Effect of labeling density and time post labeling on quality of antibody-based super resolution microscopy images

Abstract

Super resolution microscopy (SRM) has overcome the historic spatial resolution limit of light microscopy, enabling fluorescence visualization of intracellular structures and multi-protein complexes at the nanometer scale. Using single-molecule localization microscopy, the precise location of a stochastically activated population of photoswitchable fluorophores is determined during the collection of many images to form a single image with resolution of ~10-20 nm, an order of magnitude improvement over conventional microscopy. One of the key factors in achieving such resolution with single-molecule SRM is the ability to accurately locate each fluorophore while it emits photons. Image quality is also related to appropriate labeling density of the entity of interest within the sample. While ease of detection improves as entities are labeled with more fluorophores and have increased fluorescence signal, there is potential to reduce localization precision, and hence resolution, with an increased number of fluorophores that are on at the same time in the same relative vicinity. In the current work, fixed microtubules were antibody labeled using secondary antibodies prepared with a range of Alexa Fluor 647 conjugation ratios to compare image quality of microtubules to the fluorophore labeling density. It was found that image quality changed with both the fluorophore labeling density and time between completion of labeling and performance of imaging study, with certain fluorophore to protein ratios giving optimal imaging results.

Regulation of Natural Killer cell cytotoxicity by shedding of the Fc receptor CD16

“…Sample preparation can have a significant impact on the quality of acquired microscopy images. For super-resolution techniques, labelling of individual molecules with bright fluorophores is particularly important (Bittel et al, 2015). Therefore, it is crucial to optimise sample staining to ensure that the receptors of interest are brightly labelled and could be detected by single molecule localisation microscopy in high resolution.…”