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Adaptive Holographic Region of Interest Illumination with Oblique Angles for use in Single Molecule Localization Microscopy

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Abstract

To improve the localization accuracy and to reduce the overall photo-damage in Single Molecule Localization Microscopy (SMLM), we introduce a holography based Region-of-Interest (ROI) illumination with oblique angles. The ROI illumination is realized in two different application modes which offer different advantages. Both modes (A,B) are implemented within the same setup by modifying the illumination light using a phase-only spatial light modulator (SLM) twice. This allows to adaptively modify the size and the (excitation) angle of the required ROI illumination, resulting in reduced out-of focus signal and less overall phototoxicity. Mode A generates a nearly speckle free, circular ROI which can be obtained instantly (no iterative algorithm needed). Mode B allows to realize an arbitrarily shaped ROI but comes at the cost of a higher presence of speckle structures as well as in an increased calculation time of the holograms. In both cases illumination angles up to 60° with high NA objectives are realizable, enabling the effective selection of specific parts of a biological probe to be illuminated.

© 2019 SPIE/OSA

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