Nanometric axial localization of single fluorescent molecules with modulated excitation

Abstract

Strategies have been developed in LIDAR to perform distance measurements for non-coherent emission in sparse samples based on excitation modulation. Super-resolution fluorescence microscopy is also striving to perform axial localization but through entirely different approaches. Here we revisit the amplitude modulated LIDAR approach to reach nanometric localization precision and we successfully adapt it to bring distinct advantages to super-resolution microscopy. The excitation pattern is performed by interference enabling the decoupling between spatial and time modulation. The localization of a single emitter is performed by measuring the relative phase of its linear fluorescent response to the known shifting excitation field. Taking advantage of a tilted interfering configuration, we obtain a typical axial localization precision of 7.5 nm over the entire field of view and the axial capture range, without compromising on the acquisition time, the emitter density or the lateral localization precision. The interfering pattern being robust to optical aberrations, this modulated localization (ModLoc) strategy is particularly well suited for observations deep in the samples. Images performed on various biological samples show that the localization precision remains nearly constant up to several micrometers.In the presence of coherent signals, interferometry offers unmatched sensitivity for distance measurements 1 .Measuring the relative phase between the excitation in the elastically scattered or reflected signal reaches record precisions. Interferometry has thus naturally been used in some coherent microscopy configurations to obtain nanometric axial localization 2-5 . However, in the case of fluorescence microscopy which is today the most widespread technique for cell imaging such an approach remains impossible because of the non-

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