Abstract
Fluorescence superresolution (SR) microscopy, or fluorescence nanoscopy, provides nanometer scale detail of cellular structures and allows for imaging of biological processes at the molecular level. Specific SR imaging methods, such as localization-based imaging, rely on stochastic transitions between on (fluorescent) and off (dark) states of fluorophores. Imaging multiple cellular structures using multi-color imaging is complicated and limited by the differing properties of various organic dyes including their fluorescent state duty cycle, photons per switching event, number of fluorescent cycles before irreversible photobleaching, and overall sensitivity to buffer conditions. In addition, multiple color imaging requires consideration of multiple optical paths or chromatic aberration that can lead to differential aberrations that are important at the nanometer scale. Here, we report a method for sequential labeling and imaging that allows for SR imaging of multiple targets using a single fluorophore with negligible cross-talk between images. Using brightfield image correlation to register and overlay multiple image acquisitions with ~10 nm overlay precision in the x-y imaging plane, we have exploited the optimal properties of AlexaFluor647 for dSTORM to image four distinct cellular proteins. We also visualize the changes in co-localization of the epidermal growth factor (EGF) receptor and clathrin upon EGF addition that are consistent with clathrin-mediated endocytosis. These results are the first to demonstrate sequential SR (s-SR) imaging using direct stochastic reconstruction microscopy (dSTORM), and this method for sequential imaging can be applied to any superresolution technique.
Highlights
Fluorescence superresolution (SR) imaging has the potential to transform cellular imaging, providing nanometer scale detail about cellular structure and the molecular specificity of fluorescent labeling techniques
We demonstrate here a method for sequential labeling and SR imaging that utilizes a combination of photobleaching and irreversible fluorophore quenching—referred to here as “photodestruction”—to eliminate residual fluorescence signal after SR imaging
Brightfield image correlation was introduced as a means for active stabilization during SR imaging [34]; in addition to stabilization, we use this method to align the sample between imaging of different targets, a multi-target overlay is created without the need for bead registration or overlay algorithms and expedites both image acquisition and analysis
Summary
Fluorescence superresolution (SR) imaging has the potential to transform cellular imaging, providing nanometer scale detail about cellular structure and the molecular specificity of fluorescent labeling techniques. The most accessible (and arguably the highest resolution) method for fluorescence SR imaging is based on localization of single molecules; (f)PALM, (d) STORM, etc. Since single molecules can be localized with a precision much better than the diffraction limit [12], the found locations of the fluorophores that label the sample can be PLOS ONE | DOI:10.1371/journal.pone.0123941. Sequential SR Imaging of Multiple Targets Using a Single Fluorophore Since single molecules can be localized with a precision much better than the diffraction limit [12], the found locations of the fluorophores that label the sample can be PLOS ONE | DOI:10.1371/journal.pone.0123941 April 10, 2015
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