Adaptive Optics for Confocal Microscopy – CONFOC-AO

Fluorescence Fluctuations Microscopy (FFM) includes a variety of methods (FC(C)S, RICS, sFCS, PCH, etc..) mainly devoted to biology, which utilize fluorescence fluctuations to calculate the average number of molecules passing through the observation volume of a confocal microscope and obtain information on their dynamics (mobility, molecular interactions). However, in practical situations, progresses have to be made because FFM is extremely sensitive to optical aberrations caused by the lens, the laser scanning system, the support of the sample (chambered coverglass, substrate), or the sample itself (solution, culture medium, cytoplasm, nucleus, etc.). The reason for this sensitivity lies in the nature of the measurements made in FFM, i.e. fluctuations (in contrast to an image, that provides average values): see supplementary file. As a result, calculations of the concentration, diffusion, interactions, etc. depend drastically upon these aberrations: it was shown that the diffusion time measured in homogeneous solutions varies by a factor 2 between the center and the edge of the field of view (140 µm in width) of a system developed for FFM, while, on another system the brightness (signal per molecule) varies by a factor 3 (60 µm in width). The biologist partner has even observed a brighntness varying by a factor 15 across the 350 µm field of view of his commercial FFM system (also see the supplementary file)
This project aims at making a demonstrator of Adaptive Optics (AO) for FFM, based on a new function of merit, in order to measure more precisely concentration and mobility of molecules: i) in different parts of the cell (nucleus, cytoplasm , membrane); ii) at different points of the field of view, at different depths and in various environments (temperature, substrate processing); iii) on samples of different types (media, matrices, cells, tissues).
This project is based on the expertise of the LIPhy team for instrumental and methodological developments of FFM. It also benefits from the know-how of Alpao, a company specialized in AO. Within the frame of a collaborative agreement, a Deformable Mirror (DM) was put at the LIPhy disposal during 2010-2011. The Gipsa-lab partner will be in charge of controlled nanopositioning of the DM. The biologist partner, CRI U823, has the mission to identify and propose biological samples of interest to validate our proof of concept. In addition, it will conduct controlled experiments on commercial microscopes and advise the consortium regarding the implementation of an AO component on a confocal microscope in a biology laboratory.
The technological transfer of the project will be conducted by Floralis. Although the consortium has already some natural industrial partners (such as Alpao, see attached letter), it remains open to other partnerships, the goal being the development and adjustment of an AO device to the FFM market. Indeed, at present, there are, at the international level, only two companies developing AO for microscopy bioimaging, but there is no product compatible with the FFM modalities. This is why the leader of FFM microscopes, Zeiss, strongly supports our developments (see the accompanying letter).