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Clarifying Tissue Clearing

2015; Cell Press; Volume: 162; Issue: 2 Linguagem: Inglês

10.1016/j.cell.2015.06.067

1097-4172

Douglas S. Richardson, Jeff W. Lichtman,

Cell Image Analysis Techniques

Biological specimens are intrinsically three dimensional; however, because of the obscuring effects of light scatter, imaging deep into a tissue volume is problematic. Although efforts to eliminate the scatter by “clearing” the tissue have been ongoing for over a century, there have been a large number of recent innovations. This Review introduces the physical basis for light scatter in tissue, describes the mechanisms underlying various clearing techniques, and discusses several of the major advances in light microscopy for imaging cleared tissue. Biological specimens are intrinsically three dimensional; however, because of the obscuring effects of light scatter, imaging deep into a tissue volume is problematic. Although efforts to eliminate the scatter by “clearing” the tissue have been ongoing for over a century, there have been a large number of recent innovations. This Review introduces the physical basis for light scatter in tissue, describes the mechanisms underlying various clearing techniques, and discusses several of the major advances in light microscopy for imaging cleared tissue. Biologists have long appreciated that it is easier to see things in thin sections than thick volumes—hence, the pervasive use of microtomes, the indispensable tools that cut thin sections of tissue samples and provide information about cellular constituents within two-dimensional sections of biological tissues. Now, however, there is a growing trend to inquire about structure in three dimensions, requiring biologists to contend with volumes rather than sections. The need for volumetric imaging is related to the inherent three-dimensional structure of cells and organs. The nervous system is the most obvious example, given that most individual neurons extend in many directions and their true nature cannot be ascertained by a thin section. Also, much of developmental biology requires understanding morphogenesis of organs and even whole animals in the context of three dimensions. How does one obtain such three-dimensional information? One possibility is to reconstruct three-dimensional information by putting into register a series of serial thin sections. This approach is technically challenging due to loss or distortion of individual sections that become torn, folded, compressed, or stretched. With imperfect sections, the final volumetric reconstruction can be unsatisfactory. However, if done under sufficient control, serial sectioning can give rise to very useful results (Oh et al., 2014Oh S.W. Harris J.A. Ng L. Winslow B. Cain N. Mihalas S. Wang Q. Lau C. Kuan L. Henry A.M. et al.A mesoscale connectome of the mouse brain.Nature. 2014; 508: 207-214Crossref PubMed Scopus (1072) Google Scholar, Toga et al., 1997Toga A.W. Goldkorn A. Ambach K. Chao K. Quinn B.C. Yao P. Postmortem cryosectioning as an anatomic reference for human brain mapping.Comput. Med. Imaging Graph. 1997; 21: 131-141Abstract Full Text PDF PubMed Scopus (32) Google Scholar). Another possibility is to image the surface of a block of tissue and then sequentially shave off the surface. Such “blockface” methods are used in both light (Toga et al., 1994Toga A.W. Ambach K.L. Schluender S. High-resolution anatomy from in situ human brain.Neuroimage. 1994; 1: 334-344Crossref PubMed Scopus (34) Google Scholar, Tsai et al., 2009aTsai P.S. Blinder P. Migliori B.J. Neev J. Jin Y. Squier J.A. Kleinfeld D. Plasma-mediated ablation: an optical tool for submicrometer surgery on neuronal and vascular systems.Curr. Opin. Biotechnol. 2009; 20: 90-99Crossref PubMed Scopus (51) Google Scholar) and electron microscopy (Denk and Horstmann, 2004Denk W. Horstmann H. Serial block-face scanning electron microscopy to reconstruct three-dimensional tissue nanostructure.PLoS Biol. 2004; 2: e329Crossref PubMed Scopus (1022) Google Scholar, Ichimura et al., 2015Ichimura K. Miyazaki N. Sadayama S. Murata K. Koike M. Nakamura K. Ohta K. Sakai T. Three-dimensional architecture of podocytes revealed by block-face scanning electron microscopy.Sci. Rep. 2015; 5: 8993Crossref PubMed Scopus (50) Google Scholar). Blockface approaches eliminate the loss and alignment issues of sections but are destructive in the sense that, once each section is imaged, it is destroyed to reveal the next block surface. The other possibility is to image the volume without sectioning. Non-sectioning approaches avoid the demanding alignment issues, and the same tissue sample in principle can be imaged multiple times. One problem, however, with imaging a volume is that out of focus information from regions above and below the plane of focus contaminates information from any one plane. This problem led Marvin Minsky to invent the first confocal microscope to filter the out-of-focus light (Minsky, 1988Minsky M. Memoir on inventing the confocal scanning microscope.Scanning. 1988; 10: 128-138Crossref Scopus (671) Google Scholar) and led to the revolution of “optical sectioning” techniques—most notably, commercial laser-scanning confocal microscopes, laser scanning two-photon microscopy, parallelized confocal (i.e., spinning disk), computational image deconvolution methods, and lightsheet approaches (reviewed in Conchello and Lichtman, 2005Conchello J.A. Lichtman J.W. Optical sectioning microscopy.Nat. Methods. 2005; 2: 920-931Crossref PubMed Scopus (514) Google Scholar, Mertz, 2011Mertz J. Optical sectioning microscopy with planar or structured illumination.Nat. Methods. 2011; 8: 811-819Crossref PubMed Scopus (198) Google Scholar, Reynaud et al., 2008Reynaud E.G. Krzic U. Greger K. Stelzer E.H. Light sheet-based fluorescence microscopy: more dimensions, more photons, and less photodamage.HFSP J. 2008; 2: 266-275Crossref PubMed Scopus (123) Google Scholar). All of these microscopy methods provide information about single planes of a volume by minimizing contributions from other parts of the volume and do so without physical sectioning. These methods thus allow access to image data from any arbitrary thin section in a thick sample. By creating a “stack” of such optically sectioned images, a full reconstruction of the three-dimensional features of a tissue volume can be ascertained. But even with the advent of optical sectioning microcopies, there remain major obstacles facing a microscopist looking at biological tissues that are thick. First, in some tissues, a pigment gives the tissue a color (Box 1). Second, inherently fluorescent molecules may be present in the tissue or introduced during processing, giving rise to autofluorescence that masks fluorescently labeled structures of interest (Box 2). The final problem, and the one that we focus on here, is that most biological tissues have an intrinsic milky appearance. This property gives tissues the look of frosted glass or translucence. The lack of clarity undermines sharp images and becomes progressively more of an impediment the deeper one tries to look into a tissue volume.Box 1Limiting AbsorptionAbsorption of light within a sample can limit both the excitation light entering a tissue and the fluorescence emission returning to the detector. Hemoglobin, myoglobin, and melanin are the primary molecules that are responsible for visible light absorption in biological tissue. Hemoglobin is present in all vertebrate species (except the crocodile ice fish) and in many invertebrates. Hemoglobin containing red blood cells can be removed from vertebrates by perfusion of clear buffer through the circulatory system. When perfusion is not an option, treating the specimen with hydrogen peroxide to decolor hemoglobin or amino alcohol in a basic solution to elute the heme group is a possibility (Tainaka et al., 2014Tainaka K. Kubota S.I. Suyama T.Q. Susaki E.A. Perrin D. Ukai-Tadenuma M. Ukai H. Ueda H.R. Whole-body imaging with single-cell resolution by tissue decolorization.Cell. 2014; 159: 911-924Abstract Full Text Full Text PDF PubMed Scopus (268) Google Scholar). Hydrogen peroxide, however, is potentially damaging to tissue structure and may adversely affect fluorescent protein emission (Alnuami et al., 2008Alnuami A.A. Zeedi B. Qadri S.M. Ashraf S.S. Oxyradical-induced GFP damage and loss of fluorescence.Int. J. Biol. Macromol. 2008; 43: 182-186Crossref PubMed Scopus (30) Google Scholar, Steinke and Wolff, 2001Steinke H. Wolff W. A modified Spalteholz technique with preservation of the histology.Ann. Anat. 2001; 183: 91-95Crossref PubMed Scopus (42) Google Scholar). Myoglobin, the colored pigment in skeletal muscle, is also decolored by these same two treatments. Melanin, the primary pigment in hair and skin, can be avoided in albino mutant animals. In zebrafish, chemical treatment with 1-phenyl-2-thiourea or genetic modifications (the casper mutant) can be used to block the synthesis of melanin or the development of pigmented melanocytes (White et al., 2008White R.M. Sessa A. Burke C. Bowman T. LeBlanc J. Ceol C. Bourque C. Dovey M. Goessling W. Burns C.E. Zon L.I. Transparent adult zebrafish as a tool for in vivo transplantation analysis.Cell Stem Cell. 2008; 2: 183-189Abstract Full Text Full Text PDF PubMed Scopus (746) Google Scholar). One final option is to move to an area of the electromagnetic spectrum in which biological tissue does not absorb. Often referred to as the “near-infrared window,” light with wavelengths of 650–1,350 nm is very weakly absorbed by biological tissues and can be used with near IR fluorophores or two-photon microscopy. A second imaging window, in the range of 1.3–1.4 μm has recently been identified and used to image >2 mm into tissue, even passing through bone (Hong et al., 2014Hong G. Diao S. Chang J. Antaris A.L. Chen C. Zhang B. Zhao S. Atochin D.N. Huang P.L. Andreasson K.I. et al.Through-skull fluorescence imaging of the brain in a new near-infrared window.Nat. Photonics. 2014; 8: 723-730Crossref PubMed Scopus (519) Google Scholar). Unfortunately, none of these techniques address the primary hindrance to deep tissue imaging, the scatter of light in biological tissue.Box 2AutofluorescenceAutofluorescence is the background glow that results from excitation of either inherently fluorescent molecules in a sample or those introduced into the sample during its preparation. Typically, the broadband glow decreases image quality by lowering the signal-to-noise ratio across multiple fluorescence channels. Autofluorescence may arise from endogenous fluorescent biomolecules (NADPH, collagen, flavins, tyrosine, and others) or may be introduced by the formation of Schiffs bases during fixation with aldehydes (glutaraldehyde is worse than paraformaldehyde). A number of techniques reduce autofluorescence somewhat, including: bleaching with high-intensity lighting (Duong and Han, 2013Duong H. Han M. A multispectral LED array for the reduction of background autofluorescence in brain tissue.J. Neurosci. Methods. 2013; 220: 46-54Crossref PubMed Scopus (19) Google Scholar), sodium borohydride treatment to eliminate Schiffs bases (Clancy and Cauller, 1998Clancy B. Cauller L.J. Reduction of background autofluorescence in brain sections following immersion in sodium borohydride.J. Neurosci. Methods. 1998; 83: 97-102Crossref PubMed Scopus (131) Google Scholar), and spectral unmixing to remove broadband fluorescent signals (Zimmermann, 2005Zimmermann T. Spectral imaging and linear unmixing in light microscopy.Adv. Biochem. Eng. Biotechnol. 2005; 95: 245-265PubMed Google Scholar). It is important to remember that background autofluorescence is not necessarily removed by tissue clearing methods. Absorption of light within a sample can limit both the excitation light entering a tissue and the fluorescence emission returning to the detector. Hemoglobin, myoglobin, and melanin are the primary molecules that are responsible for visible light absorption in biological tissue. Hemoglobin is present in all vertebrate species (except the crocodile ice fish) and in many invertebrates. Hemoglobin containing red blood cells can be removed from vertebrates by perfusion of clear buffer through the circulatory system. When perfusion is not an option, treating the specimen with hydrogen peroxide to decolor hemoglobin or amino alcohol in a basic solution to elute the heme group is a possibility (Tainaka et al., 2014Tainaka K. Kubota S.I. Suyama T.Q. Susaki E.A. Perrin D. Ukai-Tadenuma M. Ukai H. Ueda H.R. Whole-body imaging with single-cell resolution by tissue decolorization.Cell. 2014; 159: 911-924Abstract Full Text Full Text PDF PubMed Scopus (268) Google Scholar). Hydrogen peroxide, however, is potentially damaging to tissue structure and may adversely affect fluorescent protein emission (Alnuami et al., 2008Alnuami A.A. Zeedi B. Qadri S.M. Ashraf S.S. Oxyradical-induced GFP damage and loss of fluorescence.Int. J. Biol. Macromol. 2008; 43: 182-186Crossref PubMed Scopus (30) Google Scholar, Steinke and Wolff, 2001Steinke H. Wolff W. A modified Spalteholz technique with preservation of the histology.Ann. Anat. 2001; 183: 91-95Crossref PubMed Scopus (42) Google Scholar). Myoglobin, the colored pigment in skeletal muscle, is also decolored by these same two treatments. Melanin, the primary pigment in hair and skin, can be avoided in albino mutant animals. In zebrafish, chemical treatment with 1-phenyl-2-thiourea or genetic modifications (the casper mutant) can be used to block the synthesis of melanin or the development of pigmented melanocytes (White et al., 2008White R.M. Sessa A. Burke C. Bowman T. LeBlanc J. Ceol C. Bourque C. Dovey M. Goessling W. Burns C.E. Zon L.I. Transparent adult zebrafish as a tool for in vivo transplantation analysis.Cell Stem Cell. 2008; 2: 183-189Abstract Full Text Full Text PDF PubMed Scopus (746) Google Scholar). One final option is to move to an area of the electromagnetic spectrum in which biological tissue does not absorb. Often referred to as the “near-infrared window,” light with wavelengths of 650–1,350 nm is very weakly absorbed by biological tissues and can be used with near IR fluorophores or two-photon microscopy. A second imaging window, in the range of 1.3–1.4 μm has recently been identified and used to image >2 mm into tissue, even passing through bone (Hong et al., 2014Hong G. Diao S. Chang J. Antaris A.L. Chen C. Zhang B. Zhao S. Atochin D.N. Huang P.L. Andreasson K.I. et al.Through-skull fluorescence imaging of the brain in a new near-infrared window.Nat. Photonics. 2014; 8: 723-730Crossref PubMed Scopus (519) Google Scholar). Unfortunately, none of these techniques address the primary hindrance to deep tissue imaging, the scatter of light in biological tissue. Autofluorescence is the background glow that results from excitation of either inherently fluorescent molecules in a sample or those introduced into the sample during its preparation. Typically, the broadband glow decreases image quality by lowering the signal-to-noise ratio across multiple fluorescence channels. Autofluorescence may arise from endogenous fluorescent biomolecules (NADPH, collagen, flavins, tyrosine, and others) or may be introduced by the formation of Schiffs bases during fixation with aldehydes (glutaraldehyde is worse than paraformaldehyde). A number of techniques reduce autofluorescence somewhat, including: bleaching with high-intensity lighting (Duong and Han, 2013Duong H. Han M. A multispectral LED array for the reduction of background autofluorescence in brain tissue.J. Neurosci. Methods. 2013; 220: 46-54Crossref PubMed Scopus (19) Google Scholar), sodium borohydride treatment to eliminate Schiffs bases (Clancy and Cauller, 1998Clancy B. Cauller L.J. Reduction of background autofluorescence in brain sections following immersion in sodium borohydride.J. Neurosci. Methods. 1998; 83: 97-102Crossref PubMed Scopus (131) Google Scholar), and spectral unmixing to remove broadband fluorescent signals (Zimmermann, 2005Zimmermann T. Spectral imaging and linear unmixing in light microscopy.Adv. Biochem. Eng. Biotechnol. 2005; 95: 245-265PubMed Google Scholar). It is important to remember that background autofluorescence is not necessarily removed by tissue clearing methods. This translucency is caused by light scattering. Light rays that should travel in straight lines are deviated many times as light is reflected off of molecules, membranes, organelles, and cells in the tissue. It is useful to delve a bit into the underlying physics of scattering in order to understand the kinds of strategies that have been used to clear tissues. The purpose of this short analysis is to understand that clearing tissues is not aimed at preventing scattering (only a vacuum has no scattering) but, rather, assuring that there is a high uniform density of scatterers so that lateral scattering is minimal and that all wavelengths of light pass “through” the tissue. The ways to think about light range from light as rays, light as waves, or light as photons. Although rays are easiest to talk about and photons seem closest to the true essence of light, it is the wave framework that is most helpful when thinking about light scattering. Light waves of a particular color (wavelength) vibrate at a particular frequency and have an electrical and magnetic component. The electrical component is the one that for the most part interacts with the atoms in biological tissues. The wave vibration is extraordinarily fast. For example, red light (600 nm wavelength) vibrates at the rate of 0.5 × 1015 oscillations per second. Let’s imagine this wave is a plane that passes an atom or molecule from a particular direction (plane waves are basically light that propagates in a single direction without converging or diverging, like a laser beam). As the plane wave reaches the atoms, it may impart some of its energy from its electrical component to the atom or molecule, typically to an outer electron which is more susceptible to absorbing energy than electrons closer to the nucleus or protons or neutrons in the nucleus. For most molecules, the energy absorbed by an electron is not sufficient to cause an electron to jump to a new orbital. Therefore, neither fluorescence (re-emission of lower-energy light; Lichtman and Conchello, 2005Lichtman J.W. Conchello J.A. Fluorescence microscopy.Nat. Methods. 2005; 2: 910-919Crossref PubMed Scopus (887) Google Scholar) nor ionization (removal of the electron from the atom or molecule) occurs. Rather, the light wave’s vibrational energy momentarily causes the electron to vibrate (as if it were connected to its nucleus by a spring that was stretched a bit by the incoming light wave). The electron vibration is short lived, and all of the energy absorbed is quickly released again in the form of another light wave. There are a few differences between the incoming light wave and the outgoing light wave emitted by the atom or molecule. First, the incident light is coming from a particular direction, but the outgoing light is sent in all directions as a spherical wave (see below). Thus, the light is scattered. Because the whole process occurs without any energy loss, the light is said to be elastically scattered. Elastic scattering means that the vibrational frequency (and hence the wavelength) of the scattered light is unchanged from the incoming light. The second difference is that the interaction between the incident light wave and the electron cloud of the scatterer, although brief, causes a momentary pause in the light’s progression, evidenced by the fact that the new scattered wave is delayed (usually one-half of a wavelength). The duration of the delay is only about a femtosecond (10−15 s) for visible light. But as light passes through a material, it interacts with many molecules and these little delays associated with each interaction add up. As a result, light’s propagation through the material is slowed down. This reduced velocity is the basis of the so-called refractive index of the medium (literally, the ratio of the speed of light in a vacuum divided by the speed of light in the medium). The amount of slowdown per unit volume is proportional to the density of molecules and hence the number of electrons that the light wave can interact with. But density is only one variable that affects refractive index. Some materials such as the hydrophobic molecules in the plasma membrane have electrons that are more susceptible to absorbing light energy than other molecules, such as the hydrogen atoms in water. Thus, even though the density of water surrounding a cell is higher than the density of the fatty material in cell membranes (fat, after all, floats in water), the membrane has a higher index of refraction than water (1.45 versus 1.33). Although it is commonly explained that scattering occurs due to the mismatch of the index of refractions at interfaces between different substances in a tissue, this is not the whole story. As we will explain below, scattering occurs everywhere that there are molecules, not just at sites of refractive index mismatches. It is more accurate to say that heterogeneity in the amount of scattering between different regions in biological material is actually what gives rise to the scattering and milky appearance. The light energy absorbed by an electron is re-radiated in all directions as an expanding spherical wave. This spherical wave, sometimes called a “wavelet,” is a wave that diverges from a source as if that source were a luminous object sending wave energy along the expanding surface of a sphere. The two-dimensional analog of a spherical wave would be the circular wave that originates on the surface of a pool of water from the site where a pebble is dropped. The emitted spherical wave, just like the original incident light, can and will interact with electrons in other atoms or molecules re-emerging over and over again as new spherical waves at different sites. The wave conception of light is powerful because it explains how light waves can add their amplitudes and give rise to brighter light though constructive interference when they crest or trough in phase. The intensity of light is actually the square of the summed amplitudes integrated over time, so both troughs and crests give light energy. The wave conception also explains why light may sometimes not be present if two waves reach the same place half a wavelength out of phase (i.e., while one wave is in the crest and another is in the trough at the same place). In this situation, the sum of their amplitudes is zero, and no light from those wavelets can appear at that site; this is known as destructive interference. Destructive interference explains why homogenous materials, such as air, water, and glass, appear clear even though the molecules in these substances are scattering light. In such materials, the density of scattering molecules is so high that many scatterers exist even over dimensions much smaller than the wavelength of visible light (i.e., between 400 and 700 nm). Air molecules, for example, are about 3 nm apart, and liquid water molecules are about ten times closer together (Hecht, 2001Hecht E. Optics, Fourth Edition. Addison-Wesley, 2001Google Scholar). When a plane wave of light passes through such materials, all of the molecules in a plane are set into vibration simultaneously and give rise to densely packed spherical waves. The consequence of such a high density of scatterers is nearly complete destructive interference in the axes of the plane (Figures 1A and 1B ). This cancellation occurs because when wavelets are uniformly distributed at high density, each point in the plane is bombarded by wavelets at every phase. For example, for every cresting wave, another is in the trough at the same place. As a result, the sum amplitude is zero in the plane, preventing any light from escaping in any lateral direction out of the plane (i.e., hardly any light propagates perpendicular to the direction of the impinging light wave; Figure 1B). We know, of course, that light continues in the forward direction in media like air, water, or glass without difficulty, raising the question of why the scatterers in front of each molecule don’t also give rise to destructive interference by the same argument. Distinct from the simultaneous vibrations of all the scatterers in a plane, the molecules in front of the plane are activated later, when the primary plane wave reaches them (Figures 1C and 1D). The scattered light from the molecules that were previously acted on by the plane wave reach this forward direction slightly later due to the additional half-wavelength phase delay of scattered light. The scattered light from the earlier illuminated molecules always (!) constructively interferes with the scattered light originating from molecules at more forward sites (Figure 1D). Thus, in the forward direction, the amplitudes sum constructively and light propagates. Given that dense materials like water don’t scatter in lateral directions, why do dense cellular tissues scatter? The important point here is the inhomogeneity of scatterers. The prevention of lateral scattering requires that each scattering molecule is equidistant from other scatterers at every distance in the plane. For example, in living tissue, a physiological saline solution surrounds the membrane of a cell. This means that the amount of scattering in the membrane is likely to be different than the amount of scattering in the saline solution. Thus, scattering from the water molecules near the membrane may not be completely cancelled by the scattering from the membrane itself; thus, both materials will generate light scattered perpendicular to the direction of light impinging on the sample. If a tissue is sufficiently thick, then most of the incoming light will be scattered and the tissue will behave as if it contains a multitude of little luminous sources, each sending light in every direction. This multiple scattering is the property that generates the whitish translucency of tissues. The whitish color implies that all wavelengths of visible light are scattered, and this is due to the intrinsic inhomogeneities of scatterers in the tissue. The sizes of the inhomogeneities affect the wavelength of the light that is scattered. For particles that are much smaller than the wavelength of visible light, short-wavelength light has a greater probability of being absorbed and re-emitted (in proportion to the wavelength to the fourth power, Rayleigh scattering). As a result, there is some tendency for short wavelengths to scatter more than long ones. This is sometimes mentioned as an advantage of using the long-wavelength infrared-light-based two-photon excitation on thick samples, as relatively less of the exciting light is scattered compared to visible or ultraviolet light. For particles larger than about one-tenth of the wavelength, such as organelles and large protein complexes, the wavelength dependence of scattering is not evident (Mie scattering). It has been known for more than a century that biological samples can be stably maintained in a somewhat transparent hardened material resin. Indeed, ancient flies in amber show that resins can stably maintain biological samples for 50 million years. Many of the resins used by biologists are hydrophobic, requiring that the sample be dehydrated. Following dehydration (in a series of alcohol-water mixtures with progressively less water), samples are put into solvents that dissolve lipids and act to remove one of the main sites of tissue inhomogeneity: the membranes. Canada Balsam is such a resin that provides a transparent mountant for tissue that is dehydrated and cleared with xylene. Such resins, however, are intrinsically fluorescent and are best used with absorbance dyes like the Golgi stain rather than fluorophores due to high background (Rost, 1992Rost F.W.D. Fluorescence microscopy. Cambridge University Press, 1992Google Scholar). Moreover, most fluorescent proteins require an aqueous environment, so this kind of clearing quenches the signal. At the beginning of the 20th century, Spalteholz described a clearing technique for large (entire organ and organ system) tissues using organic solvents (Spalteholz, 1914Spalteholz W. Uber das Durchsichtigmachen von menschlichen und tierischen Praparaten. S. Hierzel, 1914Google Scholar). The method was intensive, requiring various dehydration, tissue bleaching, and clearing steps. However, it produced samples that were unprecedented at the time and helped to push forward the field of anatomy (Spalteholz, 1898Spalteholz W. Hand-atlas of human anatomy.Second Edition. J.B. Lippincott, 1898Google Scholar). Unfortunately, this approach damaged the superficial few centimeters of a tissue and therefore was useful only for clearing the largest samples (Steinke and Wolff, 2001Steinke H. Wolff W. A modified Spalteholz technique with preservation of the histology.Ann. Anat. 2001; 183: 91-95Crossref PubMed Scopus (42) Google Scholar). The optical sectioning advances mentioned above (i.e., confocal, two-photon, and image deconvolution) led to fluorescence volume imaging becoming the contrast method of choice for microscopy at the end of the 20th century. Most notably, two-photon detection pushed microscopy from imaging depths of tens of microns to fractions of a millimeter (Denk et al., 1990Denk W. Strickler J.H. Webb W.W. Two-photon laser scanning fluorescence microscopy.Science. 1990; 248: 73-76Crossref PubMed Scopus (7800) Google Scholar). Genetically encoded fluorescent proteins provided a labeling method with high specificity that did not require antibodies to diffuse through the entire sample to their target and hence motivated ever deeper imaging (Chalfie et al., 1994Chalfie M. Tu Y. Euskirchen G. Ward W.W. Prasher D.C. Green fluorescent protein as a marker for gene expression.Science. 1994; 263: 802-805Crossref PubMed Scopus (5283) Google Scholar). However, the scatter of light in heterogeneous tissue remained a limiting factor for