We propose to build and use custom designed microfluidics chips that, when combined with associated optics, will greatly facilitate several modalities of fluorescence super- resolution imaging. The essential concept is the presence of a reflective, planar surface that makes an angle of near 45 degrees with respect to the cover slip normal. This reflective surface will allow a single imaging objective to also provide planar illumination at an image focal plane placed anywhere inside a mammalian cell. Since only the imaged focal plane is illuminated, the fluorescence background is greatly reduced and fluorophore longevity is improved. The dimensions of the cell accessible volumes of the chip will be designed such that cells will always locate to regions amenable for imaging by the reflected light. We will focus on three imaging modalities: 3D Single-Molecule Super-Resolution (SM-SR), extended time series, 3D imaging, and 3D Structured Illumination Microscopy (SIM). The project will focus around design of the microfluidics device and optics, and assessing the imaging performance of each of the three above mentioned imaging modalities. For each, we will compare results when using the device to conventional methods in terms of resolution, both spatial and temporal, and probe photo-stability. The developed devices and techniques will be immediately used for studying FceR1 associated signal transduction in RBL-2H3 cells, which is the central focus of the 'The New Mexico Center for Spatiotemporal Modeling of Cellular Signaling'(STMC). The STMC is an NIH funded center for systems biology, of which both senior personnel are members.
(provided by applicant): We will develop a microfluidics chip device that will be used for fluorescence imaging of fluorescently tagged proteins within single cells. The device will allow imaging at faster speed and at better resolutions than conventional microscopy approaches by making use of techniques that would otherwise have to be implemented at much greater cost or sophistication. This device could be used by many researchers as an addition to existing equipment.
|Liu, Sheng; Lidke, Keith A (2014) A multiemitter localization comparison of 3D superresolution imaging modalities. Chemphyschem 15:696-704|