Membrane nanodomains have commonly been implicated in biological processes. However, what these nanodomains are and how they participate in the processes of interest are still unclear, primarily due to challenges in probing these nanoscopic and dynamic structures in cells. Using high-throughput single particle tracking via spt-PALM and detailed trajectory analysis, here we demonstrate that membrane nanodomains associated with the small GTPase KRas could be detected and analyzed in live cells. By stochastically activating and tracking single PAmCherry1-KRas molecules on the membrane, spt-PALM yields 5,000 to 100,000 single-molecule diffusion trajectories of KRas. Analysis of these trajectories with variational Bayes SPT (vbSPT) revealed that KRas exhibits an immobile state in domains ~70 nm in size, each embedded in a larger domain (~200 nm) that confers intermediate mobility, while the rest of the membrane supports fast diffusion. By analyzing the transition kinetics among the three states, we found that KRas is continuously removed from the membrane via the immobile state and replenished to the fast state, likely coupled to internalization and recycling. Our results demonstrate the utility of high-throughput SPT in uncovering the impact of nanoscopic landscape of the membrane on the spatiotemporal dynamics and potentially multimer formation and signaling of membrane-bound biomolecules.
Single-molecule localization microscopy (SMLM) utilizes photoswitchable fluorophores to detect biological entities with 10-20 nm resolution. Multispectral superresolution microscopy (MSSRM) extends SMLM functionality by improving its spectral resolution up to 5 fold facilitating imaging of multicomponent cellular structures or signaling pathways. Current commercial fluorophores are not ideal for MSSRM as they are not designed to photoswitch and do not adequately cover the visible and far-red spectral regions required for MSSRM imaging. To obtain optimal MSSRM spatial and spectral resolution, fluorophores with narrow emission spectra and controllable photoswitching properties are necessary. Herein, a library of BODIPY-based fluorophores was synthesized and characterized to create optimal photoswitchable fluorophores for MSSRM. BODIPY was chosen as the core structure as it is photostable, has high quantum yield, and controllable photoswitching. The BODIPY core was modified through the addition of various aromatic moieties, resulting in a spectrally diverse library. Photoswitching properties were characterized using a novel polyvinyl alcohol (PVA) based film methodology to isolate single molecules. The PVA film methodology enabled photoswitching assessment without the need for protein conjugation, greatly improving screening efficiency of the BODIPY library. Additionally, image buffer conditions were optimized for the BODIPY-based fluorophores through systematic testing of oxygen scavenger systems, redox components, and additives. Through screening the photoswitching properties of BODIPY-based compounds in PVA films with optimized imaging buffer we identified novel fluorophores well suited for SMLM and MSSRM.
Cancer is a major health threat worldwide. Options for targeted cancer therapy, however, are often limited, in a large part due to our incomplete understanding of how key processes including oncogenesis and drug response are mediated at the molecular level. New imaging techniques for visualizing biomolecules and their interactions at the nanometer and single molecule scales, collectively named fluorescence nanoscopy, hold the promise to transform biomedical research by providing direct mechanistic insight into cellular processes. We discuss the principles of quantitative single-molecule localization microscopy (SMLM), a subset of fluorescence nanoscopy, and their applications to cancer biomedicine. In particular, we will examine oncogenesis and drug resistance mediated by mutant Ras, which is associated with ~1/3 of all human cancers but has remained an intractable drug target. At ~20 nm spatial and single-molecule stoichiometric resolutions, SMLM clearly showed that mutant Ras must form dimers to activate its effector pathways and drive oncogenesis. SMLM further showed that the Raf kinase, one of the most important effectors of Ras, also forms dimers upon activation by Ras. Moreover, treatment of cells expressing wild type Raf with Raf inhibitors induces Raf dimer formation in a manner dependent on Ras dimerization. Together, these data suggest that Ras dimers mediate oncogenesis and drug resistance in tumors with hyperactive Ras and can potentially be targeted for cancer therapy. We also discuss recent advances in SMLM that enable simultaneous imaging of multiple biomolecules and their interactions at the nanoscale. Our work demonstrates the power of quantitative SMLM in cancer biomedicine.
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.
KEYWORDS: Molecules, Switching, Picture Archiving and Communication System, Photons, Super resolution microscopy, Signal detection, Particles, Microscopy, Luminescence, Solids
Super resolution microscopy (SRM) has overcome the historic spatial resolution limit of light microscopy, enabling fluorescence visualization of cellular structures and multi-protein complexes at the nanometer scale. Using singlemolecule 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. However, the spectral resolution of current SRM techniques are limited by existing fluorophores with only up to four colors imaged simultaneously, limiting the number of intracellular components that can be studied in a single sample. In the current work, a library of novel BODIPY-based fluorophores was synthesized using a solid phase synthetic platform with the goal of creating a set of photoswitchable fluorophores that can be excited by 5 distinct laser lines but emit throughout the spectral range (450-850 nm) enabling multispectral super resolution microscopy (MSSRM). The photoswitching properties of all new fluorophores were quantified for the following key photoswitching characteristics: (1) the number of photons per on cycle (2) the number of on cycles (switching events), (3) the percentage of time the fluorophore spends in the fluorescent on and off states, and (4) the susceptibility of the fluorophore to photobleaching (time of last event). To ensure the accuracy of our photoswitching measurements, our methodology to detect and quantitate the photoswitching properties of individual fluorophore molecules was validated by comparing measured photoswitching properties of three commercial dyes to published results.1 We also identified two efficient methods to positionally isolate fluorophores on coverglass for screening of the BODIPY-based library.
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