Simple, Low-Cost Bayesian Super Resolution Microscopy

Abstract

Super-resolution Microscopy has made it possible to study sub-cellular structures below the resolution limit. Most super-resolution techniques, however, are costly and/or difficult to implement.We developed a low-cost super-resolution technique that allows us to observe details in the localization of rabbit polyclonal antibodies against KMP-11 protein on the membrane of Trypanosoma cruzi using equipment that is readily available in most biology labs. Also, we have confirmed the capability of our technique by imaging microtubules in Astrocytoma cell line. The technique uses an open-source algorithm based on Bayesian localization of blinking and bleaching of fluorescent molecules[1]. The process of bleaching and blinking is purely stochastic and common to all fluorescent probes[2]; therefore, the use of photoswitching dyes and specialized equipment such as EMCCDs or lasers is not necessary. Our implementation uses a standard fluorescence microscope with a mercury lamp, Alexa Fluor 594 and 488 as dyes and a standard scientific CCD.We have obtained super-resolution reconstructions of Trypanosoma cruzi with 70-nm FWHM for the cell membrane. The FWHM of microtubules was as low as 40-nm. These reconstructions exhibit much greater detail than the original fluorescence images.Our reconstructions are of inferior quality than those from other stochastic super-resolution techniques, however they can be achieved with a simple and accessible technique and standard laboratory instruments, making it suitable for both teaching and low-budget research labs.