Posted by
Reto Fiolka on
Sep 26, 2017; 2:19pm
URL: http://confocal-microscopy-list.275.s1.nabble.com/3D-structured-illumination-microscopy-with-an-axially-moving-illumination-pattern-tp7587343p7587347.html
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Dear Ian
"In traditional 3-beam £D SIM you can phase step any of the 3 beams
relative to the other two to shift the pattern axially. If you step the
central one then you just shift the pattern axially. If you shift
either of the two outer beams you shift both axially and
laterally. On the OMX Blaze setup, you focus the stripes pattern with
the exact objective focal plane by shifting one beam relative to the
other two."
To my knowledge it has not been attempted to forward a phase shift (say to the zero order alone) such that the 3D illumination pattern remains stationary on the sample while acquiring a 3D Stack. This is harder to achieve than keeping the axial illumination component shift invariant (i.e. fixed to the focal plane while acquiring a stack). You would need a very well characterized phase shifting element or alternatively shift the grating over large distances axially in image space.
However, the benefit of such a scheme would be that one could reduce the number of images for a 3D SIM reconstruction. Using three beam interference, one would need to unmix seven information bands per orientation, so 21 images per plane (3x7) would be the minimum for a reconstruction. However, the z-sampling of the data could be twice as coarse as in traditional 3D SIM. Thus for an equivalent z-step, one would need to compare 21 images to 30 images in conventional 3D SIM (two slices of 15 images).
This speed gain is known in the SIM community, but no one to my knowledge has dared to implement such a scheme. As mentioned, the axial phase stepping is harder to get right. If you do not keep the pattern perfectly stationary while acquiring an individual 3D stack, weird things can happen (one I mentioned at the end of my previous message).
The situation changes now with the idea of multifocus SIM, as you are forced into the 3D shift variant illumination regime where the 3D pattern is fixed to the sample while the multifocus system acquires a 3D stack in parallel. The advantages would be that you would be done with a minimum number of 21 images for the whole volume and you reconstruct twice the z slices from the raw data (the number of z-planes for multifocus is technologically limited). So it would have its merits to implement it that way.
I am not an expert in iterative deconvolution, so I leave it to Andrew York to comment what the minimum number of images would be for such an approach.
"It ought to be possible to collect a data set of this type on an OMX
Blaze, although the current software will not do it. The reconstruction
would also need a bit of work as you would need to fit the starting
phase for each Z position of the pattern, or calculate how the phase
shifts as you shift the pattern in Z. "
That would be great if the OMX blaze could provide the axial pattern shifts with its Galvo setup.
"> 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.
Isn't this in effect what the Abrahamsson multi focus device does?"
I tried to describe a different effect, which can arise if you attempt in a conventional 3D SIM microscope setup to acquire a z-stack while trying to keep the axial illumination fixed to the sample, but you actually slowly drift away with the pattern while acquiring the stack. This lowest order error in forwarding the zero order phase has some surprising results, it can self demodulate the axial information, but place it in the wrong place of Fourier space. It could be used in some clever ways to reduce the amount of image data, but it so far has remained a curiosity in the SIM field.
This could not happen if you have instantaneous Z stacks from the Abrahamsson device, as there is no z-stepping of the objective needed.
Best,
Reto