Very high numerical aperture three-dimensional imaging with three objectives
Huelsnitz, Thomas Pearce
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he increasing importance of three dimensional observation in biology necessitates advances in three dimensional microscopy techniques. In widefield and confocal microscopy, lateral resolution is 2 to 3 times better than axial resolution. This anisotropic resolution leads to poor three-dimensional imaging. We propose a microscope which utilizes three high numerical aperture objectives to image a sample from multiple angles and interferometrically combine the information from each objective to obtain a higher resolution and more isotropic point spread function than standard widefield and confocal techniques. The proposed microscope has a higher effective numerical aperture than standard techniques, thus it is more efficient at collecting light emitted from the sample. This improves the microscope’s signal to noise ratio and, in the context of fluorescence microscopy, reduces photobleaching. Here we introduce the theoretical framework for describing the performance of a microscope and then we apply that same reasoning to quantitatively compare our proposed microscope to existing techniques.