Multi-modal MP microscope with AFM for cell mechanical investigations

细胞与环境的机械相互作用是生物学中许多重要现象的核心。这包括具有强烈机械参与的过程，如肌肉收缩或肿瘤侵袭，以及机械刺激更微妙的领域，如胚胎发生和细胞分化，但它们在其中发挥同样重要的作用。设想中的显微镜结合了许多技术，使快速、通用、高分辨率和最小破坏性的机械表型成为可能。具体地说，显微镜包括以下形式：-共焦和多光子显微镜，-荧光寿命成像(FLIM)，-牵引力显微镜(TFM)，-原子力显微镜(AFM)，-弹性谐振器干涉应力错误复制(ERISM)，-光诱导蛋白质分子吸附的光图案化(LIMAP)。这将使得全面和同时表征1。细胞、组织和有机体的机械力使用(I)Gest教授团队开发的ERISM技术，(Ii)合成和遗传编码的张力传感器，以及(Iii)共聚焦牵引力显微镜(TFM)，包括通过多光子能力在厚样品中，2.使用AFM测量细胞、组织和有机物的硬度，3.细胞对外部施加的机械应力的机械反应，(I)使用AFM，(Ii)使用LIMAP的几何约束和生化表面属性，以及4.细胞、组织切片和有机体的3D结构及其对生化和生物力学刺激的局部生化反应(蛋白质表达水平)(使用共焦/多光子成像)。这种创新的多模式方法将允许以高分辨率和实时成像和操纵细胞力和基质硬度。因此，它将提供前所未有的实验途径，了解细胞力学和机械转导的基本过程，这对最近成立的纳米生物光子学中心至关重要，并与位于科隆大学(UoC)和外部研究机构的一些跨学科研究倡议和卓越集群的重点重叠；特别是在CECAD、合作研究中心829(“调节皮肤动态平衡的分子机制”)和临床研究单位329(“足细胞疾病的分子机制”)。此外，该设施将促进化学系内部的新合作，例如，纳米生物光子学中心与有机化学和生物化学研究所之间关于分子和基因编码的分子FRET-张力传感器的合作，或物理化学研究所关于开发用于细胞外部应激的纳米颗粒的合作。因此，显微镜将产生实质性的协同影响，影响到大学更广泛的部分和外部研究机构。

Mechanical interactions of cells with their environment are at the heart of many important phenomena in biology. This includes processes with strong mechanical involvement, like muscle contractions or tumour invasion, as well as areas where mechanical stimuli are more subtle, like embryogenesis and cell differentiation, yet where they play equally important roles.The envisioned microscope combines a number of techniques to enable fast, versatile, high-resolution, and minimally disruptive mechanical phenotyping. Specifically, the microscope comprises the following modalities:- confocal and multiphoton microscopy,- fluorescence lifetime imaging (FLIM),- traction force microscopy (TFM),- atomic force microscopy (AFM), - elastic resonator interference stress miscopy (ERISM),- photo-patterning by light-induced molecular adsorption of proteins (LIMAP).This will enable comprehensive and simultaneous characterization of1. the mechanical forces cells, tissue and organoids exert actively using (i) the ERISM technology developed in Prof Gather’s team, (ii) synthetic and genetically encoded tension sensors, and (iii) confocal traction force microscopy (TFM) including in thick specimen through multiphoton capability,2. the stiffness of cells, tissue and organoids using AFM, 3. the mechanical reaction of cells to (i) externally applied mechanical stresses using AFM, (ii) geometrical constraints and biochemical surface properties using LIMAP, and4. the 3D structure of cells, tissue slices and organoids and their local biochemical reaction (protein expression levels) to biochemical and biomechanical stimuli (using confocal/multiphoton imaging).This innovative, multi-modal approach will allow imaging and manipulation of cell forces and matrix stiffnesses with high resolution and in real-time. It will thus provide unprecedented experimental access to the processes underlying cell mechanics and mechano-transduction which is vital to the recently founded Centre for NanoBioPhotonics and overlaps with the focus of a number of interdisciplinary research initiatives and excellence clusters located at the University of Cologne (UoC) and external research institutions; in particular at CECAD, at the Collaborative Research Centre 829 (“Molecular mechanisms regulating skin homeostasis“), and at the Clinical Research Unit 329 (“Molecular mechanisms of podocyte diseases”). In addition, the facility will spur new cooperation within the Department of Chemistry, e.g. between the Centre for NanoBioPhotonics and the Institutes of Organic Chemistry and Biochemistry on molecular and genetically encoded molecular FRET-tension sensors, or the Institute of Physical Chemistry on development of nanoparticles for external stress application on cells. The microscope will thus have substantial synergetic impact that reaches into a wider part of the university and to external research institutions.