Development of 3D interferometric super-resolution methods for imaging dynamic, multi-component molecular systems, in single cells and in multi-cellular environments

摘要 了解细胞间的交流和集体细胞现象如何塑造细胞功能和细胞命运 决策需要跟踪发生在活细胞内的详细分子过程的能力， 细胞保持在其原始组织和有机体的自然环境中。尽管数量巨大 在单个分离细胞的成像方面取得了进展，探测拥挤的多细胞的要求 具有高时空分辨率和低至单分子灵敏度的系统呈现出巨大的 光学显微镜的挑战。在这里，我们建议应对这一挑战，利用最近的突破来i) 提高从微弱荧光信号中提取高分辨率信息的能力-使用3D 干涉测量法；ii)使用优化的配置对脆弱/精致的生物样品进行成像-基于选择性- 平面照明；iii)通过光学不均匀样本保持/恢复高分辨率信息- 使用自适应光学。我们假设，这三项关键技术的成功合成将创造 新的方法跨越到以前未知的时空分辨率、检测 敏感度、非侵入性和穿透深度。基于这些想法，我们提出了以下两点 具体目标：(1)开发基于可靠、无伪影的多色体三维干涉成像 光学重建，用于提高从微弱荧光中提取高分辨率信息的能力 信号；(2)实现非侵入性、无背景、长期的4D成像，在~100 nm近各向同性3D 空间分辨率，在毫秒采集时间和在大型和高度拥挤的(多)蜂窝体积上。 这些新技术将显著提高我们研究动态生物过程的能力 分子细节，在单个分离细胞中以及在完整的复杂多细胞系统中，因此具有 对生物医学学科产生广泛而直接的影响。

ABSTRACT Understanding how cell-cell communication and collective cell phenomena shape cell function and cell fate decisions requires the ability to follow the detailed molecular processes as they take place inside live cells, with the cell remaining embedded in its natural setting of the tissue and organism of origin. Although tremendous progress has been made in imaging of single isolated cells, the requirements for probing crowded multi-cellular systems with high spatio-temporal resolution and down to single-molecule sensitivity present an enormous challenge for optical microscopy. Here we propose to tackle this challenge, using recent breakthroughs to i) increase the capability of extracting high-resolution information from weak fluorescence signals – using 3D interferometry; ii) image fragile/delicate biological samples using optimized configurations - based on selective- plan illumination; iii) maintain/recover high resolution information through optically inhomogenous samples – using adaptive optics. We hypothesize that successful synthesis of these three key technologies will create new approaches that cross into previously uncharted realms of combined spatio-temporal resolution, detection sensitivity, non-invasiveness and penetration depth. Based on these ideas we propose the following two specific aims: (1) To develop multi-color volumetric 3D interferometric imaging, based on reliable, artifact-free optical reconstructions, for increasing the ability to extract high-resolution information from weak fluorescence signals; (2) To achieve non-invasive, background-free, long-term 4D imaging, at ~100nm near-isotropic 3D spatial resolution, at millisecond acquisition times and over large and highly crowded (multi)cellular volumes. The new techniques will significantly increase our abilities to interrogate dynamic biological processes with molecular detail, in single isolated cells as well as in intact complex multi-cellular systems, thus having widespread and immediate impact across biomedical disciplines.