Photoacoustic-ultrasonic dual mode microscopy with local speed-of-sound estimation

Synthetic aperture imaging and virtual point detection have been exploited to extend the depth-ofview of photoacoustic microscopy. The approach is commonly based on a constant assumed sound speed, which reduces image quality. We propose a new self-adaptive technique to estimate the speed of sound for being integrated with this hybrid strategy. It is accomplished through linear regression between the square of time-of-fight detected at individual virtual detectors and the square of their horizontal distances on the focal plane. The imaging results show our proposed method can significantly improve the lateral resolution, imaging intensity and spatial precision for inhomogeneous tissue.Research Grant G102928 with CAMBRIDGE UNIVERSITY NANJING CENTRE OF TECHNOLOGY AND INNOVATION CO LIMITE

Development of High-speed Photoacoustic Imaging technology and Its Applications in Biomedical Research

Photoacoustic (PA) tomography (PAT) is a novel imaging modality that combines the fine lateral resolution from optical imaging and the deep penetration from ultrasonic imaging, and provides rich optical-absorption–based images. PAT has been widely used in extracting structural and functional information from both ex vivo tissue samples to in vivo animals and humans with different length scales by imaging various endogenous and exogenous contrasts at the ultraviolet to infrared spectrum. For example, hemoglobin in red blood cells is of particular interest in PAT since it is one of the dominant absorbers in tissue at the visible wavelength.The main focus of this dissertation is to develop high-speed PA microscopy (PAM) technologies. Novel optical scanning, ultrasonic detection, and laser source techniques are introduced in this dissertation to advance the performance of PAM systems. These upgrades open up new avenues for PAM to be applicable to address important biomedical challenges and enable fundamental physiological studies.First, we investigated the feasibility of applying high-speed PAM to the detection and imaging of circulating tumor cells (CTCs) in melanoma models, which can provide valuable information about a tumor’s metastasis potentials. We probed the melanoma CTCs at the near-infrared wavelength of 1064 nm, where the melanosomes absorb more strongly than hemoglobin. Our high-speed PA flow cytography system successfully imaged melanoma CTCs in travelling trunk vessels. We also developed a concurrent laser therapy device, hardware-triggered by the CTC signal, to photothermally lyse the CTC on the spot in an effort to inhibit metastasis.Next, we addressed the detection sensitivity issue in the previous study. We employed the stimulated Raman scattering (SRS) effect to construct a high-repetition-rate Raman laser at 658 nm, where the contrast between a melanoma CTC and the blood background is near the highest. Our upgraded PA flow cytography successfully captured sequential images of CTCs in mouse melanoma xenograft model, with a significantly improved contrast-to-noise ratio compared to our previous results. This technology is readily translatable to the clinics to extract the information of a tumor’s metastasis risks.We extended the Raman laser technology to the field of brain functional studies. We developed a MEMS (micro-electromechanical systems) scanner for fast optical scanning, and incorporated it to a dual-wavelength functional PAM (fPAM) for high-speed imaging of cerebral hemodynamics in mouse. This fPAM system successfully imaged transient changes in blood oxygenation at cerebral micro-vessels in response to brief somatic stimulations. This fPAM technology is a powerful tool for neurological studies.Finally, we explored some approaches of reducing the size the PAM imaging head in an effort to translate our work to the field of wearable biometric monitors. To miniaturize the ultrasonic detection device, we fabricated a thin-film optically transparent piezoelectric detector for detecting PA waves. This technology could enable longitudinal studies on free-moving animals through a wearable version of PAM

Methods for optical skin clearing in molecular optical imaging in dermatology

This short review describes recent progress in using optical clearing (OC) technique in skin studies. Optical clear-ing is an efficient tool for enhancing the probing depth and data quality in multiphoton microscopy and Raman spec-troscopy. Here, we discuss the main mechanisms of OC, its safety, advantages, and limitations. The data on the OC effect on the skin water content are presented. It was demonstrated that 70% glycerol and 100% OmnipaqueTM 300 reduce the water content in the skin. Both OC agents (OCAs) significantly affect the strongly bound and weakly bound water. However, OmnipaqueTM 300 causes considerably less skin dehydration than glycerol. In addition, the results of examination of the OC effect on autofluorescence in two-photon excitation and background fluorescence in Raman scattering at different skin depths are presented. It is shown that OmnipaqueTM 300 is a promising OCA due to its ability to reduce background fluo-rescence in the upper skin layers. The possibility of multimodal imaging combining optical methods and OC technique is discussed

Submicron-resolution Photoacoustic Microscopy of Endogenous Light-absorbing Biomolecules

Photoacoustic imaging in biomedicine has the unique advantage of probing endogenous light absorbers at various length scales with a 100% relative sensitivity. Among the several modalities of photoacoustic imaging, optical-resolution photoacoustic microscopy (OR-PAM) can achieve high spatial resolution, on the order of optical wavelength, at \u3c1 mm depth in biological tissue (the optical ballistic regime). OR-PAM has been applied successfully to structural and functional imaging of blood vasculature and red blood cells in vivo. Any molecules which absorb sufficient light at certain wavelengths can potentially be imaged by PAM. Compared with pure optical imaging, which typically targets fluorescent markers, label-free PAM avoids the major concerns that the fluorescent labeling probes may disturb the function of biomolecules and may have an insufficient density. This dissertation aims to advance label-free OR-PAM to the subcellular scale. The first part of this dissertation describes the technological advancement of PAM yielding high spatial resolution in 3D. The lateral resolution was improved by using optical objectives with high numerical apertures for optical focusing. The axial resolution was improved by using broadband ultrasonic transducers for ultrasound detection. We achieved 220 nm lateral resolution in transmission mode, 0.43 µm lateral resolution in reflection mode, 7.6 µm axial resolution in normal tissue, and 5.8 µm axial resolution with silicone oil immersion/injection. The achieved lateral resolution and axial resolution were the finest reported at the time. With high-resolution in 3D, PAM was demonstrated to resolve cellular and subcellular structures in vivo, such as red blood cells and melanosomes in melanoma cells. Compared with previous PAM systems, our high-resolution PAM could resolve capillaries in mouse ears more clearly. As an example application, we demonstrated intracellular temperature imaging, assisted by fluorescence signal detection, with sub-degree temperature resolution and sub-micron lateral resolution. The second part of this dissertation describes the exploration of endogenous light-absorbing biomolecules for PAM. We demonstrated cytochromes and myoglobin as new absorption contrasts for PAM and identified the corresponding optimal wavelengths for imaging. Fixed fibroblasts on slides and mouse ear sections were imaged by PAM at 422 nm and 250 nm wavelengths to reveal cytoplasms and nuclei, respectively, as confirmed by standard hematoxylin and eosin (H&E) histology. By imaging a blood-perfused mouse heart at 532 nm down to 150 µm in depth, we derived the myocardial sheet thickness and the cleavage height from an undehydrated heart for the first time. The findings promote PAM at new wavelengths and open up new possibilities for characterizing biological tissue. Of particular interest, dual-wavelength PAM around 250 nm and 420 nm wavelengths is analogous to H&E histology. The last part of this dissertation describes the development of sectioning photoacoustic microscopy (SPAM), based on the advancement in spatial resolution and new contrasts for PAM, with applications in brain histology. Label-free SPAM, assisted by a microtome, acquires serial distortion-free images of a specimen on the surface. By exciting cell nuclei at 266 nm wavelength with high resolution, SPAM could pinpoint cell nuclei sensitively and specifically in the mouse brain section, as confirmed by H&E histology. SPAM was demonstrated to generate high-resolution 3D images, highlighting cell nuclei, of formalin-fixed paraffin-embedded mouse brains without tissue staining or clearing. SPAM can potentially serve as a high-throughput and minimal-artifact substitute for histology, probe many other biomolecules and cells, and become a universal tool for animal or human whole-organ microscopy, with diverse applications in life sciences

A Genetically-Encoded Reporter for In Vivo Imaging in Deep Tissues

Introduction. The ability to track cells in living organisms with sensitivity, accuracy and high spatial resolution would revolutionize the way we study disease. Reporter genes are valuable tools as they encode detectible products, allowing for quantitative “reporting” of cells that express them. Previously, a gene encoding Organic anion-transporting polypeptide 1a1 (Oatp1a1) was established as a magnetic resonance imaging (MRI) reporter based on its ability to take up the paramagnetic contrast agent gadolinium ethoxybenzyl diethylenetriamine pentaacetic acid (Gd-EOB-DTPA). Our objective was to assess, characterize and further develop this system for whole-body tracking of cells in vivo. Methods. Cancer cells were engineered to synthetically express Oatp1a1, or Oatp1b3, a closely related human transporter protein. In our first study, T1-weighted images of Oatp1a1-expressing primary tumours in preclinical animals were acquired before and after administration of 0.1-mmol/kg Gd-EOB-DTPA at 3-Tesla. At endpoint, heterogenous contrast enhancement patterns within the primary tumour architecture were compared to whole-tumour fluorescent histology. In the next study, T1-weighted images of Oatp1b3-expressing primary tumours, and their spontaneous metastases to the lymph nodes and lungs, were acquired before and after administration of 1-mmol/kg Gd-EOB-DTPA at 3-Tesla. In the final study, the feasibility of Oatp1b3 as a photoacoustic reporter gene was assessed by acquiring full-spectrum near infrared photoacoustic images of primary tumours in preclinical animals before and after administration of 8-mg/kg indocyanine green. Results. We were able to demonstrate the feasibility of imaging cancer cells with Oatp1a1 at 3-Tesla and 0.1 mmol/kg Gd-EOB-DTPA. Importantly, as primary tumours grew over time, heterogeneous contrast enhancement patterns that emerged near-endpoint strongly correlated to viable cell distributions on whole-tumour histology. Oatp1b3 was also shown to operate as a MRI reporter gene at 3-Tesla, based on the same principle as Oatp1a1. Impressively, single lymph node metastases and the formation of micrometastases in the lungs of preclinical animals were detected with Oatp1b3-MRI. Finally, we also demonstrated the ability of Oatp1b3 to operate as a photoacoustic reporter gene based on its ability to take up indocyanine green. Conclusion. The Oatp1 reporter gene system is a versatile imaging tool for longitudinal tracking of engineered cells in vivo with sensitivity, high resolution, and 3-dimensional spatial information

In vivo Observation of the Release of Norepinephrine and In Vivo Optical Studies on the Direct and Indirect Paths of the Striatum

This thesis focuses on my work using optical techniques to study different brain regions in vivo. The ability to optically study neurons and the circuits they comprise in vivo is an important method to better understand their role in the healthy brain and their dysfunction in disease. The first part of my thesis focuses on my work using on a collaborative project using a new optical probe to study norepinephrine synapses in vivo. In this work we were able to observe the effects of amphetamine on norepinephrine release in vivo and observed some evidence of potential silent synapses. I also describe a new method of cranial window surgery I developed for optical imaging. This technique called PHASOR, is faster, and has a higher success rate, than traditional surgical methods. The improvements demonstrated in this new surgical technique may enable more widespread use of optical imaging methods. In the second part of my thesis, I used optical techniques to study the dorsal striatum in vivo in awake behaving mice. The direct and indirect paths of the dorsal striatum play an important role in motor behavior and motor learning. Dysfunction in these paths has been implicated in motor diseases as well as in mood disorders. In this thesis, I provide a review of the anatomy and physiology of the neurons that comprise the dorsal striatum, and the circuits that they form. The next chapters describe my work using optical techniques to record from these neurons in vivo. In my first set of experiments, I recorded from the direct and indirect paths during a behavioral task of anxiety and observed differential firing depending on the anxiety state of the mouse. Finally, in a preliminary set of experiments, I record from the direct and indirect paths during tasks of motor learning. I found that both paths show changes in firing during motor learning and that these changes differ between the dorsolateral and dorsomedial striatum

Non-Destructive Viscoelasticity Microscopy: A Spectroscopic Approach using Dual Brillouin/Raman Scattering Processes

The tremendous progress in life sciences and medicine has been greatly facilitated by the development of new imaging modalities. The elastic properties of molecules, subcellular and cellular structures play a crucial role in many areas of biology and medicine. Tissue elasticity has recently been recognized as a critical regulator of cell behavior, with clear roles in embryogenesis, tissue morphogenesis and stem cell differentiation, as well as contributing to pathologies such as tumor progression, coronary artery disease and tissue scarring. This dissertation is focused on developing a novel instrumentation to image viscoelastic properties of cells and tissues using Brillouin microspectroscopy. Following design, construction and optimizations that maximize the signal quality, we obtained the highest resolution Brillouin imaging system in a confocal backscattering arrangement suitable for bio-imaging applications. Furthermore, a powerful combination of Brillouin and Raman spectroscopies has yielded a confocal microscope capable of performing simultaneous mechanical and chemical imaging in a non-invasive and noncontact manner. The novel instrument was optimized and validated for several biomedical applications. For example, we demonstrated that Brillouin spectroscopy is capable of performing in-vivo measurements of the mechanical properties of artificial biocompatible materials such as photocrosslinkable gelatin methacrylate (GelMA). With the assistance of animal models of human congenital muscular dystrophies, we show that Brillouin spectroscopy can serve as a unique diagnosis tool, which can detect differences in muscle elasticity even between very similar muscular dystrophy genotypes. We have also demonstrated that Brillouin spectroscopy is an invaluable approach in developmental biology since it is capable of making non-destructive imaging of an embryo's elasticity during its development process, which is crucial to understand the formation of many essential organs such as bone and brain. In summary, we have developed a novel instrument for biomedical imaging sensing, which is compatible with other microscopic imaging modalities and is specific to local elasticity. Numerous applications of this new technology have been explored, and the instrument’s performance was validated for several systems

Advance Nanomaterials for Biosensors

The book provides a comprehensive overview of nanostructures and methods used to design biosensors, as well as applications for these biosensor nanotechnologies in the biological, chemical, and environmental monitoring fields. Biological sensing has proven to be an essential tool for understanding living systems, but it also has practical applications in medicine, drug discovery, food safety, environmental monitoring, defense, personal security, etc. In healthcare, advancements in telecommunications, expert systems, and distributed diagnostics are challenging current delivery models, while robust industrial sensors enable new approaches to research and development. Experts from around the world have written five articles on topics including:Diagnosing and treating intraocular cancers such as retinoblastoma; Nanomedicine in cancer management; Engineered nanomaterials in osteosarcoma diagnosis and treatment; Practical design of nanoscale devices; Detect alkaline phosphatase quantitatively in clinical diagnosis; Progress in the area of non-enzymatic sensing of dual/multi biomolecules; Developments in non-enzymatic glucose and H2O2 (NEGH) sensing; Multi-functionalized nanocarrier therapies for targeting retinoblastoma; Galactose functionalized nanocarriers; Sensing performance, electro-catalytic mechanism, and morphology and design of electrode materials; Biosensors along with their applications and the benefits of machine learning; Innovative approaches to improve the NEGH sensitivity, selectivity, and stability in real-time applications; Challenges and solutions in the field of biosensors