Advanced Wound Diagnostics: Toward Transforming Wound Care into Precision Medicine.

Significance: Nonhealing wounds are an ever-growing global pandemic, with mortality rates and management costs exceeding many common cancers. Although our understanding of the molecular and cellular factors driving wound healing continues to grow, standards for diagnosing and evaluating wounds remain largely subjective and experiential, whereas therapeutic strategies fail to consistently achieve closure and clinicians are challenged to deliver individualized care protocols. There is a need to apply precision medicine practices to wound care by developing evidence-based approaches, which are predictive, prescriptive, and personalized. Recent Advances: Recent developments in "advanced" wound diagnostics, namely biomarkers (proteases, acute phase reactants, volatile emissions, and more) and imaging systems (ultrasound, autofluorescence, spectral imaging, and optical coherence tomography), have begun to revolutionize our understanding of the molecular wound landscape and usher in a modern age of therapeutic strategies. Herein, biomarkers and imaging systems with the greatest evidence to support their potential clinical utility are reviewed. Critical Issues: Although many potential biomarkers have been identified and several imaging systems have been or are being developed, more high-quality randomized controlled trials are necessary to elucidate the currently questionable role that these tools are playing in altering healing dynamics or predicting wound closure within the clinical setting. Future Directions: The literature supports the need for the development of effective point-of-care wound assessment tools, such as a platform diagnostic array that is capable of measuring multiple biomarkers at once. These, along with advances in telemedicine, synthetic biology, and "smart" wearables, will pave the way for the transformation of wound care into a precision medicine. Clinical Trial Registration number: NCT03148977.

Correction to: Feasibility Study of a Novel Protease-Activated Fluorescent Imaging System for Real-Time, Intraoperative Detection of Residual Breast Cancer in Breast Conserving Surgery.

The article Feasibility Study of a Novel Protease-Activated Fluorescent Imaging System for Real-Time, Intraoperative Detection of Residual Breast Cancer in Breast Conserving Surgery, written by Barbara L. Smith et al., was originally published electronically on the publisher's internet portal on January 2, 2020, without open access.

Feasibility Study of a Novel Protease-Activated Fluorescent Imaging System for Real-Time, Intraoperative Detection of Residual Breast Cancer in Breast Conserving Surgery.

BACKGROUND: Obtaining tumor-free margins is critical to prevent recurrence after lumpectomy for breast cancer. Unfortunately, current approaches leave positive margins that require second surgeries in 20-40% of patients. We assessed the LUM Imaging System for real-time, intraoperative detection of residual tumor. METHODS: Breast lumpectomy cavity walls and excised specimens were assessed with the LUM Imaging System after 1 mg/kg intravenous LUM015, a protease-activatable fluorescent agent. Fluorescence at potential sites of residual tumor in lumpectomy cavity walls was evaluated intraoperatively with a sterile hand-held probe, with real-time predictive results displayed on a monitor intraoperatively, and later correlated with histopathology. RESULTS: In vivo lumpectomy cavities and excised specimens were imaged after LUM015 injection in 45 women undergoing breast cancer surgery. Invasive ductal and lobular cancers and intraductal cancer (DCIS) were included. A total of 570 cavity margin surfaces in 40 patients were used for algorithm development. Image analysis and display took approximately 1 s per 2.6-cm-diameter circular margin surface. All breast cancer subtypes could be distinguished from adjacent normal tissue. For all imaged cavity surfaces, sensitivity for tumor detection was 84%. Among 8 patients with positive margins after standard surgery, sensitivity for residual tumor detection was 100%; 2 of 8 were spared second surgeries because additional tissue was excised at sites of LUM015 signal. Specificity was 73%, with some benign tissues showing elevated fluorescent signal. CONCLUSIONS: The LUM015 agent and LUM Imaging System allow rapid identification of residual tumor in the lumpectomy cavity of breast cancer patients and may reduce rates of positive margins.

Real-time, intraoperative detection of residual breast cancer in lumpectomy cavity walls using a novel cathepsin-activated fluorescent imaging system.

PURPOSE: Obtaining tumor-free surgical margins is critical to prevent recurrence in breast-conserving surgery but it remains challenging. We assessed the LUM Imaging System for real-time, intraoperative detection of residual tumor. METHODS: Lumpectomy cavity walls and excised specimens of breast cancer lumpectomy patients were assessed with the LUM Imaging System (Lumicell, Inc., Wellesley MA) with and without intravenous LUM015, a cathepsin-activatable fluorescent agent. Fluorescence at potential sites of residual tumor was evaluated with a sterile hand-held probe, displayed on a monitor and correlated with histopathology. RESULTS: Background autofluorescence was assessed in excised specimens from 9 patients who did not receive LUM015. In vivo lumpectomy cavities and excised specimens were then imaged in 15 women undergoing breast cancer surgery who received no LUM015, 0.5, or 1 mg/kg LUM015 (5 women per dose). Among these, 11 patients had invasive carcinoma with ductal carcinoma in situ (DCIS) and 4 had only DCIS. Image acquisition took 1 s for each 2.6-cm-diameter surface. No significant background normal breast fluorescence was identified. Elevated fluorescent signal was seen from invasive cancers and DCIS. Mean tumor-to-normal signal ratios were 4.70 ± 1.23 at 0.5 mg/kg and 4.22 ± 0.9 at 1.0 mg/kg (p = 0.54). Tumor was distinguished from normal tissue in pre-and postmenopausal women and readings were not affected by breast density. Some benign tissues produced fluorescent signal with LUM015. CONCLUSION: The LUM Imaging System allows rapid identification of residual tumor in the lumpectomy cavity of breast cancer patients and may reduce rates of positive margins.

A Fluorescence-Guided Laser Ablation System for Removal of Residual Cancer in a Mouse Model of Soft Tissue Sarcoma.

The treatment of soft tissue sarcoma (STS) generally involves tumor excision with a wide margin. Although advances in fluorescence imaging make real-time detection of cancer possible, removal is limited by the precision of the human eye and hand. Here, we describe a novel pulsed Nd:YAG laser ablation system that, when used in conjunction with a previously described molecular imaging system, can identify and ablate cancer in vivo. Mice with primary STS were injected with the protease-activatable probe LUM015 to label tumors. Resected tissues from the mice were then imaged and treated with the laser using the paired fluorescence-imaging/ laser ablation device, generating ablation clefts with sub-millimeter precision and minimal underlying tissue damage. Laser ablation was guided by fluorescence to target tumor tissues, avoiding normal structures. The selective ablation of tumor implants in vivo improved recurrence-free survival after tumor resection in a cohort of 14 mice compared to 12 mice that received no ablative therapy. This prototype system has the potential to be modified so that it can be used during surgery to improve recurrence-free survival in patients with cancer.

A mouse-human phase 1 co-clinical trial of a protease-activated fluorescent probe for imaging cancer.

Local recurrence is a common cause of treatment failure for patients with solid tumors. Intraoperative detection of microscopic residual cancer in the tumor bed could be used to decrease the risk of a positive surgical margin, reduce rates of reexcision, and tailor adjuvant therapy. We used a protease-activated fluorescent imaging probe, LUM015, to detect cancer in vivo in a mouse model of soft tissue sarcoma (STS) and ex vivo in a first-in-human phase 1 clinical trial. In mice, intravenous injection of LUM015 labeled tumor cells, and residual fluorescence within the tumor bed predicted local recurrence. In 15 patients with STS or breast cancer, intravenous injection of LUM015 before surgery was well tolerated. Imaging of resected human tissues showed that fluorescence from tumor was significantly higher than fluorescence from normal tissues. LUM015 biodistribution, pharmacokinetic profiles, and metabolism were similar in mouse and human subjects. Tissue concentrations of LUM015 and its metabolites, including fluorescently labeled lysine, demonstrated that LUM015 is selectively distributed to tumors where it is activated by proteases. Experiments in mice with a constitutively active PEGylated fluorescent imaging probe support a model where tumor-selective probe distribution is a determinant of increased fluorescence in cancer. These co-clinical studies suggest that the tumor specificity of protease-activated imaging probes, such as LUM015, is dependent on both biodistribution and enzyme activity. Our first-in-human data support future clinical trials of LUM015 and other protease-sensitive probes.

Controlled placement of colloidal quantum dots in sub-15 nm clusters.

We demonstrated a technique to control the placement of 6 nm-diameter CdSe and 5 nm-diameter CdSe/CdZnS colloidal quantum dots (QDs) through electron-beam lithography. This QD-placement technique resulted in an average of three QDs in each cluster, and 87% of the templated sites were occupied by at least one QD. These QD clusters could be in close proximity to one another, with a minimum separation of 12 nm. Photoluminescence measurements of the fabricated QD clusters showed intermittent photoluminescence, which indicates that the QDs were optically active after the fabrication process. This optimized top-down lithographic process is a step towards the integration of individual QDs in optoelectronic and nano-optical systems.

Biexciton quantum yield heterogeneities in single CdSe (CdS) core (shell) nanocrystals and its correlation to exciton blinking.

We explore biexciton (BX) nonradiative recombination processes in single semiconductor nanocrystals (NCs) using confocal fluorescence microscopy and second-order photon intensity correlation. More specifically, we measure the photoluminescence blinking and BX quantum yields (QYs) and study the correlation between these two measurements for single core (shell) CdSe (CdS) nanocrystals (NCs). We find that NCs with a high "on" time fraction are significantly more likely to have a high BX QY than NCs with a low "on" fraction, even though the BX QYs of NCs with a high "on" fraction vary dramatically. The BX QYs of single NCs are also weakly dependent upon excitation wavelength. The weak correlation between exciton "on" fractions and BX QYs suggests that multiple recombination processes are involved in the BX recombination. To explain our results, we propose a model that combines both trapping and an Auger mechanism for BX recombination.

Nanopatterned electrically conductive films of semiconductor nanocrystals.

We present the first semiconductor nanocrystal films of nanoscale dimensions that are electrically conductive and crack-free. These films make it possible to study the electrical properties intrinsic to the nanocrystals unimpeded by defects such as cracking and clustering that typically exist in larger-scale films. We find that the electrical conductivity of the nanoscale films is 180 times higher than that of drop-cast, microscopic films made of the same type of nanocrystal. Our technique for forming the nanoscale films is based on electron-beam lithography and a lift-off process. The patterns have dimensions as small as 30 nm and are positioned on a surface with 30 nm precision. The method is flexible in the choice of nanocrystal core-shell materials and ligands. We demonstrate patterns with PbS, PbSe, and CdSe cores and Zn(0.5)Cd(0.5)Se-Zn(0.5)Cd(0.5)S core-shell nanocrystals with a variety of ligands. We achieve unprecedented versatility in integrating semiconductor nanocrystal films into device structures both for studying the intrinsic electrical properties of the nanocrystals and for nanoscale optoelectronic applications.

Bias-stress effect in 1,2-ethanedithiol-treated PbS quantum dot field-effect transistors.

We investigate the bias-stress effect in field-effect transistors (FETs) consisting of 1,2-ethanedithiol-treated PbS quantum dot (QD) films as charge transport layers in a top-gated configuration. The FETs exhibit ambipolar operation with typical mobilities on the order of µ(e) = 8 × 10(-3) cm(2) V(-1) s(-1) in n-channel operation and µ(h) = 1 × 10(-3) cm(2) V(-1) s(-1) in p-channel operation. When the FET is turned on in n-channel or p-channel mode, the established drain-source current rapidly decreases from its initial magnitude in a stretched exponential decay, manifesting the bias-stress effect. The choice of dielectric is found to have little effect on the characteristics of this bias-stress effect, leading us to conclude that the associated charge-trapping process originates within the QD film itself. Measurements of bias-stress-induced time-dependent decays in the drain-source current (I(DS)) are well fit to stretched exponential functions, and the time constants of these decays in n-channel and p-channel operation are found to follow thermally activated (Arrhenius) behavior. Measurements as a function of QD size reveal that the stressing process in n-channel operation is faster for QDs of a smaller diameter while stress in p-channel operation is found to be relatively invariant to QD size. Our results are consistent with a mechanism in which field-induced nanoscale morphological changes within the QD film result in screening of the applied gate field. This phenomenon is entirely recoverable, which allows us to repeatedly observe bias stress and recovery characteristics on the same device. This work elucidates aspects of charge transport in chemically treated lead chalcogenide QD films and is of relevance to ongoing investigations toward employing these films in optoelectronic devices.

Exploring exciton relaxation and multiexciton generation in PbSe nanocrystals using hyperspectral near-IR probing.

Hyperspectral femtosecond transient absorption spectroscopy is employed to record exciton relaxation and recombination in colloidal lead selenide (PbSe) nanocrystals in unprecedented detail. Results obtained with different pump wavelengths and fluences are scrutinized with regard to three issues: (1) early subpicosecond spectral features due to "hot" excitons are analyzed in terms of suggested underlying mechanisms; (2) global kinetic analysis facilitates separation of the transient difference spectra into single, double, and triple exciton state contributions, from which individual band assignments can be tested; and (3) the transient spectra are screened for signatures of multiexciton generation (MEG) by comparing experiments with excitation pulses both below and well above the theoretical threshold for multiplication. For the latter, a recently devised ultrafast pump-probe spectroscopic approach is employed. Scaling sample concentrations and pump pulse intensities inversely with the extinction coefficient at each excitation wavelength overcomes ambiguities due to direct multiphoton excitation, uncertainties of absolute absorption cross sections, and low signal levels. As observed in a recent application of this method to InAs core/shell/shell nanodots, no sign of MEG was detected in this sample up to photon energy 3.7 times the band gap. Accordingly, numerous reports of efficient MEG in other samples of PbSe suggest that the efficiency of this process varies from sample to sample and depends on factors yet to be determined.

Imaging Schottky barriers and ohmic contacts in PbS quantum dot devices.

We fabricated planar PbS quantum dot devices with ohmic and Schottky type electrodes and characterized them using scanning photocurrent and photovoltage microscopies. The microscopy techniques used in this investigation allow for interrogation of the lateral depletion width and related photovoltaic properties in the planar Schottky type contacts. Titanium/QD contacts exhibited depletion widths that varied over a wide range as a function of bias voltage, while the gold/QD contacts showed ohmic behavior over the same voltage range.

Using nanowires to extract excitons from a nanocrystal solid.

Synthetic methods yielding highly uniform colloidal semiconductor nanocrystals with controlled shapes and sizes are now available for many materials. These methods have enabled geometrical control of optical properties, which are difficult or impossible to achieve in conventional bulk solids. However, incorporating nanocrystals efficiently into photodetectors remains challenging because of the low charge carrier mobilities typical of nanocrystal solids. Here we present an approach based on exciton energy transfer from CdSe/CdS core/shell nanocrystals to embedded CdSe nanowires. By combining the wide electronic tunability of nanocrystals with the excellent one-dimensional charge transport characteristics obtainable in nanowires, we are able to increase photocurrent extraction from a nanocrystal solid by 2-3 orders of magnitude. Furthermore, we correlate local device morphology with optoelectronic functionality by measuring the local photocurrent response in a scanning confocal microscope. We also discuss how nancocrystal/nanowire hybrid devices could be used in particle detector systems.

2DIR spectroscopy of human amylin fibrils reflects stable ß-sheet structure.

The aggregation of human amylin to form amyloid contributes to islet ß-cell dysfunction in type 2 diabetes. Studies of amyloid formation have been hindered by the low structural resolution or relatively modest time resolution of standard methods. Two-dimensional infrared (2DIR) spectroscopy, with its sensitivity to protein secondary structures and its intrinsic fast time resolution, is capable of capturing structural changes during the aggregation process. Moreover, isotope labeling enables the measurement of residue-specific information. The diagonal line widths of 2DIR spectra contain information about dynamics and structural heterogeneity of the system. We illustrate the power of a combined atomistic molecular dynamics simulation and theoretical and experimental 2DIR approach by analyzing the variation in diagonal line widths of individual amide I modes in a series of labeled samples of amylin amyloid fibrils. The theoretical and experimental 2DIR line widths suggest a "W" pattern, as a function of residue number. We show that large line widths result from substantial structural disorder and that this pattern is indicative of the stable secondary structure of the two ß-sheet regions. This work provides a protocol for bridging MD simulation and 2DIR experiments for future aggregation studies.

Strategies for extracting structural information from 2D IR spectroscopy of amyloid: application to islet amyloid polypeptide.

The 37-residue human islet amyloid polypeptide (hIAPP or amylin) self-assembles into fibers, the assembly of which has been associated with the disease mechanism of type II diabetes. Infrared spectroscopy in conjunction with isotope labeling is proving to be a powerful tool for studying the aggregation process of hIAPP and other amyloid forming proteins with residue specific structure and kinetic information, but the relationship between the spectroscopic observables and the structure is not fully established. We report a detailed analysis of the linear and 2D IR spectra of hIAPP fibers isotope labeled at seven different residue positions. The features of the 2D IR spectra, including the frequencies, linewidths, intensities, and polarization dependence of the diagonal and cross-peaks, rely heavily on the position of the isotope labeled residue. In order to understand how these measured parameters depend on fiber secondary and tertiary structure, we have simulated 1D and 2D IR spectra utilizing idealized structural models in addition to a recently published solid-state NMR based model of the amyloid fibril. The analysis provides a more rigorous foundation for interpreting the infrared spectra of amyloids. In addition, we demonstrate that 2D IR spectra can be employed to distinguish between residues in beta-sheets versus those in turn regions, and that transitional residues between secondary structures can be identified by the suppression of their cross-peaks in 2D IR spectra. This latter approach is not limited to amyloid fibrils and will be generally useful in identifying regions of secondary structure in proteins using 2D IR spectroscopy and isotope labeling.

Polarization shaping in the mid-IR and polarization-based balanced heterodyne detection with application to 2D IR spectroscopy.

We demonstrate amplitude, phase and polarization shaping of femtosecond mid-IR pulses using a germanium acousto-optical modulator by independently shaping the frequency-dependent amplitudes and phases of two orthogonally polarized pulses which are then collinearly overlapped using a wire-grid polarizer. We use a feedback loop to set and stabilize the relative phase of the orthogonal pulses. We have also used a wire-grid polarizer to implement polarization-based balanced heterodyne detection for improved signal-to-noise of 2D IR spectra collected in a pump-probe geometry. Applications include coherent control of molecular vibrations and improvements in multidimensional IR spectroscopy.

Two-dimensional IR spectroscopy and isotope labeling defines the pathway of amyloid formation with residue-specific resolution.

There is considerable interest in uncovering the pathway of amyloid formation because the toxic properties of amyloid likely stems from prefibril intermediates and not the fully formed fibrils. Using a recently invented method of collecting 2-dimensional infrared spectra and site-specific isotope labeling, we have measured the development of secondary structures for 6 residues during the aggregation process of the 37-residue polypeptide associated with type 2 diabetes, the human islet amyloid polypeptide (hIAPP). By monitoring the kinetics at 6 different labeled sites, we find that the peptides initially develop well-ordered structure in the region of the chain that is close to the ordered loop of the fibrils, followed by formation of the 2 parallel beta-sheets with the N-terminal beta-sheet likely forming before the C-terminal sheet. This experimental approach provides a detailed view of the aggregation pathway of hIAPP fibril formation as well as a general methodology for studying other amyloid forming proteins without the use of structure-perturbing labels.

Two-dimensional infrared spectroscopy provides evidence of an intermediate in the membrane-catalyzed assembly of diabetic amyloid.

Islet amyloid polypeptide (IAPP, also known as amylin) is responsible for pancreatic amyloid deposits in type 2 diabetes. The deposits, as well as intermediates in their assembly, are cytotoxic to pancreatic beta-cells and contribute to the loss of beta-cell mass associated with type 2 diabetes. The factors that trigger islet amyloid deposition in vivo are not well understood, but peptide membrane interactions have been postulated to play an important role in islet amyloid formation. To better understand the role of membrane interactions in amyloid formation, two-dimensional infrared (2D IR) spectroscopy was used to compare the kinetics of amyloid formation for human IAPP both in the presence and in the absence of negatively charged lipid vesicles. Comparison of spectral features and kinetic traces from the two sets of experiments provides evidence for the formation of an ordered intermediate during the membrane-mediated assembly of IAPP amyloid. A characteristic transient spectral feature is detected during amyloid formation in the presence of vesicles that is not observed in the absence of vesicles. The spectral feature associated with the intermediate raises in intensity during the self-assembly process and subsequently decays in intensity in the classic manner of a kinetic intermediate. Studies with rat IAPP, a variant that is known to interact with membranes but does not form amyloid, confirm the presence of an intermediate. The analysis of 2D IR spectra in terms of specific structural features is discussed. The unique combination of time and secondary structure resolution of 2D IR spectroscopy has enabled the time-evolution of a hIAPP intermediate to be directly monitored for the first time. The data presented here demonstrates the utility of 2D IR spectroscopy for studying membrane-catalyzed amyloid formation.

Mode selectivity with polarization shaping in the mid-IR.

We report that polarization-shaped mid-infrared (IR) pulses can be used to enhance the vibrational population of one mode over another in a coupled molecular system. A genetic algorithm and a new mid-IR polarization shaper were used to alter the relative vibrational excitation of the two carbonyl stretching modes in Mn(CO)(5)Br. One mode could be selectively enhanced over the other by 2-3 times. Control over the polarization leads to better optimization than phase-only control. Several possible mechanisms that indicate how polarization shaping leads to selective vibrational excitation are discussed using a formalism that separates polarization shaping effects on the signal strength from amplitude or phase shaping. The techniques introduced herein will have broad applications in quantum gating schemes, controlling ground state chemistry and enhancing the sensitivity of multidimensional IR and visible spectroscopies.

Spectroscopic identification of higher-order rare gas-dihalogen complexes with different geometries: He(2,3)...Br(2) and He(2,3)...ICl.

Rovibronic transitions of multiple conformers of the He(2)...(79)Br(2)(X, v'' = 0), He(3)...(79)Br(2)(X, v'' = 0), He(2)...I(35)Cl(X, v'' = 0), and He(3)...I(35)Cl(X, v'' = 0) complexes stabilized in a pulsed, supersonic expansion are observed in action spectra recorded in the B-X region of the dihalogens. In addition to features associated with He(2)...(79)Br(2) and He(2)...I(35)Cl complexes with the rare gas atoms localized in the toroidal potential well lying in a plane perpendicular to the dihalogen bond, those associated with a ground-state conformer that has one He atom localized in the toroidal potential and the other He atom localized in the linear well at the end of the dihalogen moiety are also identified. Transitions of at least three conformers of the He(3)...Br(2) complex and two conformers of the He(3)...ICl complex are also observed. The relative populations of the different conformers are found to depend on where along the supersonic expansion the spectra are recorded, and thus on the local temperature regime sampled. The He(2)...(79)Br(2) and He(2)...I(35)Cl conformers with one He atom in each well are found to be the more stable conformers.