Instant structured illumination microscopy
Due to the small size of the nematode embryo, many subcellular structures (including neurites) appear diffraction-limited. The ability to resolve structures below the diffraction limit (‘super-resolution’ microscopy) – especially tightly packed neuropil or closely spaced nuclei – would be enabling in WormGUIDES. Among the available super-resolution techniques, linear structured illumination microscopy (SIM, 1) is particularly attractive due to its speed, compatibility with standard fluorescent probes (such as GFP), and relatively low dose (~102 – 106 lower than other methods, implying far less phototoxicity). In SIM, better resolution (usually ~2x better than the diffraction limit) is obtained by multiplying excitation and emission point spread functions (PSFs). This is accomplished by exciting the sample with a series of sharp illumination patterns, recording one or more diffraction limited images, and mathematically processing these images.
We have invented an implementation of SIM that offer advantages for live, thick specimens such as worm embryos – ‘instant’ structured illumination microscopy (iSIM2). iSIM enables much more rapid imaging (10-100x improvement) than conventional SIM because only a single image is recorded and most of the image processing occurs during image formation (so less digital post-processing is needed). Additionally, and importantly for nematode embryos, a pinhole array is used to block out-of-focus fluorescence, improving SNR and SBR in these thick, densely labeled samples.
iSIM has been commercialized by Visitech International, but if you’d like to build your own, see 3.
1. Gustafsson, M. G. L. Surpassing the lateral resolution limit by a factor of two using structured illumination microscopy. (2000) J. Microsc. 198, 82-87.
2. York, A. G. et al. Instant super-resolution imaging in live cells and embryos via analog image processing. (2013) Nat Methods 10, 1122-1126.
3. Curd, A. et al. Construction of an instant structured illumination microscope. (2015) Methods 15, 30029-30023.