Optical microscopes have proven their use as a powerful tool for studying a variety of biological samples. In spite of many successes, there are still numerous obstacles limiting practical applications. Most limiting are the inherent background of physiological samples, photobleaching, and phototoxicity. To allow studies of long lasting processes such as drag delivery, three-dimensional cellular structures, embryogenesis, we have combined a technique called Single Plane Illumination Microscopy (SPIM) with Multi-Pulse Pumping with Time-Gated Detection (MPP-TGD) in order to enhance the signal relative to background. This new method allows for a decrease in light exposure times and improves image quality. This combination allows a new outlook into a variety of important, long-lasting biological processes at a level of detection previously unattainable.
Multi-pulse pumping is a burst of excitation pulses instead of a single pulse which enhances the excited state population of a long-lived label. This label is chosen so that its lifetime is at least 5 times longer than that of typical autofluorescence. The pulse separation within the burst is chosen so that it is at least 5 times shorter than the lifetime of the label. In this case only the population of the fluorescent label is increased and the background remains the same. By subtracting the image acquired with the burst from an image with a single pulse, we were able to increase the signal-to-background ratio of about 100 fold.
KEYWORDS: Molecular assembly, Real time imaging, Molecules, Optical microscopy, Image resolution, Ultrafast imaging, Super resolution, In vitro testing, In vivo imaging, Biomedical optics
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.
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