Secretin-Activated Brown Fat Mediates Prandial Thermogenesis to Induce Satiation.

The molecular mediator and functional significance of meal-associated brown fat (BAT) thermogenesis remains elusive. Here, we identified the gut hormone secretin as a non-sympathetic BAT activator mediating prandial thermogenesis, which consequentially induces satiation, thereby establishing a gut-secretin-BAT-brain axis in mammals with a physiological role of prandial thermogenesis in the control of satiation. Mechanistically, meal-associated rise in circulating secretin activates BAT thermogenesis by stimulating lipolysis upon binding to secretin receptors in brown adipocytes, which is sensed in the brain and promotes satiation. Chronic infusion of a modified human secretin transiently elevates energy expenditure in diet-induced obese mice. Clinical trials with human subjects showed that thermogenesis after a single-meal ingestion correlated with postprandial secretin levels and that secretin infusions increased glucose uptake in BAT. Collectively, our findings highlight the largely unappreciated function of BAT in the control of satiation and qualify BAT as an even more attractive target for treating obesity.

Controlling the sound of light: photoswitching optoacoustic imaging.

Optoacoustic (photoacoustic) imaging advances allow high-resolution optical imaging much deeper than optical microscopy. However, while label-free optoacoustics have already entered clinical application, biological imaging is in need of ubiquitous optoacoustic labels for use in ways that are similar to how fluorescent proteins propelled optical microscopy. We review photoswitching advances that shine a new light or, in analogy, 'bring a new sound' to biological optoacoustic imaging. Based on engineered labels and novel devices, switching uses light or other energy forms and enables signal modulation and synchronous detection for maximizing contrast and detection sensitivity over other optoacoustic labels. Herein, we explain contrast enhancement in the spectral versus temporal domains and review labels and key concepts of switching and their properties to modulate optoacoustic signals. We further outline systems and applications and discuss how switching can enable optoacoustic imaging of cellular or molecular contrast at depths and resolutions beyond those of other optical methods.

A submicrometre silicon-on-insulator resonator for ultrasound detection.

Ultrasound detectors use high-frequency sound waves to image objects and measure distances, but the resolution of these readings is limited by the physical dimensions of the detecting element. Point-like broadband ultrasound detection can greatly increase the resolution of ultrasonography and optoacoustic (photoacoustic) imaging1,2, but current ultrasound detectors, such as those used for medical imaging, cannot be miniaturized sufficiently. Piezoelectric transducers lose sensitivity quadratically with size reduction3, and optical microring resonators4 and Fabry-Pérot etalons5 cannot adequately confine light to dimensions smaller than about 50 micrometres. Micromachining methods have been used to generate arrays of capacitive6 and piezoelectric7 transducers, but with bandwidths of only a few megahertz and dimensions exceeding 70 micrometres. Here we use the widely available silicon-on-insulator technology to develop a miniaturized ultrasound detector, with a sensing area of only 220 nanometres by 500 nanometres. The silicon-on-insulator-based optical resonator design provides per-area sensitivity that is 1,000 times higher than that of microring resonators and 100,000,000 times better than that of piezoelectric detectors. Our design also enables an ultrawide detection bandwidth, reaching 230 megahertz at -6 decibels. In addition to making the detectors suitable for manufacture in very dense arrays, we show that the submicrometre sensing area enables super-resolution detection and imaging performance. We demonstrate imaging of features 50 times smaller than the wavelength of ultrasound detected. Our detector enables ultra-miniaturization of ultrasound readings, enabling ultrasound imaging at a resolution comparable to that achieved with optical microscopy, and potentially enabling the development of very dense ultrasound arrays on a silicon chip.

A protease-activated, near-infrared fluorescent probe for early endoscopic detection of premalignant gastrointestinal lesions.

Fluorescence imaging is currently being actively developed for surgical guidance; however, it remains underutilized for diagnostic and endoscopic surveillance of incipient colorectal cancer in high-risk patients. Here we demonstrate the utility and potential for clinical translation of a fluorescently labeled cathepsin-activated chemical probe to highlight gastrointestinal lesions. This probe stays optically dark until it is activated by proteases produced by tumor-associated macrophages and accumulates within the lesions, enabling their detection using an endoscope outfitted with a fluorescence detector. We evaluated the probe in multiple murine models and a human-scale porcine model of gastrointestinal carcinogenesis. The probe provides fluorescence-guided surveillance of gastrointestinal lesions and augments histopathological analysis by highlighting areas of dysplasia as small as 400 µm, which were visibly discernible with significant tumor-to-background ratios, even in tissues with a background of severe inflammation and ulceration. Given these results, we anticipate that this probe will enable sensitive fluorescence-guided biopsies, even in the presence of highly inflamed colorectal tissue, which will improve early diagnosis to prevent gastrointestinal cancers.

Merged Molecular Switches Excel as Optoacoustic Dyes: Azobenzene-Cyanines Are Loud and Photostable NIR Imaging Agents.

Optoacoustic (or photoacoustic) imaging promises micron-resolution noninvasive bioimaging with much deeper penetration (>cm) than fluorescence. However, optoacoustic imaging of enzyme activity would require loud, photostable, NIR-absorbing molecular contrast agents, which remain unknown. Most organic molecular contrast agents are repurposed fluorophores, with severe shortcomings of photoinstability or phototoxicity under optoacoustic imaging, as consequences of their slow S1→S0 electronic relaxation. We now report that known fluorophores can be rationally modified to reach ultrafast S1→S0 rates, without much extra molecular complexity, simply by merging them with molecular switches. Here, we merge azobenzene switches with cyanine dyes to give ultrafast relaxation (<10 ps, >100-fold faster). Without even adapting instrument settings, these azohemicyanines display outstanding improvements in signal longevity (>1000-fold increase of photostability) and signal loudness (>3-fold even at time zero). We show why this simple but unexplored design strategy can still offer stronger performance in the future, and can also increase the spatial resolution and the quantitative linearity of photoacoustic response over extended longitudinal imaging. By bringing the world of molecular switches and rotors to bear on problems facing optoacoustic agents, this practical strategy will help to unleash the full potential of optoacoustic imaging in fundamental studies and translational uses.

Towards in vivo characterization of thyroid nodules suspicious for malignancy using multispectral optoacoustic tomography.

PURPOSE: Patient-tailored management of thyroid nodules requires improved risk of malignancy stratification by accurate preoperative nodule assessment, aiming to personalize decisions concerning diagnostics and treatment. Here, we perform an exploratory pilot study to identify possible patterns on multispectral optoacoustic tomography (MSOT) for thyroid malignancy stratification. For the first time, we directly correlate MSOT images with histopathology data on a detailed level. METHODS: We use recently enhanced data processing and image reconstruction methods for MSOT to provide next-level image quality by means of improved spatial resolution and spectral contrast. We examine optoacoustic features in thyroid nodules associated with vascular patterns and correlate these directly with reference histopathology. RESULTS: Our methods show the ability to resolve blood vessels with diameters of 250 µm at depths of up to 2 cm. The vessel diameters derived on MSOT showed an excellent correlation (R2-score of 0.9426) with the vessel diameters on histopathology. Subsequently, we identify features of malignancy observable in MSOT, such as intranodular microvascularity and extrathyroidal extension verified by histopathology. Despite these promising features in selected patients, we could not determine statistically relevant differences between benign and malignant thyroid nodules based on mean oxygen saturation in thyroid nodules. Thus, we illustrate general imaging artifacts of the whole field of optoacoustic imaging that reduce image fidelity and distort spectral contrast, which impedes quantification of chromophore presence based on mean concentrations. CONCLUSION: We recommend examining optoacoustic features in addition to chromophore quantification to rank malignancy risk. We present optoacoustic images of thyroid nodules with the highest spatial resolution and spectral contrast to date, directly correlated to histopathology, pushing the clinical translation of MSOT.

Short-wavelength optoacoustic spectroscopy based on water muting.

Infrared (IR) optoacoustic spectroscopy can separate a multitude of molecules based on their absorption spectra. However, the technique is limited when measuring target molecules in aqueous solution by strong water absorption at IR wavelengths, which reduces detection sensitivity. Based on the dependence of optoacoustic signal on the temperature of the probed medium, we introduce cooled IR optoacoustic spectroscopy (CIROAS) to mute water contributions in optoacoustic spectroscopy. We showcase that spectral measurements of proteins, lipids, and glucose in the short-wavelength IR region, performed at 4 °C, lead to marked sensitivity improvements over conventional optoacoustic or IR spectroscopy. We elaborate on the dependence of optoacoustic signals on water temperature and demonstrate polarity changes in the recorded signal at temperatures below 4 °C. We further elucidate the dependence of the optoacoustic signal and the muting temperature on sample concentration and demonstrate that changes in these dependences enable quantification of the solute concentration. We discuss how CIROAS may enhance abilities for molecular sensing in the IR.

Targeted Hsp70 fluorescence molecular endoscopy detects dysplasia in Barrett's esophagus.

PURPOSE: The incidence of esophageal adenocarcinoma (EAC) has been increasing for decades without significant improvements in treatment. Barrett's esophagus (BE) is best established risk factor for EAC, but current surveillance with random biopsies cannot predict progression to cancer in most BE patients due to the low sensitivity and specificity of high-definition white light endoscopy. METHODS: Here, we evaluated the membrane-bound highly specific Hsp70-specific contrast agent Tumor-Penetrating Peptide (Hsp70-TPP) in guided fluorescence molecular endoscopy biopsy. RESULTS: Hsp70 was significantly overexpressed as determined by IHC in dysplasia and EAC compared with non-dysplastic BE in patient samples (n = 12) and in high-grade dysplastic lesions in a transgenic (L2-IL1b) mouse model of BE. In time-lapse microscopy, Hsp70-TPP was rapidly taken up and internalized  by human BE dysplastic patient-derived organoids. Flexible fluorescence endoscopy of the BE mouse model allowed a specific detection of Hsp70-TPP-Cy5.5 that corresponded closely with the degree of dysplasia but not BE. Ex vivo application of Hsp70-TPP-Cy5.5 to freshly resected whole human EAC specimens revealed a high (> 4) tumor-to-background ratio and a specific detection of previously undetected tumor infiltrations. CONCLUSION: In summary, these findings suggest that Hsp70-targeted imaging using fluorescently labeled TPP peptide may improve tumor surveillance in BE patients.

Wide-Field Mid-Infrared Hyperspectral Imaging by Snapshot Phase Contrast Measurement of Optothermal Excitation.

Vibrational microscopy methods based on Raman scattering or infrared absorption provide a label-free approach for chemical-contrast imaging, but employ point-by-point scanning and impose a compromise between the imaging speed and field-of-view (FOV). Optothermal microscopy has been proposed as a promising imaging modality to avoid this compromise, although at restrictively small FOVs capable of imaging only few cells. Here, we present wide-field optothermal mid-infrared microscopy (WOMiM) for wide-field chemical-contrast imaging based on snapshot pump-probe detection of optothermal signal, using a custom-made condenser-free phase contrast microscopy to capture the phase change of samples after mid-infrared irradiation. We achieved chemical contrast for FOVs up to 180 µm in diameter, yielding 10-fold larger imaging areas than the state-of-the-art, at imaging speeds of 1 ms/frame. The maximum possible imaging speed of WOMiM was determined by the relaxation time of optothermal heat, measured to be 32.8 µs in water, corresponding to a frame rate of ∼30 kHz. This proof-of-concept demonstrates that vibrational imaging can be achieved at an unprecedented imaging speed and large FOV with the potential to significantly facilitate label-free imaging of cellular dynamics.

Individual transducer impulse response characterization method to improve image quality of array-based handheld optoacoustic tomography.

The physical properties of each transducer element play a vital role in the quality of images generated in optoacoustic (photoacoustic) tomography using transducer arrays. Thorough experimental characterization of such systems is often laborious and impractical. A shortcoming of the existing impulse response correction methods, however, is the assumption that all transducers in the array are identical and therefore share one electrical impulse response (EIR). In practice, the EIRs of the transducer elements in the array vary, and the effect of this element-to-element variability on image quality has not been investigated so far, to the best of our knowledge. We hereby propose a robust EIR derivation for individual transducer elements in an array using sparse measurements of the total impulse response (TIR) and by solving the linear system for temporal convolution. Thereafter, we combine a simulated spatial impulse response with the derived individual EIRs to obtain a full characterization of the TIR, which we call individual synthetic TIR. Correcting for individual transducer responses, we demonstrate significant improvement in isotropic resolution, which further enhances the clinical potential of array-based handheld transducers.

A Low-Cost Optoacoustic Sensor for Environmental Monitoring.

Attention to Black Carbon (BC) has been rising due to its effects on human health as well its contribution to climate change. Measurements of BC are challenging, as currently used devices are either expensive or impractical for continuous monitoring. Here, we propose an optoacoustic sensor to address this problem. The sensor utilizes a novel ellipsoidal design for refocusing the optoacoustic signal with minimal acoustic energy losses. To reduce the cost of the system, without sacrificing accuracy, an overdriven laser diode and a Quartz Tuning Fork are used as the light source and the sound detector, respectively. The prototype was able to detect BC particles and to accurately monitor changes in concentration in real time and with very good agreement with a reference instrument. The response of the sensor was linearly dependent on the BC particles concentration with a normalized noise equivalent absorption coefficient (NNEA) for soot equal to 7.39 × 10-9 W cm-1 Hz-1/2. Finally, the prototype was able to perform NO2 measurements, demonstrating its ability to accurately monitor both particulate and gaseous pollutants. The proposed sensor has the potential to offer a significant economic impact for BC environmental measurements and source appointment technologies.

Challenging a Preconception: Optoacoustic Spectrum Differs from the Optical Absorption Spectrum of Proteins and Dyes for Molecular Imaging.

Optoacoustic (photoacoustic) imaging has seen marked advances in detection and data analysis, but there is less progress in understanding the photophysics of common optoacoustic contrast agents. This gap blocks the development of novel agents and the accurate analysis and interpretation of multispectral optoacoustic images. To close it, we developed a multimodal laser spectrometer (MLS) to enable the simultaneous measurement of optoacoustic, absorbance, and fluorescence spectra. Herein, we employ MLS to analyze contrast agents (methylene blue, rhodamine 800, Alexa Fluor 750, IRDye 800CW, and indocyanine green) and proteins (sfGFP, mCherry, mKate, HcRed, iRFP720, and smURFP). We found that the optical absorption spectrum does not correlate with the optoacoustic spectrum for the majority of the analytes. We determined that for dyes, the transition underlying an aggregation state has more optoacoustic signal generation efficiency than the monomer transition. For proteins we found a favored optoacoustic relaxation that stems from the neutral or zwitterionic chromophores and unreported photoswitching behavior of tdTomato and HcRed. We then crystalized HcRed in its photoswitch optoacoustic state, confirming structurally the change in isomerization with respect to HcReds' fluorescence state. Finally, on the example of the widely used label tdTomato and the dye indocyanine green, we show the importance of correct photophysical (e.g., spectral and kinetic) information as a prerequisite for spectral-unmixing for in vivo imaging.

NeuBtracker-imaging neurobehavioral dynamics in freely behaving fish.

A long-standing objective in neuroscience has been to image distributed neuronal activity in freely behaving animals. Here we introduce NeuBtracker, a tracking microscope for simultaneous imaging of neuronal activity and behavior of freely swimming fluorescent reporter fish. We showcase the value of NeuBtracker for screening neurostimulants with respect to their combined neuronal and behavioral effects and for determining spontaneous and stimulus-induced spatiotemporal patterns of neuronal activation during naturalistic behavior.

Propagation of structured light through tissue-mimicking phantoms.

Optical interrogation of tissues is broadly considered in biomedical applications. Nevertheless, light scattering by tissue limits the resolution and accuracy achieved when investigating sub-surface tissue features. Light carrying optical angular momentum or complex polarization profiles, offers different propagation characteristics through scattering media compared to light with unstructured beam profiles. Here we discuss the behaviour of structured light scattered by tissue-mimicking phantoms. We study the spatial and the polarization profile of the scattered modes as a function of a range of optical parameters of the phantoms, with varying scattering and absorption coefficients and of different lengths. These results show the non-trivial trade-off between the advantages of structured light profiles and mode broadening, stimulating further investigations in this direction.

Low-cost single-point optoacoustic sensor for spectroscopic measurement of local vascular oxygenation.

Optical sensors developed for the assessment of oxygen in tissue microvasculature, such as those based on near-infrared spectroscopy, are limited in application by light scattering. Optoacoustic methods are insensitive to light scattering, and therefore, they can provide higher specificity and accuracy when quantifying local vascular oxygenation. However, currently, to the best of our knowledge, there is no low-cost, single point, optoacoustic sensor for the dedicated measurement of oxygen saturation in tissue microvasculature. This work introduces a spectroscopic optoacoustic sensor (SPOAS) for the non-invasive measurement of local vascular oxygenation in real time. SPOAS employs continuous wave laser diodes and measures at a single point, which makes it low-cost and portable. The SPOAS performance was benchmarked using blood phantoms, and it showed excellent linear correlation (R2=0.98) with a blood gas analyzer. Subsequent measurements of local vascular oxygenation in living mice during an oxygen stress test correlated well with simultaneous readings from a reference instrument.

Targeting Endothelin Receptors in a Murine Model of Myocardial Infarction Using a Small Molecular Fluorescent Probe.

The endothelin (ET) axis plays a pivotal role in cardiovascular diseases. Enhanced levels of circulating ET-1 have been correlated with an inferior clinical outcome after myocardial infarction (MI) in humans. Thus, the evaluation of endothelin-A receptor (ETAR) expression over time in the course of myocardial injury and healing may offer valuable information toward the understanding of the ET axis involvement in MI. We developed an approach to track the expression of ETAR with a customized molecular imaging probe in a murine model of MI. The small molecular probe based on the ETAR-selective antagonist 3-(1,3-benzodioxol-5-yl)-5-hydroxy-5-(4-methoxyphenyl)-4-[(3,4,5-trimethoxyphenyl)methyl]-2(5H)-furanone (PD156707) was labeled with fluorescent dye, IRDye800cw. Mice undergoing permanent ligation of the left anterior descending artery (LAD) were investigated at day 1, 7, and 21 post surgery after receiving an intravenous injection of the ETAR probe. Cryosections of explanted hearts were analyzed by cryotome-based CCD, and fluorescence reflectance imaging (FRI) and fluorescence signal intensities (SI) were extracted. Fluorescence-mediated tomography (FMT) imaging was performed to visualize probe distribution in the target region in vivo. An enhanced fluorescence signal intensity in the infarct area was detected in cryoCCD images as early as day 1 after surgery and intensified up to 21 days post MI. FRI was capable of detecting significantly enhanced SI in infarcted regions of hearts 7 days after surgery. In vivo imaging by FMT localized enhanced SI in the apex region of infarcted mouse hearts. We verified the localization of the probe and ETAR within the infarct area by immunohistochemistry (IHC). In addition, neovascularized areas were found in the affected myocardium by CD31 staining. Our study demonstrates that the applied fluorescent probe is capable of delineating ETAR expression over time in affected murine myocardium after MI in vivo and ex vivo.

Optical and Optoacoustic Imaging.

The present chapter summarizes progress with optical methods that go beyond human vision. The focus is on two particular technologies: fluorescence molecular imaging and optoacoustic (photoacoustic) imaging. The rationale for the selection of these two methods is that in contrast to optical microscopy techniques, both fluorescence and optoacoustic imaging can achieve large fields of view, i.e., spanning several centimeters in two or three dimensions. Such fields of views relate better to human vision and can visualize large parts of tissue, a necessary premise for clinical detection. Conversely, optical microscopy methods only scan millimeter-sized dimensions or smaller. With such operational capacity, optical microscopy methods need to be guided by another visualization technique in order to scan a very specific area in tissue and typically only provide superficial measurements, i.e., information from depths that are of the order of 0.05-1 mm. This practice has generally limited their clinical applicability to some niche applications, such as optical coherence tomography of the retina. On the other hand, fluorescence molecular imaging and optoacoustic imaging emerge as more global optical imaging methods with wide applications in surgery, endoscopy, and non-invasive clinical imaging, as summarized in the following. The current progress in this field is based on a volume of recent review and other literature that highlights key advances achieved in technology and biomedical applications. Context and figures from references from the authors of this chapter have been used here, as it reflects our general view of the current status of the field.

Optoacoustic mesoscopy shows potential to increase accuracy of allergy patch testing.

BACKGROUND: Differentiation between irritant and allergic skin reactions in epicutaneous patch testing is based largely on subjective clinical criteria, with the risk of high intraobserver and interobserver variability. Novel dermatological imaging using optoacoustic mesoscopy allows quantitative three-dimensional assessment of microvascular biomarkers. OBJECTIVES: We investigated the potential of optoacoustic imaging to improve the precision of patch test evaluation. METHODS: Sixty-nine test reactions and 48 healthy skin sections in 52 patients with suspected type IV allergy were examined using raster-scan optoacoustic mesoscopy. RESULTS: We identified biomarkers from the optoacoustic images. Allergic reactions were associated with higher fragmentation of skin vasculature than irritant reactions (19.5 ± 9.7 vs 14.3 ± 3.7 fragments/100 pixels2 ; P < .05), as well as lower ratio of low- to high-frequency acoustic signals (1.6 ± 0.5 vs 2.0 ± 0.6, P < .05). Allergic reactions graded "++" showed higher vessel fragmentation than reactions graded "+" (25.4 ± 13.2 vs 17.1 ± 6.5 fragments/100 pixels2 ; P < .05). A linear model combining the biomarkers fragmentation and frequency ratio could differentiate allergic from irritant test reactions with an area under the receiving operator characteristic curve of 0.80 (95% confidence interval 0.64-0.91), reaching a sensitivity of 81% and specificity of 63%. CONCLUSIONS: Optoacoustic mesoscopy shows potential to help in differentiating between allergic and irritant test reactions based on novel biomarkers that may reflect vasodilation, vessel tortuosity, and edema.

Sensitivity Enhanced Photoacoustic Imaging Using a High-Frequency PZT Transducer with an Integrated Front-End Amplifier.

Photoacoustic (PA) imaging is a hybrid imaging technique that can provide both structural and functional information of biological tissues. Due to limited permissible laser energy deposited on tissues, highly sensitive PA imaging is required. Here, we developed a 20 MHz lead zirconium titanate (PZT) transducer (1.5 mm × 3 mm) with front-end amplifier circuits for local signal processing to achieve sensitivity enhanced PA imaging. The electrical and acoustic performance was characterized. Experiments on phantoms and chicken breast tissue were conducted to validate the imaging performance. The fabricated prototype shows a bandwidth of 63% and achieves a noise equivalent pressure (NEP) of 0.24 mPa/√Hz and a receiving sensitivity of 62.1 µV/Pa at 20 MHz without degradation of the bandwidth. PA imaging of wire phantoms demonstrates that the prototype is capable of improving the detection sensitivity by 10 dB compared with the traditional transducer without integrated amplifier. In addition, in vitro experiments on chicken breast tissue show that structures could be imaged with enhanced contrast using the prototype and the imaging depth range was improved by 1 mm. These results demonstrate that the transducer with an integrated front-end amplifier enables highly sensitive PA imaging with improved penetration depth. The proposed method holds the potential for visualization of deep tissue structures and enhanced detection of weak physiological changes.

Photocontrollable Proteins for Optoacoustic Imaging.

Photocontrollable proteins revolutionized life-science imaging due to their contribution to subdiffraction-resolution optical microscopy. They might have yet another lasting impact on photo- or optoacoustic imaging (OA). OA combines optical contrast with ultrasound detection enabling high-resolution real-time in vivo imaging well-beyond the typical penetration depth of optical methods. While OA already showed numerous applications relying on endogenous contrast from blood hemoglobin or lipids, its application in the life-science was limited by a lack of labels overcoming the strong signal from the aforementioned endogenous absorbers. Here, a number of recent studies showed that photocontrollable proteins provide the means to overcome this barrier eventually enabling OA to image small cell numbers in a complete organism in vivo. In this Feature article, we introduce the key photocontrollable proteins, explain the basic concepts, and highlight achievements that have been already made.