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Structured illumination microscopy SIM is a wide-field technique in fluorescence microscopy that provides fast data acquisition and two-fold resolution improvement beyond the Abbe limit. We observed a further resolution improvement using the nonlinear emission response of a fluorescent protein. We demonstrated a two-beam nonlinear structured illumination microscope by introducing only a minor change into the system used for linear SIM LSIM. To achieve the required nonlinear dependence in nonlinear SIM NL-SIM we exploited the photoswitching of the recently introduced fluorophore Kohinoor. It is particularly suitable due to its positive contrast photoswitching characteristics. Contrary to other reversibly photoswitchable fluorescent proteins which only have high photostability in living cells, Kohinoor additionally showed little degradation in fixed cells over many switching cycles.



Autor: Hui-Wen Lu-Walther, Wenya Hou, Martin Kielhorn, Yoshiyuki Arai, Takeharu Nagai, Michael M. Kessels, Britta Qualmann, Rainer Heint

Fuente: http://plos.srce.hr/



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