Posted by
Thomas Oldershaw on
URL: http://confocal-microscopy-list.275.s1.nabble.com/3D-structured-illumination-microscopy-with-an-axially-moving-illumination-pattern-tp7587343.html
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Dear list,
I am involved in developing a three-beam 3DSIM system and have noticed that in every system I've read about, the pattern is kept fixed in relation to the objective. However, in Gustafsson et al.'s 2008 article on 3D structured illumination microscopy [1], it is stated that:
"either the axial functions I_m are also purely harmonic, or, when the three- dimensional data are acquired as a sequence of two-dimensional images with different focus, the illumination pattern is maintained fixed in relation to the focal plane of the microscope, not in relation to the object."
This is justified by showing that under this arrangement the axial illumination pattern acts to multiply through with the point spread function, effectively providing increased axial support to the overall optical transfer function 'for free' (hence why only five phase steps are required for unmixing despite seven points in the illumination structure being present).
My question relates to the situation in which the pattern remains fixed in relation to the object, but with purely harmonic axial functions I_m. Based on my limited understanding, this would seem to reduce the axial situation to that of the lateral pattern, requiring phase steps to separate out the components. Indeed, equation 4 in that paper seems to show that the axial illumination pattern does not multiply the point spread function and thereby does not give the increased axial support to the OTF 'for free'. Is this correct? If we were to build a system in which the pattern remained fixed in relation to the object would we need seven images per slice? Would we even have doubled axial resolution when we do this?
This situation seems similar to that of the multifocus SIM recently published by Abrahamsson et al. [2]. Here the authors were unable to design a 3D reconstruction routine for a similar situation in which the object remains fixed, but say that such a scheme should be possible.
Thank you for any clarity anyone can provide,
Thomas
[1] Gustafsson et al. Three-dimensional resolution doubling in wide-field fluorescence microscopy by structured illumination. Biophysical Journal 94 4957-4970 (2008).
https://doi.org/10.1529/biophysj.107.120345[2] Abrahamsson et al. Multifocus structured illumination microscopy for fast volumetric super-resolution imaging. Biomedical Optics Express 8 4135-4140 (2017).
https://doi.org/10.1364/BOE.8.004135