Multimodal imaging approach to monitor browning of adipose tissue in vivo.

The discovery that white adipocytes can undergo a browning process to become metabolically active beige cells has attracted significant interest in the fight against obesity. However, the study of adipose browning has been impeded by a lack of imaging tools that allow longitudinal and noninvasive monitoring of this process in vivo. Here, we report a preclinical imaging approach to detect development of beige adipocytes during adrenergic stimulation. In this approach, we expressed near-infrared fluorescent protein, iRFP720, driven under an uncoupling protein-1 (Ucp1) promoter in mice by viral transduction, and used multispectral optoacoustic imaging technology with ultrasound tomography (MSOT-US) to assess adipose beiging during adrenergic stimulation. We observed increased photoacoustic signal at 720 nm, coupled with attenuated lipid signals in stimulated animals. As a proof of concept, we validated our approach against hybrid positron emission tomography combined with magnetic resonance (PET/MR) imaging modality, and quantified the extent of adipose browning by MRI-guided segmentation of 2-deoxy-2-18F-fluoro-d-glucose uptake signals. The browning extent detected by MSOT-US and PET/MR are well correlated with Ucp1 induction. Taken together, these systems offer great opportunities for preclinical screening aimed at identifying compounds that promote adipose browning and translation of these discoveries into clinical studies of humans.

cDNA microarray assays to evaluate immune responses following intracranial injection of baculoviral vectors in non-human primates.

Recombinant insect baculoviral vectors efficiently transduce several types of cells in the brain and can possibly be used in gene therapy for brain disorders. However, together with contaminating insect cell proteins, they trigger immune responses that might damage host brain cells. To substantially reduce unwanted immune responses due to the insect cell impurities, we purified and concentrated baculoviral vectors by combining an ion-exchange membrane chromatography method with high-speed centrifugation and demonstrated reduced immune responses of the vector preparations in the mouse brain. To verify the suitability of using these viral vectors for gene therapy strategies in the brain, we evaluated immune reactions and vector toxicity upon acute administration of baculoviral vectors into the brains of cynomolgus macaques. We demonstrated that the virus inoculation caused no abnormalities to non-human primates but induced host anti-viral responses in the brain. With global cellular gene expression profiling, using cDNA microarray technology, we detected that the affected genes were mainly associated with innate immunity, involving the genes of RIG-1-like receptor signaling pathway as the major interferon production pathway. These findings in non-human primates, which share close physiological and genomic similarities with man, may better mimic the responses to baculoviral transduction in the human brain and should be useful in guiding rational therapeutic applications of baculoviral vectors to treat brain disorders.

Hybrid Photoacoustic Ultrasound Imaging System for Cold-Induced Vasoconstriction and Vasodilation Monitoring.

Lewis hunting reaction refers to the alternating cold-induced vasoconstriction and dilation in extremities, whose underlying mechanism is complex. While numerous studies reported this intriguing phenomenon by measuring cutaneous temperature fluctuation under cold exposure, few of them tracked peripheral vascular responses in real-time, lacking a non-invasive and quantitative imaging tool. To better monitor hunting reaction and diagnose relevant diseases, we developed a hybrid photoacoustic ultrasound (PAUS) tomography system to monitor finger vessels' dynamic response to cold, together with simultaneous temperature measurement. We also came out a standard workflow for image analysis with self-defined indices. In the small cohort observational study, vascular changes in the first cycle of hunting reaction were successfully captured by the image series and quantified. Time difference between vasodilation and temperature recovery was noticed and reported for the first time, thanks to the unique capability of the PAUS imaging system in real-time and continuous vascular monitoring. The developed imaging system and indices enabled more objective and quantitative monitoring of peripheral vascular activities, indicating its great potential in numerous clinical applications.

Diffuse reflectance spectroscopy and imaging for non-invasive objective assessment of genitourinary syndrome of menopause: a pilot study.

The genitourinary symptom of menopause (GSM) affects up to 65% of women, resulting in symptoms such as vulvovaginal dryness, discomfort, and dysuria, which significantly impacts quality of life. The current assessment methods rely on subjective questionnaires that can be influenced by individual differences, as well as invasive measurements that are time-consuming and not easily accessible. In this study, we explore the potential of a non-invasive and objective assessment tool called diffuse reflectance spectroscopy and imaging (DRSI) to evaluate tissue chromophores, including water, lipid, oxyhemoglobin, and deoxyhemoglobin. These measurements provide information about moisture content, lipid levels, oxygen saturation, and blood fraction, which can serve as surrogate markers for genital estrogen levels. Our findings reveal distinct differences in these chromophores among pre, peri, and postmenopausal subjects. By using lipid and blood fraction tissue chromophores in a K-Nearest Neighbour classifier model, we achieved a prediction accuracy of 65% compared to vaginal maturation index (VMI) that is clinically used to assess estrogen-related hormonal changes. When age was included as the third feature, the accuracy increased to 78%. We believe that by refining the study protocol and configuring the fiber probe to examine tissue chromophores both in the superficial vulva skin for epidermal water content and the deeper layers, DRSI has the potential to provide objective diagnosis and aid in monitoring the treatment outcome of GSM.

Ultrasound-guided photoacoustic (US-PA) tomography of the breast: Biochemical differentiation using intrinsic tissue markers-lipids, collagen and hemoglobin with histopathologic correlation.

In this pilot study, we investigated the utility of handheld ultrasound-guided photoacoustic (US-PA) imaging probe for analyzing ex-vivo breast specimens obtained from female patients who underwent breast-conserving surgery (BCS). We aimed to assess the potential of US-PA in detecting biochemical markers such as collagen, lipids, and hemoglobin, and compare these findings with routine imaging modalities (mammography, ultrasound) and histopathology results, particularly across various breast densities. Twelve ex-vivo breast specimens were obtained from female patients with a mean age of 59.7 ± 9.5 years who underwent BCS. The tissues were illuminated using handheld US-PA probe between 700 and 1100 nm across all margins and analyzed for collagen, lipids, and hemoglobin distribution. The obtained results were compared with routine imaging and histopathological assessments. Our findings revealed that lipid intensity and distribution decreased with increasing breast density, while collagen exhibited an opposite trend. These observations were consistent with routine imaging and histopathological analyses. Moreover, collagen intensity significantly differed (P < 0.001) between cancerous and normal breast tissue, indicating its potential as an additional biomarker for risk stratification across various breast conditions. The study results suggest that a combined assessment of PA biochemical information, such as collagen and lipid content, superimposed on grey-scale ultrasound findings could aid in distinguishing between normal and malignant breast conditions, as well as assist in BCS margin assessment. This underscores the potential of US-PA imaging as a valuable tool for enhancing breast cancer diagnosis and management, offering complementary information to existing imaging modalities and histopathology.

Tumor tropism of intravenously injected human-induced pluripotent stem cell-derived neural stem cells and their gene therapy application in a metastatic breast cancer model.

Human pluripotent stem cells can serve as an accessible and reliable source for the generation of functional human cells for medical therapies. In this study, we used a conventional lentiviral transduction method to derive human-induced pluripotent stem (iPS) cells from primary human fibroblasts and then generated neural stem cells (NSCs) from the iPS cells. Using a dual-color whole-body imaging technology, we demonstrated that after tail vein injection, these human NSCs displayed a robust migratory capacity outside the central nervous system in both immunodeficient and immunocompetent mice and homed in on established orthotopic 4T1 mouse mammary tumors. To investigate whether the iPS cell-derived NSCs can be used as a cellular delivery vehicle for cancer gene therapy, the cells were transduced with a baculoviral vector containing the herpes simplex virus thymidine kinase suicide gene and injected through tail vein into 4T1 tumor-bearing mice. The transduced NSCs were effective in inhibiting the growth of the orthotopic 4T1 breast tumor and the metastatic spread of the cancer cells in the presence of ganciclovir, leading to prolonged survival of the tumor-bearing mice. The use of iPS cell-derived NSCs for cancer gene therapy bypasses the sensitive ethical issue surrounding the use of cells derived from human fetal tissues or human embryonic stem cells. This approach may also help to overcome problems associated with allogeneic transplantation of other types of human NSCs.

Biochemical "decoding" of breast ultrasound images with optoacoustic tomography fusion: First-in-human display of lipid and collagen signals on breast ultrasound.

To date, studies which utilized ultrasound (US) and optoacoustic tomography (OT) fusion (US-OT) in biochemical differentiation of malignant and benign breast conditions have relied on limited biochemical data such as oxyhaemoglobin (OH) and deoxyhaemoglobin (DH) only. There has been no data of the largest biochemical components of breast fibroglandular tissue: lipid and collagen. Here, the authors believe the ability to image collagen and lipids within the breast tissue could serve as an important milestone in breast US-OT imaging with many potential downstream clinical applications. Hence, we would like to present the first-in-human US-OT demonstration of lipid and collagen differentiation in an excised breast tissue from a 38-year-old female.

Photoacoustic Tomography Appearance of Fat Necrosis: A First-in-Human Demonstration of Biochemical Signatures along with Histological Correlation.

A 50-year-old woman with no past medical history presented with a left anterior chest wall mass that was clinically soft, mobile, and non-tender. A targeted ultrasound (US) showed findings suggestive of a lipoma. However, focal "mass-like" nodules seen within the inferior portion suggested malignant transformation of a lipomatous lesion called for cross sectional imaging, such as MRI or invasive biopsy or excision for histological confirmation. A T1-weighted image demonstrated a large lipoma that has a central fat-containing region surrounded by an irregular hypointense rim in the inferior portion, confirming the benignity of the lipoma. An ultrasound-guided photoacoustic imaging (PA) of the excised specimen to derive the biochemical distribution demonstrated the "mass-like" hypoechoic regions on US as fat-containing, suggestive of benignity of lesion, rather than fat-replacing suggestive of malignancy. The case showed the potential of PA as an adjunct to US in improving the diagnostic confidence in lesion characterization.

Biophotonic technologies for assessment of breast tumor surgical margins-A review.

Breast conserving surgery (BCS) offering similar surgical outcomes as mastectomy while retaining breast cosmesis is becoming increasingly popular for the management of early stage breast cancers. However, its association with reoperation rates of 20% to 40% following incomplete tumor removal warrants the need for a fast and accurate intraoperative surgical margin assessment tool that offers cellular, structural and molecular information of the whole specimen surface to a clinically relevant depth. Biophotonic technologies are evolving to qualify as such an intraoperative tool for clinical assessment of breast cancer surgical margins at the microscopic and macroscopic scale. Herein, we review the current research in the application of biophotonic technologies such as photoacoustic imaging, Raman spectroscopy, multimodal multiphoton imaging, diffuse optical imaging and fluorescence imaging using medically approved dyes for breast cancer detection and/or tumor subtype differentiation toward intraoperative assessment of surgical margins in BCS specimens, and possible challenges in their route to clinical translation.

Circulating microRNA breast cancer biomarker detection in patient sera with surface plasmon resonance imaging biosensor.

In this article, we report for the first time, the detection of circulating miRNA as a breast cancer biomarker in patient sera using surface plasmon resonance imaging biosensor. The advantage of this approach lies in the rapid, label-free and sensitive detection. The sensor excites plasmonic resonance on the gold sensor surface and specific DNA-miRNA molecular bindings elucidate responses in the plasmonic resonance image. Experiments of detecting synthetic miRNA molecules (miR-1249) were performed and the sensor resolution was found to be 63.5 nM. The sensor was further applied to screen 17 patient serum samples from National Cancer Centre Singapore and Tan Tock Seng Hospital. Sensor intensity response was found to differ by 20% between malignant and benign cases and thus forms, a potential and an important metric in distinguishing benignity and malignancy.

Acoustic resolution photoacoustic microscopy based on microelectromechanical systems scanner.

Photoacoustic microscopy (PAM) can be classified as optical resolution (OR)-PAM and acoustic resolution (AR)-PAM depending on the type of resolution achieved. Using microelectromechanical systems (MEMS) scanner, high-speed OR-PAM system was developed earlier. Depth of imaging limits the use of OR-PAM technology for many preclinical and clinical imaging applications. Here, we demonstrate the use of a high-speed MEMS scanner for AR-PAM imaging. Lateral resolution of 84 µm and an axial resolution of 27 µm with ~2.7 mm imaging depth was achieved using a 50 MHz transducer-based AR-PAM system. Use of a higher frequency transducer at 75 MHz has further improved the resolution characteristics of the system with a reduction in imaging depth and a lateral resolution of 53 µm and an axial resolution of 18 µm with ~1.8 mm imaging depth was achieved. Using the two-axis MEMS scanner a 2 × 2 .5 mm2 area was imaged in 3 seconds. The capability of achieving acoustic resolution images using the MEMS scanner makes it beneficial for the development of high-speed miniaturized systems for deeper tissue imaging.

Optoacoustic characterization of breast conserving surgery specimens - A pilot study.

In this pilot study, we tested an ultrasound-guided optoacoustic tomography (US-OT) two-dimensional (2D) array scanner to understand the optoacoustic patterns of excised breastconserving surgery (BCS) specimens. We imaged 14 BCS specimens containing malignant tumors at eight wavelengths spanning 700-1100 nm. Spectral unmixing across multiple wavelengths allowed for visualizing major intrinsic chromophores in the breast tissue including hemoglobin and lipid up to a depth of 7 mm. We identified less/no lipid signals within the tumor and intense deoxy-hemoglobin (Hb) signals on the rim of the tumor as unique characteristics of malignant tumors in comparison to no tumor region. We also observed continuous broad lipid signals as features of negative margins and compromised lipid signals interrupted by vasculature as features of positive margins. These differentiating patterns can form the basis of US-OT to be explored as an alternate, fast and efficient intraoperative method for evaluation of tumor resection margins.

Ultrasound Guided Optoacoustic Tomography in Assessment of Tumor Margins for Lumpectomies.

PURPOSE: To determine the accuracy of a handheld ultrasound-guided optoacoustic tomography (US-OT) probe developed for human deep-tissue imaging in ex vivo assessment of tumor margins postlumpectomy. METHODS: A custom-built two-dimensional (2D) US-OT-handheld probe was used to scan 15 lumpectomy breast specimens. Optoacoustic signals acquired at multiple wavelengths between 700 and 1100 nm were reconstructed using model linear algorithm, followed by spectral unmixing for lipid and deoxyhemoglobin (Hb). Distribution maps of lipid and Hb on the anterior, posterior, superior, inferior, medial, and lateral margins of the specimens were inspected for margin involvement, and results were correlated with histopathologic findings. The agreement in tumor margin assessment between US-OT and histopathology was determined using the Bland-Altman plot. Accuracy, sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of margin assessment using US-OT were calculated. RESULTS: Ninety margins (6 × 15 specimens) were assessed. The US-OT probe resolved blood vessels and lipid up to a depth of 6 mm. Negative and positive margins were discriminated by marked differences in the distribution patterns of lipid and Hb. US-OT assessments were concordant with histopathologic findings in 87 of 89 margins assessed (one margin was uninterpretable and excluded), with diagnostic accuracy of 97.9% (kappa = 0.79). The sensitivity, specificity, PPV, and NPV were 100% (4/4), 97.6% (83/85), 66.7% (4/6), and 100% (83/83), respectively. CONCLUSION: US-OT was capable of providing distribution maps of lipid and Hb in lumpectomy specimens that predicted tumor margins with high sensitivity and specificity, making it a potential tool for intraoperative tumor margin assessment.

High-speed simultaneous multiscale photoacoustic microscopy.

Photoacoustic microscopy (PAM) is a fast-growing biomedical imaging technique that provides high-resolution in vivo imaging beyond the optical diffusion limit. Depending on the scalable lateral resolution and achievable penetration depth, PAM can be classified into optical resolution PAM (OR-PAM) and acoustic resolution PAM (AR-PAM). The use of a microelectromechanical systems (MEMS) scanner has improved OR-PAM imaging speed significantly and is highly beneficial in the development of miniaturized handheld devices. The shallow penetration depth of OR-PAM limits the use of such devices for a wide range of clinical applications. We report the use of a high-speed MEMS scanner for both OR-PAM and AR-PAM. A high-speed, wide-area scanning integrated OR-AR-PAM system combining MEMS scanner and raster mechanical movement was developed. A lateral resolution of 5 µm and penetration depth ∼0.9-mm in vivo was achieved using OR-PAM at 586 nm, whereas a lateral resolution of 84 µm and penetration depth of ∼2-mm in vivo was achieved using AR-PAM at 532 nm.

A review of clinical photoacoustic imaging: Current and future trends.

Photoacoustic imaging (or optoacoustic imaging) is an upcoming biomedical imaging modality availing the benefits of optical resolution and acoustic depth of penetration. With its capacity to offer structural, functional, molecular and kinetic information making use of either endogenous contrast agents like hemoglobin, lipid, melanin and water or a variety of exogenous contrast agents or both, PAI has demonstrated promising potential in a wide range of preclinical and clinical applications. This review provides an overview of the rapidly expanding clinical applications of photoacoustic imaging including breast imaging, dermatologic imaging, vascular imaging, carotid artery imaging, musculoskeletal imaging, gastrointestinal imaging and adipose tissue imaging and the future directives utilizing different configurations of photoacoustic imaging. Particular emphasis is placed on investigations performed on human or human specimens.

Functionalised iron oxide nanoparticles for multimodal optoacoustic and magnetic resonance imaging.

The novel attachment of the optoacoustic (OA) molecules indocyanine green (ICG) and Flamma®774 to the core of an iron oxide (Fe3O4) nanoparticle has resulted in the facile synthesis of a multimodal imaging probe for both multispectral optoacoustic tomography (MSOT) imaging and magnetic resonance imaging (MRI). The nanoparticles have been analysed structurally, optically and magnetically to demonstrate the multimodal characteristics. The OA analysis of the dyes ICG and Flamma®774 showed that they have absorbance at the near IR wavelengths of 790 and 780 nm, respectively, when conjugated to an iron oxide core. These wavelengths are ideal for spectral unmixing of the probe intensity from any endogenous contrast, such as oxy-(HbO2) and deoxy-hemoglobin (Hb). MRI showed that citrate capped Fe3O4 exhibited a good r2 contrast of 230 mM-1 s-1, which is in line with literature values. Upon optoacoustic dye modification, the r2 relaxivity coefficient is comparable with that of Flamma®774 iron oxide nanoparticles (FeO-774) with r2 = 212 mM-1 s-1, showing that an OA dye attachment can have little to no effect on the MRI contrast. Indocyanine green functionalised iron oxide (FeO-ICG) nanoparticles showed an r2 contrast that was dramatically reduced with r2 = 5 mM-1 s-1. These results indicate that the facile synthesis of an effective dual modality MRI-MSOT probe can be developed using an iron oxide core and simple ligand coordination chemistry using an optoacoustic dye.

SERS-based detection of haptoglobin in ovarian cyst fluid as a point-of-care diagnostic assay for epithelial ovarian cancer.

PURPOSE: To evaluate haptoglobin (Hp) in ovarian cyst fluid as a diagnostic biomarker for epithelial ovarian cancers (EOCs) using surface-enhanced Raman spectroscopy (SERS)-based in vitro diagnostic assay for use in an intraoperative setting. METHODS: SERS-based method was used to detect and quantify Hp in archived ovarian cyst fluids collected from suspicious ovarian cysts and differentiate benign tumors from EOCs. The diagnostic performance of SERS-based assay was verified against the histopathology conclusions and compared with the results of CA125 test and frozen sections. RESULTS: Hp concentration present in the clinical cyst fluid measured by SERS was normalized to 3.3 mg/mL of standard Hp. Normalized mean values for patients with benign cysts were 0.65 (n=57) and malignant cysts were 1.85 (n=54), demonstrating a significantly (P<0.01) higher Hp in malignant samples. Verified against histology, Hp measurements using SERS had a sensitivity of 94% and specificity of 91%. Receiver operating characteristic curve analysis of SERS-based Hp measurements resulted in area under the curve of 0.966±0.03, establishing the robustness of the method. CA125 test on the same set of patients had a sensitivity of 85% and specificity of 90%, while frozen section analysis on 65 samples had 100% sensitivity and specificity. CONCLUSION: With a total execution time of <10 minutes and consistent performance across different stages of cancer, the SERS-based Hp detection assay can serve as a promising intra-operative EOC diagnostic test.

Photoacoustic microscopy for evaluating combretastatin A4 phosphate induced vascular disruption in orthotopic glioma.

The use of an optical resolution photoacoustic microscopy (OR-PAM) system to evaluate the vascular disruptive effect of combretastatin A4 Phosphate (CA4P) on a murine orthotopic glioma with intact skull is described here. Second generation optical-resolution photoacoustic microscopy scanner with a 532 nm pulsed diode-pumped solid-state laser that specifically matches the absorption maximum of hemoglobin in tissues was used to image orthotopic glioma inoculated in mouse brain. Two-dimensional maps of brain vasculature with a lateral resolution of 5 µm and a depth of 700 µm at a field of view 5 × 4 mm were acquired on normal brain and glioma brain. Longitudinal imaging of the brain pre- and post-administration of CA4P, a FDA approved drug for solid tumors, enabled the monitoring of hemodynamic changes in tumor vasculature revealing the well documented vascular shutdown and recovery associated with this drug. Our study marks the beginning of potential prospects of this technology as an imaging tool for preclinical and clinical study of pathologies characterized by changes in the vasculature.

Noninvasive Anatomical and Functional Imaging of Orthotopic Glioblastoma Development and Therapy using Multispectral Optoacoustic Tomography.

PURPOSE: Here we demonstrate the potential of multispectral optoacoustic tomography (MSOT), a new non-invasive structural and functional imaging modality, to track the growth and changes in blood oxygen saturation (sO2) in orthotopic glioblastoma (GBMs) and the surrounding brain tissues upon administration of a vascular disruptive agent (VDA). METHODS: Nude mice injected with U87MG tumor cells were longitudinally monitored for the development of orthotopic GBMs up to 15 days and observed for changes in sO2 upon administration of combretastatin A4 phosphate (CA4P, 30 mg/kg), an FDA approved VDA for treating solid tumors. We employed a newly-developed non-negative constrained approach for combined MSOT image reconstruction and unmixing in order to quantitatively map sO2 in whole mouse brains. RESULTS: Upon longitudinal monitoring, tumors could be detected in mouse brains using single-wavelength data as early as 6 days post tumor cell inoculation. Fifteen days post-inoculation, tumors had higher sO2 of 63 ± 11% (n = 5, P < .05) against 48 ± 7% in the corresponding contralateral brain, indicating their hyperoxic status. In a different set of animals, 42 days post-inoculation, tumors had lower sO2 of 42 ± 5% against 49 ± 4% (n = 3, P < .05) in the contralateral side, indicating their hypoxic status. Upon CA4P administration, sO2 in 15 days post-inoculation tumors dropped from 61 ± 9% to 36 ± 1% (n = 4, P < .01) within one hour, then reverted to pre CA4P treatment values (63 ± 6%) and remained constant until the last observation time point of 6 hours. CONCLUSION: With the help of advanced post processing algorithms, MSOT was capable of monitoring the tumor growth and assessing hemodynamic changes upon administration of VDAs in orthotopic GBMs.