Optical hyperspectral imaging in microscopy and spectroscopy – a review of data acquisition

Rather than simply acting as a photographic camera capturing two‐dimensional (x, y) intensity images or a spectrometer acquiring spectra (λ), a hyperspectral imager measures entire three‐dimensional (x, y, λ) datacubes for multivariate analysis, providing structural, molecular, and functional information about biological cells or tissue with unprecedented detail. Such data also gives clinical insights for disease diagnosis and treatment. We summarize the principles underpinning this technology, highlight its practical implementation, and discuss its recent applications at microscopic to macroscopic scales.

Datacube acquisition strategies in hyperspectral imaging x, y, spatial coordinates; λ, wavelength.     

Wearable scanning photoacoustic brain imaging in behaving rats

A wearable scanning photoacoustic imaging (wPAI) system is presented for noninvasive brain study in behaving rats. This miniaturized wPAI system consists of four pico linear servos and a single transducer‐based PAI probe. It has a dimension of 50 mm × 35 mm × 40 mm, and a weight of 26 g excluding cablings. Phantom evaluation shows that wPAI achieves a lateral resolution of ～0.5 mm and an axial resolution of ～0.1 mm at a depth of up to 11 mm. Its imaging ability is also tested in a behaving rat, and the results indicate that wPAI is able to image blood vessels at a depth of up to 5 mm with intact scalp and skull. With its noninvasive, deep penetration, and functional imaging ability in behaving animals, wPAI can be used for behavior, cognition, and preclinical brain disease studies.

     

Molecular probes for fluorescent sensing of metal ions in non-mammalian organisms

While metal ions play an important role in the proper functioning of all life, many questions remain unanswered about exactly how different metals contribute to health and disease. The development of fluorescent probes, which respond to metals, has allowed greater understanding of the cellular location, concentration and speciation of metals in living systems, giving a new appreciation of their function. While the focus of studies using these fluorescent tools has largely been on mammalian organisms, there has been relatively little application of these powerful tools to other organisms. In this review, we highlight recent examples of molecular fluorophores, which have been applied to sensing metals in non-mammalian organisms.     

Optical emission of 223Radium: in vitro and in vivo preclinical applications

²²³Radium (²²³Ra) is widely used in nuclear medicine to treat patients with osseous metastatic prostate cancer. In clinical practice ²²³Ra cannot be imaged directly; however, gamma photons produced by its short‐lived daughter nuclides can be captured by conventional gamma cameras. In this work, we show that ²²³Ra and its short‐lived daughter nuclides can be detected with optical imaging techniques. The light emission of ²²³Ra was investigated in vitro using different setups in order to clarify the mechanism of light production. The results demonstrate that the luminescence of the ²²³Ra chloride solution, usually employed in clinical treatments, is compatible with Cerenkov luminescence having an emission spectrum that is almost indistinguishable from CR one. This study proves that luminescence imaging can be successfully employed to detect ²²³Ra in vivo in mice by imaging whole body ²²³Ra biodistribution and more precisely its uptake in bones.      

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。     

In vivo quantification of renal function in mice using clinical gamma cameras

IntroductionIn preclinical research, the growing number of transgenic models has led to the need for renal-function studies in mice. Many efforts have been made to develop dedicated SPECT systems for rodents, but their availability is limited due to high capital costs. The aim of this work is to demonstrate the feasibility of mouse renal imaging by using an inexpensive alternative based on clinical gamma-cameras.MethodsA healthy mouse was scanned 3 h after injection of 6 mCi of Dimercaptosuccinic acid (DMSA) labeled with 99mTc by using a single-head gamma-camera in conjunction with a dedicated pinhole collimator. List-mode data were binned to emulate multiple injections of 1 mCi, 0.1 mCi and 0.01 mCi of 99mTc-DMSA and 6-min ventral and dorsal planar images were acquired and SPECT imaging (60 projection images acquired over 60 min) was performed. An optimization of the protocols in terms of injected activity, time scan, renal cortex uniformity and cortex-to-pelvis contrast was carried out.ResultsThe appropriate protocols were an injected activity of 0.6 mCi, combined with duration of scanning of 1 min for planar and 60 min for SPECT imaging. Our results were validated through the relative quantification of renal function, which showed that both kidneys contributed equally to the total function. They showed that functional structures of the mouse kidneys can be visually distinguished as easily as in human studies.ConclusionsOur findings showed the feasibility of conducting quantitative DMSA SPECT studies of anesthetized mice on clinical gamma cameras.     

Intraoperative multispectral and hyperspectral label‐free imaging: A systematic review of in vivo clinical studies

Multispectral and hyperspectral imaging (HSI) are emerging optical imaging techniques with the potential to transform the way surgery is performed but it is not clear whether current systems are capable of delivering real‐time tissue characterization and surgical guidance. We conducted a systematic review of surgical in vivo label‐free multispectral and HSI systems that have been assessed intraoperatively in adult patients, published over a 10‐year period to May 2018. We analysed 14 studies including 8 different HSI systems. Current in‐vivo HSI systems generate an intraoperative tissue oxygenation map or enable tumour detection. Intraoperative tissue oxygenation measurements may help to predict those patients at risk of postoperative complications and in‐vivo intraoperative tissue characterization may be performed with high specificity and sensitivity. All systems utilized a line‐scanning or wavelength‐scanning method but the spectral range and number of spectral bands employed varied significantly between studies and according to the system's clinical aim. The time to acquire a hyperspectral cube dataset ranged between 5 and 30 seconds. No safety concerns were reported in any studies. A small number of studies have demonstrated the capabilities of intraoperative in‐vivo label‐free HSI but further work is needed to fully integrate it into the current surgical workflow.      

多光子显微成像技术在脑部疾病研究中的应用

由于多光子显微技术具有高时空分辨率、低损伤性、可对活体长时间成像等特点,近年来已被广泛应用于生物医学等领域,并且在多种疾病诊断中展现出巨大的应用潜力.尤其是在脑部疾病的研究中,利用多光子成像技术可实现对复杂神经网络的研究,包括对脑部神经细胞、血管、肿瘤等进行实时成像并研究各自之间的相互作用,能进一步揭示脑疾病的发病机制并指导检测治疗方法的开发.本文简要介绍了多光子成像技术的基本原理及特点,总结了其在阿尔茨海默病、脑中风、脑肿瘤等多种脑部疾病中的应用,详细阐述了近年来利用多光子成像技术在脑部疾病研究中所获得的成果,并对多光子成像技术的发展前景进行了展望,预期其在脑部疾病的研究中将发挥更大的作用.     

Gold nanoclusters as novel optical probes for in vitro and in vivo fluorescence imaging

Fluorescent probes play an important role in the development of fluorescence-based imaging techniques for life sciences research. Gold nanoclusters (AuNCs) are a novel type of fluorescent nanomaterials which have attracted great interest in recent years. Composed of only a few atoms, these ultrasmall AuNCs exhibit quantum confinement effects and molecule-like properties. Fluorescent AuNCs have an attractive set of features including ultrasmall size, good biocompatibility and photostability, and tunable emission in the red to near-infrared spectral region, which make them promising as fluorescent labels for biological imaging. Examples of their application include live cell labeling, cancer cell targeting, cellular apoptosis monitoring, and in vivo tumor imaging. Here, we present a brief overview of recent advances in utilizing these emissive ultrasmall AuNCs as optical probes for in vitro and in vivo fluorescence imaging.     

Quantitative measurement of serotonin synthesis and sequestration in individual live neuronal cells

Synthesis and subsequent sequestration into vesicles are essential steps that precede neurotransmitter exocytosis, but neither the total neurotransmitter content nor the fraction sequestered into vesicles have been measured in individual live neurons. We use multiphoton microscopy to directly observe intracellular and intravesicular serotonin in the serotonergic neuronal cell line RN46A. We focus on how the relationship between synthesis and sequestration changes as synthesis is up-regulated by differentiation or down-regulated by chemical inhibition. Temperature-induced differentiation causes an increase of about 60% in the total serotonin content of individual cells, which goes up to about 10 fmol. However, the number of vesicles per cell increases by a factor of four and the proportion of serotonin sequestered inside the vesicles increases by a factor of five. When serotonin synthesis is inhibited in differentiated cells and the serotonin content goes down to the level present in undifferentiated cells, the sequestered proportion still remains at this high level. The total neurotransmitter content of a cell is, thus, an unreliable indicator of the sequestered amount.     

Novel biomedical applications of Cerenkov radiation and radioluminescence imaging

The main goals of this review is to provide an up-to-date account of the different uses of Cerenkov radiation (CR) and radioluminescence imaging for pre-clinical small animal imaging. We will focus on new emerging applications such as the use of Cerenkov imaging for monitoring radionuclide and external radiotherapy in humans. Another novel application that will be described is the monitoring of radiochemical synthesis using microfluidic chips.Several pre-clinical aspects of CR will be discussed such as the development of 3D reconstruction methods for Cerenkov images and the use of CR as excitation source for nanoparticles or for endoscopic imaging.We will also include a discussion on radioluminescence imaging that is a more general method than Cerenkov imaging for the detection using optical methods of alpha and gamma emitters.     

Label‐free in vivo cellular‐level detection and imaging of apoptosis

Cell death plays a critical role in health and homeostasis as well as in the pathogenesis and treatment of a broad spectrum of diseases and can be broadly divided into two main categories: apoptosis, or programmed cell death, and necrosis, or acute cell death. While these processes have been characterized extensively in vitro, label‐free detection of apoptosis and necrosis at the cellular level in vivo has yet to be shown. In this study, for the first time, fluorescence lifetime imaging microscopy (FLIM) of intracellular reduced nicotinamide adenine dinucleotide (NADH) was utilized to assess the metabolic response of in vivo mouse epidermal keratinocytes following induction of apoptosis and necrosis. Results show significantly elevated levels of both the mean lifetime of NADH and the intracellular ratio of protein bound‐to‐free NADH in the apoptotic compared to the necrotic tissue. In addition, the longitudinal profiles of these two cell death processes show remarkable differences. By identifying and extracting these temporal metabolic signatures, apoptosis in single cells can be studied in native tissue environments within the living organism.

     

OxLDL-targeted iron oxide nanoparticles for in vivo MRI detection of perivascular carotid collar induced atherosclerotic lesions in ApoE-deficient mice

Atherosclerotic disease is a leading cause of morbidity and mortality in developed countries, and oxidized LDL (OxLDL) plays a key role in the formation, rupture, and subsequent thrombus formation in atherosclerotic plaques. In the current study, anti-mouse OxLDL polyclonal antibody and nonspecific IgG antibody were conjugated to polyethylene glycol-coated ultrasmall superparamagnetic iron oxide (USPIO) nanoparticles, and a carotid perivascular collar model in apolipoprotein E-deficient mice was imaged at 7.0 Tesla MRI before contrast administration and at 8 h and 24 h after injection of 30 mg Fe/kg. The results showed MRI signal loss in the carotid atherosclerotic lesions after administration of targeted anti-OxLDL-USPIO at 8 h and 24 h, which is consistent with the presence of the nanoparticles in the lesions. Immunohistochemistry confirmed the colocalization of the OxLDL/macrophages and iron oxide nanoparticles. The nonspecific IgG-USPIO, unconjugated USPIO nanoparticles, and competitive inhibition groups had limited signal changes (p < 0.05). This report shows that anti-OxLDL-USPIO nanoparticles can be used to directly detect OxLDL and image atherosclerotic lesions within 24 h of nanoparticle administration and suggests a strategy for the therapeutic evaluation of atherosclerotic plaques in vivo.     

综述——基于纳米材料的生物检测与医学诊断技术：功能化量子点在肿瘤活体成像中的应用

量子点表面经生物分子或药物分子修饰而具有生物功能．功能化量子点具有独特的光学性质和生物相容性,在生物医学光学诊断和治疗领域具有广泛的应用．本文简要介绍了功能化量子点制备及修饰方法,综合评述了量子点在肿瘤活体诊断和治疗中的应用,包括活体淋巴结成像、血管动态成像、肿瘤成像和抗肿瘤药物示踪等,讨论了功能化量子点在肿瘤活体诊断和治疗中的应用前景以及面临的挑战．     

近红外标记的维甲酸类造影剂用于骨肉瘤成像的研究

目的：开发一种具有＂找寻、治疗、可视＂功能的生物造影剂。方法：采用化学合成的方法得到近红外标记的维甲酸类造影剂,并进行骨肉瘤细胞的体外结合试验;皮下接种裸鼠,构建骨肉瘤的异种移植模型,持续10d对裸鼠进行体内光学成像,观察药物在体内的重新分布,并最终用免疫组织化学法对成像结果进行验证。结果：体外细胞结合试验证明,合成的维甲酸造影剂可以很好的与人的骨肉瘤细胞相结合,进而内化。近红外光学成像表明,该造影剂可用于骨肉瘤的早期和晚期诊断。全身成像显示了在肿瘤和肝脏的高信号强度。正电子发射断层显像（PET）显示肿瘤部位具有较高水平的18F-FDG代谢。剂量增加反应和毒性试验表明,高剂量的维甲酸造影剂必然与其全身毒性息息相关。免疫组化染色显示,发光组织中肿瘤细胞呈阳性。结论：合成的近红外标记的维甲酸造影剂可以用于检测人类骨肿瘤的异种移植,实现个体化分子诊疗的同时减少全身毒性。     

近红外标记的维甲酸类造影剂用于骨肉瘤成像的研究

目的:开发一种具有"找寻、治疗、可视"功能的生物造影剂。方法:采用化学合成的方法得到近红外标记的维甲酸类造影剂,并进行骨肉瘤细胞的体外结合试验;皮下接种裸鼠,构建骨肉瘤的异种移植模型,持续10d对裸鼠进行体内光学成像,观察药物在体内的重新分布,并最终用免疫组织化学法对成像结果进行验证。结果:体外细胞结合试验证明,合成的维甲酸造影剂可以很好的与人的骨肉瘤细胞相结合,进而内化。近红外光学成像表明,该造影剂可用于骨肉瘤的早期和晚期诊断。全身成像显示了在肿瘤和肝脏的高信号强度。正电子发射断层显像(PET)显示肿瘤部位具有较高水平的18F-FDG代谢。剂量增加反应和毒性试验表明,高剂量的维甲酸造影剂必然与其全身毒性息息相关。免疫组化染色显示,发光组织中肿瘤细胞呈阳性。结论:合成的近红外标记的维甲酸造影剂可以用于检测人类骨肿瘤的异种移植,实现个体化分子诊疗的同时减少全身毒性。     

High-field MR spectroscopy in the multiparametric MRI evaluation of breast lesions

IntroductionThe aim of this work was to assess the role of 3T-MR spectroscopy (MRS) in the multi-parametric MRI evaluation of breast lesions, using a pattern-recognition based classification method.Methods291 patients (301 lesions, median 2.3 cm³) were enrolled in the study (age 18–85 y, mean 54.2 y). T1-TSE (TR/TE = 400/10 ms) and T2-STIR imaging (TR/TE = 5000/60 ms), dynamic-contrast-enhanced MRI (DCE-MRI), apparent diffusion coefficient (ADC) (b = 0–800 s/mm²), and single-voxel MRS (10 × 10 × 10 mm³, PRESS, TR/TE = 3000 ms/135 ms) were performed by means of a 3T scanner. MRS results were accepted if the FWHM of the water peak was ⩽45 Hz. Total choline (tCho) was considered detected if the signal-to-noise ratio (SNR) of the 3.2 ppm peak was ⩾2. A classifier-based analysis (support-vector-machines, SVM) was performed with 4-dimensional vectors including type of margin, DCE-MRI kinetic curve type, ADC mean value, and tCho SNR. A comparison with 3-dimensional vectors (without tCho SNR) was used to assess MRS impact on sensitivity, specificity, and positive-negative predictive values (PPV-NPV) for malignancy.Results228 lesions (180 malignant/48 benign) showed acceptable spectral quality. Comparison of classification results with histopathological examination of surgical specimens showed sensitivity = 93.7%, specificity = 84.9%, PPV = 95.2%, NPV = 81.5% without the inclusion of MRS in the SVM analysis. When MRS was included, the figures increased to 95.1%, 90.7%, 97.2%, and 85.0%, respectively.ConclusionsInclusion of 3T-MRS in the multi-parametric MRI evaluation of breast lesions improved the performance of the SVM-based classifier, showing a possible role of high-field MR spectroscopy in the differential diagnosis between benign and malignant breast lesions. Further research is however needed to confirm this initial evidence.     

激光散斑成像技术在脑科学研究中的应用

激光散斑衬比成像(laser speckle contrast imaging,LSCI)是一种非扫描式实时血流动力学成像技术,具有高分辨率、快速实时成像、非接触、仪器结构较简单等优势.尽管由于深度分辨率的限制,LSCI主要用于浅表组织测量,但其在神经疾病、皮肤病等领域的基础研究及临床应用中展现出良好的应用潜力.本文简要介绍了激光散斑衬比成像技术的基本原理与技术进展,综述其在脑卒中、吸毒成瘾、阿尔茨海默病等脑疾病及其他脑科学应用中的研究进展,并展望其发展前景.     

Functional imaging in radiation therapy planning for head and neck cancer

Functional imaging and its application to radiotherapy (RT) is a rapidly expanding field with new modalities and techniques constantly developing and evolving. As technologies improve, it will be important to pay attention to their implementation. This review describes the main achievements in the field of head and neck cancer (HNC) with particular remarks on the unsolved problems.