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Live Cell Multicolor Structured Illumination Microscope (SIM)

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Live Cell Multicolor Structured Illumination Microscope (SIM)
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Live TIRF SIM of actin in Jurkat cell

The structured illumination microscope (SIM) at the Advanced Imaging Center (AIC) of HHMI Janelia Campus is capable of three-dimensional two-color super-resolution imaging, with 488- and 560-nm excitation, at a high speed by using a programmable liquid crystal spatial light modulator and a fast-switching variable retarder to change the grating pattern rapidly [3].  This system allows its user to achieve SIM imaging speed currently not available on commercially available systems – up to 3 optical sections/second/color in 3D mode or 6 time points in TIRF mode.

In addition to achieving a lateral resolution of 100–130 nm and axial resolution of 360–400 nm, the 3D- and TIRF-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 same idea is behind the well-known moiré pattern — the coarse pattern that appears when two much finer patterns overlap and is therefore much easier to see than either of the original patterns. In the context of microscopy, think of one of the original patterns as some undetectable super-resolution sample structures and the other pattern as a designed illumination pattern. The resultant moiré pattern is much coarser than the sample structure and thus readily detectable by a microscope. In addition, it contains encoded information about the normally unresolvable sample structures that can be mathematically decoded to make those structures effectively resolvable.

 

Figure 1: The basis of structured illumination. (Left) Moiré fringes (dark bands) form between two periodic patterns. (Right) The cyan circle (I) indicates the “observable region” of spatial frequencies in a conventional microscope. The three blue dots represent the Fourier components of a sinusoidal illumination pattern; they cannot be beyond the cyan circle because the formation of the illumination pattern by the objective lens is also subject to the same resolution limit. (II) Illuminating the sample with structured light extends the observable region in (I) to contain the spatial frequencies within two offset regions (violet). (III) Repeating structured illumination in two other directions can isotropically extend the radius of the observable region of frequencies by a factor of 2.

One of the strengths 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 no special consideration needed in sample preparation. Any fluorophore that is good for confocal or widefield fluorescence microscopy is also good for SIM.

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 of three pattern angles and three pattern phases for two-dimensional SIM or 15 images per Z slice of three pattern angles and five pattern phases for three-dimensional SIM. Such requirement in most commercially available SIM systems severely compromises the temporal resolution needed to perform live cell imaging.

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 parts. This feature greatly speeds up the imaging process (up to 3 optical sections/second/color in 3D mode or 6 time points in TIRF mode), making the system in the AIC one of the fastest SIM systems in the world.

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 fluorophore choices, makes our SIM ideally suited for observing macromolecular interactions and subcellular structure in live cell. Our SIM system is housed within a temperature and CO2 controlled incubator.

Live 3D-SIM imaging of HeLa cell stained with MitoTracker Green, a mitochondria-specific dye. Each 3D volume was acquired in 25 sec, with 35 msec per exposure. Colors blue→green→yellow→orange→red indicate depth from 0 to 6 µm. Video adapted from Shao, L., et al. Nat Meth 2011.

Live 3D-SIM imaging of U2OS cell labeled with myosin IIA-mEmerald (green) and alpha-actinin-mCherry (magenta). Sample prepared by Dylan Burnette, Lippincott-Schwartz Group, NICHD/NIH

vimentin FRAP

Live TIRF-SIM imaging of RPE cells labeled with vimentin-mEmerald. The spatial light modulator can be programmed to excite a selected region of interest (square box at the beginning of the video) and thus allow fluorescence recovery after photobleaching (FRAP).

SIM Strengths

  • X-Y-Z resolution of 110 × 110 × 360 nm for 488nm excitation in the 3D mode; lateral resolution of 100 nm in the TIRF mode (or 82 nm if NA 1.7 objective is used)
  • No special sample preparation or fluorophore requirements
  • As fast as 3 sections/sec/color in 3D mode,  or 6 frames/sec in TIRF-SIM mode
  • 2-color 3D live cell imaging capability

SIM Limitations

  • Post-processing of images is required for reconstruction.
  • Samples for 3D SIM should be thinner than 12 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.

Excitation

  • 488 nm
  • 560 nm

Detection

  • Green
  • Orange/Red

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)