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Abstract：
Though microscopy is most often intended as a technique for providing qualitative assessment of cellular and subcellular properties, when coupled with other instruments such as wavelength selectors, lasers, photoelectric devices and computers, it can perform a wide variety of quantitative measurements, which are demanding in establishing relationships between the properties and structures of biological material in all their spatial and temporal complexities. These combinations of instruments are a powerful approach to improve non-destructive investigations of cellular and subcellular properties (both physical and chemical) at a macromolecular scale resolution. Since many subcellular compartments in living cells are characterized by structurally organized molecules, this review deals with three advanced microscopy techniques well-suited for these kind of investigations, i.e., microspectrophotometry (MSP), super-resolution localization microscopy (SRLM) and holotomographic microscopy (HTM). These techniques can achieve an insight view into the role intracellular molecular organizations such as photoreceptive and photosynthetic structures and lipid bodies play in many cellular processes as well as their biophysical properties. Microspectrophotometry uses a set-up based on the combination of a wide-field microscope and a polychromator, which allows the measurement of spectroscopic features such as absorption spectra. Super resolution localization microscopy combines dedicated optics and sophisticated software algorithms to overcome the diffraction limit of light and allow the visualization of subcellular structures and dynamics in greater detail with respect to conventional optical microscopy. Holotomographic microscopy combines holography and tomography techniques into a single microscopy set-up, and allows 3D reconstruction by means of the phase separation of biomolecule condensates. This review is organized in sections, which for each technique describe some general aspects, a peculiar theoretical aspect, a specific experimental configuration and examples of applications (fish and algae photoreceptors, single labeled proteins and endocellular aggregates of lipids).
摘要：
虽然显微镜通常是作为一种技术来提供细胞和亚细胞性质的定性评估，当与波长选择器等其他仪器耦合时，激光,光电设备和计算机，它可以进行各种各样的定量测量，它们要求在生物材料的所有时空复杂性中建立性质和结构之间的关系。这些仪器的组合是以大分子尺度分辨率改进细胞和亚细胞性质(物理和化学)的非破坏性研究的有力方法。由于活细胞中许多亚细胞区室的特征是结构上有组织的分子，这篇综述涉及三种非常适合此类研究的先进显微镜技术，即，显微分光光度法（MSP），超分辨率定位显微镜（SRLM）和全息显微镜（HTM）。这些技术可以深入了解细胞内分子组织，例如感光和光合结构以及脂质在许多细胞过程中的作用及其生物物理特性。显微分光光度法使用基于宽视场显微镜和多色仪的组合的设置，它允许测量光谱特征，如吸收光谱。超分辨率定位显微镜结合了专用光学和复杂的软件算法，以克服光的衍射极限，并允许相对于传统光学显微镜更详细地可视化亚细胞结构和动力学。全息显微镜将全息和断层扫描技术结合到一个单一的显微镜设置，并允许通过生物分子缩合物的相分离进行3D重建。这次审查是按部分组织的，对于每种技术都描述了一些一般方面，一个特殊的理论方面，特定的实验配置和应用示例(鱼类和藻类光感受器，单一标记的蛋白质和脂质的细胞聚集体)。
