Enhancing spatial and temporal resolution for isotropic volumetric imaging and 3D cell tracking

增强各向同性体积成像和 3D 细胞跟踪的空间和时间分辨率

• 批准号: BB/L018039/1
• 负责人: James McGinty
• 金额: $ 17.9万
• 依托单位: Imperial College London
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2014
• 资助国家: 英国
• 起止时间: 2014 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FL018039%2F1
• 关键词: Enhancing; spatial; temporal; resolution; isotropic

Optical microscopy is ubiquitous in biological sciences with fluorescence microscopy in particular being utilised to map specific labelled proteins and/or structures. While the majority of such research is performed on populations of cells growing on glass slides, increasingly there is an appreciation that more realistic environments are required to obtain relevant data on biological processes, and ultimately this means using live biological models. A range of small, optically accessible, organisms (e.g. zebrafish, nematode worms, etc) provide convenient live samples for studying biological processes in vivo. Such samples are inherently three-dimensional (3-D), zebrafish being <1 mm in diameter when under 16 days old, and therefore require 3-D discrimination to provide unambiguous positional/structural information. Most 3-D microscopy is undertaken with laser scanning microscopes that scan a spot of light through the sample, building up a map of fluorescence intensity point by point. Such scanning microscopes are typically optimised for higher magnifications (i.e. small fields of view), suffer from unequal resolution (transverse better than axial) and require significant financial investment (e.g. often >£150K). An alternative method of acquiring 3-D data is optical projection tomography (OPT), the optical equivalent to X-ray computed tomography, which can be implemented on a standard wide-field imaging microscope and can provide 3-D imaging at a fraction of the cost of point scanning systems. In OPT, wide-field images (either fluorescence or transmitted light) of a rotating sample are acquired at different orientations. These images can be used to reconstruct the 3-D distribution of fluorescence/absorption. The standard approach to OPT imposes three key constraints: the requirement that at least the front half of the sample must be 'in focus', that the whole sample must stay in the field of view throughout the acquisition to prevent artefacts in the reconstruction process and that the sample must be non-scattering (i.e. transparent). The first two constraints limit the achievable spatial resolution, since they require the numerical aperture (NA) of imaging system to be small. This limit can be overcome by scanning the imaging lens, and therefore the focal plane, through the rotating sample while acquiring the angularly-resolved images. This produces an 'in focus' image of the whole sample that is superimposed on an out of focus "back-ground" signal that can be removed during image processing. We propose to extend this approach to yet higher resolution imaging of selected sub-volumes within the sample by incorporating a lateral scanning microscope stage to allow the motion of a "volume of interest" (VOI) inside a larger specimen (e.g. an organ) to be maintained in focus as the sample rotates. This VOI can then be modelled as a detailed structure within a larger 'unstructured' volume, to permit high resolution reconstruction without artefacts associated with parts of the sample entering/leaving the field of view. This would permit isotropic high resolution 3-D imaging of, e.g. immune cell distribution in specific organs in live zebrafish, which is currently not possible using the standard commercially available instruments.To address the limits to temporal resolution, we would investigate a novel "orthogonal scanning approach", acquiring sequential images at right-angles with respect to each other, such that the 3-D structure/location of features within the sample could be determined much faster than the rotation period. This could be applied, e.g. to follow cell migration within a live zebrafish. Finally we will extend this system to simultaneously acquire two images at different wavelengths of light using a commercially available spectral image splitter. By analysing these two wavelength channels we will be able to indirectly probe the signalling events that control the immune response and occur within cells.

光学显微镜在生物科学中是普遍存在的，荧光显微镜特别用于绘制特定标记的蛋白质和/或结构。虽然大多数此类研究是在载玻片上生长的细胞群上进行的，但越来越多的人认识到，需要更真实的环境来获得生物过程的相关数据，最终这意味着使用活的生物模型。一系列小的、光学可接近的生物体（例如斑马鱼、蠕虫）为研究体内生物过程提供了方便的活样品。这些样本本质上是三维的（3-D），斑马鱼在16天以下时直径<1 mm，因此需要3-D辨别以提供明确的位置/结构信息。大多数3D显微镜都是使用激光扫描显微镜进行的，激光扫描显微镜可以扫描穿过样本的光点，逐点建立荧光强度图。这种扫描显微镜通常针对更高的放大率（即小视场）进行优化，具有不相等的分辨率（横向优于轴向），并且需要大量的财务投资（例如，通常>£ 150 K）。获取3D数据的另一种方法是光学投影断层扫描（OPT），它是X射线计算机断层扫描的光学等效物，可以在标准的宽视场成像显微镜上实现，并且可以以点扫描系统的一小部分成本提供3D成像。在OPT中，旋转样品的宽视场图像（荧光或透射光）在不同的方向上采集。这些图像可用于重建荧光/吸收的3-D分布。OPT的标准方法施加了三个关键约束：要求至少样品的前半部分必须“对焦”，整个样品必须在整个采集过程中保持在视场中以防止重建过程中的伪影，以及样品必须是非散射的（即透明的）。前两个约束限制了可实现的空间分辨率，因为它们要求成像系统的数值孔径（NA）很小。通过在获取角分辨图像的同时扫描成像透镜并因此扫描焦平面穿过旋转样品，可以克服该限制。这产生了整个样品的“聚焦”图像，该图像叠加在可以在图像处理期间去除的失焦“背景”信号上。我们建议将这种方法扩展到更高分辨率的成像的选定的子体积内的样品，通过合并一个横向扫描显微镜阶段，以允许运动的“感兴趣的体积”（VOI）内的一个较大的标本（例如器官），以保持在焦点作为样品旋转。然后，该VOI可以被建模为更大的“非结构化”体积内的详细结构，以允许高分辨率重建，而没有与进入/离开视场的样本的部分相关联的伪影。这将允许各向同性的高分辨率3-D成像，例如活斑马鱼中特定器官中的免疫细胞分布，这是目前使用标准市售仪器不可能的。为了解决时间分辨率的限制，我们将研究一种新的“正交扫描方法”，以相对于彼此成直角的方式获取序列图像，使得可以比旋转周期快得多地确定样本内特征的3-D结构/位置。这可以应用于例如跟踪活斑马鱼内的细胞迁移。最后，我们将扩展这个系统，同时获得两个图像在不同波长的光使用市售的光谱图像分离器。通过分析这两个波长通道，我们将能够间接探测控制免疫反应和细胞内发生的信号事件。

项目成果

Accelerated Optical Projection Tomography Applied to In Vivo Imaging of Zebrafish.

• DOI: 10.1371/journal.pone.0136213
• 发表时间: 2015
• 期刊: PloS one
• 影响因子: 3.7
• 作者: Correia T;Lockwood N;Kumar S;Yin J;Ramel MC;Andrews N;Katan M;Bugeon L;Dallman MJ;McGinty J;Frankel P;French PM;Arridge S
• 通讯作者: Arridge S

Visualising apoptosis in live zebrafish using fluorescence lifetime imaging with optical projection tomography to map FRET biosensor activity in space and time.

• DOI: 10.1002/jbio.201500258
• 发表时间: 2016-04
• 期刊: Journal of biophotonics
• 影响因子: 2.8
• 作者: Andrews N;Ramel MC;Kumar S;Alexandrov Y;Kelly DJ;Warren SC;Kerry L;Lockwood N;Frolov A;Frankel P;Bugeon L;McGinty J;Dallman MJ;French PM
• 通讯作者: French PM

OPTiM: Optical projection tomography integrated microscope using open-source hardware and software.

• DOI: 10.1371/journal.pone.0180309
• 发表时间: 2017
• 期刊: PloS one
• 影响因子: 3.7
• 作者: Watson T;Andrews N;Davis S;Bugeon L;Dallman MD;McGinty J
• 通讯作者: McGinty J