1064 nm acoustic resolution photoacoustic microscopy

Photoacoustic imaging is a noninvasive imaging technique having the advantages of high‐optical contrast and good acoustic resolution at improved imaging depths. Light transport in biological tissues is mainly characterized by strong optical scattering and absorption. Photoacoustic microscopy is capable of achieving high‐resolution images at greater depth compared to conventional optical microscopy methods. In this work, we have developed a high‐resolution, acoustic resolution photoacoustic microscopy (AR‐PAM) system in the near infra‐red (NIR) window II (NIR‐II, eg, 1064 nm) for deep tissue imaging. Higher imaging depth is achieved as the tissue scattering at 1064 nm is lesser compared to visible or near infrared window‐I (NIR‐I). Our developed system can provide a lateral resolution of 130 μm, axial resolution of 57 μm, and image up to 11 mm deep in biological tissues. This 1064‐AR‐PAM system was used for imaging sentinel lymph node and the lymph vessel in rat. Urinary bladder of rat filled with black ink was also imaged to validate the feasibility of the developed system to study deeply seated organs.      

Three-dimensional, tomographic super-resolution fluorescence imaging of serially sectioned thick samples

Three-dimensional fluorescence imaging of thick tissue samples with near-molecular resolution remains a fundamental challenge in the life sciences. To tackle this, we developed tomoSTORM, an approach combining single-molecule localization-based super-resolution microscopy with array tomography of structurally intact brain tissue. Consecutive sections organized in a ribbon were serially imaged with a lateral resolution of 28 nm and an axial resolution of 40 nm in tissue volumes of up to 50 μm×50 μm×2.5 μm. Using targeted expression of membrane bound (m)GFP and immunohistochemistry at the calyx of Held, a model synapse for central glutamatergic neurotransmission, we delineated the course of the membrane and fine-structure of mitochondria. This method allows multiplexed super-resolution imaging in large tissue volumes with a resolution three orders of magnitude better than confocal microscopy.     

Acoustic resolution photoacoustic microscopy based on microelectromechanical systems scanner

Photoacoustic microscopy (PAM) can be classified as optical resolution (OR)‐PAM and acoustic resolution (AR)‐PAM depending on the type of resolution achieved. Using microelectromechanical systems (MEMS) scanner, high‐speed OR‐PAM system was developed earlier. Depth of imaging limits the use of OR‐PAM technology for many preclinical and clinical imaging applications. Here, we demonstrate the use of a high‐speed MEMS scanner for AR‐PAM imaging. Lateral resolution of 84 μm and an axial resolution of 27 μm with ~2.7 mm imaging depth was achieved using a 50 MHz transducer‐based AR‐PAM system. Use of a higher frequency transducer at 75 MHz has further improved the resolution characteristics of the system with a reduction in imaging depth and a lateral resolution of 53 μm and an axial resolution of 18 μm with ~1.8 mm imaging depth was achieved. Using the two‐axis MEMS scanner a 2 × 2 .5 mm² area was imaged in 3 seconds. The capability of achieving acoustic resolution images using the MEMS scanner makes it beneficial for the development of high‐speed miniaturized systems for deeper tissue imaging.      

一体化的光声-荧光显微镜用于活体肿瘤成像

报道了一种利用单一波长激发的同时产生光声和荧光信号的显微成像系统,本成像系统具有超高的成像分辨率(<6μm)。借助外源的造影剂在近红外的吸收特性,利用光声-荧光显微成像系统对活体肿瘤进行光声/荧光成像。实验结果表明,光声-荧光显微镜在早期肿瘤的成像和检测等方面具有潜在的应用价值。因此,通过研究和选择适当的双模态造影剂,该系统在不同病理模型中可以提供更准确的组织信息及生理参数。     

In vivo study of rat cortical hemodynamics using a stereotaxic‐apparatus‐compatible photoacoustic microscope

Brain imaging is an important technique in cognitive neuroscience. In this article, we designed a stereotaxic‐apparatus‐compatible photoacoustic microscope for the studies of rat cortical hemodynamics. Compared with existing optical resolution photoacoustic microscopy (ORPAM) systems, the probe owns feature of fast, light and miniature. In this microscope, we integrated a miniaturized ultrasound transducer with a center frequency of 10 MHz to detect photoacoustic signals and a 2‐dimensional (2D) microelectromechanical system (MEMS) scanner to achieve raster scanning of the optical focus. Based on phantom evaluation, this imaging probe has a high lateral resolution of 3.8 μm and an effective imaging domain of 2 × 2 mm². Different from conventional ORPAMs, combining with standard stereotaxic apparatus enables broad studies of rodent brains without any motion artifact. To show its capability, we successfully captured red blood cell flow in the capillary, monitored the vascular changes during bleeding and blood infusion and visualized cortical hemodynamics induced by middle cerebral artery occlusion.      

Functional photoacoustic microscopy for high-resolution and noninvasive in vivo imaging

Although optical absorption is strongly associated with the physiological status of biological tissue, existing high-resolution optical imaging modalities, including confocal microscopy, two-photon microscopy and optical coherence tomography, do not sense optical absorption directly. Furthermore, optical scattering prevents these methods from imaging deeper than approximately 1 mm below the tissue surface. Here we report functional photoacoustic microscopy (fPAM), which provides multiwavelength imaging of optical absorption and permits high spatial resolution beyond this depth limit with a ratio of maximum imaging depth to depth resolution greater than 100. Reflection mode, rather than orthogonal or transmission mode, is adopted because it is applicable to more anatomical sites than the others. fPAM is demonstrated with in vivo imaging of angiogenesis, melanoma, hemoglobin oxygen saturation (sO2) of single vessels in animals and total hemoglobin concentration in humans.     

Fast, three-dimensional super-resolution imaging of live cells

We report super-resolution fluorescence imaging of live cells with high spatiotemporal resolution using stochastic optical reconstruction microscopy (STORM). By labeling proteins either directly or via SNAP tags with photoswitchable dyes, we obtained two-dimensional (2D) and 3D super-resolution images of living cells, using clathrin-coated pits and the transferrin cargo as model systems. Bright, fast-switching probes enabled us to achieve 2D imaging at spatial resolutions of ～25 nm and temporal resolutions as fast as 0.5 s. We also demonstrated live-cell 3D super-resolution imaging. We obtained 3D spatial resolution of ～30 nm in the lateral direction and ～50 nm in the axial direction at time resolutions as fast as 1-2 s with several independent snapshots. Using photoswitchable dyes with distinct emission wavelengths, we also demonstrated two-color 3D super-resolution imaging in live cells. These imaging capabilities open a new window for characterizing cellular structures in living cells at the ultrastructural level.     

Optical imaging fiber-based live bacterial cell array biosensor

A live cell array biosensor was fabricated by immobilizing bacterial cells on the face of an optical imaging fiber containing a high-density array of microwells. Each microwell accommodates a single bacterium that was genetically engineered to respond to a specific analyte. A genetically modified Escherichia coli strain, containing the lacZ reporter gene fused to the heavy metal-responsive gene promoter zntA, was used to fabricate a mercury biosensor. A plasmid carrying the gene coding for the enhanced cyan fluorescent protein (ECFP) was also introduced into this sensing strain to identify the cell locations in the array. Single cell lacZ expression was measured when the array was exposed to mercury and a response to 100nM Hg(2+) could be detected after a 1-h incubation time. The optical imaging fiber-based single bacterial cell array is a flexible and sensitive biosensor platform that can be used to monitor the expression of different reporter genes and accommodate a variety of sensing strains.     

多尺度的光声显微镜用于癌细胞及实体瘤成像

多尺度显微成像系统(M-PAM)被发展,并被用于成像从癌细胞到实体肿瘤的多尺度生物结构.该装置由二维运动平台,扫描振镜,物镜,聚焦超声换能器组成,其横向分辨率达到3 μm.结果显示该系统可以对体外培养黑色素瘤细胞与体内的黑色素瘤进行无标记成像.基于具有靶向性的探针,M-PAM系统可以对体外培养的U87-MG肿瘤细胞以及体内U87-MG实体肿瘤进行成像.综上所述,M-PAM系统将是研究肿瘤的有力工具.     

基于直线电机的大视场快速光声显微成像系统

光声成像技术是近年来发展的一种新型的无损医学成像技术,它是以脉冲激光作为激发源,以检测的声信号为信息载体,通过相应的图像重建算法重建组织内部结构和功能信息的成像方法。该方法结合了光学成像和声学成像的特点,可提供深层组织高分辨率和高对比度的组织层析图像,在生物医学临床诊断以及在体成像领域具有广泛的应用前景。目前光声成像的扫描方式主要有基于步进电机扫描方式和基于振镜的扫描方式,本文针对目前步进电机扫描速度慢(10 mm×10 mm;0.001帧/s),振镜扫描范围小(1 mm2)的不足,发展了基于直线电机扫描的大视场快速光声显微成像系统。同一条扫描线过程中直线电机速度最高可达200 mm/s。该技术采用逐线采集光声信号的方式,比逐点采集光声信号的步进电机快800倍。该系统对10 mm×10 mm全场扫描的扫描速度为0.8帧/s。最大可扫描视场范围可以达到50 mm×50 mm。大视场快速光声显微成像系统的发展将为生物医学提供新的成像工具。     

Microscopic optical projection tomography in vivo

We describe a versatile optical projection tomography system for rapid three-dimensional imaging of microscopic specimens in vivo. Our tomographic setup eliminates the in xy and z strongly asymmetric resolution, resulting from optical sectioning in conventional confocal microscopy. It allows for robust, high resolution fluorescence as well as absorption imaging of live transparent invertebrate animals such as C. elegans. This system offers considerable advantages over currently available methods when imaging dynamic developmental processes and animal ageing; it permits monitoring of spatio-temporal gene expression and anatomical alterations with single-cell resolution, it utilizes both fluorescence and absorption as a source of contrast, and is easily adaptable for a range of small model organisms.     

Dual-objective STORM reveals three-dimensional filament organization in the actin cytoskeleton

By combining astigmatism imaging with a dual-objective scheme, we improved the image resolution of stochastic optical reconstruction microscopy (STORM) and obtained <10-nm lateral resolution and <20-nm axial resolution when imaging biological specimens. Using this approach, we resolved individual actin filaments in cells and revealed three-dimensional ultrastructure of the actin cytoskeleton. We observed two vertically separated layers of actin networks with distinct structural organizations in sheet-like cell protrusions.     

Three-dimensional resolution doubling in wide-field fluorescence microscopy by structured illumination

Structured illumination microscopy is a method that can increase the spatial resolution of wide-field fluorescence microscopy beyond its classical limit by using spatially structured illumination light. Here we describe how this method can be applied in three dimensions to double the axial as well as the lateral resolution, with true optical sectioning. A grating is used to generate three mutually coherent light beams, which interfere in the specimen to form an illumination pattern that varies both laterally and axially. The spatially structured excitation intensity causes normally unreachable high-resolution information to become encoded into the observed images through spatial frequency mixing. This new information is computationally extracted and used to generate a three-dimensional reconstruction with twice as high resolution, in all three dimensions, as is possible in a conventional wide-field microscope. The method has been demonstrated on both test objects and biological specimens, and has produced the first light microscopy images of the synaptonemal complex in which the lateral elements are clearly resolved.     

Comparison of pulsed photothermal radiometry,optical coherence tomography and ultrasound for melanoma thickness measurement in PDMS tissue phantoms

Melanoma accounts for 75% of all skin cancer deaths. Pulsed photothermal radiometry (PPTR), optical coherence tomography (OCT) and ultrasound (US) are non‐invasive imaging techniques that may be used to measure melanoma thickness, thus, determining surgical margins. We constructed a series of PDMS tissue phantoms simulating melanomas of different thicknesses. PPTR, OCT and US measurements were recorded from PDMS tissue phantoms and results were compared in terms of axial imaging range, axial resolution and imaging time. A Monte Carlo simulation and three‐dimensional heat transfer model was constructed to simulate PPTR measurement. Experimental results show that PPTR and US can provide a wide axial imaging range (75 μm–1.7 mm and 120–910 μm respectively) but poor axial resolution (75 and 120 μm respectively) in PDMS tissue phantoms, while OCT has the most superficial axial imaging range (14–450 μm) but highest axial resolution (14 μm). The Monte Carlo simulation and three‐dimensional heat transfer model give good agreement with PPTR measurement. PPTR and US are suited to measure thicker melanoma lesions (<$>><$>400 μm), while OCT is better to measure thin melanoma lesions (<$><<$>400 μm). (© 2011 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Axial Resolution Enhancement by 4Pi Confocal Fluorescence Microscopy with Two-Photon Excitation

Confocal fluorescence microscopy and two-photon microscopy have become important techniques for the three-dimensional imaging of intact cells. Their lateral resolution is about 200–300 nm for visible light, whereas their axial resolution is significantly worse. By superimposing the spherical wave fronts from two opposing objective lenses in a coherent fashion in 4Pi microscopy, the axial resolution is greatly improved to ～100 nm. In combination with specific tagging of proteins or other cellular structures, 4Pi microscopy enables a multitude of molecular interactions in cell biology to be studied. Here, we discuss the choice of appropriate fluorescent tags for dual-color 4Pi microscopy and present applications of this technique in cellular biophysics. We employ two-color fluorescence detection of actin and tubulin networks stained with fluorescent organic dyes; mitochondrial networks are imaged using the photoactivatable fluorescent protein EosFP. A further example concerns the interaction of nanoparticles with mammalian cells.     

Miniature probe for dual‐modality photoacoustic microscopy and white‐light microscopy for image guidance: A prototype toward an endoscope

In this study, a novel photoacoustic microscopy (PAM) probe integrating white‐light microscopy (WLM) modality that provides guidance for PAM imaging and complementary information is implemented. One single core of an imaging fiber bundle is employed to deliver a pulsed laser for photoacoustic excitation for PAM mode, which provides high resolution with deep penetration. Meanwhile, for WLM mode, the imaging fiber bundle is used to transmit two‐dimensional superficial images. Lateral resolution of 7.2 μm for PAM is achieved. Since miniature components are used, the probe diameter is only 1.7 mm. Imaging of phantom and animals in vivo is conducted to show the imaging capability of the probe. The probe has several advantages by introducing the WLM mode, such as being able to conveniently identify regions of interest and align the focus for PAM mode. The prototype of an endoscope shows potential to facilitate clinical photoacoustic endoscopic applications.     

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.     

Volumetric imaging of erythrocytes using label‐free multiphoton photoacoustic microscopy

Photoacoustic microscopy (PAM) is an imaging modality well suited to mapping vasculature and other strong absorbers in tissue. However, one of the primary drawbacks to PAM when used for high‐resolution imaging is the relatively poor axial resolution due to the inverse dependence on the transducer bandwidth. While submicron lateral resolution PAM can be achieved by tightly focusing the excitation light, the axial resolution is fundamentally limited to 10s of microns for typical transducer frequencies. Here we present a multiphoton PAM technique called transient absorption ultrasonic microscopy (TAUM), which results in a completely optically resolved voxel with an experimentally measured axial resolution of 1.5 microns. This technique is demonstrated by imaging individual red blood cells in three dimensions in blood smear and ex vivo tissues. To the best of our knowledge, this is the first demonstration of fully resolved, volumetric photoacoustic imaging of erythrocytes. (© 2014 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Molecular imaging of the embryonic heart: Fables and facts on 3D imaging of gene expression patterns

Molecular imaging, which is the three-dimensional (3D) visualization of gene expression patterns, is indispensable for the study of the function of genes in cardiac development. The instrumentation, as well as the development of specific contrast agents for molecular imaging, has shown spectacular advances in the last decade. In this review, the spatial resolutions, contrast agents, and applications of these imaging methods in the field of cardiac embryology are discussed. Apart from 3D reconstructions from histological sections, not many of these methods have been applied in embryological research. This review shows that, for most methods, neither the spatial resolutions nor the specificity and applicability of the contrast agents are adequate for the reliable imaging of specific gene expression at the microscopic resolution required for embryological studies of small organs like the developing heart. Although a 3D reconstruction from sections will always suffer from imperfections, the resulting reconstructions meet the aim of most biological studies, especially since the original microscopic images are linked. With respect to imaging of gene expression, only histological sections and laser scanning microscopy provide the required resolution and specificity at the tissue and cellular level. Episcopic fluorescence image capturing and optical projection tomography are being used for microscopic phenotyping and lineage analysis, and both show potential for detailed molecular imaging. Other methods can be used very efficiently in rapid evaluation of biological experiments and high-throughput screens of large-scale gene expression profiling efforts when high spatial resolution is not required.