Objectives and optics for are rapidly evolving to allow wider and wider FOVs for two photon laser scanning microscopes (2PLSM). This development introduces a new challenge: point-by- point raster scanning over the entirety of the FOV requires the experimenter to sacrifice either single-cell sensitivity (because of faster scanning rates required to cover the larger field in the same amount of time) or temporal resolution (because of scanning at the same speed and thus taking longer to cover the FOV), or both. This tradeoff will be a major pain point for neuroscience labs moving to wide-FOV 2PLSM, and a major hindrance to harvesting the scientific benefits of wide-FOV 2PLSM. Current 2PLSM systems use two scanners in series; either two galvanometer based scanners or an 8-12kHz resonant scanner paired with a galvanometer scanner. Such scanner combinations allow addressable X-Y point-by-point laser scanning at video rates for small FOV. For larger FOV it is desirable to divide the FOV into smaller regions of interest to achieve higher scanning rates that retain cellular resolution and sensitivity. The work described in this proposal will produce a nove 2PLSM three scanner hardware solution that will help address this challenge. Combining a resonant (R) scanner with a galvanometer scanner (G) pair provides a means to perform faster scans in arbitrary rectangular regions of interest within a FOV. Vidrio Technologies' patent pending RGG scanning assembly will be a compact, plug-and-play system that will easily integrate into existing 2PLSM systems. The software required to drive this new scanner assembly will be added to Vidrio's existing and popular 2PLSM imaging software ScanImage. The multi-region of interest imaging approach we describe, is designed to scale up with the rapidly expanding reach of 2PLSM cellular resolution brain activity imaging thus providing a long term solution for large FOV imaging.
Two-photon laser scanning microscopy has revolutionized neuroscience by allowing three-dimensional imaging with cellular resolution within the brain, an impact recently complemented by rapid developments in fluorescent biochemical reporter molecules. Optical engineers and scientists have been recently developing new lenses to expand two-photon imaging beyond the millimeter barrier, thus enabling the imaging of multiple cortical columns that is required for studying integrative brain functions. Traditional two-photon laser scanning would become very slow if scanned over such new large fields of view; we propose to develop a new scanning device and system to image rapidly in selectable smaller subregions of interest within these large new optical fields, allowing scientists to continue looking at fast activity in single cells and synapses in the brain.
| Sofroniew, Nicholas James; Flickinger, Daniel; King, Jonathan et al. (2016) A large field of view two-photon mesoscope with subcellular resolution for in vivo imaging. Elife 5: |
