Re: 3D structured illumination microscopy with an axially moving illumination pattern

Posted by Reto Fiolka on
URL: http://confocal-microscopy-list.275.s1.nabble.com/3D-structured-illumination-microscopy-with-an-axially-moving-illumination-pattern-tp7587343p7587344.html

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
Post images on http://www.imgur.com and include the link in your posting.
*****

Dear Thomas

If the axial pattern is fixed to the sample, then its "sidebands" need to be actively unmixed, i.e. you need additional phase steps along the axial pattern orientation and you would need to solve the algebraic equation system and move the sidebands to their true location in Fourier space numerically.

You can think of it as the same as 2D SIM (phase stepping, unmixing, shifting), now just applied to all three dimensions. Therefore all information gets passed through the conventional, widefield 3D OTF. This means that 3D Nyquist sampling for conventional 3D microscopy is sufficient for the raw data. In normal 3D SIM with axial self modulation, you already have to apply a higher axial sampling for the final SIM resolution.

You are correct that for Sara's Multifocus SIM you would need axial phase steps to unmix the axial information components. A conventional grating based setup can not do that (it would need to shift the grating axially over large distances in image space or have a phase shifting element for the zero diffraction order). Therefore a proper 3D reconstruction was not attempted in that work.

Such a system has to my best knowledge not been realized yet, but would unlock the full potential of multifocus 3D SIM.

Reto

PS: an interesting situation occurs if you shift the axial illumination pattern at a different rate than you make z steps in a normal 3D SIM microscope. The axial information, while being the same Fourier coefficients, would be shifted to other areas in 3D Fourier space than what the normal self demodulation would do.