| Autor: |
Bittel AM; Department of Biomedical Engineering, Oregon Health and Science University, Portland, Oregon 97201., Saldivar IS; Department of Biomedical Engineering, Oregon Health and Science University, Portland, Oregon 97201., Dolman N; Molecular Probes Labeling and Detection Technologies, Thermo Fisher Scientific, Eugene, Oregon 97402., Nickerson A; Department of Biomedical Engineering, Oregon Health and Science University, Portland, Oregon 97201., Lin LJ; Department of Biomedical Engineering, Oregon Health and Science University, Portland, Oregon 97201., Nan X; Department of Biomedical Engineering, Oregon Health and Science University, Portland, Oregon 97201.; Knight Cancer Institute, Oregon Health and Science University, Portland, Oregon 97201.; Oregon Center for Spatial Systems Biology, Oregon Health and Science University, Portland, Oregon 97201., Gibbs SL; Department of Biomedical Engineering, Oregon Health and Science University, Portland, Oregon 97201.; Knight Cancer Institute, Oregon Health and Science University, Portland, Oregon 97201.; Oregon Center for Spatial Systems Biology, Oregon Health and Science University, Portland, Oregon 97201. |
| Abstrakt: |
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. |
