Heintzmann, R., Jovin, T.M. & Cremer, C. Saturated patterned excitation microscopy-a concept for optical resolution improvement. J. Opt. Soc. Am. A. Opt. Image Sci. Vis. 19, 1599-1609

Universität Heidelberg, Heidelburg, Baden-Württemberg, Germany
Journal of the Optical Society of America A (Impact Factor: 1.56). 09/2002; 19(8):1599-609. DOI: 10.1364/JOSAA.19.001599
Source: PubMed


The resolution of optical microscopy is limited by the numerical aperture and the wavelength of light. Many strategies for improving resolution such as 4Pi and I5M have focused on an increase of the numerical aperture. Other approaches have based resolution improvement in fluorescence microscopy on the establishment of a nonlinear relationship between local excitation light intensity in the sample and in the emitted light. However, despite their innovative character, current techniques such as stimulated emission depletion (STED) and ground-state depletion (GSD) microscopy require complex optical configurations and instrumentation to narrow the point-spread function. We develop the theory of nonlinear patterned excitation microscopy for achieving a substantial improvement in resolution by deliberate saturation of the fluorophore excited state. The postacquisition manipulation of the acquired data is computationally more complex than in STED or GSD, but the experimental requirements are simple. Simulations comparing saturated patterned excitation microscopy with linear patterned excitation microscopy (also referred to in the literature as structured illumination or harmonic excitation light microscopy) and ordinary widefield microscopy are presented and discussed. The effects of photon noise are included in the simulations.

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Available from: Christoph Cremer, Feb 27, 2015
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    • "Generally, achieving two dimensional resolution improvement requires nine images, whereas three dimensional improvement requires fifteen. It is possible to improve on this using non-linear methods (linear means that intensity in is proportional to intensity out, which is no longer true when the sample saturates), which use saturation to create illumination patterns that contain higher frequencies than the base frequency (Heintzmann et al., 2002; Gustafsson, 2005). However, while this method has been demonstrated in cells (Rego et al., 2012), it is highly challenging and not currently available commercially. "
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    • "They include photoactivated localization microscopy (PALM) [37] and stochastic optical reconstruction microscopy (STORM) [38]. Apart from localization microscopy, stimulated emission depletion (STED) microscopy [39], a method based on RESOLFT technique [40] [41], is another commonly used method for exploring the distribution of membrane proteins. These new imaging techniques are commonly known as super-resolution microscopy. "
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    • "(Saturated) structured-illumination microscopy [(S)SIM] has been developed by Gustafsson (2005) and Heintzmann et al. (2002). In (S)SIM, samples are illuminated by an excitation light, which has a periodical spatial pattern, typically a sinusoidal pattern (Figure 1B). "
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