Toward drift-free high-throughput nanoscopy through adaptive intersection maximization.

Single-molecule localization microscopy (SMLM) often suffers from suboptimal resolution due to imperfect drift correction. Existing marker-free drift correction algorithms often struggle to reliably track high-frequency drift and lack the computational efficiency to manage large, high-throughput localization datasets. We present an adaptive intersection maximization-based method (AIM) that leverages the entire dataset's information content to minimize drift correction errors, particularly addressing high-frequency drift, thereby enhancing the resolution of existing SMLM systems. We demonstrate that AIM can robustly and efficiently achieve an angstrom-level tracking precision for high-throughput SMLM datasets under various imaging conditions, resulting in an optimal resolution in simulated and biological experimental datasets. We offer AIM as one simple, model-free software for instant resolution enhancement with standard CPU devices.

Self-enhancing sono-inks enable deep-penetration acoustic volumetric printing.

Volumetric printing, an emerging additive manufacturing technique, builds objects with enhanced printing speed and surface quality by forgoing the stepwise ink-renewal step. Existing volumetric printing techniques almost exclusively rely on light energy to trigger photopolymerization in transparent inks, limiting material choices and build sizes. We report a self-enhancing sonicated ink (or sono-ink) design and corresponding focused-ultrasound writing technique for deep-penetration acoustic volumetric printing (DAVP). We used experiments and acoustic modeling to study the frequency and scanning rate-dependent acoustic printing behaviors. DAVP achieves the key features of low acoustic streaming, rapid sonothermal polymerization, and large printing depth, enabling the printing of volumetric hydrogels and nanocomposites with various shapes regardless of their optical properties. DAVP also allows printing at centimeter depths through biological tissues, paving the way toward minimally invasive medicine.

Epinephrine-induced Effects on Cerebral Microcirculation and Oxygenation Dynamics Using Multimodal Monitoring and Functional Photoacoustic Microscopy.

BACKGROUND: The administration of epinephrine after severe refractory hypotension, shock, or cardiac arrest restores systemic blood flow and major vessel perfusion but may worsen cerebral microvascular perfusion and oxygen delivery through vasoconstriction. The authors hypothesized that epinephrine induces significant microvascular constriction in the brain, with increased severity after repetitive dosing and in the aged brain, eventually leading to tissue hypoxia. METHODS: The authors investigated the effects of intravenous epinephrine administration in healthy young and aged C57Bl/6 mice on cerebral microvascular blood flow and oxygen delivery using multimodal in vivo imaging, including functional photoacoustic microscopy, brain tissue oxygen sensing, and follow-up histologic assessment. RESULTS: The authors report three main findings. First, after epinephrine administration, microvessels exhibited severe immediate vasoconstriction (57 ± 6% of baseline at 6 min, P < 0.0001, n = 6) that outlasted the concurrent increase in arterial blood pressure, while larger vessels demonstrated an initial increase in flow (108 ± 6% of baseline at 6 min, P = 0.02, n = 6). Second, oxyhemoglobin decreased significantly within cerebral vessels with a more pronounced effect in smaller vessels (microvessels to 69 ± 8% of baseline at 6 min, P < 0.0001, n = 6). Third, oxyhemoglobin desaturation did not indicate brain hypoxia; on the contrary, brain tissue oxygen increased after epinephrine application (from 31 ± 11 mmHg at baseline to 56 ± 12 mmHg, 80% increase, P = 0.01, n = 12). In the aged brains, microvascular constriction was less prominent yet slower to recover compared to young brains, but tissue oxygenation was increased, confirming relative hyperoxia. CONCLUSIONS: Intravenous application of epinephrine induced marked cerebral microvascular constriction, intravascular hemoglobin desaturation, and paradoxically, an increase in brain tissue oxygen levels, likely due to reduced transit time heterogeneity.

Multiscale photoacoustic tomography using reversibly switchable thermochromics.

Significance: Based on acoustic detection of optical absorption, photoacoustic tomography (PAT) allows functional and molecular imaging beyond the optical diffusion limit with high spatial resolution. However, multispectral functional and molecular PAT is often limited by decreased spectroscopic accuracy and reduced detection sensitivity in deep tissues, mainly due to wavelength-dependent optical attenuation and inaccurate acoustic inversion. Aim: Previous work has demonstrated that reversible color-shifting can drastically improve the detection sensitivity of PAT by suppressing nonswitching background signals. We aim to develop a new color switching-based PAT method using reversibly switchable thermochromics (ReST). Approach: We developed a family of ReST with excellent water dispersion, biostability, and temperature-controlled color changes by surface modification of commercial thermochromic microcapsules with the hydrophilic polysaccharide alginate. Results: The optical absorbance of the ReST was switched on and off repeatedly by modulating the surrounding temperature, allowing differential photoacoustic detection that effectively suppressed the nonswitching background signal and substantially improved image contrast and detection sensitivity. We demonstrate reversible thermal-switching imaging of ReST in vitro and in vivo using three PAT modes at different length scales. Conclusions: ReST-enabled PAT is a promising technology for high-sensitivity deep tissue imaging of molecular activity in temperature-related biomedical applications, such as cancer thermotherapy.

Glassfrogs conceal blood in their liver to maintain transparency.

Transparency in animals is a complex form of camouflage involving mechanisms that reduce light scattering and absorption throughout the organism. In vertebrates, attaining transparency is difficult because their circulatory system is full of red blood cells (RBCs) that strongly attenuate light. Here, we document how glassfrogs overcome this challenge by concealing these cells from view. Using photoacoustic imaging to track RBCs in vivo, we show that resting glassfrogs increase transparency two- to threefold by removing ~89% of their RBCs from circulation and packing them within their liver. Vertebrate transparency thus requires both see-through tissues and active mechanisms that "clear" respiratory pigments from these tissues. Furthermore, glassfrogs' ability to regulate the location, density, and packing of RBCs without clotting offers insight in metabolic, hemodynamic, and blood-clot research.

Intraperitoneal administration for sustained photoacoustic contrast agent imaging.

Photoacoustic (PA) imaging at 1064 nm in the second near-infrared (NIR-II) has attracted recent attention. We recently reported a surfactant-based formulation of a NIR-II dye (BIBDAH) for NIR-II PA contrast. Here, we investigated BIBDAH as a NIR-II PA contrast agent for longitudinal preclinical PA imaging. When administered to mice by the conventional intravenous (I.V.) route, BIBDAH was rapidly cleared from circulation, as indicated by a decrease in NIR-II absorption in sampled plasma. Conversely, when mice were injected with BIBDAH by the intraperitoneal (I.P.) route, peak NIR-II absorption levels in plasma were lower initially, but there was a sustained dye presence that resulted in a more consistent concentration of dye in plasma over 2 days. Increasing the I.P. injection dose and volume resulted in increased NIR-II area under the curve (AUC) in serum. Bimodal PA and ultrasound imaging reflected these results, showing a rapid decrease in PA signal in blood with I.V. administration, but permitting sustained imaging with I.P. administration. These results show that I.P. administration can be considered as an administration route in preclinical animal studies for improved longitudinal observation with more consistent contrast signal intensity.

High-speed wide-field photoacoustic microscopy using a cylindrically focused transparent high-frequency ultrasound transducer.

Combining focused optical excitation and high-frequency ultrasound detection, optical-resolution photoacoustic microscopy (OR-PAM) can provide micrometer-level spatial resolution with millimeter-level penetration depth and has been employed in a variety of biomedical applications. However, it remains a challenge for OR-PAM to achieve a high imaging speed and a large field of view at the same time. In this work, we report a new approach to implement high-speed wide-field OR-PAM, using a cylindrically-focused transparent ultrasound transducer (CFT-UT). The CFT-UT is made of transparent lithium niobate coated with indium-tin-oxide as electrodes. A transparent cylindrical lens is attached to the transducer surface to provide an acoustic focal line with a length of 9 mm. The excitation light can pass directly through the CFT-UT from the above and thus enables a reflection imaging mode. High-speed imaging is achieved by fast optical scanning of the focused excitation light along the CFT-UT focal line. With the confocal alignment of the optical excitation and acoustic detection, a relatively high detection sensitivity is maintained over the entire scanning range. The CFT-UT-based OR-PAM system has achieved a cross-sectional frame rate of 500 Hz over the scanning range of 9 mm. We have characterized the system's performance on phantoms and demonstrated its application on small animal models in vivo. We expect the new CFT-UT-based OR-PAM will find matched biomedical applications that need high imaging speed over a large field of view.

Detection of weak optical absorption by optical-resolution photoacoustic microscopy.

Optical-resolution photoacoustic microscopy (OR-PAM) is one of the major implementations of photoacoustic (PA) imaging. With tightly focused optical illumination and high-frequency ultrasound detection, OR-PAM provides micrometer-level resolutions as well as high sensitivity to optical absorption contrast. Traditionally, it is assumed that the detected PA signal in OR-PAM has a linear dependence on the target's optical absorption coefficient, which is the basis for quantitative functional and molecular PA imaging. In this paper, we demonstrate that, due to the limited detection bandwidth and detection view, OR-PAM can have a strong nonlinear dependence on the optical absorption, especially for weak optical absorption (<10 cm-1). We have investigated the nonlinear dependence in OR-PAM using numerical simulations, analyzed the underlining mechanisms, proposed potential solutions, and experimentally confirmed the results on phantoms. This work may correct a traditional misunderstanding of the OR-PAM signals and improve quantitative accuracy for functional and molecular applications.

Photoacoustic imaging of 3D-printed vascular networks.

Thrombosis in the circulation system can lead to major myocardial infarction and cardiovascular deaths. Understanding thrombosis formation is necessary for developing safe and effective treatments. In this work, using digital light processing (DLP)-based 3D printing, we fabricated sophisticatedin vitromodels of blood vessels with internal microchannels that can be used for thrombosis studies. In this regard, photoacoustic microscopy (PAM) offers a unique advantage for label-free visualization of the 3D-printed vessel models, with large penetration depth and functional sensitivity. We compared the imaging performances of two PAM implementations: optical-resolution PAM and acoustic-resolution PAM, and investigated 3D-printed vessel structures with different patterns of microchannels. Our results show that PAM can provide clear microchannel structures at depths up to 3.6 mm. We further quantified the blood oxygenation in the 3D-printed vascular models, showing that thrombi had lower oxygenation than the normal blood. We expect that PAM can find broad applications in 3D printing and bioprinting forin vitrostudies of various vascular and other diseases.

High-speed functional photoacoustic microscopy using a water-immersible two-axis torsion-bending scanner.

Optical-resolution photoacoustic microscopy (OR-PAM) can provide functional, anatomical, and molecular images at micrometer level resolution with an imaging depth of less than 1 mm in tissue. However, the imaging speed of traditional OR-PAM is often low due to the point-by-point mechanical scanning and cannot capture time-sensitive dynamic information. In this work, we demonstrate a recent effort in improving the imaging speed of OR-PAM, using a newly developed water-immersible two-axis scanner. Driven by water-compatible electromagnetic actuation force, the new scanning mirror employs a novel torsion-bending mechanism to achieve fast 2D scanning. The torsion scanning along the fast-axis works in the resonant model, and the bending scanning along the slow-axis operate at the quasi-static mode. The scanning speed and scanning range along the two axes can be independently adjusted. Steered by the two-axis torsion-bending scanning mirror immersed in water, the focused excitation light and the generated acoustic wave can be confocally aligned over the entire imaging area. Thus, a high imaging speed can be achieved without sacrificing the detection sensitivity. Equipped with the torsion-bending scanner, the high-speed OR-PAM system has achieved a cross-sectional frame rate of 400 Hz, and a volumetric imaging speed of 1 Hz over a field of view of 1.5 × 2.5 mm2. We have also demonstrated high-speed OR-PAM of the hemodynamic changes in response to pharmaceutical and physiological challenges in small animal models in vivo. We expect the torsion-bending scanner based OR-PAM will find matched biomedical studies of tissue dynamics.

Structural and Functional Imaging of the Retina in Central Retinal Artery Occlusion - Current Approaches and Future Directions.

Central retinal artery occlusion (CRAO) is a form of acute ischemic stroke which affects the retina. Intravenous thrombolysis is emerging as a compelling therapeutic approach. However, it is not known which patients may benefit from this therapy because there are no imaging modalities that adequately distinguish viable retina from irreversibly infarcted retina. The inner retina receives arterial supply from the central retinal artery and there is robust collateralization between this circulation and the outer retinal circulation, provided by the posterior ciliary circulation. Fundus photography can show canonical changes associated with CRAO including a cherry-red spot, arteriolar boxcarring and retinal pallor. Fluorescein angiography provides 2-dimensional imaging of the retinal circulation and can distinguish a complete from a partial CRAO as well as central versus peripheral retinal non-perfusion. Transorbital ultrasonography may assay flow through the central retinal artery and is useful in the exclusion of other orbital pathology that can mimic CRAO. Optical coherence tomography provides structural information on the different layers of the retina and exploratory work has described its utility in determining the time since onset of ischemia. Two experimental techniques are discussed. 1) Retinal functional imaging permits generation of capillary perfusion maps and can assay retinal oxygenation and blood flow velocity. 2) Photoacoustic imaging combines the principles of optical excitation and ultrasonic detection and - in animal studies - has been used to determine the retinal oxygen metabolic rate. Future techniques to determine retinal viability in clinical practice will require rapid, easily used, and reproducible methods that can be deployed in the emergency setting.

Deep image prior for undersampling high-speed photoacoustic microscopy.

Photoacoustic microscopy (PAM) is an emerging imaging method combining light and sound. However, limited by the laser's repetition rate, state-of-the-art high-speed PAM technology often sacrifices spatial sampling density (i.e., undersampling) for increased imaging speed over a large field-of-view. Deep learning (DL) methods have recently been used to improve sparsely sampled PAM images; however, these methods often require time-consuming pre-training and large training dataset with ground truth. Here, we propose the use of deep image prior (DIP) to improve the image quality of undersampled PAM images. Unlike other DL approaches, DIP requires neither pre-training nor fully-sampled ground truth, enabling its flexible and fast implementation on various imaging targets. Our results have demonstrated substantial improvement in PAM images with as few as 1.4 % of the fully sampled pixels on high-speed PAM. Our approach outperforms interpolation, is competitive with pre-trained supervised DL method, and is readily translated to other high-speed, undersampling imaging modalities.

Photoacoustic imaging of in vivo hemodynamic responses to sodium nitroprusside.

The in vivo hemodynamic impact of sodium nitroprusside (SNP), a widely used antihypertensive agent, has not been well studied. Here, we applied functional optical-resolution photoacoustic microscopy (OR-PAM) to study the hemodynamic responses to SNP in mice in vivo. As expected, after the application of SNP, the systemic blood pressure (BP) was reduced by 53%. The OR-PAM results show that SNP induced an arterial vasodilation of 24% and 23% in the brain and skin, respectively. A weaker venous vasodilation of 9% and 5% was also observed in the brain and skin, respectively. The results show two different types of blood oxygenation response. In mice with decreased blood oxygenation, the arterial and venous oxygenation was respectively reduced by 6% and 13% in the brain, as well as by 7% and 18% in the skin. In mice with increased blood oxygenation, arterial and venous oxygenation was raised by 4% and 22% in the brain, as well as by 1% and 9% in the skin. We observed venous change clearly lagged the arterial change in the skin, but not in the brain. Our results collectively show a correlation among SNP induced changes in systemic BP, vessel size and blood oxygenation.

Reconstructing Undersampled Photoacoustic Microscopy Images Using Deep Learning.

One primary technical challenge in photoacoustic microscopy (PAM) is the necessary compromise between spatial resolution and imaging speed. In this study, we propose a novel application of deep learning principles to reconstruct undersampled PAM images and transcend the trade-off between spatial resolution and imaging speed. We compared various convolutional neural network (CNN) architectures, and selected a Fully Dense U-net (FD U-net) model that produced the best results. To mimic various undersampling conditions in practice, we artificially downsampled fully-sampled PAM images of mouse brain vasculature at different ratios. This allowed us to not only definitively establish the ground truth, but also train and test our deep learning model at various imaging conditions. Our results and numerical analysis have collectively demonstrated the robust performance of our model to reconstruct PAM images with as few as 2% of the original pixels, which can effectively shorten the imaging time without substantially sacrificing the image quality.

A near-infrared genetically encoded calcium indicator for in vivo imaging.

While calcium imaging has become a mainstay of modern neuroscience, the spectral properties of current fluorescent calcium indicators limit deep-tissue imaging as well as simultaneous use with other probes. Using two monomeric near-infrared (NIR) fluorescent proteins (FPs), we engineered an NIR Förster resonance energy transfer (FRET)-based genetically encoded calcium indicator (iGECI). iGECI exhibits high levels of brightness and photostability and an increase up to 600% in the fluorescence response to calcium. In dissociated neurons, iGECI detects spontaneous neuronal activity and electrically and optogenetically induced firing. We validated the performance of iGECI up to a depth of almost 400 µm in acute brain slices using one-photon light-sheet imaging. Applying hybrid photoacoustic and fluorescence microscopy, we simultaneously monitored neuronal and hemodynamic activities in the mouse brain through an intact skull, with resolutions of ~3 µm (lateral) and ~25-50 µm (axial). Using two-photon imaging, we detected evoked and spontaneous neuronal activity in the mouse visual cortex, with fluorescence changes of up to 25%. iGECI allows biosensors and optogenetic actuators to be multiplexed without spectral crosstalk.

In vivo photoacoustic imaging dynamically monitors the structural and functional changes of ischemic stroke at a very early stage.

Ischemic stroke (IS) is one of the leading causes of death and accounts for 85% of stroke cases. Since the symptoms are not obvious, diagnosis of IS, particularly at an early stage, is a great challenge. Photoacoustic imaging combines high sensitivity of optical imaging and fine resolution of ultrasonography to non-invasively provide structural and functional information of IS. Methods: We adopted three rapid photoacoustic imaging systems with varying characteristics, including a portable handheld photoacoustic system, high-sensitivity bowl-shaped array photoacoustic computed tomography (PACT), and high-resolution photoacoustic microscopy (PAM) to assess the stereoscopic and comprehensive pathophysiological status of IS at an early stage. Two representative models of IS, referring to photothrombosis and middle cerebral artery occlusion (MCAO) models, were established to verify the feasibility of photoacoustic imaging detection. Results: Non-invasive, rapid PACT of the IS model in mouse provided structural information of the brain lesion, achieving early disease identification (5 min after the onset of disease). Moreover, it was able to dynamically reflect disease progression. Quantitative high-resolution PAM allowed observation of pathological changes in the microvascular system of mouse brain. In terms of functional imaging, significant differences in oxygen saturation (sO2) levels between infarcted and normal areas could be observed by PACT, permitting effective functional parameters for the diagnosis of IS. Conclusions: We used PACT to perform full-view structural imaging and functional imaging of sO2 in IS at the macroscopic level, and then observed the microvascular changes in the infarcted area at the microscopic level by using PAM. This work may provide new tools for the early diagnosis of IS and its subsequent complications as well as assessment of disease progression.

Simultaneous photoacoustic imaging of intravascular and tissue oxygenation.

Hypoxia, a low tissue oxygenation condition caused by insufficient oxygen supply, leads to potentially irreversible tissue damage, such as brain infarction during stroke. Intravascular oxygenation has long been used by photoacoustic imaging, among other imaging modalities, to study hypoxia. However, intravascular oxygenation describes only the oxygen supply via microcirculation, which does not directly reflect the amount of free oxygen available for metabolism in the interstitial fluid. Therefore, to fully understand hypoxia, it is highly desirable to monitor blood oxygenation as well as tissue oxygenation during the same biological process. In this work, by combining high-resolution photoacoustic microscopy (PAM) and a novel bioreducible N-oxide-based hypoxia-sensitive probe HyP-650, we have demonstrated simultaneous imaging of intravascular oxygenation and tissue hypoxia. We have established detailed chemical, optical, and photoacoustic properties of HyP-650 for hypoxic activation in vitro and in living cells. We have also performed PAM on hindlimb ischemia models and tumor-bearing mice to study the correlation between intravascular oxygenation and tissue oxygenation at various hypoxic levels. We expect that Hyp-650 enhanced photoacoustic imaging will find a variety of applications in brain and cancer research.

Potential tumor suppressing role of microRNA-545 in epithelial ovarian cancer.

Epithelial ovarian cancer (EOC) is the most common type of ovarian cancer, which exhibits invasive traits. MicroRNAs (miRNAs/miRs) have been demonstrated to serve important functions in the pathogenesis of EOC. However, the function of miR-545 in EOC remains unknown. In the present study, the function of miR-545 in EOC was analyzed and it was identified that miR-545 is downregulated in EOC tissues and cell lines. Additionally, a low level of miR-545 expression was associated with a low survival rate of patients with EOC. Furthermore, overexpression of miR-545 inhibited cell growth and promoted apoptosis. Suppression of miR-545 promoted cell growth and inhibited apoptosis. Additionally, the RAC-γ serine/threonine-protein kinase gene was targeted by miR-545. Thus, it may be concluded that miR-545 exhibited antitumor traits in EOC.

Noninvasive photoacoustic and fluorescent tracking of optical dye labeled T cellular activities of diseased sites at new depth.

The migration of immune cells is crucial to the immune response. Visualization of these processes has previously been limited because of the imaging depth. We developed a deep-penetrating, sensitive and high-resolution method to use fast photoacoustic tomography (PAT) to image the dynamic changes of T cells in lymph node and diseases at new depth (up to 9.5 mm). T cells labeled with NIR-797-isothiocyanate, an excellent near-infrared photoacoustic and fluorescent agent, were intravenously injected to the mice. We used fluorescence imaging to determine the location of T cells roughly and photoacoustic imaging is used to observe T-cell responses in diseased sites deeply and carefully. The dynamic changes of T cells in lymph node, acute disease (bacterial infection) and chronic disease (tumor) were observed noninvasively by photoacoustic and fluorescence imaging at different time points. T cells accumulated gradually and reached a maximum at 4 hours and declined afterwards in lymph node and bacterial infection site. At tumor model, T cells immigrated to the tumor with a maximum at 12 hours. Our study can not only provide a new observing method for immune activities tracking, but also enable continuous monitoring for therapeutic interventions.