Interferometer-based structured-illumination microscopy utilizing complementary phase relationship through constructive and destructive image detection by two cameras

Keck Advanced Microscopy Laboratory, Department of Biochemistry and Biophysics, University of California, San Francisco, California 94158, USA.
Journal of Microscopy (Impact Factor: 2.33). 04/2012; 246(3):229-36. DOI: 10.1111/j.1365-2818.2012.03604.x
Source: PubMed


In an interferometer-based fluorescence microscope, a beam splitter is often used to combine two emission wavefronts interferometrically. There are two perpendicular paths along which the interference fringes can propagate and normally only one is used for imaging. However, the other path also contains useful information. Here we introduced a second camera to our interferometer-based three-dimensional structured-illumination microscope (I(5)S) to capture the fringes along the normally unused path, which are out of phase by π relative to the fringes along the other path. Based on this complementary phase relationship and the well-defined phase interrelationships among the I(5)S data components, we can deduce and then computationally eliminate the path length errors within the interferometer loop using the simultaneously recorded fringes along the two imaging paths. This self-correction capability can greatly relax the requirement for eliminating the path length differences before and maintaining that status during each imaging session, which are practically challenging tasks. Experimental data is shown to support the theory.

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Available from: Lin Shao, Jan 22, 2015
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    ABSTRACT: Optical microscopy plays an essential role in biological studies due to its capability and compatibility of non-contact, minimally invasive observation and measurement of live specimens. However, the conventional optical microscopy only has a spatial resolution about 200 nm due to the Abbe diffraction limit, and also lacks the ability of three-dimensional imaging. Super-resolution far-field optical microscopy based on special illumination schemes has been dramatically developed over the last decade. Among them, only the structured illumination microscopy (SIM) is of wide-field geometry that enables it easily compatible with the conventional optical microscope. In this article, the principle of SIM was introduced in terms of point spread function (PSF) and optical transform function (OTF) of the optical system. The SIM for super-resolution (SIM-SR) proposed by Gustafsson et al. and the SIM for optical sectioning (SIM-OS) proposed by Neil et al. are the most popular ones in the research community of microscopy. They have the same optical configuration, but with different data post-processing algorithms. We mathematically described the basic theories for both of the SIMs, respectively, and presented some numerical simulations to show the effects of super-resolution and optical sectioning. Various approaches to generation of structured illumination patterns were reviewed. As an example, a SIM system based on DMD-modulation and LED-illumination was demonstrated. A lateral resolution of 90 nm was achieved with gold nano-particles. The optical sectioning capability of the microscope was demonstrated with Golgi-stained mouse brain neurons, and the sectioning strength of 930 nm was obtained.
    Full-text · Article · Apr 2014 · Chinese Science Bulletin