Superresolution imaging system on innovative localization microscopy technique with commonly using dyes and CMOS camera

摘要

Optical methods for study biological tissue and cell at micro- and nanoscale level step now over diffraction limit. Really it is single molecule localization techniques that achieve the highest spatial resolution. One of those techniques, called bleaching/blinking assisted localization microscopy (BaLM) relies on the intrinsic bleaching and blinking behavior characteristic of commonly used fluorescent probes. This feature is the base of BaLM image series acquisition and data analysis. In our work blinking of single fluorescent spot against a background of others comes to light by subtraction of time series successive frames. Then digital estimation gives the center of the spot as a point of fluorescent molecule presence, which transfers to other image with higher resolution according to accuracy of the center localization. It is a part of image with improved resolution. This approach allows overlapping fluorophores and not requires single photon sensitivity, so we use 8,8 megapixel CMOS camera with smallest (1.55 um) pixel size. This instrumentation on the base of Zeiss Axioscope 2 FS MOT allows image transmission from object plane to matrix on a scale less than 100 nm/pixel using 20x-objective, thereafter the same resolution and 5 times more field of view as compared to EMCCD camera with 6 um pixel size. To optimize excitation light power, frame rate and gain of camera we have made appropriate estimations taking into account fluorophores behaviors features and equipment characteristics. Finely we have clearly distinguishable details of the sample in the processed field of view.