Live 2D SIM of α-actinin in mouse embryonic fibroblasts (MEF).
The structured illumination microscope (SIM) at the Advanced Imaging Center (AIC) of HHMI at Janelia is capable of three-dimensional multi-color super-resolution imaging at a high speed through a combination of rapidly programmable liquid crystal devices, spatial light modulator and a flexible grating pattern generation algorithm to shift and rotate the grating pattern rapidly [3]. This system thus allows its user to achieve SIM imaging speed currently not available on commercially available systems: It is capable of delivering up to 6 optical sections/microns/second.
In addition to achieving a lateral resolution of 110 nm and an axial resolution of 360 nm (for excitation at 488 nm), the 3D-SIM system at AIC is a balanced approach, among the super-resolution imaging methods, between the gain in a high spatial resolution and a relatively high speed for studying live cell dynamics.
The Unique Capabilities of Live cell SIM at the AIC
Structured illumination microscopy is an imaging method capable of doubling the spatial resolution of conventional widefield fluorescence microscopy by using spatially structured illumination light. The idea is use the interference effect when fine structures overlap, which produces the moiré fringes which produces pattern much larger than the actual structures that create it, thus making it possible for conventional microscope to detect. The moiré thus contain otherwise undetectable high-resolution information which can then be extracted to reconstruct an extended-resolution final image.
The strength of SIM is that it is accommodating to fluorophores choice. Unlike its other nanoscopy counterparts such as localization super-resolution microscopy or stimulated emission depletion microscopy, there is little special consideration needed in sample preparation. Samples used for confocal or widefield fluorescence microscopy can be easily adapted for SIM imaging.
One drawback of conventional SIM is its image acquisition speed. In order to obtain enough information for the subsequent image reconstruction, SIM must take 9 widefield images (shifting the structure illumination pattern in three angles and three phases) for two-dimensional SIM or up to 15 images for three-dimensional SIM. Such requirement in most commercially available SIM systems severely compromises the temporal resolution needed to perform live cell imaging. In addition, conventional SIM does not provide as robust an improvement on axial resolution as it does on lateral resolution.
What sets the AIC SIM microscope apart from its commercial counterparts is the use of the spatial light modulator that is capable of rapidly delivering the desired illumination pattern without having to physically move any microscope component. This feature greatly speeds up the imaging process (up to 6 optical sections/microns/second), making the system in the AIC one of the fastest SIM systems in the world. In addition, the AIC SIM is fully equipped to perform total internal reflection fluorescence TIRF-SIM at up to 10 frames/sec, providing an exquisitely detailed and high contrast look at dynamic processes within ~100 nm or so of the surfaces of cells.
In the TIRF-SIM mode, the system uses a different SLM patterns and a different demagnification factor from the SLM to the sample, compared to the 3D-SIM. Two high NA objectives are available for TIRF-SIM. The objective with NA 1.49 needs only the common microscope coverslips and immersion oil, and can achieve the lateral resolution of ~100 nm. The objective of NA 1.70 requires special immersion oil (RI = 1.79) and special coverslips, and can achieve a lateral resolution of 82 nm.
These unique strengths, coupled with the inherently accommodating nature of SIM in terms of fluorophores choice, makes our 3D-SIM ideally suited for observing macromolecular interactions and subcellular structure in live cell. Our SIM system is housed within a temperature controlled, CO2 incubator, making it ideal for most live cell work.
Live 3D-SIM imaging of HeLa cell stained with MitoTracker Green, a mitochondria-specific dye. Each 3D volume was acquired in 20.33 sec, with 35 msec per exposure. Figure adapted from Shao, L., et al. Nat Meth 2011.
SIM Strengths
• X-Y-Z resolution of 110 X 110 X 360nm for 488nm excitation in the 3D mode, XY resolution of 82 X 82nm in the TIRF mode with 1.7 NA objective lens.
• No special sample preparation requirements
• No special fluorescent probe requirement
• Imaging ~2-10 sections/sec in 3D-SIM mode, ~10 frames/sec in TIRF-SIM mode
• 2-color 3D live cell imaging possible at multiple time points (up to ~50 time points)
SIM Limitations
• Post-processing of images is required for reconstruction.
• Samples should be thinner than 10 µm.
• The SIM reconstruction algorithm suffers from motion artifacts, if the sample moves while the illumination pattern changes. However, motion less than 100 nm/s is well tolerated [4].
• Photobleaching and phototoxicity can severely restrict imaging time. The lattice light sheet microscope can serve a possible alternative if necessary.
Excitation
• 405 nm, 488 nm, 560 nm (currently available)
• 445 nm and 647 nm (to be added in the near future)
Detection
• 2 channels
Further Reading
1. Gustafsson, M. G. (2000). Surpassing the lateral resolution limit by a factor of two using structured illumination microscopy. Journal of Microscopy, 198(Pt 2), 82–87.
2. Gustafsson, M. G. L. et al. Three-dimensional resolution doubling in wide-field fluorescence microscopy by structured illumination. Biophys J 94, 4957–4970 (2008).
3. Fiolka, R., Shao, L., Rego, E. H., Davidson, M. W. & Gustafsson, M. G. L. Time-lapse two-color 3D imaging of live cells with doubled resolution using structured illumination. Proc Natl Acad Sci USA 109, 5311–5315 (2012).
4. Shao, L., Kner, P., Rego, E. H. & Gustafsson, M. G. L. Super-resolution 3D microscopy of live whole cells using structured illumination. Nat Meth 8, 1044–1046 (2011).
5. Kner, P., Chhun, B. B., Griffis, E. R., Winoto, L., & Gustafsson, M. G. L. (2009). Super-resolution video microscopy of live cells by structured illumination. Nature Methods, 6(5), 339–342.
6. Schermelleh, L., Heintzmann, R. & Leonhardt, H. A guide to super-resolution fluorescence microscopy. J Cell Biol 190, 165–175 (2010).
7. Agard, David: Structured Illumination Microscopy (YouTube Video).
8. Beach, J.R., Shao, L., Remmert K., Li, D., Betzig, E., & Hammer, J.A. Non-muscle myosin II isoforms coassemble in living cells. Curr. Biol., 24, 1160-1166 (2014)
9. Burnette, D.T., Shao, L., Ott, C., Pasapera A.M., Fischer, R.S., Baird, M.A., Loughian, C.D., Delance-Ayari, H., Paszek, M. J., Davidson, M.W., Betzig, E., & Lippincott-Schwartz, J., J. Cell Biol. 205, 83-96 2014)