A method of laser dark-field illumination for equal-probability excitation and photobleaching of randomly oriented fluorescent molecules

Abstract

Calculations have been done that allow developing a new type of illuminators optimized for dark-field microscopy and fluorescent nanoscopy, a new type of ultrahigh-resolution microscopy for which we have patent priority, and its later analogs. The concept proposed here substantiates the feasibility of uniformly illuminating different parts of an object to ensure equal probability of excitation and, in due time, simultaneous photobleaching of fluorescent molecules despite the random orientation of their light-absorbing oscillators. The latter is important because non-bleached fluorophores that accumulate in the specimen produce background noise and make it too difficult to locate the centers of spots produced by new fluorescent molecules.