Principle of structured illumination fluorescence microscopy: Illumination with a known pattern (“resolvable moire fringe”) leads to the generation of beat signals in the resulting fluorescence image, transforming signals below the diffraction limit to above the diffraction limit.
A Revolution in Optical Microscopy
Research at the level of single cells and cellular organelles makes a key conundrum very clear, i.e. the fact that our ability to visualize subcellular events (e.g. tracking of HIV-1 inside cells) in all 3 dimensions is greatly limited by the spatial resolution provided by conventional optical microscopes. The size of most viruses, for example, is substantially below the resolution limit of traditional optical microscopes. Over the last few years, however, dramatic progress has been made in optical superresolution microscopy. - In essence, all the optical superresolution techniques developed over the last couple of years rely on schemes that make use of our ability to control molecular emission properties in the far-field. They can be roughly separated into approaches that either try to locate individual fluorescent molecules with very high precision or approaches that reduce the probe volume by exploiting specific molecular processes. Through funding from the National Science Foundation (Major Research Instrumentation Program) we were able to help develop a commercial prototype of a widefield superresolution microscope: the OMX v2.0, based on widefield structured illumination microscopy. This type of superresolution microscopy was first demonstrated in Prof. John Sedat’s laboratory at the University of California, San Francisco (see e.g.: Gustafsson MGL, Shao L, Carlton PM, Wang CJR, Golubovskaya IN, Cande WZ, Agard DA, Sedat JW: "Three-dimensional Resolution Doubling in Widefield Fluorescence Microscopy by Structured Illumination", Biophys. J. 2008, 94:4957-4970; Schermelleh L et al.: "Subdiffraction Multicolor Imaging of the Nuclear Periphery with 3D Structured Illumination Microscopy", Science 2008, 320:1332-1336). The very first commercial prototype of this instrument was recently installed in our labs at the Center for Biophotonics in Sacramento, CA, in March 2008.
Structured Illumination - the Principle behind OMX
Structured illumination microscopy acquires high-resolution images of fluorescent samples by exciting them with a sinusoidal illumination pattern rather than uniform illumination. No special fluorescent probes are required for this type of superresolution microscopy, but they might be beneficial in enhancing its ultimately achievable resolution even further. In structured illumination microscopy, a periodic illumination pattern is created in the focal plane and then moved across the sample laterally and at different angles (see figure in inset).
Typically about 15 images have to be acquired in order to reconstruct a single high-resolution image per vertical plane. This scheme works because sample features with higher spatial frequencies than the illumination pattern are modulated by the pattern and result in beat frequencies that fall within the transfer function of the microscope (see the schematic representation in the figure in the inset). Since the periodicity of the illumination pattern is known, its effects on the image can be calculated and high-resolution images can be reconstructed. By moving the sample through the illumination pattern in the vertical direction, high-resolution images of the entire sample in all three dimensions are obtained.
Typically, a spatial resolution of approximately 100 nm laterally and 200 nm vertically can be achieved, but if nonlinear illumination conditions are used, e.g. by saturating the fluorescence emission, this form of super-resolution microscopy is virtually unlimited in its resolving power (Gustafsson: "Nonlinear structured-illumination microscopy: Wide-field fluorescence imaging with theoretically unlimited resolution", Proc. Natl. Acad. Sci. USA 2005, 102:13081-13086).
The example in the image to the right demonstrates the gain in resolution obtained by structured illumination microscopy. Panel A is an conventional fluorescence micrograph of 50 nm diameter fluorescent beads. Panel B shows the same image obtained with the OMX v2.0. The lower panels show a similar comparison based on the example of imaging the fibronectin network formed by rat chondrocytes grown in culture. Panel C is the conventional light micrograph, while Panel D shows the gain achieved in resolution when imaging the same sample with structured illumination microscopy.
Most recently, we have managed - for the first time - to image fenestrations in liver sinusoidal endothelial cells by 3D structured illumination microscopy. Fenestrations are collections of pores that stretch through the entire cytoplasm from the upper to the lower membrane. These fenestrae permit blood plasma and small particles, such as lipoproteins, but also viruses to pass through these cells. The size of the fenestrae, however, has so far prohibited their study by optical microscopy. The images to the right compare typical images obtained after staining the cells with a lipophilic fluorophore (CellMask Orange, Invitrogen) and then imaging them with either diffraction-limited fluorescence deconvolution microscopy (images on the left) vs. 3D structured illumination microscopy (images on the right). Note the higher degree of detail in the 3D-SIM image.
Publications in this area from our group
- V.C. Cogger, G.P. McNerney, T. Nyunt, L. DeLeve, P. McCourt, B. Smedsrød, D.G. Le Couteur, and T.R. Huser,
- "Three-dimensional, structured illumination microscopy of liver sinusoidal endothelial cell fenestrations", J. Struct. Biol. 171, 382 - 388 (2010)
- T. Huser, “Nano-Biophotonics: new tools for chemical nano-analytics”, Curr. Opin. Chem. Biol. 12, 497–504 (2008)