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Abstract
Wide-field fluorescence microscopy, while much faster than confocal microscopy, suffers from a lack of optical sectioning and poor axial resolution. 3D structured illumination microscopy (SIM) has been demonstrated to provide optical sectioning and to double the resolution limit both laterally and axially, but even with this the axial resolution is still worse than the lateral resolution of unmodified wide-field microscopy. Interferometric schemes using two high numerical aperture objectives, such as 4Pi confocal and I5M microscopy, have improved the axial resolution beyond that of the lateral, but at the cost of a significantly more complex optical setup. Here, we theoretically and numerically investigate a simpler dual-objective scheme which we propose can be easily added to an existing 3D-SIM microscope, providing lateral and axial resolutions in excess of 125 nm with conventional fluorophores and without the need for interferometric detection.
Highlights
Over the past three decades, there has been a considerable effort to improve the spatial resolution of fluorescence microscopy, with the majority of advances being focussed on improving the lateral resolution [1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16]
The main difficulty arising in implementing Type C 4Pi or I5S microscopy, in which both illumination and emission light are made to interfere, is ensuring that the path lengths from sample to beamsplitter in the interferometric arms are equal to within the coherence length of the fluorescence light
We have investigated the possibility of further axial resolution enhancement in 3D-structured illumination microscopy (SIM) by reflecting the central beam and have provided methods for countering the existance of holes in the optical transfer function (OTF) support
Summary
Over the past three decades, there has been a considerable effort to improve the spatial resolution of fluorescence microscopy, with the majority of advances being focussed on improving the lateral resolution [1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16]. In a conventional 3D-SIM system, the illumination is generated by three interfering laser beams produced by the 0 and ±1 diffraction orders of a grating All that remains is to unmix laterally, by applying lateral phase shifts to the illumination pattern, producing as many bands of information as there are columns of components in the illumination Fourier domain distribution In this case, while there are seven components in total (magenta points in Fig. 1(c)), only five phase steps are required.
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