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Downloaded from http://cshprotocols.cshlp.org/ at PENN STATE UNIV on September 15, 2016 - Published by Cold Spring Harbor Laboratory Press Topic Introduction Light-Sheet-Based Fluorescence Microscopy for ThreeDimensional Imaging of Biological Samples Jim Swoger, Francesco Pampaloni, and Ernst H.K. Stelzer In modern biology, most optical imaging technologies are applied to two-dimensional cell culture systems; that is, they are used in a cellular context that is defined by hard and flat surfaces. However, a physiological context is not found in single cells cultivated on coverslips. It requires the complex three-dimensional (3D) relationship of cells cultivated in extracellular matrix (ECM) gels, tissue sections, or in naturally developing organisms. In fact, the number of applications of 3D cell cultures in basic research as well as in drug discovery and toxicity testing has been increasing over the past few years. Unfortunately, the imaging of highly scattering multicellular specimens is still challenging. The main issues are the limited optical penetration depth, the phototoxicity, and the fluorophore bleaching. Light-sheet-based fluorescence microscopy (LSFM) overcomes many drawbacks of conventional fluorescence microscopy by using an orthogonal/azimuthal fluorescence arrangement with independent sets of lenses for illumination and detection. The basic idea is to illuminate the specimen from the side with a thin light sheet that overlaps with the focal plane of a wide-field fluorescence microscope. Optical sectioning and minimal phototoxic damage or photobleaching outside a small volume close to the focal plane are intrinsic properties of LSFM. We discuss the basic principles of LSFM and methods for the preparation, embedding, and imaging of 3D specimens used in the life sciences in an implementation of LSFM known as the single (or selective) plane illumination microscope (SPIM). TRADITIONAL OPTICAL IMAGING TECHNIQUES An understanding of modern developmental biology requires us to monitor the complex 3D relationship of cells, whether cultivated (e.g., in an ECM-based gel), in developing embryos, or in tissue sections (Pampaloni et al. 2007). However, the observation and the optical manipulation of multicellular biological specimens remains a challenge, principally for two reasons: • Such specimens are optically dense. They scatter and absorb light; thus, the delivery of the probing light and the collection of the signal light tend to become inefficient. • In addition to the fluorophores, many endogenous biochemical compounds absorb light and suffer phototoxicity, which can induce damage or can kill the specimen (Pampaloni et al. 2007). The situation is particularly dramatic in confocal fluorescence microscopy. Even when only a single plane is observed, the entire specimen is illuminated. Recording stacks of images along the optical z-axis thus illuminates the entire specimen once for each plane. Hence, cultured cells are illuminated 10–20 times and fish embryos are illuminated 100–300 times more often than they are observed. As we will see in the next section, this can be avoided by a simple change to the optical arrangement. Adapted from Imaging: A Laboratory Manual (ed. Yuste). CSHL Press, Cold Spring Harbor, NY, USA, 2011. © 2014 Cold Spring Harbor Laboratory Press Cite this introduction as Cold Spring Harb Protoc; doi:10.1101/pdb.top080168 1 Downloaded from http://cshprotocols.cshlp.org/ at PENN STATE UNIV on September 15, 2016 - Published by Cold Spring Harbor Laboratory Press J. Swoger et al. Traditionally, biological imaging is dominated by wide-field imaging techniques, in which the full field of view is captured in a single exposure. Although invaluable for two-dimensional applications, conventional wide-field fluorescence microscopy provides no optical sectioning. This means that any image recorded in a particular plane in a thick specimen will contain both the in-focus and the out-offocus signals from the fluorophores above and below the plane of focus. This is the fundamental limitation of conventional wide-field fluorescence light microscopy and makes the construction of quantitative 3D models even of moderately thick specimens difficult, if not impossible, without some form of further innovation. One obvious way to avoid the problem of out-of-focus light in wide-field fluorescence microscopy is to physically section the sample so that material that would be a source of out-of-focus light is physically removed. Traditionally, the sample is mechanically cut into thin slices, which are treated as two-dimensional specimens, whose images can be computationally assembled into a 3D reconstruction. However, because it is destructive, the technique is not useful for the live time-lapse imaging that is essential in the modern life sciences. In addition, physical sectioning is a low-throughput technique that is not suitable for automated scanning of many specimens. Over the last few decades, confocal fluorescence microscopy (Pawley 2006) has become a standard tool for biological imaging in which 3D data sets are required. This is a technique whereby, through the use of a confocal pinhole, images containing mainly in-focus information are acquired. Therefore, an optical rather than a physical sectioning of the specimen is achieved. By using confocal fluorescence microscopes to localize fluorophore distributions in three dimensions, studies of complex biological structures can be performed with fixed and live samples. Although confocal fluorescence microscopy does provide 3D voxel data sets, the resolution along the axial direction of a confocal fluorescence microscope is always lower than along the lateral directions (Pawley 2006). This is because of the physics of diffraction. This resolution anisotropy can be reduced by working with high-numericalaperture (NA) objective lenses. However, in practical lens design, a very large NA is accompanied by a small working distance (generally substantially less than a millimeter). In addition to the mechanical limitations imposed by the working distance of the lens, the image quality in confocal data stacks degenerates rapidly with depth because of the high optical scattering caused by the refractive index heterogeneity of thick biological specimens. Taken together, these properties limit the suitability of confocal fluorescence microscopy for imaging thick 3D samples. An alternative to confocal fluorescence microscopy that provides optically sectioned data sets through a different physical mechanism is multiphoton microscopy (Denk et al. 1990; Konig 2000). Instead of using a pinhole to discriminate against out-of-focus light, nonlinear interactions between the excitation light and the fluorophores are used to ensure that only molecules exposed to very high illumination intensities (i.e., those located directly in the focal plane) are excited. The resulting optically sectioned images produced by multiphoton microscopy are, in many respects, similar to those recorded with confocal fluorescence microscopy. However, because of the longer wavelengths used, the resolution is considerably worse along all three dimensions than in a confocal or a conventional fluorescence microscope (Stelzer and Lindek 1994). Several other imaging methods have been used successfully in developmental biology. Optical coherence tomography, a technique based on interferometric detection of low-coherence illumination, has received considerable attention in the past decade as a technique for noninvasive biological imaging. Magnetic resonance imaging (MRI; Bamforth et al. 2011; Metscher 2011; Ruffins and Jacobs 2011), ultrasound imaging (Foster and Brown 2011), and microcomputed tomography (μCT; Quintana and Sharpe 2011) all allow imaging of relatively large samples and are, therefore, suitable for imaging the later stages of development of specimens such as mouse embryos. Although, in many ways, these are ideal methods for imaging a range of samples, a common drawback is that they cannot produce images using the wide range of highly specific chemical and genetically encoded fluorophores that have become invaluable in light microscopy (Lichtman and Conchello 2005). Optical projection tomography (OPT; Gu et al. 2011; Quintana and Sharpe 2011) is one of the few optical imaging techniques suitable for developmental biology that is capable of operating with both fluorescence and absorption contrasts. 2 Cite this introduction as Cold Spring Harb Protoc; doi:10.1101/pdb.top080168 Downloaded from http://cshprotocols.cshlp.org/ at PENN STATE UNIV on September 15, 2016 - Published by Cold Spring Harbor Laboratory Press LSFM for 3D Imaging PRINCIPLES OF LSFM The basic idea of LSFM is to use a light sheet that illuminates the specimen from the side and that overlaps with the focal plane of a wide-field fluorescence microscope (Huisken et al. 2004). In contrast to an epifluorescence arrangement, which uses the same lens, a light-sheet-based orthogonal/ azimuthal fluorescence arrangement uses two independent lenses for illumination and detection. An LSFM system is able to provide optical sectioning without phototoxic damage or photobleaching outside of a small volume located close to the focal plane. LSFM takes advantage of modern chargecoupled device (CCD) camera technologies to generate images with a signal-to-noise ratio (SNR) that, for a given fluorophore, objective lens, and specimen, is at least one order of magnitude better than that of a confocal fluorescence microscope (Keller et al. 2008). (Note that good-quality confocal fluorescence images have an SNR between 10 and 40 [given by the square root of the number of detected photons; see the Scientific Volume Imaging-Wiki, http://support.svi.nl/wiki/WikiWikiWeb]. CCD cameras used in fluorescence microscopy have an SNR between 100 and 1000 [defined as the square root of the full-well capacity]. For example, the ORCA-AG [Hamamatsu] has a full-well capacity of 18,000 electrons, which gives an SNR of 130.) The idea of using light-sheet illumination for microscopy has occurred to various groups at different times and has been implemented in a number of configurations. These are summarized in Table 1. All of these techniques intrinsically provide optical sectioning and, except for the original implementation of ultramicroscopy (Siedentopf and Zsigmondy 1903), which works with incoherent illumination and scattered light contrast, all rely on laser illumination focused in one dimension to excite fluorescence. Here, we refer to an instrument known as a single (or selective) plane illumination microscope (SPIM). The optical properties of LSFM can be calculated by following the approach in Engelbrecht et al. (2007) and Stelzer and Lindek (1994). In SPIM, the sample is illuminated by a sheet of laser light. A laser array, in combination with dichroic mirrors and an acousto-optical tunable filter (AOTF), is used to provide a variety of wavelengths for the excitation of different fluorescent dyes (Fig. 1A). Cylindrical optics (Huisken et al. 2004; Greger et al. 2007) or a scanning mechanism in the digital scanned laser light-sheet fluorescence microscopy (DSLM) (Keller et al. 2008) is used to focus a collimated laser beam along one dimension, thereby creating a single plane of light. The specimen is embedded in a cylinder of agarose or other mounting gel, which is generally immersed in an aqueous medium. The fluorescence emitted from the narrow volume of the specimen that is excited by the light TABLE 1. Some methods that use LSFM Name Distinctions/features Reference(s) Scattering ultramicroscopya Developed to study subwavelength colloidal particles (these are illuminated from the side by an incoherent illumination) Point-scanning device using an orthogonal or, in general, azimuthal optical arrangement Applied to fixed, chemically cleared samples Developed for imaging very large and chemically cleared organs, such as the guinea pig cochlea Used in microbial oceanography Developed for live, fast, multiple-view and multiple-dye imaging of embryos, cell clusters, and single cells Illumination optics mechanically coupled to detection objective lens Siedentopf and Zsigmondy 1903 Confocal θ fluorescence microscopya Fluorescence ultramicroscopy Orthogonal-plane fluorescence optical sectioning device Thin laser light-sheet microscopy SPIM, scanned light-sheet microscopy (DSLM) Objective-coupled planar illumination microscopy Stelzer and Lindek 1994 Dodt et al. 2007 Voie et al. 1993; Buytaert and Dirckx 2009 Fuchs et al. 2002 Huisken et al. 2004; Keller et al. 2008 Holekamp et al. 2008; Turaga and Holy 2008 The first two examples (original ultramicroscopy and confocal θ fluorescence microscopy) are forerunner techniques exploiting the idea of side illumination of the specimen. a Cite this introduction as Cold Spring Harb Protoc; doi:10.1101/pdb.top080168 3 Downloaded from http://cshprotocols.cshlp.org/ at PENN STATE UNIV on September 15, 2016 - Published by Cold Spring Harbor Laboratory Press J. Swoger et al. A Camera Detection TL FW OL LA AOTF BEO S CO Light-sheet illumination B C Single-plane acquisition Conventional and confocal Illumination Light-sheet microscopy Detection 3D data acquisition Light-sheet microscopy Detection Single view i) Illumination Multiview Photodamage 0° ii) Specimen 60° Advantages of the light-sheet-based approach: i) Intrinsic optical sectioning, i.e., capability of 3D imaging ii) Reduction of photodamage in 3D image stacks by a factor of n, where n is equal to the number of planes in the stack 120° Improved axial resolution reconstruction of large specimen FIGURE 1. Principles of light-sheet-based microscopy. (A) The overall layout of an SPIM. The emission from a laser array (LA) of various wavelengths for exciting fluorescence is combined into a single beam. An acousto-optic tunable filter (AOTF) is used to select the wavelengths and the intensity of the excitation beam. The beam expanding and cylindrical optics (BEO and CO) create the light sheet that illuminates the sample (S). Detection is through a wide-field fluorescence microscope consisting of an objective lens (OL), a filter wheel (FW) that contains filters for rejection of scattered excitation light and background fluorescence, a tube lens (TL), and a camera. (B) Comparison of sample illumination and fluorescence detection in conventional/confocal microscopy and in LSFM. (i) The entire region of interest in the specimen is illuminated in conventional/confocal microscopy, although only a single plane in the specimen is being observed. (ii) In contrast, no photodamage is inflicted outside the in-focus plane of the detection system in the LSFM. (C ) Three-dimensional imaging in LSFM is performed by moving the specimen step by step through the light sheet while recording two-dimensional images. In multiple-view imaging, the same volume inside the specimen or even inside the entire specimen is recorded along several angles. The resulting multiple-view information is combined into a single image stack by a fusion algorithm. (B,C, Adapted, with permission, from Keller and Stelzer 2008.) sheet is imaged onto a camera using standard fluorescence microscopy optics: an objective lens, a filter wheel, and a tube lens (Fig. 1A). The sample can be moved along the detection axis (i.e., perpendicular to the plane of the light sheet) and imaged in a stepwise fashion to generate a 3D data set. The SPIM setup usually includes a mechanism for rotating the specimen, so that its orientation can be optimized for imaging in a particular experiment or for acquiring image stacks from multiple directions (Fig. 1C). With an SPIM, optical sectioning is obtained in a direct and efficient way. The sectioning capability is defined by the thickness of the light sheet (typically 2–6 µm, depending on the extent of the field of 4 Cite this introduction as Cold Spring Harb Protoc; doi:10.1101/pdb.top080168 Downloaded from http://cshprotocols.cshlp.org/ at PENN STATE UNIV on September 15, 2016 - Published by Cold Spring Harbor Laboratory Press LSFM for 3D Imaging view). By illuminating only the plane of interest, no out-of-focus light is created, and there is no need for a confocal pinhole for the discrimination of this background (Fig. 1Bi). Therefore, photobleaching and photodamage are less than with confocal or wide-field fluorescence microscopy, in which the complete specimen is illuminated even when observing only a single plane (Fig. 1Bii). Objective lenses with a long working distance and a relatively low NA can be used for detection when imaging large specimens (a few millimeters). The lateral resolution is defined by the NA of the detection objective lens, whereas the axial resolution of the system is determined by the light-sheet thickness, when it is thinner than the axial detection resolution. The combination of optical sectioning, long working distances, and minimal photodamage make LSFM ideal for imaging of samples typically of interest in developmental and 3D multicellular biology. A schematic of an LSFM setup can be found in Keller et al. (2011). One of the advantages of an SPIM is that the sample can be rotated and can be observed from multiple orientations (Fig. 1C). Three-dimensional stacks can be acquired from different directions, and the images can be computationally fused into a single representation of the sample (Swoger et al. 2007; Verveer et al. 2007). This has the advantage of improving the resolution in small transparent specimens and of improving the uniformity of image quality in larger specimens for which absorption and/or scattering are significant limitations to imaging. Examples are provided in the next section. SPIM APPLICATIONS Figure 2 illustrates the effect of multiple-view image fusion on a grain of paper mulberry pollen. The same grain of pollen has been imaged along 18 directions, two of which are shown in Figure 2, A and B. Note that although the light-sheet illumination improves the axial resolution over wide-field or confocal fluorescence microscopy with the same detection objective lens, the resolution is anisotropic. This is indicated schematically by the red ellipses: These point-spread functions (PSFs) are narrowest along the direction with the highest resolution. In the image shown in Figure 2A, the resolution is good vertically but poor horizontally; in Figure 2B, which was acquired with the sample oriented at 90˚ to the orientation in Figure 2A, the situation is the reverse. By recording 18 three-dimensional data sets (i.e., a rotation of the sample in 20˚ steps), aligning them, and taking the average, the image shown in Figure 2C is obtained. It can be seen that the resolution (the PSF is represented by the red circle) is quite high along all directions and approximately equal to the resolution along the best axis in either Figure 2, A or B. A further improvement in resolution and contrast can be obtained by using a multiple-view image deconvolution (Verveer et al. 2007) rather than the simple average used in Figure 2C. The result is illustrated in Figure 2D, in which the interior details and the quasispherical surface layer or sporoderm of the pollen grain can be clearly seen. If the sample is nearly optically transparent (e.g., the pollen grain in Fig. 2) and the different multiple-view data sets overlap, then the multiple-view reconstruction compensates for the anisotropy FIGURE 2. SPIM of paper mulberry pollen, multiview reconstruction. Slices from volumetric data stacks of an autofluorescent grain of paper mulberry pollen are imaged with an SPIM. (A,B) Single-view images along orthogonal directions. (C ) Combination of 18 views by averaging. (D) Combination of the same 18 views as in (C ) by multipleview deconvolution. Scale bar, 5 µm. (Image acquisition by Klaus Greger, EMBL, Heidelberg.) Cite this introduction as Cold Spring Harb Protoc; doi:10.1101/pdb.top080168 5 Downloaded from http://cshprotocols.cshlp.org/ at PENN STATE UNIV on September 15, 2016 - Published by Cold Spring Harbor Laboratory Press J. Swoger et al. in each single view and provides an isotropic resolution. In the case of a more translucent or opaque sample (e.g., the Drosophila melanogaster embryo in Fig. 3), additional views provide information about regions of the sample that is not visible in a single view. Here, the multiple-view reconstruction combines the information into a complete image of the sample. It can be seen in the single-view images (Fig. 3A,C) that some regions are not well resolved (arrowheads) or are entirely missing (arrows) because they were imaged through thick optically scattering tissue. When a multiple-view reconstruction is performed (Fig. 3B,D) the processing combines the best-resolved features from each individual view into a single 3D representation with uniform high-resolution coverage of the sample. Imaging 3D Cell Biology Specimens with a SPIM Increasingly, cell biologists appreciate that conventional two-dimensional cell cultures do not provide a physiological environment for the cell (Pampaloni et al. 2007; Mazzoleni et al. 2009). The surfaces of plastic and glass substrates are flat and rigid. In contrast, a tissue is a 3D soft-matter structure. There are many instances in which cells have drastically different phenotypes in two dimensions than in three dimensions. For example, fibroblasts cultured in two dimensions have a flat shape dissimilar from the bipolar/stellate shape found in tissue. Strikingly, culturing fibroblasts in 3D collagen induces their tissue-specific phenotype (Beningo et al. 2004; Rhee et al. 2007; Rhee and Grinnell 2007). It is currently acknowledged that establishing 3D cell–cell interactions and using ECMs or 3D gels reduces the gap between cell culture and real tissue. There are strong indications that toxicity assays based on 3D cultures of human cells provide more accurate results than two-dimensional cultures (Pampaloni et al. 2009). Three-dimensional cultures are promising for toxicity screening of chemicals and to sort out toxic substances at early stages of drug discovery. Imaging of fluorophore-tagged targets provides the specificity that is required to perform detailed studies at the molecular level in live 3D cultures. Novel advanced fluorescence imaging technologies are required to study the behavior of living 3D cultures. LSFM provides an excellent tool to study the live behavior of 3D cellular systems with high spatial resolution and for long periods of time. Light-sheet illumination provides optical sectioning deep inside large 3D tissue. The extremely low illumination level induces minimum photobleaching and photodamage. Embedding in agarose is a well-established approach to stably mount specimens for imaging with an SPIM. The agarose concentration used is 0.5%–1% (w/v) in buffer or culture medium. Agarose is a nearly ideal embedding medium because it is optically clear and introduces very limited aberrations. Agarose is nontoxic and, in most cases, does not interfere with the physiology of a living specimen. For detailed protocols for embedding and imaging with an SPIM, see Imaging Cellular Spheroids with a FIGURE 3. SPIM of a D. melanogaster embryo. Maximum-value projections through volumetric data stacks of a fruit fly embryo with the trachea labeled with green fluorescent protein. (A) Single-view data set, projected along the dorsoventral axis. (B) Multiple-view fusion of 12 views similar to that shown in (A) but taken with different orientations of the sample. (C,D) Projections corresponding to those in (A) and (B) but along the lateral axis of the embryo. Scale bar, 100 µm. (Sample courtesy of Ferenc Jankovics and Damian Brunner, EMBL, Heidelberg; image acquisition by Klaus Greger, EMBL, Heidelberg.) 6 Cite this introduction as Cold Spring Harb Protoc; doi:10.1101/pdb.top080168 Downloaded from http://cshprotocols.cshlp.org/ at PENN STATE UNIV on September 15, 2016 - Published by Cold Spring Harbor Laboratory Press LSFM for 3D Imaging Single (Selective) Plane Illumination Microscope (Swoger et al. 2014a) and Imaging MDCK Cysts with a Single (Selective) Plane Illumination Microscope (Swoger et al. 2014b). SUMMARY We have described the SPIM, a microscope that uses the principles of LSFM for imaging 3D specimens, such as are commonly studied in developmental biology and in 3D cell culturing. The principal advantages of SPIM over techniques such as wide-field or confocal microscopy are as follows: • • • reduced photobleaching and phototoxicity the ability to achieve high resolution with long working distance objectives, which allows imaging of thick 3D specimens the option of achieving high isotropic resolution via multiple-view reconstructions Autofluorescent pollen grains and genetically labeled D. melanogaster embryos were used to show the advantages of multiview reconstructions, which (to the best of our knowledge) is not a technique available in systems other than SPIM. The ability of SPIM to generate high-resolution images of live samples as they develop over time will lead to novel types of experiments in developmental biology and 3D cell cultures. REFERENCES Bamforth SD, Schneider JE, Bhattacharya S. 2011. High-throughput analysis of mouse embryos by magnetic resonance imaging. In Imaging in developmental biology: A laboratory manual (ed. J Sharpe, RO Wong). 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High-resolution three-dimensional imaging of large specimens with light sheet-based microscopy. Nat Methods 4: 311–313. Voie AH, Burns DH, Spelman FA. 1993. Orthogonal-plane fluorescence optical sectioning: 3-dimensional imaging of macroscopic biological specimens. J Microsc 170: 229–236. Cite this introduction as Cold Spring Harb Protoc; doi:10.1101/pdb.top080168 Downloaded from http://cshprotocols.cshlp.org/ at PENN STATE UNIV on September 15, 2016 - Published by Cold Spring Harbor Laboratory Press Light-Sheet-Based Fluorescence Microscopy for Three-Dimensional Imaging of Biological Samples Jim Swoger, Francesco Pampaloni and Ernst H.K. Stelzer Cold Spring Harb Protoc; doi: 10.1101/pdb.top080168 Email Alerting Service Subject Categories Receive free email alerts when new articles cite this article - click here. Browse articles on similar topics from Cold Spring Harbor Protocols. 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