The long standing unmet need of optical microscopy has been imaging subcellular structures with nanometer precision with speed that will allow following physiological processes in real time. Herein we presenting a new approach (multi-pulse pumping with time-gated detection; MPP-TGD) to increase image resolution and most importantly to significantly improve imaging speed. Alternative change from single pulse to multiple-pulse excitation within continuous excitation trace (in interleave excitation mode) allows for the instantaneous and specific increase (many-folds) in the intensity of subwavelength sized object labeled with long-lived probes. This permits for quick localization of the object. Such intensity change (blinking) on demand can be done with MHz frequency allowing for ultrafast point localization several hundred folds faster than localization based on single molecule blinking. Much higher speed for super-resolution imaging will pave the way for obtaining real time functional information and probing structural rearrangements at the nanometer scale in-vitro and in-vivo. This will have a critical impact on many biomedical applications and enhance our understanding of many cellular functions.
We use the microtubules as a model biological system with our new approach to studying microtubule dynamics in real time. The recent work based on single molecule localization microscopy (SMLM) (Mikhaylova et al., 2015) clearly indicates that microtubules are ~25 nm diameter hollow biopolymers that are organized in a closely spaced (about 20-70 nm apart) microtubule bundles. These structures are organized differently between axons and dendrites and their precise organization in different cell compartments is not completely understood.
Zhangatay Nurekeyev, Hung Doan, Zygmunt Gryczynski, Rafal Fudala, Ignacy Gryczynski, Julian Borejdo, Dorota Stankowska, and Sergei Dzyuba, "Multi-pulse based approach to study dynamics of molecular assemblies with subwavelength resolution (Conference Presentation)," Proc. SPIE 10500, Single Molecule Spectroscopy and Superresolution Imaging XI, 105000H (Presented at SPIE BiOS: January 28, 2018; Published: 14 March 2018); https://doi.org/10.1117/12.2299436.5751631618001.
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