Optical sectioning microscopy with planar or structured illumination

Department of Biomedical Engineering, Boston University, Boston, Massachusetts, USA.
Nature Methods (Impact Factor: 32.07). 09/2011; 8(10):811-9. DOI: 10.1038/nmeth.1709
Source: PubMed


A key requirement for performing three-dimensional (3D) imaging using optical microscopes is that they be capable of optical sectioning by distinguishing in-focus signal from out-of-focus background. Common techniques for fluorescence optical sectioning are confocal laser scanning microscopy and two-photon microscopy. But there is increasing interest in alternative optical sectioning techniques, particularly for applications involving high speeds, large fields of view or long-term imaging. In this Review, I examine two such techniques, based on planar illumination or structured illumination. The goal is to describe the advantages and disadvantages of these techniques.

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    • "Other fluorescence signals coming from the TPE volume of the TFMPEM, except for the confined region where the pattern can be imaged well, are rejected as background noise. The structured illumination has provided an optical sectioning ability even in widefield one-photon excitation microscopy [13,16,25]. Similar to the structured illumination approach, the TFMPEM with the NSIM can achieve a better axial resolution than conventional TFMPEM alone. "
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    ABSTRACT: In this study, the light diffraction of temporal focusing multiphoton excitation microscopy (TFMPEM) and the excitation patterning of nonlinear structured-illumination microscopy (NSIM) can be simultaneously and accurately implemented via a single high-resolution digital micromirror device. The lateral and axial spatial resolutions of the TFMPEM are remarkably improved through the second-order NSIM and projected structured light, respectively. The experimental results demonstrate that the lateral and axial resolutions are enhanced from 397 nm to 168 nm (2.4-fold) and from 2.33 μm to 1.22 μm (1.9-fold), respectively, in full width at the half maximum. Furthermore, a three-dimensionally rendered image of a cytoskeleton cell featuring ~25 nm microtubules is improved, with other microtubules at a distance near the lateral resolution of 168 nm also able to be distinguished.
    Biomedical Optics Express 08/2014; 5(8). DOI:10.1364/BOE.5.002526 · 3.65 Impact Factor
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    • "In recent years, selective-plane illumination microscopy (SPIM) has been rediscovered in the context of embryo development (Mertz, 2011; Tomer et al., 2011; Weber and Huisken, 2011) and physiology (Huisken et al., 2004; Verveer et al., 2007; Keller and Dodt, 2012). In this imaging configuration, optical sectioning is performed through side-on illumination of the sample by a thin (micrometer-thick) laser sheet, whereas fluorescence photons are collected by a camera whose optical axis is orthogonal to the illumination plane. "
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    ABSTRACT: The optical transparency and the small dimensions of zebrafish at the larval stage make it a vertebrate model of choice for brain-wide in-vivo functional imaging. However, current point-scanning imaging techniques, such as two-photon or confocal microscopy, impose a strong limit on acquisition speed which in turn sets the number of neurons that can be simultaneously recorded. At 5 Hz, this number is of the order of one thousand, i.e., approximately 1–2% of the brain. Here we demonstrate that this limitation can be greatly overcome by using Selective-plane Illumination Microscopy (SPIM). Zebrafish larvae expressing the genetically encoded calcium indicator GCaMP3 were illuminated with a scanned laser sheet and imaged with a camera whose optical axis was oriented orthogonally to the illumination plane. This optical sectioning approach was shown to permit functional imaging of a very large fraction of the brain volume of 5–9-day-old larvae with single-or near single-cell resolution. The spontaneous activity of up to 5,000 neurons was recorded at 20 Hz for 20–60 min. By rapidly scanning the specimen in the axial direction, the activity of 25,000 individual neurons from 5 different z-planes (approximately 30% of the entire brain) could be simultaneously monitored at 4 Hz. Compared to point-scanning techniques, this imaging strategy thus yields a 20-fold increase in data throughput (number of recorded neurons times acquisition rate) without compromising the signal-to-noise ratio (SNR). The extended field of view offered by the SPIM method allowed us to directly identify large scale ensembles of neurons, spanning several brain regions, that displayed correlated activity and were thus likely to participate in common neural processes. The benefits and limitations of SPIM for functional imaging in zebrafish as well as future developments are briefly discussed.
    Frontiers in Neural Circuits 04/2013; 7(Suppl 1). DOI:10.3389/fncir.2013.00065 · 3.60 Impact Factor
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    • "Techniques like SPIM (Dodt et al., 2007; Keller and Stelzer, 2008), generally called light-sheet microscopy techniques, are gaining popularity because of the high efficiency in accessing volumetric information of the specimen while minimizing photo-bleaching and energy load (Verveer et al., 2007, Holekamp et al., 2008). The advantages of SPIM over confocal was thoroughly discussed (Mertz, 2011). A further development of the SPIM method was proposed by the application of Bessel beams to light sheet microscopy by Planchon et al. (2011). "
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    ABSTRACT: Driven by the biological sciences, there is an increased need for imaging modalities capable of live cell imaging with high spatial and temporal resolution. To achieve this goal in a comprehensive manner, three-dimensional acquisitions are necessary. Ideal features of a modern microscope system should include high imaging speed, high contrast ratio, low photo-bleaching and photo-toxicity, good resolution in a 3D context, and mosaic acquisition for large samples. Given the importance of collecting data in live sample further increases the technical challenges required to solve these issues. This work presents a practical version of a microscopy method, Selective Plane Illumination Microscopy re-introduced by Huisken et al. (Science2004,305,1007-1009). This method is gaining importance in the biomedical field, but its use is limited by difficulties associated with unconventional microscope design which employs two objectives and a particular kind of sample preparation needed to insert the sample between the objectives. Based on the selective plane illumination principle but with a design similar to the Total Internal Reflection Fluorescence microscope, Dunsby (Dunsby, Opt Express2008,16,20306-20316) demonstrated the oblique plane microscope (OPM) using a single objective which uses conventional sample preparation protocols. However, the Dunsby instrument was not intended to be part of a commercial microscope. In this work, we describe a system with the advantages of OPM and that can be used as an adaptor to commonly used microscopes, such as IX-71 Olympus, simplifying the construction of the OPM and increasing performance of a conventional microscope. We named our design inclined selective plane illumination microscope (iSPIM). Microsc. Res. Tech. 2012. © 2012 Wiley Periodicals, Inc.
    Microscopy Research and Technique 11/2012; 75(11):1461-6. DOI:10.1002/jemt.22089 · 1.15 Impact Factor
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