High resolution fluorescence stereomicroscope for 3D imaging

在现代细胞和分子生物学实验室中，对蛋白质定位和细胞表型的仔细观察被广泛用于深入了解特定基因在生物体中的作用。因此，近年来，光学显微镜技术在生命科学研究中经历了显著的卷土重来。在处理较大的标本时，直立或倒置的显微镜不足以提供足够的景深。为此，研究人员需要一个高分辨率的体视显微镜。此外，随着各种荧光蛋白质标签的出现，高分辨率荧光立体显微镜的出现将使我们能够以更高的效率检测在较大样本中表达的蛋白质融合，如花粉管或胚珠。这里提出的设备首先是为了满足我们日益增长和仍未得到满足的需求：1)解剖和观察来自突变的透明组织；2)荧光应用，不涉及通过软件分析进行复杂的重建，但在受精研究中需要更大的景深，如GFP荧光或苯胺蓝染色的花粉管；3)更高的吞吐量，在定位或蛋白质相互作用研究中快速定位GFP或YFP标记的蛋白质。这对于通过微粒子轰击洋葱表皮或叶片农业渗透进行瞬时表达后快速定位表达荧光标记的融合蛋白的细胞尤其重要；4)通过透射、反射或荧光立体显微镜观察较大样品，即使在极低的放大倍数下，由于景深较差，这些样品也无法在正常的立式或倒置显微镜中整体观察到。

In modern cell and molecular biology laboratories, careful observations of protein localization and cellular phenotypes are intensively used to gain insight into the role of specific genes in an organism. Therefore, in recent years, optical microscopy techniques have experienced a remarkable comeback in life science research. When dealing with larger specimens, upright or inverted microscopes are not adequate to provided sufficient depth of field. For this, researchers need a high resolution stereomicroscope. Furthermore, with the advent of various fluorescent protein tags, the availability of a high resolution fluorescent stereomicroscope will enable us to detect with higher efficiency protein fusions expressed in larger specimens, like pollen tubes or ovules. The equipment proposed here is intended firstly to cover our increasing and still unmet needs in: 1) dissection and observation of cleared tissues from mutants; 2) fluorescence applications that do not involve elaborate reconstructions through software analysis but that demand an increased depth of field like GFP fluorescent or aniline blue stained pollen tubes in fertilization studies; 3) higher throughput fast localization of GFP or YFP tagged protein in localization or protein-protein interaction studies. This will be particularly important for the rapid localization of cells expressing the fluorescent-tagged fusion proteins following transient expression through micro particle bombardment of onion epidermis or leaf agroinfiltration; 4) transmitted, reflected, or fluorescent light stereomicroscopy of larger samples that cannot be observed as a whole in a normal upright or inverted microscope due to poor depth of field, even at very low magnification.