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

光学显微镜在生物科学中普遍存在，特别是荧光显微镜被用来绘制特定的标记蛋白质和/或结构。虽然大多数这类研究是在玻片上生长的细胞群体上进行的，但越来越多的人认识到，需要更现实的环境来获得有关生物过程的相关数据，最终这意味着使用活的生物模型。一系列可光学接触的小型生物(如斑马鱼、线虫等)为研究体内的生物过程提供了方便的活体样本。这种样品天生就是三维的，16天以下的斑马鱼直径为1毫米，因此需要三维分辨才能提供明确的位置/结构信息。大多数3-D显微镜是用激光扫描显微镜扫描样品中的一个光点，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倍数(即小视场)进行了优化，分辨率不同(横向比轴向更好)，并且需要大量的资金投资(例如，经常是&gt；GB 150K)。另一种获取三维数据的方法是光学投影层析成像(OPT)，这是一种光学上相当于X射线计算机层析成像的方法，可以在标准的广域成像显微镜上实现，可以以点扫描系统的一小部分成本提供三维成像。在光学光学成像仪中，旋转样品的广视场图像(荧光或透射光)在不同的方向上被获取。这些图像可以用来重建荧光/吸收的三维分布。OPT的标准方法施加了三个关键限制：要求样品的至少前半部分必须是“焦点”的，整个样品必须在整个采集过程中保持在视野中，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。这一限制可以通过扫描成像透镜，从而通过旋转的样品扫描焦平面，同时获取角度分辨率的图像来克服。这会产生整个样本的“对焦”图像，该图像叠加在可以在图像处理过程中去除的散焦“背景”信号上。我们建议将这种方法扩展到更高分辨率的样本内选定的子体积的成像，方法是结合横向扫描显微镜工作台，允许在样本旋转时保持较大样本(例如器官)内的“感兴趣体积”(VOI)的运动。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，而不会出现与进入/离开视野的部分样本相关联的伪影。这将允许对活斑马鱼特定器官中免疫细胞分布的各向同性高分辨率三维成像，这是目前使用标准商业仪器无法实现的。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”，获取彼此成直角的序列图像，以便可以比旋转周期快得多地确定样品中特征的三维结构/位置。这可以应用于跟踪活斑马鱼内的细胞迁移。最后，我们将使用商用的光谱图像分离器将该系统扩展到同时获取不同波长的两幅图像。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并发生在细胞内的信号事件。

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.