Fluorescence microscopy has poor spatial resolution (~200nm for the best confocal microscope) owing to the diffraction limit, but provides exquisite dynamical information on live cells. With the development of novel fluorescent sensors and molecules in the past decade, the usage of fluorescence microscopy (especially confocal microscopy) for live-cell studies has greatly increased. The goal of our project is to increase the resolution of confocal imaging techniques to a few tens of nanometers in spatial resolution, using a new microscopy technique that we termed """"""""Difference Deconvolution Microscopy (DDM)"""""""" Furthermore, we plan to increase the temporal resolution of DDM beyond that of traditional scanning confocal microscopy, so we can access even faster dynamical information than currently possible using a standard scanning confocal microscope. We believe DDM, when successfully developed, will increase dramatically the resolution and capability of fluorescence microscopy, particularly for applications in live- cell imaging where laser power needs to be sufficiently low to avoid cellular damage and the time resolution must be sufficiently fast to resolve the dynamics of interest. The goal of this project is to increase the resolution of confocal imaging techniques to the tens of nanometers in length scale and with sub-millisecond time resolution for live-cell imaging, using a new microscopy method that we termed """"""""Difference Deconvolution Microscopy (DDM)"""""""". DDM uses comparable experimental parameters (e.g. similar laser powers) as confocal microscopy, and thus is inherently suited to use with fragile samples, such as live mammalian cells. ? ? ? ?
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