Super-Resolution Optical Microscopy
Although the resolution of a light microscope is fundamentally limited by diffraction to about half of the wavelength of light, in recent years several techniques have been developed that can overcome this limitation in fluorescence microscopy. We are aiming to implement several of these techniques and apply them to biological samples and polymers.
Structured Illumination Microscopy (SIM)
Structured illumination microscopy is based on the projection of a fine grating structure onto the sample. When the high-frequency sample structure is multiplied with a high-frequency grating pattern, low-frequency information appears in the form of Moiré fringes. These fringes transfer otherwise unobservable high-frequency information about the sample into a lower-frequency region that is observable through a microscope. The final image is reconstructed from a series of images where the pattern is moved and rotated between the images, typically from a total of 9-15 images. No unusual properties are required from the sample or the fluorophore compared to conventional fluorescence microscopy. The resolution improvement is limited to a factor of two, but we are hoping to extend this technique to saturated structured illumination microscopy (SSIM) using photoswitchable fluorophores, where the resolution is limited only by the signal-to-noise ratio.
Figure 1: Structured illumination microscopy. (a) A grating in the beam path diffracts the excitation beam, and the diffracted beams are focused to the back focal plane of the objective. The beams interfere in the sample and create a sinusoidal pattern (b, dashed line). When the illumination intensity is no longer linearly dependent on the excitation intensity, the effective excitation pattern is no longer sinusoidal (b, solid line). (c) Moiré fringes: when two high-frequency patterns are multiplied, Moiré fringes appear. While SIM is capable of resolving structures half the size of the diffraction-limited resolution, SSIM allows features much smaller than this to be resolved.
Figure 2: GFP-stained mitochondria in COS-7 cells imaged with conventional wide-field and structured illumination microscopy. (Sample from Laura Osellame and Mike Ryan, La Trobe University.)
We are also in the process of developing photoactivated localisation microscopy (PALM), scanning near field optical microscopy (SNOM) and Stimulated Emission Depletion (STED) Microscopy.
- L.M. Hirvonen & T.A. Smith, “Imaging on the Nanoscale: Super-Resolution Fluorescence Microscopy”, Aust. J. Chem. 63, 1-5, (2010)
- L.M. Hirvonen, K. Wicker, O. Mandula and R. Heintzmann, “Structured illumination microscopy of a living cell”, European Biophysics Journal 38 (6), pp. 807-812 (DOI: 10.1007/s00249-009-0501-6)
- L. Hirvonen, O. Mandula, K. Wicker and R. Heintzmann”Structured illumination microscopy using photoswitchable fluorescent proteins”, Proc. SPIE, Vol. 6861, 68610L (2008); doi:10.1117/12.763021