Light-Sheet Microscopy for Whole-Brain Imaging

Chapter 3

Light-Sheet Microscopy for Whole-Brain Imaging Monika Pawłowska, Marzena Stefaniuk, Diana Legutko and Leszek Kaczmarek

Abstract The brain is a complex system of interconnected and interacting structures. To fully understand its architecture, we need an imaging method that is capable of imaging the entire brain at sufficient resolution to capture single cells and projections and discriminate between cells with different functions. This has been made possible by the combination of light-sheet fluorescence microscopy with optical tissue clearing. In this chapter, we briefly describe the currently known methods of tissue clearing, discussing the advantages and disadvantages of different approaches. Next, we explain the principles of light-sheet fluorescence microscopy focusing on its application, the whole-brain imaging. We discuss the technical challenges specific to this approach, in particular those resulting from high refractive index of cleared tissue as well as from the large size of collected data. Finally, we list some of the recent papers which make use of light-sheet imaging of cleared tissue.

3.1 Introduction Brain orchestrates the action of the whole body. Therefore by its nature, it has to be complex and highly organized. Brain contains billions of cells that are densely packed and riddled with a dense network of blood vessels that provide oxygen and nutrients. Not only the location of a cell in a specific brain region is important, but most importantly its position in a particular region or within a particular brain network M. Pawłowska (B) · M. Stefaniuk · D. Legutko · L. Kaczmarek Nencki Institute of Experimental Biology, Polish Academy of Sciences, 3 Pasteur Street, 02-093 Warsaw, Poland e-mail: [email protected] M. Stefaniuk e-mail: [email protected] D. Legutko e-mail: [email protected] L. Kaczmarek e-mail: [email protected] © Springer Nature Singapore Pte Ltd. 2019 F.-J. Kao et al. (eds.), Advanced Optical Methods for Brain Imaging, Progress in Optical Science and Photonics 5, https://doi.org/10.1007/978-981-10-9020-2_3

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matters a lot. That is why brain as an organ should be studied in an intact form and at the same time in sufficient resolution to capture single cells and projections. Currently, high-resolution imaging of large volumes of tissue is achievable only with optical microscopy. However, light scattering limits imaging deep into the tissue, even if multiphoton microscopy is used. Imaging of the whole intact brain at cellular resolution has been made possible only recently by advances in two fields: tissue clearing and light-sheet microscopy. Cleared-tissue imaging with a light-sheet microscope dates back to the work of Voie and co-workers [1] and is based on ideas that are more than a hundred years old [2, 3]. However, most of the field developed in the last ten years, starting with the work of Dodt and co-workers [4]. This review concentrates on the history and latest developments in the field of whole-brain imaging using light-sheet microscopy. First, it provides a short summary of tissue clearing methods. Note that a comprehensive description of the chemical processing of brain tissues for large-volume, high-resolution optical imaging can be found in Chap. 15 of this book. Next, light-sheet microscopy is described in more detail. Further, the challenges related to the processing and analysis of large image data are discussed. Finally, we give examples of the latest applications of light-sheet microscopy in neurobiology.

3.2 Tissue Clearing During the last ten years, many tissue-clearing protocols have been developed. So far, none of them has proved to be universally superior. Some approaches are more suitable for preserving fine structure, some for clearing entire brains of mice and other animals, such as rats. Also, some protocols can be applied to other organs, including specimens of already fixed human tissue. Below, we briefly describe different tissueclearing approaches. More information can be found in several reviews [5–8]. Light scattering in brain tissue is caused by the different refractive indices (RIs) of its major chemical components: water, proteins and lipids. Therefore, one of the ways of understanding the different properties of various tissue-clearing methods is looking at their influence on the lipid membranes. Briefly, light scattering caused by lipid membranes can be reduced through three different mechanisms (Fig. 3.1a). The first approach is dissolving and removing the lipids, replacing them with a medium, the RI of which is similar to that of the remaining tissue, i.e. around 1.48. This medium can be either a solution containing urea, sugars and detergents, like in Scale [9], CUBIC [10] and similar methods, or RapiClear or refractive index matching solution (RIMS) reagents, like in CLARITY and PACT/PARS, respectively [11, 12]. All these methods cause some degree of sample swelling. Examples of rat brains cleared with CLARITY and CUBIC are shown in Fig. 3.1b. Another approach relies on removing water and dissolving lipids using hydrophobic solvents. The remaining tissue is shrunken and dense, with refractive index above 1.5. The first versions of this method suffered from strong quenching of fluorescence of green fluorescent protein (GFP), a widely used molecular marker [4]. Since

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Fig. 3.1 Brain optical clearing methods a Optical clearing methods: group I—based on dissolving and removal of lipids (i.e. CLARITY or CUBIC), group II—based on dissolving lipids (i.e. BABB, uDISCO, iDISCO) and group III—cell membrane permeabilization (SeeDB or Scale). b Clearing of rat brain using CLARITY and CUBIC. Note expansion of the brain and uneven clearing of different brain structures. c Clearing of mouse brains using BABB, iDISCO and uDISCO and before clearing (PBS). Note shrinkage of brains and light amber colour of cleared tissue

then, the method has been improved by careful choice of solvents, as well as pHmaintenance and prevention of oxidation [13, 14]. The solvent-based clearing techniques are robust and applicable to a wide range of tissues, including entire adult rat brain, which is difficult to clear due to its size and degree of myelination [15]. On the other hand, the high RI and corrosive properties of the solvents make it difficult to find suitable imaging optics. Examples of mice brains cleared using FluoClearBABB, uDISCO and iDISCO are shown in Fig. 3.1c. The third approach is based on a permeabilization of the membrane without disrupting its structure in order to preserve cell morphology. However, this leaves more residual scattering so that methods of this class, such as SeeDB [16] and ScaleS [17], are more suitable for super-resolution than whole-brain imaging. One of the challenges with clearing large samples is the penetration of the clearing reagents. This can be improved by delivering the reagents through perfusion rather than simple immersion. Perfusion-assisted clearing has been demonstrated in combination of most of the approaches mentioned above [12, 14, 18, 19]. Although numerous transgenic animal models are now available, many experiments benefit from the flexibility offered by immunolabeling and other staining methods. Several clearing protocols have been shown to be compatible with immunolabeling protocols [10, 11, 20]. Notably, staining and imaging of the entire mouse brain hemisphere have been demonstrated [21]. However, usually long incubation

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times and large quantities of antibodies are required, which limits the usefulness of this approach. A factor that can improve the quality of imaging of cleared tissue is the right choice of the fluorophore. Shorter wavelengths are more strongly absorbed and scattered in the tissue [15]. Moreover, tissue autofluorescence is strong in the blue-green spectrum. Consequently, fluorophores in the red, or even better, the far-red spectrum help to reduce background [20]. Finally, one has to bear in mind that the chosen method has to be specially tailored both for the experiment and for the organ that is to be cleared. Brain differs in its structure from other organs. High-fat content strongly influences its light scattering properties. On the other hand, it is not heme-rich like kidney or spleen. Also, not all techniques that have been successfully used for mouse brain are also suitable for rat brain tissue (see [15]). Depending on the clearing techniques, as mentioned, brain might expand or shrink which influences the image resolution. This property has been exploited in so-called expansion microscopy [22]. On the other hand, shrunken samples are easier to image considering the limited working distance of existing microscope lenses [14].

3.3 Whole-Brain Imaging with Light-Sheet Microscopy Optical tissue clearing significantly reduces light scattering in the tissue, thus increasing the imaging depth by an order of magnitude or more. This in turn creates a need for new imaging methods. Although whole-brain imaging with a scanning microscope is in principle possible, provided that the microscope is equipped with a longworking-distance objective, the long imaging time makes this approach impractical. Wide-field microscopy, on the other hand, enables imaging of the entire field of view at once, but has no mechanism for rejecting out-of-focus fluorescence and thus is limited only to thin specimens. Light-sheet fluorescence microscopy (LSFM) overcomes these disadvantages, enabling high-resolution, rapid imaging of thick intact specimens. This is achieved by separating the detection and the illumination axes. In light-sheet microscopy, the illuminating beam enters the sample at right angle to detection axis (see an example set-up in Fig. 3.2). Furthermore, it is not focused to a point, but shaped into a flat, elongated “light sheet” that coincides with the focal plane of the detection objective. This way, only one plane of the specimen is illuminated—hence another name of this method, selective plane illumination microscopy or SPIM [23]. In order to image the entire volume, the specimen is translated along the direction of the optical axis. This configuration significantly reduces out-of-focus fluorescence and enables imaging deep inside intact specimens. Additionally, an entire plane is imaged at once, so collecting a three-dimensional image stack requires scanning in only one dimension instead of three like in conventional laser scanning microscopy.

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Fig. 3.2 Light-sheet microscope set-up. The excitation laser beam is split in two to enable dualsided illumination. Next, the beam is expanded with a telescope and shaped into a light sheet with a cylindrical lens. The light sheet is focused on a sample with the illumination objectives. The sample is immersed in a chamber filled with a RI–matched liquid. The illuminated plane of the sample is imaged on a high-resolution camera with a detection objective placed perpendicularly to the illumination axis. A 3D translation stage moves the sample in order to image the entire volume

3.3.1 Light-Sheet Microscope Configuration and Parameters There are two ways of creating a light sheet that illuminates the specimen, a stationary light sheet, formed by a cylindrical lens and a scanned light sheet [24, 25], where a laser beam is formed into a line and scanned in one direction across the focal plane. The latter approach, known as digital scanned laser light-sheet fluorescence microscopy (DSLM), can be combined with passing the collected fluorescence through a physical slit [26] or with a virtual slit, so-called rolling shutter, available in some newer scientific CMOS cameras [27] to achieve confocal line detection. This approach further increases image contrast at the cost of somewhat, increased set-up complexity. The resolution of a light-sheet microscope is determined by several factors. The lateral resolution is theoretically limited by the numerical aperture of the detection objective. However, in order to achieve this limit, it is necessary to use a highresolution camera with sufficiently small pixel size. The axial resolution of an objective lens scales with the square of NA, so for low NAs it is much worse than the lateral resolution. This is a problem when imaging thick samples, since long-workingdistance objectives typically have lower NAs. However, in a light-sheet microscope the axial point spread function is determined not only but NA, but also by light-sheet thickness. It has been shown that depending on NA, the axial resolution of a lightsheet microscope is two to three times better than that of a confocal or two-photon microscope using the same imaging lens [28].

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The light-sheet thickness in the centre of the field of view is determined by numerical aperture of the illuminating beam. However, due to light diffraction the beam gets thicker nearer to the edges of the field of view so that for bigger samples requiring a large field of view, such as whole rodent brains, it is not possible to keep it both thin and uniform across the entire field of view. Several solutions to this problem have been suggested. One of them is scanning the light-sheet focus along the illumination axis in order to achieve more uniform illumination. The use of structured illumination, such as Bessel beams [29], or extended focusing [30] has been also proposed, but seems to be limited to small specimens, such as single cells. Typical light-sheet thickness in set-ups suited for whole-brain imaging is a few micrometres [21]. In the basic light-sheet microscope layout, the distance between the light sheet and the imaging objective is fixed. The cleared sample is immersed in a RI-matched liquid so that the optical distance stays the same as the sample is moved towards the objective. However, in practice refractive index variations within the cleared sample can cause defocusing and have to be compensated by corrections of either the light sheet or the imaging objective position [31, 32]. Finally, even cleared tissue is not completely free from scattering. In LSFM, this leads to characteristic stripe-shaped artefacts. They can be reduced by pivoting the light sheet [33] or removed during image processing [34, 35].

3.3.2 Cleared-Tissue-Optimized Objectives When a long-working-distance dry objective is used for imaging in immersion, like it was done in the original ultramicroscope set-up, its working distance increases and numerical aperture decreases proportionally to the refractive index of the liquid. However, the decreasing NA is not the only factor limiting resolution in the approach. As it has been shown by Silvestri and co-workers [36], refractive index mismatch causes defocusing and the appearance of sidelobes in the point spread function. This effect gets stronger as NA of the imaging objective increases. In consequence, the application of dry objectives for cleared-tissue imaging is limited to low-NA, low magnification objectives. As an answer to this problem, several cleared-tissue-optimized objectives have been developed recently [37]. For example, the CLARITY-optimized light-sheet microscopy (COLM) set-up developed by Tomer and co-workers [31] makes use of CLARITY-optimized Olympus objectives with 8-mm-working distance. However, these objectives remain costly and, in many cases, limited only to some classes of clearing methods. Specifically, multi-immersion objectives compatible both with the hydrogel-based and water-based methods (RI around 1.48) and the solvent-based methods (RI around 1.55) are to our knowledge currently offered only by one vendor.

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3.3.3 Commercial and Self-built Set-ups Since tissue-clearing methods are relatively new, few commercial light-sheet set-ups optimized for cleared-tissue imaging are currently available on the market. Moreover, some of them work as an extension of existing set-up that was originally designed for other purposes, such as confocal imaging or light-sheet imaging of small, water-based organisms. This causes limitations, mostly of the specimen size. In consequence, a significant part of published work is done using self-built set-ups [15, 38–40]. Self-built set-ups have the advantage of being more flexible and extendable. The latter is an advantage since tissue clearing is still a rapidly developing field, and new methods appear that might be using, e.g. a different immersion liquid with different properties. Moreover, such a set-up can be also significantly cheaper, especially if standard components such as lasers and cameras are already available in the laboratory. A simple, low-cost self-built set-up can be the best way to conduct preliminary experiments before deciding whether tissue clearing is a suitable method for a given experiment. In the field of developmental biology, light-sheet imaging has been made significantly more accessible thanks to so-called open set-ups. These set-ups have not only been described in scientific journals [41, 42], but are accompanied by Websites containing additional material, parts list, technical drawings for the custom-made set-up as well as software plug-ins that enable the operation of a SPIM set-up using the open-source µManager application [43]. The authors of this review have adapted the OpenSPIM design for cleared-organs imaging [15]. For this, water-immersion objectives were replaced with long-workingdistance 4X-magnification air objectives placed outside the chamber. The beamshaping telescope magnification was increased in order to illuminate the entire field of view of about 3 × 3 mm. For more uniform illumination of large samples, the set-up was expanded to include double-sided illumination. Same as the original setup, the OpenSPIM for brains uses a static light-sheet generated with a cylindrical lens. We have demonstrated that already this simple, compact set-up is sufficient for cellular resolution whole-brain imaging.

3.4 Image Data Analysis The capability of rapid imaging of large volumes creates a new challenge: the size of data that has to be acquired, stored and analysed also increases rapidly. A data set containing single-channel imaging of an entire mouse brain has a size between tens of gigabytes to a few terabytes, depending on resolution [31]. Therefore, one of the first decisions to make before attempting a whole-brain imaging is the choice of the right resolution for the experiment: sufficient for extracting the features of interest, but as small as possible to limit the size of the data sets to be analysed. It has

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been shown that cell counting and projection tracing in the entire mouse brain can be performed after imaging with small magnifications in the 1.2X–2X range [21, 44]. Whole-brain imaging at higher magnification requires acquiring several 3D tiles that have to be stitched before further processing. The free, open-source Terastitcher software enables stitching of arbitrarily big data sets already on workstations with limited (