A deconvolution Light-Sheet Microscope for Mesoscopic Tissue Imaging

光片荧光显微镜（LSFM）已成为生物医学研究的支柱。虽然LSFM可以达到超分辨率，从而在单细胞或小动物中实现极快的成像速度，但同样重要的领域是对大型动物、组织切片甚至整个器官进行细胞分辨率成像，从而可以快速重建整个器官的三维结构。这被称为介观成像。它通常需要对组织切片或器官进行化学处理，使其具有光学透明度，这一过程称为清除。在其显微镜核心设施IMCES （https://imces.uk-essen.de）中，埃森大学医院多年来一直在进行介观LSFM，并在实验动物的许多不同器官和人体组织中开发了先进的成像技术。因此，在生理器官功能、免疫排斥、自身免疫、中风、心肌梗死和癌症发展等多个领域获得了高度原创的见解。此外，已经制定了用于器官清除的新的无毒实验室规程，这使得现有仪器的使用大大增加。我们现在正在达到现有技术的极限，这种技术阻碍我们获得更深入的科学理解，尽管用我们的方法基本上是可能的。这些限制集中在我们的LSFM设置的次优图像质量上，这妨碍了高级图像分析，从而阻碍了生物学洞察力。通过应用一种新型的反褶积LSFM，首次可以获得介观LSFM数据的图像质量，从而可以应用复杂的图像重建和真正的反褶积方法。这是通过光学仪器的基本改进来实现的，以获得关于光片位置和质量的精确信息，从而获得整个照明区域内的点扩展函数。此外，创新和专有的图像重建算法用于去噪和反卷积图像，以及实现在符合人类视觉的一幅图像中从微弱的细胞过程到非常明亮的细胞体的极大范围的像素强度的表示。该手术产生的图像质量远远优于以前的技术，并将使生理和病理器官功能领域的全新见解成为可能。它也可能对临床诊断产生深远的影响，至少对像淋巴结这样的人体小器官。

Light Sheet Fluorescence Microscopy (LSFM) has become a mainstay for biomedical research. While LSFM can reach superresolution and thereby extremely rapid imaging speeds in single cells or small animals, an equally important realm is the imaging of larger animals, tissue sections or even entire organs with cellular resolution, thereby allowing to quickly reconstruct whole organs in 3-D. This is called mesoscopic imaging. It typically requires the chemical treatment of tissue sections or organs to make them optically transparent, a process termed clearing. Within its microscopy core facility IMCES (https://imces.uk-essen.de) the University Hospital Essen is performing mesoscopic LSFM since many years and has developed advanced imaging in many different organs of experimental animals and also human tissue. Thereby, highly original insights have been gained in diverse areas such as physiological organ function, immune rejection, autoimmunity, stroke, myocardial infarction and cancer development. Also new non-toxic laboratory protocols for organ clearing have been developed which have led to a strong increase in the use of the available instrumentation. We are now reaching the limits of the available technology that inhibit us from achieving a deeper scientific understanding, even though it would principally be possible with our approaches. These limits are centered around suboptimal image quality of our LSFM setup that precludes advanced image analysis and hence biological insight. With a new type of deconvolution LSFM that is applied for, here, it would be possible for the first time to obtain an image quality for mesoscopic LSFM data that allows to apply sophisticated methods of image reconstruction and true deconvolution. This is achieved by fundamental improvements in the optical instrument to obtain precise information on light sheet position and quality and hence the point spread function within the entire illumination zone. In addition, innovative and proprietary image reconstruction algorithms are used to de-noise and deconvolve images as well as to achieve the representation of an extremely large spread of pixel intensities from faint cellular processes to very bright cell bodies in one image that fits human vision. The image quality resulting from this operation is far superior over previous technology and will enable totally new insights into the areas of physiological and pathological organ function. It might also have a profound impact on clinical diagnostics, at least of small human organs such as lymph nodes.