Introduction

The STED (Stimulated Emission Depletion) microscope allows the image of a biological sample to be obtained by virtually decomposing the biological sample in regions from which an optical signal is recorded. By recomposing all the signals from the different regions it is possible to reconstruct a very detailed image (in super-resolution) of the sample. Each optical signal includes a main component emitted by the markers present in the region in question and excited by a first light beam and a spurious component (background noise), emitted by other markers of the same chemical species but excited undesirably by a second beam of light. For this purpose, the method according to this invention uses a synchronous detection system to reliably estimate, and subsequently eliminate, the unwanted background noise generated by the direct excitation of the fluorescent species with the second light beam (called STED beam).

Technical features

Synchronous detection is performed pixel by pixel, with periodic disruption of the excitation beam. This interruption can be achieved by using the modulation characteristics of the laser source that generates the light beam, or external devices such as acusto-optical modulators (AOM), fast mechanical shutters, electro-optical modulators (EOM) or acusto-adjustable filtersoptical (AOTF). The optical signal received from the sample is then detected synchronously with the timing of the  excitation beam and is then used for the background noise estimation. Thanks to fast excitation beam modulation, all unwanted signal fluctuations, such as photobleaching of fluorescent chemical species or sample displacement, are illuminated, thus obtaining a true noise estimate. This invention can be applied to any existing STED microscope, regardless of the type of excitation source and the STED source. In the case of pulsed lasers, the excitation beam break frequency shall be chosen taking into account the excitation beam pulse interval; In particular, it should be chosen so as to be much lower than the pulse frequency of the excitation beam. TLR 4

Possible Applications

• Imaging methods and more specifically high-resolution, high-speed microscopy methods that can resolve details below the Abbe diffraction limit
• Study of subcellular architectures and dynamics by exploiting the non-linear fluorophore response commonly used for the marking of biological samples

Advantages

• Considerably reduced background noise
• The risks associated with dead times of the detection apparatus are considerably reduced
• Method applicable in all STED microscopy configurations