Integrating photoacoustic microscopy with other imaging technologies for multimodal imaging

As a hybrid optical microscopic imaging technology, photoacoustic microscopy images the optical absorption contrasts and takes advantage of low acoustic scattering of biological tissues to achieve high-resolution anatomical and functional imaging. When combined with other imaging modalities, photoacoustic microscopy-based multimodal technologies can provide complementary contrast mechanisms to reveal complementary information of biological tissues. To achieve intrinsically and precisely registered images in a multimodal photoacoustic microscopy imaging system, either the ultrasonic transducer or the light source can be shared among the different imaging modalities. These technologies are the major focus of this minireview. It also covered the progress of the recently developed penta-modal photoacoustic microscopy imaging system featuring a novel dynamic focusing technique enabled by OCT contour scan.     

Biofunctionalized Prussian Blue Nanoparticles for Multimodal Molecular Imaging Applications

Multimodal, molecular imaging allows the visualization of biological processes at cellular, subcellular, and molecular-level resolutions using multiple, complementary imaging techniques. These imaging agents facilitate the real-time assessment of pathways and mechanisms in vivo, which enhance both diagnostic and therapeutic efficacy. This article presents the protocol for the synthesis of biofunctionalized Prussian blue nanoparticles (PB NPs) - a novel class of agents for use in multimodal, molecular imaging applications. The imaging modalities incorporated in the nanoparticles, fluorescence imaging and magnetic resonance imaging (MRI), have complementary features. The PB NPs possess a core-shell design where gadolinium and manganese ions incorporated within the interstitial spaces of the PB lattice generate MRI contrast, both in T₁ and T₂-weighted sequences. The PB NPs are coated with fluorescent avidin using electrostatic self-assembly, which enables fluorescence imaging. The avidin-coated nanoparticles are modified with biotinylated ligands that confer molecular targeting capabilities to the nanoparticles. The stability and toxicity of the nanoparticles are measured, as well as their MRI relaxivities. The multimodal, molecular imaging capabilities of these biofunctionalized PB NPs are then demonstrated by using them for fluorescence imaging and molecular MRI in vitro.     

活体动物体内光学成像技术的研究进展

生物发光和荧光成像作为近年来新兴的活体动物体内光学成像技术,以其操作简便及直观性成为研究小动物活体成像的一种理想方法,在生命科学研究中得以不断发展。利用这种成像技术,可以直接实时观察标记的基因及细胞在活体动物体内的活动及反应。利用光学标记的转基因动物模型可以研究疾病的发生发展过程,进行药物研究及筛选等。本文综述了现有活体动物体内光学成像技术的原理、应用领域及发展前景,比较了生物发光与几种荧光技术的不同特点和应用。     

光学透明技术在植物多尺度成像中的应用

光学透明技术是一种通过各种化学试剂,将原本不透明的生物样本实现透明化,并在光学显微镜下深度成像的技术。结合多种光学显微成像新技术,光学透明技术可对整个组织进行成像和三维重建,深度剖析生物体内部空间特征与形成机制。近年来,多种植物光学透明技术和多尺度成像技术被陆续研发,并取得了丰硕的研究成果。该文综述了生物体光学透明技术的基本原理和一些新技术,重点介绍基于光学透明技术开发的新型成像方法及其在植物成像与细胞生物学中的应用,为后续植物整体、组织或器官的透明、成像与三维重构及功能研究提供理论依据和技术支持。     

Metal-based environment-sensitive MRI contrast agents

Interactions of paramagnetic metal complexes with their biological environment can modulate their magnetic resonance imaging (MRI) contrast–enhancing properties in different ways, and this has been widely exploited to create responsive probes that can provide biochemical information. We survey progress in two rapidly growing areas: the MRI detection of biologically important metal ions, such as calcium, zinc, and copper, and the use of transition metal complexes as smart MRI agents. In both fields, new imaging technologies, which take advantage of other nuclei (¹⁹F) and/or paramagnetic contact shift effects, emerge beyond classical, relaxation-based applications. Most importantly, in vivo imaging is gaining ground, and the promise of molecular MRI is becoming reality, at least for preclinical research.     

光学分子影像技术及其在药物研发领域的应用

光学分子影像技术是一种发展迅速的生物医学影像技术,能够利用生物发光技术或荧光蛋白等,对生物体内特定的生物过程进行无创的定性或定量研究。应用该技术可以对药物进行筛选,选取具有潜在治疗效果的药物进行后续研究,而终止对可能无效药物的研究,同时可以评价药物对肿瘤的代谢、增殖、血管形成、凋亡和组织乏氧等方面的影响。本文主要介绍光学分子影像技术及其在药物研发,尤其是抗肿瘤药物研发领域的应用。     

Mass spectrometry images acylcarnitines,phosphatidylcholines, and sphingomyelin in MDA-MB-231 breast tumor models

The lipid compositions of different breast tumor microenvironments are largely unknown due to limitations in lipid imaging techniques. Imaging lipid distributions would enhance our understanding of processes occurring inside growing tumors, such as cancer cell proliferation, invasion, and metastasis. Recent developments in MALDI mass spectrometry imaging (MSI) enable rapid and specific detection of lipids directly from thin tissue sections. In this study, we performed multimodal imaging of acylcarnitines, phosphatidylcholines (PC), a lysophosphatidylcholine (LPC), and a sphingomyelin (SM) from different microenvironments of breast tumor xenograft models, which carried tdTomato red fluorescent protein as a hypoxia-response element-driven reporter gene. The MSI molecular lipid images revealed spatially heterogeneous lipid distributions within tumor tissue. Four of the most-abundant lipid species, namely PC(16:0/16:0), PC(16:0/18:1), PC(18:1/18:1), and PC(18:0/18:1), were localized in viable tumor regions, whereas LPC(16:0/0:0) was detected in necrotic tumor regions. We identified a heterogeneous distribution of palmitoylcarnitine, stearoylcarnitine, PC(16:0/22:1), and SM(d18:1/16:0) sodium adduct, which colocalized primarily with hypoxic tumor regions. For the first time, we have applied a multimodal imaging approach that has combined optical imaging and MALDI-MSI with ion mobility separation to spatially localize and structurally identify acylcarnitines and a variety of lipid species present in breast tumor xenograft models.     

Fast x-ray fluorescence microtomography of hydrated biological samples

Metals and metalloids play a key role in plant and other biological systems as some of them are essential to living organisms and all can be toxic at high concentrations. It is therefore important to understand how they are accumulated, complexed and transported within plants. In situ imaging of metal distribution at physiological relevant concentrations in highly hydrated biological systems is technically challenging. In the case of roots, this is mainly due to the possibility of artifacts arising during sample preparation such as cross sectioning. Synchrotron x-ray fluorescence microtomography has been used to obtain virtual cross sections of elemental distributions. However, traditionally this technique requires long data acquisition times. This has prohibited its application to highly hydrated biological samples which suffer both radiation damage and dehydration during extended analysis. However, recent advances in fast detectors coupled with powerful data acquisition approaches and suitable sample preparation methods can circumvent this problem. We demonstrate the heightened potential of this technique by imaging the distribution of nickel and zinc in hydrated plant roots. Although 3D tomography was still impeded by radiation damage, we successfully collected 2D tomograms of hydrated plant roots exposed to environmentally relevant metal concentrations for short periods of time. To our knowledge, this is the first published example of the possibilities offered by a new generation of fast fluorescence detectors to investigate metal and metalloid distribution in radiation-sensitive, biological samples.     

膨胀显微成像技术的原理及应用

膨胀显微成像技术（expansion microscopy,ExM）是一种新型超分辨成像技术。该技术借助可膨胀水凝胶均匀地物理放大生物样本,在常规光学成像条件下实现超分辨成像。ExM适用于细胞、组织切片等多种类型生物样本。蛋白质、核酸、脂质等生物大分子均可借助ExM进行超分辨成像。ExM可与共聚焦显微镜、光片显微镜、超高分辨显微镜联合使用,进一步提高成像分辨率。近年来,多种从基础ExM拓展而来的衍生技术进一步促进了该技术的实际应用。本文综述了ExM及其衍生技术的基本原理、ExM与不同成像技术联用的研究进展及ExM在不同类型生物样本中的应用进展,并对ExM技术的发展前景做出展望。     

Biological applications of X-ray fluorescence microscopy: exploring the subcellular topography and speciation of transition metals

Synchrotron X-ray fluorescence microscopy (SXRF) is a microanalytical technique for the quantitative mapping of elemental distributions. Among currently available imaging modalities, SXRF is the only technique that is compatible with fully hydrated biological samples such as whole cells or tissue sections, while simultaneously offering trace element sensitivity and submicron spatial resolution. Combined with the ability to provide information regarding the oxidation state and coordination environment of metal cations, SXRF is ideally suited to study the intracellular distribution and speciation of trace elements, toxic heavy metals and therapeutic or diagnostic metal complexes.     

Inorganic nanoparticles for multimodal molecular imaging

Multimodal molecular imaging can offer a synergistic improvement of diagnostic ability over a single imaging modality. Recent development of hybrid imaging systems has profoundly impacted the pool of available multimodal imaging probes. In particular, much interest has been focused on biocompatible, inorganic nanoparticle-based multimodal probes. Inorganic nanoparticles offer exceptional advantages to the field of multimodal imaging owing to their unique characteristics, such as nanometer dimensions, tunable imaging properties, and multifunctionality. Nanoparticles mainly based on iron oxide, quantum dots, gold, and silica have been applied to various imaging modalities to characterize and image specific biologic processes on a molecular level. A combination of nanoparticles and other materials such as biomolecules, polymers, and radiometals continue to increase functionality for in vivo multimodal imaging and therapeutic agents. In this review, we discuss the unique concepts, characteristics, and applications of the various multimodal imaging probes based on inorganic nanoparticles.     

The role of secondary ion mass spectrometry (SIMS) in biological microanalysis: technique comparisons and prospects.

The virtues and limitations of SIMS ion microscopy are compared with other spectroscopic techniques applicable to biological microanalysis, with a special emphasis on techniques for elemental localization in biological tissue (electron, X-ray, laser, nuclear, ion microprobes). Principal advantages of SIMS include high detection sensitivity, high depth resolution, isotope specificity, and possibilities for three-dimensional imaging. Current limitations, especially in comparison to X-ray microanalysis, center on lateral spatial resolution and quantification. Recent SIMS instrumentation advances involving field emission liquid metal ion sources and laser post-ionization will help to minimize these limitations in the future. The molecular surface analysis capabilities of static SIMS, especially with the new developments in commercial time-of-flight spectrometers, are promising for application to biomimetic, biomaterials, and biological tissue or cell surfaces. However, the direct microchemical imaging of biomolecules in tissue samples using SIMS will be hindered by limited concentrations, small analytical volumes, and the inefficiencies of converting surface molecules to structurally significant gas phase ions. Indirect detection using elemental or isotopically tagged molecules, however, shows considerable promise for molecular imaging studies using SIMS ion microscopy.     

Acoustic-based chemical tools for profiling the tumor microenvironment

Acoustic-based imaging modalities (e.g. ultrasonography and photoacoustic imaging) have emerged as powerful approaches to noninvasively visualize the interior of the body due to their biocompatibility and the ease of sound transmission in tissue. These technologies have recently been augmented with an array of chemical tools that enable the study and modulation of the tumor microenvironment at the molecular level. In addition, the application of ultrasound and ultrasound-responsive materials has been used for drug delivery with high spatiotemporal control. In this review, we highlight recent advances (in the last 2–3 years) in acoustic-based chemical tools and technologies suitable for furthering our understanding of molecular events in complex tumor microenvironments.     

Fluorescence molecular imaging of small animal tumor models

In vivo imaging of molecular events in small animals has great potential to impact basic science and drug development. For this reason, several imaging technologies have been adapted to small animal research, including X-ray, magnetic resonance, and radioisotope imaging. Despite this plethora of visualization techniques, fluorescence imaging is emerging as an important alternative because of its operational simplicity, safety, and cost-effectiveness. Fluorescence imaging has recently become particularly interesting because of advances in fluorescent probe technology, including targeted fluorochromes as well as fluorescent "switches" sensitive to specific biochemical events. While past biological investigations using fluorescence have focused on microscopic examination of ex vivo, in vitro, or intravital specimens, techniques for macroscopic fluorescence imaging are now emerging for in vivo molecular imaging applications. This review illuminates fluorescence imaging technologies that hold promise for small animal imaging. In particular we focus on planar illumination techniques, also known as Fluorescence Reflectance Imaging (FRI), and discuss its performance and current use. We then discuss fluorescence molecular tomography (FMT), an evolving technique for quantitative three-dimensional imaging of fluorescence in vivo. This technique offers the promise of non-invasively quantifying and visualizing specific molecular activity in living subjects in three dimensions.     

Optical imaging and tumor angiogenesis

Tumor angiogenesis is essential for tumor growth and progression. Therefore, targeting tumor blood vessels is a promising approach for cancer therapy. Angiogenesis, the formation of blood vessels, is a multistep process, and strongly influenced by the microenvironment. There are no in vitro assays that can resemble this dynamic process in vivo. For this reason, animal models and imaging technologies are critical for studying tumor angiogenesis, identifying therapeutic targets as well as validating the targets. Non-invasive molecular imaging in animal models presents an unprecedented opportunity and ability for us to perform repetitive observations and analysis of the biological processes underlying tumor angiogenesis and tumor progression in living animals in real time. As we gain a better understanding of the fundamental molecular nature of cancer, these techniques will be an important adjunct in translating the knowledge into clinical practice. This important information may elucidate how the tumor blood vessels behave and respond to certain treatments and therapies.     

Proteome imaging: a closer look at life's organization

Imaging the proteome is a term that is used in many different contexts. The term implies that the entire cohort of proteins and their modifications are visualized. This unfortunately is not the case. In this mini-review, a concise overview is provided on different imaging technologies that are currently used to investigate the structure, function and dynamics of proteins and their organization. These techniques have been selected for review based on the unique insights they provide in subsets of the proteome. These techniques have been illustrated with practical examples of their merits. Mass spectrometry-based imaging technologies are playing a key role in proteome research and have been reviewed in more detail. They hold the promise of detailed molecular insight in the spatial organization of living system.     

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.     

In vivo molecular and single cell imaging

Molecular imaging is used to improve the disease diagnosis, prognosis, monitoring of treatment in living subjects. Numerous molecular targets have been developed for various cellular and molecular processes in genetic, metabolic, proteomic, and cellular biologic level. Molecular imaging modalities such as Optical Imaging, Magnetic Resonance Imaging (MRI), Positron Emission Tomography (PET), Single Photon Emission Computed Tomography (SPECT), and Computed Tomography (CT) can be used to visualize anatomic, genetic, biochemical, and physiologic changes in vivo. For in vivo cell imaging, certain cells such as cancer cells, immune cells, stem cells could be labeled by direct and indirect labeling methods to monitor cell migration, cell activity, and cell effects in cell-based therapy. In case of cancer, it could be used to investigate biological processes such as cancer metastasis and to analyze the drug treatment process. In addition, transplanted stem cells and immune cells in cell-based therapy could be visualized and tracked to confirm the fate, activity, and function of cells. In conventional molecular imaging, cells can be monitored in vivo in bulk non-invasively with optical imaging, MRI, PET, and SPECT imaging. However, single cell imaging in vivo has been a great challenge due to an extremely high sensitive detection of single cell. Recently, there has been great attention for in vivo single cell imaging due to the development of single cell study. In vivo single imaging could analyze the survival or death, movement direction, and characteristics of a single cell in live subjects. In this article, we reviewed basic principle of in vivo molecular imaging and introduced recent studies for in vivo single cell imaging based on the concept of in vivo molecular imaging.     

Micro-chemical imaging of cesium distribution in Arabidopsis thaliana plant and its interaction with potassium and essential trace elements

Cesium as an alkali element exhibits a chemical reactivity similar to that of potassium, an essential element for plants. It has been suggested that Cs phytotoxicity might be due either to its competition with potassium to enter the plant, resulting in K starvation, or to its intracellular competition with K binding sites in cells. Such elemental interactions can be evidenced by chemical imaging, which determines the elemental distributions. In this study, the model plant Arabidopsis thaliana was exposed to 1 mM cesium in the presence (20 mM) or not of potassium. The quantitative imaging of Cs and endogenous elements (P, S, Cl, K, Ca, Mn, Fe, and Zn) was carried out using ion beam micro-chemical imaging with 5 microm spatial resolution. Chemical imaging was also evidenced by microfocused synchrotron-based X-ray fluorescence (microXRF) which presents a better lateral resolution (<1 microm) but is not quantitative. Cesium distribution was similar to potassium which suggests that Cs can compete with K binding sites in cells. Cesium and potassium were mainly concentrated in the vascular system of stems and leaves. Cs was also found in lower concentration in leaves mesophyll/epidermis. This late representing the larger proportion in mass, mesophyll/epidermis can be considered as the major storage site for cesium in A. thaliana. Trichomes were not found to accumulate cesium. Interestingly, increased Mn, Fe, and Zn concentrations were observed in leaves at high chlorosis. Mn and Fe increased more in the mesophyll than in veins, whereas zinc increased more in veins than in the mesophyll suggesting a tissue specific interaction of Cs with these trace elements homeostasis. This study illustrates the sensitivity of ion beam microprobe and microfocused synchrotron-based X-ray fluorescence to investigate concentrations and distributions of major and trace elements in plants. It also shows the suitability of these analytical imaging techniques to complement biochemical investigations of metallic stress in plants.     

In vivo molecular imaging using nanomaterials: General in vivo characteristics of nano-sized reagents and applications for cancer diagnosis (Review)

Abstract

Nanoparticles present a new collection of contrast agents for the field of in vivo molecular imaging. This review focuses on promising molecular imaging probes for optical and magnetic resonance imaging based on four representative nanomaterial(s) platforms: quantum dots, upconversion phosphors, superparamagnetic iron oxides, and dendrimer-based agents. Quantum dots are extremely efficient fluorescent nanoparticles with size-tunable emission properties, enabling high sensitivity and greater depth penetration. Their heavy metal composition and long retention in the body, however, pose concerns for clinical translational applications. Upconversion phosphors generate excellent signal-to-background contrast because they emit light with higher energy than the excitation photons and autofluorescence signals. For MRI, iron oxide particles also generate excellent signal and have been used in liver imaging and for cell tracking studies. As they are metabolized through endogenous iron salvage pathways, they have already been introduced as clinical contrast agents. Lastly, dendrimers, a ‘soft’ nanoparticle, can be used as a structural basis for the attachment of small molecule imaging agents and/or targeting groups. This array of nanoparticles should offer insights into the uses and potentials of nanoparticles for the molecular imaging.