Structured illumination for thicker samples

[Andrew York]

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 Thought this might be of interest to the list. Please forgive the
shameless self-promotion.

 We made a new kind of structured illumination microscope (SIM):
http://dx.doi.org/10.1038/nmeth.2025 <http://code.google.com/p/msim/>
(Until the article shows up on pubmed:
http://dl.dropbox.com/u/38049396/nmeth.2025.pdf )

Rough specifications:

3D superresolution (145 nm transverse, 400 nm axial resolution)
Standard fluorescent probes like GFP
Works in thick samples (>50 microns)
1 2D slice per second
Live-cell compatible (tens or hundreds of 3D volumes)
Low photobleaching (similar to a spinning disk confocal)
50x50 micron field of view

 We tried to combine the sectioning of a spinning-disk microscope with the
resolution-doubling of traditional SIM. So far our users seem pleased with
performance in embryos and tissue sections.

 I have very little experience with commercial microscopes, so I'm
particularly interested how we compare to existing, proven technology. Has
anyone gotten good results more than 10 microns deep with a commercial or
homebuilt SIM? In zebrafish embryos, we seem to take better pictures than a
commercial point-scanning confocal at similar depth, although I'm not sure
how to compare the two microscopes in a way that's truly fair.

 Not sure how popular it's going to be, but if you're interested in using
the scope, contact our lab at the NIH:
http://www.nibib.nih.gov/Research/Intramural/HighResolutionOpticalImaging

 It's actually reasonably straightforward to build one of your own. Let me
know if you're interested in building one, I might be able to help out. The
software is all open-source, too:
http://code.google.com/p/msim/

Links to the supplementary material (movies!):

Supplementary Text and Figures
(2M)<http://www.nature.com/nmeth/journal/vaop/ncurrent/extref/nmeth.2025-S1.pdf>
Supplementary Figures 1-10 and Supplementary Notes 1-4

Supplementary Video 1
(11M)<http://www.nature.com/nmeth/journal/vaop/ncurrent/extref/nmeth.2025-S2.gif>
Multifocal excitation (left), pinholing, scaling (middle) and summing
process (right). Data are from the 120-frame acquisition displayed in
Figure 1; only the top-right region of the field is displayed.

Supplementary Video 2
(3M)<http://www.nature.com/nmeth/journal/vaop/ncurrent/extref/nmeth.2025-S3.gif>
Dual-color MSIM stack of a fixed cell, to accompany Figure 2. Green, Alexa
Fluor 488-immunolabeled microtubules; magenta, Mitotracker Red-labeled
mitochondria.

Supplementary Video 3
(10M)<http://www.nature.com/nmeth/journal/vaop/ncurrent/extref/nmeth.2025-S4.gif>
Dual-color wide-field stack of a fixed cell, to accompany Figure 2. Green,
Alexa Fluor 488-immunolabeled microtubules; magenta, Mitotracker
Red-labeled mitochondria. The illumination dose is the same as in
Supplementary Video 2.

Supplementary Video 4
(10M)<http://www.nature.com/nmeth/journal/vaop/ncurrent/extref/nmeth.2025-S5.mov>
A 3D rendering of the zebrafish tissue section shown in Figure 3. The
three-dimensional volumetric rendering and video were made from the MSIM
stack of two-dimensional images using Imaris software version 7.3
(Bitplane). Display brightness and contrast were adjusted to emphasize the
full dynamic range of the intensities in the images.

Supplementary Video 5
(13M)<http://www.nature.com/nmeth/journal/vaop/ncurrent/extref/nmeth.2025-S6.mov>
MSIM z stack of the zebrafish tissue section shown in Figure 3.

Supplementary Video 6
(9M)<http://www.nature.com/nmeth/journal/vaop/ncurrent/extref/nmeth.2025-S7.mov>
Confocal z stack (taken on a Zeiss 510) of zebrafish tissue shown in
Supplementary Figure 8.

Supplementary Video 7
(8M)<http://www.nature.com/nmeth/journal/vaop/ncurrent/extref/nmeth.2025-S8.mov>
MSIM maximum intensity projections of the zebrafish dataset described in
Figure 4.

Supplementary Video 8
(3M)<http://www.nature.com/nmeth/journal/vaop/ncurrent/extref/nmeth.2025-S9.mov>
MSIM z stack of the first timepoint shown in Figure 4.

Supplementary Video 9
(1M)<http://www.nature.com/nmeth/journal/vaop/ncurrent/extref/nmeth.2025-S10.mov>
A 3D rendering of the inset shown in Figure 4b. The three-dimensional
volumetric rendering and video were made from the MSIM stack of
two-dimensional images using ImageJ.

Supplementary Video 10
(10M)<http://www.nature.com/nmeth/journal/vaop/ncurrent/extref/nmeth.2025-S11.mov>
MSIM time series of the indicated slice, to accompany data in Supplementary
Figure 9.

Supplementary Video 11
(6M)<http://www.nature.com/nmeth/journal/vaop/ncurrent/extref/nmeth.2025-S12.gif>
MSIM maximum intensity projections of the two-color cellular dataset
described in Figure 5. Scale bar, 5 μm.

Supplementary Software
(18M)<http://www.nature.com/nmeth/journal/vaop/ncurrent/extref/nmeth.2025-S13.zip>
Processing code for constructing resolution-doubled images from raw MSIM
data. (A bit outdated; see code.google.com/p/msim for the most recent code).