Based on the fact that the absorption of light is a molecular-orientation sensitive process, fluorescence microscopy has been recently completed by a technique called angle-resolved linear dichroism. By analyzing the fluorescence emission response with respect to the polarization orientation of the exciting light, this technique allows retrieving orientation information of an ensemble of fluorescent molecules, namely the average orientation angle and the amplitude of the angular fluctuations around this average. In this PhD thesis, we implement new methods and instrumentation tools able to improve the robustness and speed of the polarization resolved data analysis, the rate of the data acquisition, and at last to explore the possibility to record molecular 3D orientation information. A scheme able to monitor the real-time orientation properties of fluorescent lipid probes is proposed using a high-speed spinning disk coupled to camera imaging, combined with fast switching of the polarization state by an electro optical modulator. A new data processing method is developed which considerably improves the speed and the precision of the retrieved information by investigating the sources of bias and uncertainty due to noise and instrumentation factors. The technique has been successfully tested on giant unilamellar vesicles and on living cells labeled with different fluorescent lipid probes, DiIC18 and di-8-ANEPPQ. It was able to acquire precise molecular orientation images at full frame rates in the range of one frame per second. At last in order to probe unambiguously the 3D orientation information of an ensemble of molecules, a new method is proposed and supported by simulations, based on the out-of-plane tuning of the excitation polarization realized in the focusing volume by coherently summing linearly and radially polarized fields.
