Localisation microscopy of individual molecules allows one to bypass the diffraction limit, revealing cellular organisation on a nanometric scale. This method, which relies on spatial analysis of the signal emitted by molecules, is often limited to the observation of biological objects at shallow depths, or with very few aberrations. The introduction of a temporal parameter into the localisation process through a time-modulated excitation was recently proposed to address these limitations. This method, called ModLoc, is demonstrated here with an alternative flexible strategy. In this implementation, to encode the time-modulated excitation a digital micromirror device (DMD) is used in combination with a fast demodulation approach, and provides a twofold enhancement in localisation precision. Layout: Nowadays, we can use an optical microscope to observe how proteins are organised in 3D within a cell at the nanoscale. By carefully controlling the emission of molecules in both space and time, we can overcome the limitations set by the diffraction limit. This allows us to pinpoint the exact location of molecules more precisely. However, the usual spatial analysis method limits observations to shallow depths or causing low distortion of optical waves. To overcome these restrictions, a recent approach introduces a temporal element to the localisation process. This involves changing the illumination over time to enhance the precision of localisation. This method, known as ModLoc, is showcased here using a flexible and alternative strategy. In this setup, a matrix of micrometric mirrors, working together with a fast demodulation optical module, is used to encode and decode the time-modulated information. This combination results in a twofold improvement in localisation precision.
Keywords: DMD; fluorescence; microscopy; single-molecule localisation; structured excitation.
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