Quantitative optical microscopy for aberrating and scattering biological media – MICROSCATTAB

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 most of these methods have been concerned with compensating low-order aberrations that 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 this goal, 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.

This project relies on the long-year expertise in FFM techniques of the two partners: the Grenoble group has in particular implemented adaptive optics in a modal approach to compensate optical aberrations; the Göttingen group has a very strong experience in the field of single-molecule spectroscopy, optical modelling and superresolution microscopy.