[54] and computational chemistry, which predict that replacement of the bridging xanthene oxygen atom with less electron rich groups such as boron,^{[55, 56]} dimethylsilicon (SiMe₂),^[57] germanium,^[58] tin,^[58] phosphinate,^[59–62] phosphine oxide,^[63] sulfone,^[64] or ketone (C=O)^{[65, 66]} functionalities would significantly decrease the HOMO-LUMO energy gap of the resulting dye. Indeed, heteroatom substitution was found to yield xanthene derivatives with absorbance ranging from 650 nm (Si-rhodamines) to 850 nm (Ketone rhodamines).

SiMe₂ substitution yields dyes that display excellent fluorescence brightness and are on the blue edge of the NIR range (λ_abs ≈ 650 nm). Analogs of these dyes bearing azetidine auxochromes have been extensively investigated as probes for confocal and super resolution microscopy. ^{[42, 44, 67–69]}

While Si-rhodamines are excellent fluorophores they lack significant PA output. Conversely, more red-shifted rhodamines such the C=O bridged (Ke) rhodamines, KR-1 (Figure 3a), introduced by Ivanic and Schnermann,^[65] have low quantum yield with a maximal absorbance beyond 850 nm, indicating their potential for PAI. Work from Zhou and Miller^[66] demonstrated the ability of Ke-rhodamines to produce robust PA signal ( Figure 3a–c).^[66] Utilization of julolidine auxochromes improved the chemical stability of this dye scaffold by providing protection from nucleophilic attack at the C=O bridge. This improvement carried over to a complex media such as defibrinated sheep’s blood where KeJuR was compared to ICG and showed 10-fold higher signal after unmixing (Figure 3d). This study highlights how the introduction of a bridging atom modifications can open avenues for the development of diverse xanthene-based PA probes with applications in multicolor PA imaging experiments using multispectral optoacoustic tomography (MSOT), pairing well with existing NIR contrast agents like ICG.

Figure 3.

a) Structures of Ketone bridged rhodamines KR-1 and KeJuR. b) Plot of relative PA intensity for 50 μM of the indicated dye in DPBS (pH 7.2, 1% DMSO). c) Plot of PA signal (ex: 860 nm) vs. KeJuR concentration. d) 3D rendering of a tissue phantom containing KeJuR (magenta) or ICG (green) dissolved in defibrinated sheep blood, with x-y and x-z visualizations. Adapted with permission from ref ^[66]. Copyright 2022, Royal Society of Chemistry.

2.2. Auxochrome Modified Xanthenes – Beyond NIR-I

While heteroatom substitution alone can now yield xanthene dyes with absorbance above 800 nm, additional strategies are required to push farther into the shortwave infrared (SWIR, 1000–1700 nm) region. Recent endeavors have explored the utilization of dyes tuned to the SWIR region in an effort to maximize tissue penetration and signal-to-noise ratio for deep-tissue imaging. In other dye classes, such as donor-acceptor-donor (D-A-D) dyes (CH1055, Figure 4a),^{[71, 72]} extending the pi-conjugation or adjusting the donor-acceptor strength of terminal auxochromes, ^[73–75] can be independently or synergistically employed to achieve significant bathochromic shifts. Building on this approach, Delcamp, Scott and colleagues^[76–78] employed dimethyl aniline modified indolizine heterocycles at the C’3- and C’6- positions to yield xanthene-based dyes that absorb at >900 nm and emit in the SWIR window (7-DMA-Ri, 1092 nm, 1256 nm, Figure 4a). Liu and colleagues^[79] demonstrated how julolidine styryl auxochromes could shift both absorbance and emission to the SWIR range (VIX-4, λ_abs = 1028 nm, λ_em = 1210 nm, Figure 4a). Applying this strategy for PAI, Scott and colleagues^[70] used Pd-catalyzed direct C-H arylation to install thienylpiperidine donors onto the xanthene core at the C’3- and C’6-positions to give XanthCR-880 (Figure 4a). The thiophene donor, reinforced by the terminal piperidines, enhanced the push-pull character of the dye and amplified intramolecular charge transfer. Furthermore, cation delocalization occurred through an extended pi-system compared to traditional xanthenes resulting in a 300 nm bathochromic shift compared to rhodamine B. The observed weak fluorescence and broad full-width at half maximum absorbance indicated significant vibronic relaxation. Additionally, XanthCR-880 exhibited commendable photo- and chemical- stability, along with a detectable PA signal at a depth of 4 cm through swine tissue, (Figure 4b) underscoring its potential for deep-tissue imaging.

Figure 4.

a) Structures of D-A-D dye CH1055 and SWIR absorbing xanthenes with auxochrome modifications. b) Integrated photoacoustic signal of XanthCR-880 (20 μM) in PBS with 0.1% SDS overlaid with swine tissue with a thickness of 1, 2, 3, or 4 cm. Adapted with permission from ref ^[70]. Copyright 2021, American Chemical Society.

3. Xanthenes in Molecular PAI

Analyte-responsive probes that target disease-relevant analytes are highly valuable tools for fundamental science as well as clinical applications. In the context of small molecule dyes, this involves incorporation of chemoselective functional groups, which upon reaction leads to an uncaging of the chromophore to provide a change in signal.^{[22, 23]} Although these ABS approaches for PAI have been more commonly applied using BODIPY and hemicyanine scaffolds, xanthenes show significant promise when tuned for PA applications, as evidenced by their widespread application in similar contexts in fluorescence imaging using spiroring opening (Figure 2b).^{[23, 40, 80, 81]}

3.1. Detection of Hypochlorous Acid

Hypochlorous acid (HOCl) is a ROS produced by the enzyme myeloperoxidase (MPO).^{[83, 84]} HOCl is among the strongest endogenous oxidizing agents and used by macrophage cells to kill pathogens, and as such HOCl is strongly correlated with inflammation.^[85] In addition, MPO has been found to be overexpressed specifically in acute myeloid leukemia (AML),^[84] and HOCl levels are significantly increased compared to other cell or cancer types with levels reported at 2–15 μM.^[83] The chemical reactivity of HOCl makes it an ideal target for detection via spiroring opening using the xanthene scaffold. One approach to accomplish this goal employs a mercaptomethyl group at the 2-position of the pendant phenyl ring, to create a spirocyclic thioether.^{[51, 86]} In the presence of HOCl, the sulfur is oxidized, and the C’9-S bond is broken, reestablishing conjugation, and leading to production of a species capable of absorbing NIR light (Figure 5a). In 2019, Urano and colleagues^[82] detailed a Si-rhodamine, 2-Me wsSiNQ660, bearing an aromatic ring on each nitrogen auxochrome to improve PA signal (Figure 5b). The installation of the aromatic auxochrome quenched radiative decay through twisted intramolecular charge transfer (TICT)^[87] leading to a 4.5-fold increase in PA intensity compared to the parent dye. 2-Me wsSiNQ660 was functionalized with the spirocyclic thioether motif to give PA-MMSiNQ (Figure 5a), which showed good selectivity for HOCl in vitro and allowed for detection of subcutaneously injected HOCl in a mouse (Figure 5c). While this represented an elegant application of the spiroring opening to detect HOCl, the absorbance maximum of PA-MMSiNQ (λ_abs = 660 nm) is not well suited for commercial instrumentation (660–1300 nm).

Figure 5.

a) General scheme for the reaction of HOCl with a spirocyclic thioether functionalized rhodamines (PA-MMSiNQ and SNR₇₀₀-HOCl) to produce PA active chromophores. b) Structures of 2-Me SiR650 and 2-Me wsSiNQ660. c) Photoacoustic imaging of subcutaneously injected HOCl in mice using PA-MMSiNQ. d) Structures of NR₇₀₀ and SNR₇₀₀ e-g) Spectrally unmixed PA images (Hb = blue, HbO₂ = red, and SNR₇₀₀-HOCl = green) were acquired from nude mice following subcutaneous injection on the right flank. Representative cross-sectional images (top) are shown from animals injected with SNR₇₀₀-HOCl (e), SNR₇₀₀- HOCl immediately followed by HOCl (f), or saline (g). Arrows indicate the site of injection. h) Quantified PA signal after multispectral unmixing within regions of interest from three subsequent cross-sectional images, spaced 1 mm apart, in each mouse from panel e (n=3 mice), f (n=4 mice), and g (n=3 mice). *** indicates a p-value of <0.001.The color scheme used in the PA images is as follows: blue represents hemoglobin, red represents oxyhemoglobin, and green represents activated SNR₇₀₀-HOCl. Adapted with permission from refs ^[82] and ^[62]. Copyright 2019, American Chemical Society, and copyright 2023, John Wiley and Sons, respectively.

Recent work from our lab^[62] investigated the ability of phosphinate containing-xanthenes (termed Nebraska Red or NR dyes) to produce PA signal, and we found that this simple modification leads to dyes well-suited for PAI with analogues absorbing >700 nm. The phosphinate ethyl ester-containing rhodamine, NR₇₀₀, (Figure 5d) was identified as our lead dye with the highest PA signal. Due to hydrolysis of the phosphinate ethyl ester group of NR₇₀₀ in water, we employed a single-atom substitution of P=O to P=S, inspired by ATP-γS,^{[88, 89]} to yield a thiophosphinate analogue, SNR₇₀₀ (Figure 5d). SNR₇₀₀ was further functionalized with the spirocyclic thioether to yield SNR₇₀₀-HOCl (Figure 5a). SNR₇₀₀ and SNR₇₀₀-HOCl displayed a 3.6-fold longer half-life of hydrolysis compared to NR₇₀₀ making them hydrolytically stable over the timescale needed for PAI while retaining similar photophysical properties to NR₇₀₀. SNR₇₀₀-HOCl displayed excellent sensitivity and selectivity for HOCl while providing a 2.2-fold stronger PA signal than PA-MMSiNQ. This allowed for imaging through 2.9 cm of tissue, and detection of exogenous HOCl in live mice using PAI (Figure 5e–h). SNR₇₀₀-HOCl carries the additional benefit of being cell permeable, allowing for the potential detection of intracellular HOCl in AML.

3.2. Detection of Nitric Oxide

Nitric oxide (NO) has important roles in control of vasodilation,^[90] neurotransmission,^[91] inflammation,^[92] and cancer.^[93–95] NO is an interesting biological target since it displays concentration-dependent effects on cancer progression. At elevated levels of NO synthase expression, high concentrations of NO exhibit protective effects against cancer progression.

Conversely, once the presence of tumor-associated macrophages is established, low steady-state NO levels emerge which correlates with poorer prognosis.^{[36, 96, 97]} The nuanced involvement of NO in such critical biological phenomena underscores its significance and the complexity of its regulatory functions in cancer biology.

Following the initial report of thienylpiperdine modified xanthenes,^[70] Scott and colleagues employed both auxochrome and pendant ring modifications to design a ratiometric NO nanosensor with absorbance in the NIR-I/SWIR region.^[38] Employing computational modeling and rational design they enhanced the pi-conjugation, increased donor strength, and bolstered the rigidity of the donor moiety using dibenzazepine donors and thiophene (SCR-1), thienothiophene (SCR-2), or bithiophene (SCR-3) linkers for an extended pi-system (Figure 6a).

Figure 6.

a) Structures of dyes SCR-1, SCR-2, SCR-3, and SWIR reference dye IR-1061. b) Reaction scheme of PA active NO sensor SCR-NO with NO to give a PA-inactive product, yielding a “turn-off” response. Representative cross-sectional SWIR PA images of the liver from mice treated with (c) saline or (d) acetaminophen (APAP) to induce DILI. Signals from IR-1061 (reference signal) and SCR-NO (probe signal) are shown in green and blue, respectively. Adapted with permission from ref ^[38]. Copyright 2023, John Wiley and Sons.

To capitalize on the SWIR properties of these dyes, the carboxylate on the pendant ring was modified with an NO-responsive o-phenylenediamine trigger to give SCR-NO which primarily exists in the closed state upon reaction with NO (Figure 6b). The authors cleverly crafted a multidye system using IR-1061 as a SWIR reference dye, that was co-encapsulated with SCR-NO in DSPE-PEG to make the ratiometric nanosensor, rNP-NO. The spectral separation of SCR-NO and IR-1061 allowed for ratiometric detection of NO measured using the ratio of PA signal from SCR-NO divided by that of IR-1061. The authors used rNP-NO to study a drug-induced liver injury (DILI) model. While the reference dye signal was visible in the liver of both the control and the DILI model, SCR-NO signal was only detectable in the control mice indicating NO synthase activity is upregulated in DILI (Figure 6c,d).

4. Hemi-Xanthenes

While classical xanthene dyes are characterized by the heteroatom bridged anthracene structure with a push-pull system distribution between the C’3 and C’6 positions, recent efforts have resulted in a new type of asymmetric xanthene-based dye with a push-pull system connected by C’4 and C’6 that we refer to here as hemi-xanthenes (hemi-rhodols or hemi-rhodamines, Figure 7a). This alteration effectively elongates the pi-system leading to red-shifted dyes. The exploration of various donor motifs has resulted in the design of NIR-absorbing/emitting, and SWIR-absorbing hemi-xanthenes.^[98–101] These dyes not only preserve the biocompatibility of xanthenes, but also the spiroring opening feature, enabling design of analyte-responsive PA probes.

Figure 7.

a) General structures of hemixanthene dyes and examples of C’4 terminal auxochromes. b) Structures of Rhodol-PA and turnover product Rhodol-NIR. c) PA imaging upon tail vein injection of Rhodol-PA. Representative PA images of HT-29 and MDA-MB-231 tumor-bearing mice at different time points. Dotted lines indicated the tumor area. Scale bars = 2 mm. (d) Time-dependent PA intensity variations of HT-29 and MDA-MB-231 tumor-bearing mice. (e) In vivo PA spectra from both HT-29 and MDA-MB-231 tumor-bearing mice 5 h post injection. Adapted with permission from ref ^[100]. Copyright 2019, Royal Society of Chemistry.

4.1. Detection of Human NAD(P)H: quinone oxidoreductase isozyme 1 (hNQO1)

Human NAD(P)H: quinone oxidoreductase isozyme 1 (hNQO1) is a well-recognized cancer biomarker, exhibiting overexpression in various cancer types, including non-small cell lung cancer, colorectal, liver, and breast cancer.^[102] This enzyme reduces quinones, quinone epoxides, and aromatic nitro compounds through the utilization of NADH or NADPH as a cofactor and is ubiquitously expressed in cells. Cancer-associated hNQO1 levels are significantly elevated (2- to 50-fold higher) compared to healthy tissue^[102] and the strategic installation of an hNQO1-responsive motif can enable selective activation of a probe in cancer cells.

Jiang and collaborators^{[100, 103–105]} developed a NIR absorbing hemi-rhodol dye termed Rhodol-NIR (Figure 7a) by introducing a 2,6-difluorophenol auxochrome at the C’4 position, resulting in broad absorbance peaking at 630 nm. The authors observed that the deprotonated phenol state of the dye assumed the zwitterionic open state, while acetylation of the phenolic hydroxyl favored the spirocyclic closed form. Capitalizing on this insight, the researchers capped the phenolic hydroxyl with the hNQO1 substrate Q3, creating Rhodol-PA (Figure 7b). In response to hNQO1 activity, Rhodol-PA undergoes ring opening, resulting in a remarkable 110-fold increase signal at 680 nm (tail of dye absorbance). The authors were able to leverage this tail signal to detect hNQO1 at 680 nm in HT-29 cells and in vivo using PAI and NIR fluorescence microscopy. The specificity for hNQO1 was affirmed by comparing the contrast in hNQO1-positive HT-29 tumors versus hNQO1-negative MDA-MB-231 tumors (Figure 7c–e).

4.2. Detection of Cu²⁺

Dysregulation of copper (I) and (II) (Cu¹⁺, Cu²⁺) levels are linked to Alzheimer’s^[103–105] and Wilson’s disease.^{[106, 107]} Elevated Cu²⁺ in Alzheimer’s is believed to promote protein aggregation, contributing to plaque and tangle formation, while in Wilson’s disease, Cu¹⁺ accumulation can result in chronic liver damage. Consequently, Cu is a valuable molecular target for imaging. However, traditional quantification methods like ICP-MS lack spatiotemporal resolution and are highly invasive, preventing real-time monitoring of Cu levels in the native environment.

To address this need, Chen and colleagues^[99] devised a Cu²⁺-responsive hemi-rhodamine derivative of NRh1 (Figure 7a). Following established strategies, a Cu-responsive spirolactam gate was incorporated at the C’9 position to selectively open in response to Cu²⁺, giving NRh (Figure 8b).

Figure 8.

a) Structures of the Cu²⁺-responsive probe NRh, PA active product NRh1, and reference dye IR820. b) Representative PAI at 716 nm (NRh1) and 834 nm (IR820) after subcutaneous injection of nanoprobe NRh-IR-NM with Cu²⁺ along with ultrasound (US) imaging background with 2 mm pork tissue overlay. Adapted with permission from ref ^[99]. Copyright 2019, American Chemical Society.

NRh was coencapsulated with a nonresponsive reference dye, IR820, in a nanoparticle, giving the nanoprobe NRh-IR-NM. With the nanoprobe at hand, the authors selectively targeted Cu²⁺ in both in vitro and in vivo settings, noting minimal alterations in the reference dye signal in response to Cu²⁺. Utilizing the ratio of PA_NRh1/PA_IR820, a negative control, saline injection, and mice injected with exogenous Cu²⁺ could be distinguished, even when imaging through an additional 2 mm of pork tissue (Figure 8b).

4.3. Detection of carbon monoxide

Carbon monoxide (CO) participates in various physiological processes, including anti-inflammatory responses, neurotransmission, vascular smooth muscle function, and vasodilation.^{[108, 109]} Deviations from normal CO levels can lead to serious conditions such as respiratory diseases, Alzheimer’s disease, and hypertension, making CO an interesting analyte to target with deep-tissue imaging approaches. Yoon, Lin, and colleagues^[98] designed hemi-xanthene dyes with wavelengths extending into the SWIR through computational and experimental analysis of various xanthene donors. They found that increasing the donor strength of xanthene auxochromes through terminal heterocyclic amines led to increased intramolecular charge transfer and substantial bathochromic shifts with absorption reaching beyond 900 nm. The lead dye, designated GX-5, (Figure 7a) allowed for excellent imaging of mouse vasculature by PAI. Subsequently, the authors extended their focus beyond untargeted vasculature imaging. Employing the spiroring opening strategy, they engineered GX-5-CO to selectively target CO (Figure 9a). After in vitro experiments confirmed appropriate selectivity and sensitivity to CO, the authors sought to study induced hypertension in mice using angiotensin II (Figure 9b). Satisfyingly, GX-5-CO provided a clear turn-on PA signal allowing for detection in vasculature of hypertensive mice, whereas control and amlodipine, valsartan, and hydrochlorothiazide treated mice (treatment, Figure 9d) exhibited minimal GX-5-CO signal in the vasculature. (Figure 9c and d) Thus, GX-5-CO serves as an additional illustration of the diverse ways in which the xanthene scaffold can be tailored to target a range of biological analytes using PA signal as a readout.

Figure 9.

a) General scheme of GX-5-CO reaction with CO to give the PA active dye GX-5. b) Protocol of inducing and treating hypertension (DBP: Diastolic blood pressure, SBP: systolic blood pressure). c) PA intensity at different time points post tail vein injection comparing control (yellow), treatment with amlodipine, valsartan, and hydrochlorothiazide to decrease BP (green), and hypertensive mice (pink). d) Time-dependent NIR-II PA imaging comparing control, treatment, and hypertensive mice. Adapted with permission from ref ^[98]. Copyright 2023, John Wiley and Sons.

5. Summary and Outlook

In this Concept, we present approaches to fine-tune xanthene dyes for effective use in PAI in vivo. We focused on how different bridging heteroatom substitutions and careful auxochrome design can 1) decrease the HOMO-LUMO energy gap through electron withdrawing C’10 bridging motifs, 2) extend the pi-conjugated system, 3) improve intramolecular charge transfer, using a D-A-D-like design, and 4) increase vibronic coupling leading to more nonradiative relaxation. Additionally, we have underscored the adaptability of the xanthene scaffold for development of analyte-responsive PA probes, showcasing a diverse array of detectable analytes facilitated by the C’9 spiroring opening strategy. Furthermore, the positional shift of the push-pull system from the symmetric C’3/C’6 to the asymmetric C’4/C’6 combination yielding hemi-xanthenes, and their unique applications were described.

PAI is an emerging field, and we are confident that ongoing advancements will enable minimally invasive monitoring of disease, capitalizing on the deep-tissue penetration capabilities offered by PAI. Coupling ABS approaches with new dye scaffolds, ^[110–113] more sensitive instrumentation, and better image processing and unmixing will allow for more accurate determination of disease-relevant analytes and enzymatic activities, providing increased insight into the individualized biochemistry of diseased tissue. Chemists can contribute to this goal through the development of new and improved ABS approaches to enhance and expand the repertoire of detectable analytes and enzyme activities. Additionally, rational approaches to increase PA signal from small molecule dyes are needed as current probe design generally relies on repurposed fluorescent dyes.^{[114, 115]} A combination of advances in these two areas will ultimately move us towards analyte-responsive PA probes that could be used to inform precision medicine efforts to individually tailor treatments to the unique biochemistry of a patient. We envision that xanthene dyes will play a pivotal role in this pursuit.

Biographies

Frederik Brøndsted received in BS in biochemistry from West Virginia Wesleyan College in 2019. He is currently a Ph.D. candidate at the University of Virginia. His research focuses on the rational development of photoacoustic small-molecule probes.

Cliff I. Stains obtained his PhD degree with Indraneel Ghosh (University of Arizona) followed by postdoctoral studies with Barbara Imperiali (MIT). He is currently an Associate Professor of Chemistry at the University of Virginia. His research program focuses on developing new probes for disease processes as well as targeted therapeutics.