| Popis: |
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. |
