Quantitative optical microscopy for aberrating and scattering biological media

今天，生物学中的光学显微镜沿着两个主要方向发展：提高成像质量，特别是在分辨率和3D视场方面；以及提高相关测量的内容和数量，如分子浓度和动力学。为了获得后一种信息，一种非常成功和成熟的技术是荧光涨落显微镜(FFM)，它是经典荧光相关光谱(FCS)的推广，它利用了不同的显微技术。在此背景下，MICROSCATTAB项目的主要目标是：i)了解光散射和光学像差如何干扰荧光涨落显微镜进行的测量；ii)开发新的方案来校正这些影响。过去，自适应光学(AO)方法已被成功地用于显微镜校正样品引起的像差，但这些方法中的大多数都涉及补偿在相当透明的样品中或在浅聚焦深度处出现的低阶像差。然而，当聚焦到样品的深处(几十到几百微米)时，会出现具有复杂相位结构的大幅度像差，光散射急剧增加。在我们的项目中，我们将研究如何考虑这些影响，以及什么是最有效的FFM校正方案。为了实现这一点，我们将对像差/散射模体进行实验测量，并结合实验的分析和数值模拟。然后将所得到的波前校正方案应用于生物组织模型中的FFM。

Optical microscopy in biology develops today along two major directions: improving imaging quality, especially in terms of resolution and 3D field of view; and improving content and quantitativeness of associated measurements, such as molecular concentration and dynamics. For obtaining the latter information, a very successful and well-established technique is Fluorescence Fluctuation Microscopy (FFM), which is a generalisation of classical Fluorescence Correlation Spectroscopy (FCS) that exploits different microscopy techniques. Within this context, the main goals of the MICROSCATTAB project are: i) to understand how light scattering and optical aberrations disturb measurements performed with Fluorescence Fluctuation Microscopy; ii) to develop new schemes to correct for these effects. In the past, Adaptive Optics (AO) approaches have been successfully used in microscopy for correcting sample-induced aberrations, but the majority of these methods have been concerned with compensating low-order aberrations which arise in rather transparent samples or at shallow focusing depth. However, when focusing deep into a specimen (tens to hundreds of µm), large-amplitude aberrations with complex phase structure arise, and light scattering dramatically increases. In our project, we will investigate how to take these effects into account, and what are the most efficient correction schemes for FFM. For achieving this, we will perform experimental measurements on aberrating/scattering phantoms combined with analytical and numerical modelling of the experiments. The resulting wavefront correction schemes will then be applied to FFM in a biological tissue model.