Oxygen Tomography by Čerenkov-Excited Phosphorescence during External Beam Irradiation

The efficacy of radiation therapy depends strongly on tumor oxygenation during irradiation. However, current techniques to measure this parameter in vivo do not facilitate routine monitoring in patients. Herein, we demonstrate a noninvasive method for tomographic imaging of oxygen partial pressure (pO2 ) in deep tissue using the phosphorescence decay of an oxygen-sensitive probe excited by Čerenkov radiation induced by external beam radiotherapy. Tissue-simulating scattering phantoms (60 mm diameter with a 20 mm anomaly) containing platinum(II)-G4 (PtG4), a dendritic porphyrin-based phosphor, whose phosphorescence is quenched in the presence of oxygen, were irradiated with a clinical linear accelerator. The emitted phosphorescence was measured at various positions on the phantom boundary using a spectrograph coupled to an intensified charge-coupled device (ICCD). At each position, PtG4 phosphorescence decay curves were measured by synchronizing the ICCD to the linear accelerator pulses. Tomographic images of phosphorescence yield and lifetime were recovered for phantoms with homogenous PtG4 concentrations and heterogeneous pO2 . Since PtG4 lifetime is strongly and predictably dependent on pO 2 through the Stern-Volmer relationship, tomographic images of pO 2 were also reported, and showed excellent agreement with independent oxygenation measurements. Translating this approach to the clinic could facilitate direct sensing of pO2 during radiotherapy

More Homogeneous Capillary Flow and Oxygenation in Deeper Cortical Layers Correlate with Increased Oxygen Extraction

Our understanding of how capillary blood flow and oxygen distribute across cortical layers to meet the local metabolic demand is incomplete. We addressed this question by using two-photon imaging of resting-state microvascular oxygen partial pressure (PO2) and flow in the whisker barrel cortex in awake mice. Our measurements in layers I-V show that the capillary red-blood-cell flux and oxygenation heterogeneity, and the intracapillary resistance to oxygen delivery, all decrease with depth, reaching a minimum around layer IV, while the depth-dependent oxygen extraction fraction is increased in layer IV, where oxygen demand is presumably the highest. Our findings suggest that more homogeneous distribution of the physiological observables relevant to oxygen transport to tissue is an important part of the microvascular network adaptation to local brain metabolism. These results will inform the biophysical models of layer-specific cerebral oxygen delivery and consumption and improve our understanding of the diseases that affect cerebral microcirculation

Tumor Blood Flow Differs between Mouse Strains: Consequences for Vasoresponse to Photodynamic Therapy

Fluctuations in tumor blood flow are common and attributed to factors such as vasomotion or local vascular structure, yet, because vessel structure and physiology are host-derived, animal strain of tumor propagation may further determine blood flow characteristics. In the present report, baseline and stress-altered tumor hemodynamics as a function of murine strain were studied using radiation-induced fibrosacomas (RIF) grown in C3H or nude mice. Fluctuations in tumor blood flow during one hour of baseline monitoring or during vascular stress induced by photodynamic therapy (PDT) were measured by diffuse correlation spectroscopy. Baseline monitoring revealed fluctuating tumor blood flow highly correlated with heart rate and with similar median periods (i.e., ∼9 and 14 min in C3H and nudes, respectively). However, tumor blood flow in C3H animals was more sensitive to physiologic or stress-induced perturbations. Specifically, PDT-induced vascular insults produced greater decreases in blood flow in the tumors of C3H versus nude mice; similarly, during baseline monitoring, fluctuations in blood flow were more regular and more prevalent within the tumors of C3H mice versus nude mice; finally, the vasoconstrictor L-NNA reduced tumor blood flow in C3H mice but did not affect tumor blood flow in nudes. Underlying differences in vascular structure, such as smaller tumor blood vessels in C3H versus nude animals, may contribute to strain-dependent variation in vascular function. These data thus identify clear effects of mouse strain on tumor hemodynamics with consequences to PDT and potentially other vascular-mediated therapies

Synthesis of Phosphorescent Asymmetrically π‑Extended Porphyrins for Two-Photon Applications

Significant effort has been directed in recent years toward porphyrins with enhanced two-photon absorption (2PA). However, the properties of their triplet states, which are central to many applications, have rarely been examined in parallel. Here we report the synthesis of asymmetrically π-extended platinum­(II) and palladium­(II) porphyrins, whose 2PA into single-photon-absorbing states is enhanced as a result of the broken center-of-inversion symmetry and whose triplet states can be monitored by room-temperature phosphorescence. 5,15-Diaryl-<i>syn</i>-dibenzoporphyrins (DBPs) and <i>syn</i>-dinaphthoporphyrins (DNPs) were synthesized by [2 + 2] condensation of the corresponding dipyrromethanes and subsequent oxidative aromatization. Butoxycarbonyl groups on the <i>meso</i>-aryl rings render these porphyrins well-soluble in a range of organic solvents, while 5,15-<i>meso</i>-aryl substitution causes minimal nonplanar distortion of the macrocycle, ensuring high triplet emissivity. A <i>syn</i>-DBP bearing four alkoxycarbonyl groups in the benzo rings and possessing a large static dipole moment was also synthesized. Photophysical properties (2PA brightness and phosphorescence quantum yields and lifetimes) of the new porphyrins were measured, and their ground-state structures were determined by DFT calculations and/or X-ray analysis. The developed synthetic methods should facilitate the construction of π-extended porphyrins for applications requiring high two-photon triplet action cross sections

Synthesis and Calibration of Phosphorescent Nanoprobes for Oxygen Imaging in Biological Systems

Oxygen measurement by phosphorescence quenching [1, 2] consists of the following steps: 1) the probe is delivered into the medium of interest (e.g. blood or interstitial fluid); 2) the object is illuminated with light of appropriate wavelength in order to excite the probe into its triplet state; 3) the emitted phosphorescence is collected, and its time course is analyzed to yield the phosphorescence lifetime, which is converted into the oxygen concentration (or partial pressure, pO2). The probe must not interact with the biological environment and in some cases to be 4) excreted from the medium upon the measurement completion. Each of these steps imposes requirements on the molecular design of the phosphorescent probes, which constitute the only invasive component of the measurement protocol. Here we review the design of dendritic phosphorescent nanosensors for oxygen measurements in biological systems. The probes consist of Pt or Pd porphyrin-based polyarylglycine (AG) dendrimers, modified peripherally with polyethylene glycol (PEG's) residues. For effective two-photon excitation, termini of the dendrimers may be modified with two-photon antenna chromophores, which capture the excitation energy and channel it to the triplet cores of the probes via intramolecular FRET (Förster Resonance Energy Transfer). We describe the key photophysical properties of the probes and present detailed calibration protocols

Two-photon phosphorescence lifetime microscopy of retinal capillary plexus oxygenation in mice

Impaired oxygen delivery and/or consumption in the retinal tissue underlies the pathophysiology of many retinal diseases. However, the essential tools for measuring oxygen concentration in retinal capillaries and studying oxygen transport to retinal tissue are still lacking. We show that two-photon phosphorescence lifetime microscopy can be used to map absolute partial pressures of oxygen (pO2) in the retinal capillary plexus. Measurements were performed at various retinal depths in anesthetized mice under systemic normoxic and hyperoxic conditions. We used a newly developed two-photon phosphorescent oxygen probe, based on a two-photon absorbing platinum tetraphthalimidoporphyrin, and commercially available optics without correction for optical aberrations of the eye. The transverse and axial distances within the tissue volume were calibrated using a model of the eye's optical system. We believe this is the first demonstration of in vivo depth-resolved imaging of pO2 in retinal capillaries. Application of this method has the potential to advance our understanding of oxygen delivery on the microvascular scale and help elucidate mechanisms underlying various retinal diseases.R01 NS091230 - NINDS NIH HHS; R01 AA027097 - NIAAA NIH HHS; R00 AG042026 - NIA NIH HHS; P01 NS055104 - NINDS NIH HHS; R01 MH111359 - NIMH NIH HHShttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC6278707/Published versio

Stabilizing g-States in Centrosymmetric Tetrapyrroles: Two-Photon-Absorbing Porphyrins with Bright Phosphorescence

Using time-dependent density functional theory (TDDFT) and sum-overstates (SOS) formalism, we predicted significant stabilization of 2P-active g-states in a compact fully symmetric porphyrin, in which all four pyrrolic fragments are fused with phathalimide residues via the β-carbon positions. The synthesis of a soluble, nonaggregating meso-unsubstituted tetraarylphthalimidoporphyrin (TAPIP) was then developed, and the spectroscopic measurements confirmed that a strongly 2P-active state in this porphyrin is stabilized below the B (Soret) state level. Single-crystal X-ray analysis revealed near-ideally planar geometry of the TAPIP macrocycle, while its tetra-meso-arylated analogue (meso-Ar4TAPIP) was found to be highly saddled. Consistent with these structural features, Pt meso-Ar4TAPIP phosphoresces rather weakly (ϕphos = 0.05 in DMF at 22 °C), while both Pt and Pd complexes of TAPIP are highly phosphorescent (ϕphos = 0.45 and 0.23, respectively). In addition PdTAPIP exhibits non-negligible thermally activated (E-type) delayed fluorescence (ϕfl(d) ∼ 0.012). Taken together, these photophysical properties make metal complexes of meso-unsubstituted tetaarylphthalimidoporphyrins the brightest 2P-absorbing phosphorescent chromophores known to date

Stabilizing G-States In Centrosymmetric Tetrapyrroles: Two-Photon-Absorbing Porphyrins With Bright Phosphorescence

Using time-dependent density functional theory (TDDFT) and sum-overstates (SOS) formalism, we predicted significant stabilization of 2P-active g-states in a compact fully symmetric porphyrin, in which all four pyrrolic fragments are fused with phathalimide residues via the β-carbon positions. The synthesis of a soluble, nonaggregating meso-unsubstituted tetraarylphthalimidoporphyrin (TAPIP) was then developed, and the spectroscopic measurements confirmed that a strongly 2P-active state in this porphyrin is stabilized below the B (Soret) state level. Single-crystal X-ray analysis revealed near-ideally planar geometry of the TAPIP macrocycle, while its tetra-meso-arylated analogue (meso-Ar4TAPIP) was found to be highly saddled. Consistent with these structural features, Pt meso-Ar4TAPIP phosphoresces rather weakly (φphos = 0.05 in DMF at 22 °C), while both Pt and Pd complexes of TAPIP are highly phosphorescent (φphos = 0.45 and 0.23, respectively). In addition PdTAPIP exhibits non-negligible thermally activated (E-type) delayed fluorescence (φfl(d) ∼ 0.012). Taken together, these photophysical properties make metal complexes of meso-unsubstituted tetaarylphthalimidoporphyrins the brightest 2P-absorbing phosphorescent chromophores known to date

Two-photon absorbing phosphorescent metalloporphyrins: effects of π-extension and peripheral substitution

The ability to form triplet excited states upon two-photon excitation is important for several applications of metalloporphyrins, including two-photon phosphorescence lifetime microscopy (2PLM) and two-photon photodynamic therapy (PDT). Here we analyzed one-photon (1P) and degenerate two-photon (2P) absorption properties of several phosphorescent Pt (II) porphyrins, focusing on the effects of aromatic π-extension and peripheral substitution on triplet emissivity and two-photon absorption (2PA). Our 2PA measurements for the first time made use of direct time-resolved detection of phosphorescence, having the ability to efficiently reject laser background through microsecond time gating. π-Extension of the porphyrin macrocycle by way of syn-fusion with two external aromatic fragments, such as in syn-dibenzo- (DBP) and syn-dinaphthoporphyrins (DNP), lowers the symmetry of the porphyrin skeleton. As a result, DBPs and DNPs exhibit stronger 2PA into the one-photon-allowed B (Soret) and Q states than fully symmetric (D4h) nonextended porphyrins. However, much more 2P-active states lie above the B state and cannot be accessed due to the interfering linear absorption. Alkoxycarbonyl groups (CO2R) in the benzo-rings dramatically enhance 2PA near the B state level. Time-dependent density functional theory (TDDFT) calculations in combinations with the sum-over-states (SOS) formalism revealed that the enhancement is due to the stabilization of higher-lying 2P-active states, which are dominated by the excitations involving orbitals extending onto the carbonyl groups. Furthermore, calculations predicted even stronger stabilization of the 2P-allowed gerade-states in symmetric Pt octaalkoxycarbonyl-tetrabenzoporphyrins. Experiments confirmed that the 2PA cross-section of PtTBP(CO2Bu)8 near 810 nm reaches above 500 GM in spite of its completely centrosymmetric structure. Combined with exceptionally bright phosphorescence (ϕphos = 0.45), strong 2PA makes Pt(II) complexes of π-extended porphyrins a valuable class of chromophores for 2P applications. Another important advantage of these porphyrinoids is their compact size and easily scalable synthesis

Oxyphor 2P: A High-Performance Probe for Deep-Tissue Longitudinal Oxygen Imaging

Quantitative imaging of oxygen distributions in tissue can provide invaluable information about metabolism in normal and diseased states. Two-photon phosphorescence lifetime microscopy (2PLM) has been developed to perform measurements of oxygen in vivo with micron-scale resolution in 3D; however, the method’s potential has not yet been fully realized due to the limitations of current phosphorescent probe technology. Here, we report a new sensor, Oxyphor 2P, that enables oxygen microscopy twice as deep (up to 600 μm below the tissue surface) and with ∼60 times higher speed than previously possible. Oxyphor 2P allows longitudinal oxygen measurements without having to inject the probe directly into the imaged region. As proof of principle, we monitored oxygen dynamics for days following micro-stroke induced by occlusion of a single capillary in the mouse brain. Oxyphor 2P opens up new possibilities for studies of tissue metabolic states using 2PLM in a wide range of biomedical research areas