Device and programs for investigation of biological objects autofluorescence

A method of fluorescent sub-diffractional microscopy is proposed based on using object dyeing by special dye, which is able to bind to some object structures and initially not absorb light used for excitation of fluorescence. As a result of some processes small part of molecules is activated, that is converted to molecules able to absorb light and fluoresce sufficiently long to be registered by a sensitive video camera as separate spots covering tens of pixels of camera matrix. Then registered molecules must be photobleached or transformed to nonfluorescing state by other influence and new portion of molecules is activated. This circle of «activation» - «fluorescence excitation and registration» - «photobleaching» may be repeated thousands times. The accuracy of center spots determination may be up to 2 nm. A collection of all calculated centers of spots in all registered frames may be used for reconstruction of object image with resolution not limited by diffraction. A method of improving visibility of spots produced by single molecules in object having autofluorescence is described. © 2012 Pleiades Publishing, Ltd.