Chemical Probes for Visualizing Intact Animal and Human Brain Tissue
2017; Elsevier BV; Volume: 24; Issue: 6 Linguagem: Inglês
10.1016/j.chembiol.2017.05.015
ISSN2451-9456
AutoresH. M. Lai, Wai‐Lung Ng, Steve Gentleman, Wutian Wu,
Tópico(s)Neurogenesis and neuroplasticity mechanisms
ResumoNewly developed tissue clearing techniques can be used to render intact tissues transparent. When combined with fluorescent labeling technologies and optical sectioning microscopy, this allows visualization of fine structure in three dimensions. Gene-transfection techniques have proved very useful in visualizing cellular structures in animal models, but they are not applicable to human brain tissue. Here, we discuss the characteristics of an ideal chemical fluorescent probe for use in brain and other cleared tissues, and offer a comprehensive overview of currently available chemical probes. We describe their working principles and compare their performance with the goal of simplifying probe selection for neuropathologists and stimulating probe development by chemists. We propose several approaches for the development of innovative chemical labeling methods which, when combined with tissue clearing, have the potential to revolutionize how we study the structure and function of the human brain. Newly developed tissue clearing techniques can be used to render intact tissues transparent. When combined with fluorescent labeling technologies and optical sectioning microscopy, this allows visualization of fine structure in three dimensions. Gene-transfection techniques have proved very useful in visualizing cellular structures in animal models, but they are not applicable to human brain tissue. Here, we discuss the characteristics of an ideal chemical fluorescent probe for use in brain and other cleared tissues, and offer a comprehensive overview of currently available chemical probes. We describe their working principles and compare their performance with the goal of simplifying probe selection for neuropathologists and stimulating probe development by chemists. We propose several approaches for the development of innovative chemical labeling methods which, when combined with tissue clearing, have the potential to revolutionize how we study the structure and function of the human brain. In order to understand how the brain functions and malfunctions, we must first understand its composition and fine structure, as well as how all its pieces come together (Sporns, 2013Sporns O. The human connectome: origins and challenges.NeuroImage. 2013; 80: 53-61Crossref PubMed Scopus (141) Google Scholar). It is increasingly recognized that such a connectivity map must be not only globally complete but also exhaustively precise in the finest details. We have come a long way in mapping the anatomical connectivity of the human brain by physically sectioning tissue and tracing the fibers through sections (Shibata et al., 2014Shibata S. Komaki Y. Seki F. Inouye M.O. Nagai T. Okano H. Connectomics: comprehensive approaches for whole-brain mapping.Microscopy (Oxf). 2014; 64: 57-67Crossref PubMed Scopus (2) Google Scholar, Micheva et al., 2010Micheva K.D. Busse B. Weiler N.C. O'Rourke N. Smith S.J. Single-synapse analysis of a diverse synapse population: proteomic imaging methods and markers.Neuron. 2010; 68: 639-653Abstract Full Text Full Text PDF PubMed Scopus (179) Google Scholar); however, to understand how our brain functions, a large number of brains under different pathophysiological states will have to be mapped in great detail (Kopell et al., 2014Kopell N.J. Gritton H.J. Whittington M.A. Kramer M.A. Beyond the connectome: the dynome.Neuron. 2014; 83: 1319-1328Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar). This goal requires a major development of techniques that enable peering into a working brain and intact brain tissue. Recently, a number of techniques have emerged that take a first step by turning intact brain tissue transparent (Susaki and Ueda, 2016Susaki E.A. Ueda H.R. Whole-body and whole-organ clearing and imaging techniques with single-cell resolution: toward organism-level systems biology in mammals.Cell Chem. Biol. 2016; 23: 137-157Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). The next major step is to pair these tissue clearing methods with appropriate labeling techniques that work in human brain tissue where genetic, viral, and toxin tracers are not applicable (Marx, 2016Marx V. Optimizing probes to image cleared tissue.Nat. Methods. 2016; 13: 205-209Crossref PubMed Scopus (5) Google Scholar). The shortcomings of commonly used immunostaining procedures have necessitated the development of a different strategy for visualizing human brain tissue. Here, chemical labeling methods represent a uniquely reliable and feasible approach for transparent brain tissue labeling. In this review, we hope to provide a chemical probe guide for biologists and pathologists working with tissue clearing techniques, as well as raise awareness of the need for more chemical probe development within the chemical biology community. Thus, we first provide a brief overview of the physicochemical processes involved in tissue clearing and how this affects the choice of the most suitable chemical probes, followed by a discussion of how to design an ideal fluorescent probe for labeling of the cleared tissues. We then describe the currently available chemical probes for brain visualization, as well as strategies to expand their utility. The vast array of genetic and viral labeling tools applicable only to rodents can be found in other recent reviews (Susaki and Ueda, 2016Susaki E.A. Ueda H.R. Whole-body and whole-organ clearing and imaging techniques with single-cell resolution: toward organism-level systems biology in mammals.Cell Chem. Biol. 2016; 23: 137-157Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar, Lerner et al., 2016Lerner T.N. Ye L. Deisseroth K. Communication in neural circuits: tools, opportunities, and challenges.Cell. 2016; 164: 1136-1150Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar) and are not further discussed here. The first step toward three-dimensional (3D) brain visualization is tissue clearing, a process that alters the physicochemical properties of tissues to achieve optical transparency. Therefore, the choice of tissue clearing strategy will have major implications for selecting a specific chemical probe. We do not go into the question of how tissues clearing methods work here, as these topics have been reviewed recently (Susaki and Ueda, 2016Susaki E.A. Ueda H.R. Whole-body and whole-organ clearing and imaging techniques with single-cell resolution: toward organism-level systems biology in mammals.Cell Chem. Biol. 2016; 23: 137-157Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar, Treweek and Gradinaru, 2016Treweek J.B. Gradinaru V. Extracting structural and functional features of widely distributed circuits with single cell resolution via tissue clearing and delivery vectors.Curr. Opin. Biotechnol. 2016; 40: 193-207Crossref PubMed Scopus (5) Google Scholar, Tainaka et al., 2016Tainaka K. Kuno A. Kubota S.I. Murakami T. Ueda H.R. Chemical principles in tissue clearing and staining protocols for whole-body cell profiling.Annu. Rev. Cell Dev. Biol. 2016; 32: 713Crossref PubMed Scopus (28) Google Scholar). Rather, here we present an overview of the methods and how they may affect chemical probe development. Overall, tissue clearing methods can be conceptually unified as a single, simple workflow (Figure 1). In brief, the tissue has to be immersed into a final refractive index homogenizing medium before optical sectioning microscopy. Alterations in the physicochemical properties of tissues induced by these optical clearing methods can alter the effectiveness of chemical probes. Therefore, when choosing the tissue clearing method and chemical probes, the user needs to check for compatibility between the two (Table S1). Overall, major sources of incompatibilities are due to changes in the structure of the tissue itself, interference of the interactions between the small-molecule probe and its desired binding targets, alterations in the fluorescent properties of the tissue, and alterations in the fluorescent properties of the small-molecule probe. Some harsh treatment conditions in tissue clearing can lead to the destruction of certain tissue components. In delipidation-assisted tissue clearing methods, the tissue itself is deprived of most of its lipids (Chung et al., 2013Chung K. Wallace J. Kim S. Kalyanasundaram S. Andalman A.S. Davidson T.J. Mirzabekov J.J. Zalocusky K.A. Mattis J. Denisin A.K. et al.Structural and molecular interrogation of intact biological systems.Nature. 2013; 497: 332-337Crossref PubMed Scopus (604) Google Scholar, Susaki et al., 2014Susaki E.A. Tainaka K. Perrin D. Kishino F. Tawara T. Watanabe T.M. Yokoyama C. Onoe H. Eguchi M. Yamaguchi S. et al.Whole-brain imaging with single-cell resolution using chemical cocktails and computational analysis.Cell. 2014; 157: 726-739Abstract Full Text Full Text PDF PubMed Scopus (321) Google Scholar, Murray et al., 2015Murray E. Cho J. Goodwin D. Ku T. Swaney J. Kim S. Choi H. Park Y. Park J. Hubbert A. et al.Simple, scalable proteomic imaging for high-dimensional profiling of intact systems.Cell. 2015; 163: 1500-1514Abstract Full Text Full Text PDF PubMed Google Scholar), and is therefore incompatible with dyes that specifically label lipid membranes and lipid droplets, unless the lipid molecules contain aldehyde-crosslinkable groups and are present in abundance within the brain tissue (Murray et al., 2015Murray E. Cho J. Goodwin D. Ku T. Swaney J. Kim S. Choi H. Park Y. Park J. Hubbert A. et al.Simple, scalable proteomic imaging for high-dimensional profiling of intact systems.Cell. 2015; 163: 1500-1514Abstract Full Text Full Text PDF PubMed Google Scholar). Furthermore, prolonged treatment of tissue at high temperatures or denaturing conditions can destroy unstable biomolecules, most notably RNAs (Murray et al., 2015Murray E. Cho J. Goodwin D. Ku T. Swaney J. Kim S. Choi H. Park Y. Park J. Hubbert A. et al.Simple, scalable proteomic imaging for high-dimensional profiling of intact systems.Cell. 2015; 163: 1500-1514Abstract Full Text Full Text PDF PubMed Google Scholar). In these cases, the probe-binding targets are lost and specific measures are needed to preserve them (Sylwestrak et al., 2016Sylwestrak E.L. Rajasethupathy P. Wright M.A. Jaffe A. Deisseroth K. Multiplexed intact-tissue transcriptional analysis at cellular resolution.Cell. 2016; 164: 792-804Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar) to allow staining after the clearing process. Tissue clearing methods can also alter the target conformation, resulting in either no chemical probe binding or nonspecific binding. For example, using denaturing detergents, such as SDS (Chung et al., 2013Chung K. Wallace J. Kim S. Kalyanasundaram S. Andalman A.S. Davidson T.J. Mirzabekov J.J. Zalocusky K.A. Mattis J. Denisin A.K. et al.Structural and molecular interrogation of intact biological systems.Nature. 2013; 497: 332-337Crossref PubMed Scopus (604) Google Scholar, Murray et al., 2015Murray E. Cho J. Goodwin D. Ku T. Swaney J. Kim S. Choi H. Park Y. Park J. Hubbert A. et al.Simple, scalable proteomic imaging for high-dimensional profiling of intact systems.Cell. 2015; 163: 1500-1514Abstract Full Text Full Text PDF PubMed Google Scholar), urea (Hama et al., 2015Hama H. Hioki H. Namiki K. Hoshida T. Kuroka H. Kurokawa H. Ishidate F. Kaneka T. Akagi T. Saito T. et al.ScaleS: an optical clearing palette for biological imaging.Nat. Neurosci. 2015; 18: 1518-1529Crossref PubMed Google Scholar), or organic solvents (Ertürk et al., 2012Ertürk A. Mauch C.P. Hellal F. Fӧrstner F. Keck T. Becker K. Jӓhrling N. Steffens H. Richter M. Hübener M. et al.Three-dimensional imaging of the unsectioned adult spinal cord to assess axon regeneration and glial responses after injury.Nat. Med. 2012; 18: 166-171Crossref Scopus (161) Google Scholar), may disrupt the protein structures required for efficient binding of ligand dyes and result in no staining. Delipidation methods can lead to loss of the hydrophobic environment required for the proper folding of some proteins, most notably transmembrane proteins, where successful labeling has yet to be reported. With the use of tissue clearing agents, it is also possible that a slight reduction of the dye's affinity toward its target can translate into preferential binding to other tissue components and nonspecific labeling. Finally, the infiltration of tissues with a fixative such as glutaraldehyde or formaldehyde can lead to nonspecific labeling of amine-, hydrazide-, and oxime-containing probes, as the aldehyde groups or imines so formed might not be adequately quenched. Another obstacle to brain visualization is significant tissue autofluorescence due to the abundance of lipofuscin and pigmented substances with an absorption maximum in the blue-green region of the spectrum, and tissue clearing techniques that use glutaraldehyde or tissue dehydration can exacerbate the situation. So far, chemical attempts to minimize or remove autofluorescence have been either ineffective or led to excessive destruction of tissues or antigens, thus outweighing the benefits (Murray et al., 2015Murray E. Cho J. Goodwin D. Ku T. Swaney J. Kim S. Choi H. Park Y. Park J. Hubbert A. et al.Simple, scalable proteomic imaging for high-dimensional profiling of intact systems.Cell. 2015; 163: 1500-1514Abstract Full Text Full Text PDF PubMed Google Scholar, Treweek et al., 2015Treweek J.B. Chan K.Y. Flytzanis N.C. Yang B. Deverman B.E. Greenbaum A. Lignell A. Xiao C. Cai L. Ladinsky M.S. et al.Whole-body tissue stabilization and selective extractions via tissue-hydrogel hybrids for high-resolution intact circuit mapping and phenotyping.Nat. Protoc. 2015; 10: 1860-1896Crossref PubMed Google Scholar). In our experience, the prolonged treatment of tissues with SDS at high temperatures (55°C) resulted in reduced autofluorescence (Liu et al., 2016Liu A.K. Lai H.M. Chang R.C. Gentleman S.M. Free-of-acrylamide sodium doceyl sulphate (SDS)-based Tissue Clearing (FASTClear): a novel protocol of tissue clearing for three-dimensional visualisation of human brain tissues.Neuropathol. Appl. Neurobiol. 2016; https://doi.org/10.1111/nan.12361Crossref Scopus (11) Google Scholar), however we also observed a plateau for clearing of archived tissues fixed in formalin for a long period of time, indicating that autofluorescence for those samples cannot be entirely eliminated. Bleaching by methanolic hydrogen peroxide reduces autofluorescence in archived tissues (Renier et al., 2014Renier N. Wu Z. Simon D.J. Yang J. Ariel P. Tessier-Lavigne M. iDISCO: a simple, rapid method to immunolabel large tissue samples for volume imaging.Cell. 2014; 159: 896-910Abstract Full Text Full Text PDF PubMed Scopus (236) Google Scholar) but comes with a higher risk of antigen or tissue destruction. Given all these considerations, it is best to combine clearing methods that reduce autofluorescence with the use probes with red-shifted excitation/emission spectra to avoid spectral overlap between tissue components and the probes. The tissue clearing methods can also have an effect on the fluorescent properties of the small-molecule probes. For example, pH fluctuations in refractive index homogenizing media could lead to loss of fluorescence, which is especially problematic with organic solvent-based clearing methods where buffer systems are not well established, and result in conflicting reports on which fluorophores are compatible with the same method (Renier et al., 2014Renier N. Wu Z. Simon D.J. Yang J. Ariel P. Tessier-Lavigne M. iDISCO: a simple, rapid method to immunolabel large tissue samples for volume imaging.Cell. 2014; 159: 896-910Abstract Full Text Full Text PDF PubMed Scopus (236) Google Scholar, Becker et al., 2012Becker K. Jährling N. Saghafi S. Weiler R. Dodt H.U. Chemical clearing and dehydration of GFP expressing mouse brains.PLoS One. 2012; 7: e33916Crossref PubMed Scopus (0) Google Scholar). Attempts to stabilize pH using sodium ethoxide or trimethylamine (Schwarz et al., 2015Schwarz M.K. Scherbarth A. Sprengel R. Engelhardt J. Theer P. Giese G. Fluorescent-protein stabilization and high-resolution imaging of cleared, intact mouse brains.PLoS One. 2015; 10: e0124650Crossref PubMed Scopus (35) Google Scholar) can lead to tissue or dye destruction due to their reactive properties. In aqueous-based refractive index homogenizing media, some quenching of fluorescence may occur by direct chemical reaction or fluorescent energy transfer to surrounding molecules. Not all effects of tissue clearing methods are detrimental. The delipidation of tissues essentially permeabilizes the tissue and facilitates the diffusion of hydrophilic probes, thus increasing labeling efficiency (Chung et al., 2013Chung K. Wallace J. Kim S. Kalyanasundaram S. Andalman A.S. Davidson T.J. Mirzabekov J.J. Zalocusky K.A. Mattis J. Denisin A.K. et al.Structural and molecular interrogation of intact biological systems.Nature. 2013; 497: 332-337Crossref PubMed Scopus (604) Google Scholar, Lai et al., 2016Lai H.M. Liu A.K.L. Ng W.L. DeFelice J. Lee W.S. Li H. Li W. Ng H.M. Chang R.C.C. Lin B. et al.Rationalization and validation of an acrylamide-free procedure in three-dimensional histological imaging.PLoS One. 2016; 11: e0158628Crossref PubMed Scopus (12) Google Scholar). Certain amino alcohols as well as denaturants can decolorize endogenous chromophores and fluorophores (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 (128) Google Scholar), widening the spectral window of probe choices. Prolonged incubation of long-formaldehyde-fixed archival human brain tissues at high temperatures and slight alkaline pH can facilitate the decomposition of N,N′-methylene bridges, which in turn reduces the diffusion restriction and can potentially open up binding sites for dyes (Evers et al., 2011Evers D.L. Fowler C.B. Cunningham B.R. Mason J.T. O'Leary T.J. The effect of formaldehyde fixation on RNA. Optimization of formaldehyde adduct removal.J. Mol. Diagn. 2011; 13: 282-288Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar). Although refractive index matching media can quench fluorescence, with appropriate formulation it can also be used to preserve fluorescence and minimize bleaching. Apart from the impact of tissue clearing processes, the quality of staining is also determined by the penetration depth of the dye as well as the homogeneity of the staining, a challenge unique to intact bulky tissues. Permeabilization of the tissue with detergent opens up transcellular pathways of diffusion, which are especially important for hydrophilic, cell-impermeant stains. The effect of permeabilization usually rapidly plateaus for commonly used detergents such as Triton X-100 and SDS, and further permeabilization efforts are usually fruitless. The diffusion-convection model best describes the penetration of probes in a bulky tissue, with diffusion restriction (Li et al., 2015Li J. Czajkowsky D.M. Li X. Shao Z. Fast immune-labeling by electrophoretically driven infiltration for intact tissue imaging.Sci. Rep. 2015; 5: 10640Crossref PubMed Google Scholar, Liu et al., 2016Liu A.K. Lai H.M. Chang R.C. Gentleman S.M. Free-of-acrylamide sodium doceyl sulphate (SDS)-based Tissue Clearing (FASTClear): a novel protocol of tissue clearing for three-dimensional visualisation of human brain tissues.Neuropathol. Appl. Neurobiol. 2016; https://doi.org/10.1111/nan.12361Crossref Scopus (11) Google Scholar), convection restriction (Yang et al., 2014Yang B. Treweek J.B. Kulkarni R.P. Deverman B.E. Chen C. Lubeck E. Shah S. Cai L. Gradinaru V. Single-cell phenotyping within transparent intact tissue through whole-body clearing.Cell. 2014; 158: 945-958Abstract Full Text Full Text PDF PubMed Scopus (293) Google Scholar, Lai et al., 2016Lai H.M. Liu A.K.L. Ng W.L. DeFelice J. Lee W.S. Li H. Li W. Ng H.M. Chang R.C.C. Lin B. et al.Rationalization and validation of an acrylamide-free procedure in three-dimensional histological imaging.PLoS One. 2016; 11: e0158628Crossref PubMed Scopus (12) Google Scholar), and the "sink" effect (Renier et al., 2014Renier N. Wu Z. Simon D.J. Yang J. Ariel P. Tessier-Lavigne M. iDISCO: a simple, rapid method to immunolabel large tissue samples for volume imaging.Cell. 2014; 159: 896-910Abstract Full Text Full Text PDF PubMed Scopus (236) Google Scholar, Murray et al., 2015Murray E. Cho J. Goodwin D. Ku T. Swaney J. Kim S. Choi H. Park Y. Park J. Hubbert A. et al.Simple, scalable proteomic imaging for high-dimensional profiling of intact systems.Cell. 2015; 163: 1500-1514Abstract Full Text Full Text PDF PubMed Google Scholar, Liu et al., 2016Liu A.K. Lai H.M. Chang R.C. Gentleman S.M. Free-of-acrylamide sodium doceyl sulphate (SDS)-based Tissue Clearing (FASTClear): a novel protocol of tissue clearing for three-dimensional visualisation of human brain tissues.Neuropathol. Appl. Neurobiol. 2016; https://doi.org/10.1111/nan.12361Crossref Scopus (11) Google Scholar) as the three major variables that govern the penetration depth of a probe. The diffusion of chemical probes in fully permeabilized tissues is unrestricted (Li et al., 2015Li J. Czajkowsky D.M. Li X. Shao Z. Fast immune-labeling by electrophoretically driven infiltration for intact tissue imaging.Sci. Rep. 2015; 5: 10640Crossref PubMed Google Scholar). However, convection restriction has to be considered for hydrogel-embedded tissues, and increasing the hydrogel porosity may improve probe penetration (Yang et al., 2014Yang B. Treweek J.B. Kulkarni R.P. Deverman B.E. Chen C. Lubeck E. Shah S. Cai L. Gradinaru V. Single-cell phenotyping within transparent intact tissue through whole-body clearing.Cell. 2014; 158: 945-958Abstract Full Text Full Text PDF PubMed Scopus (293) Google Scholar). The more significant obstacle to probe penetration are the sinks, any sites that consume the probe along its diffusion path, regardless of whether the binding is specific or not (Liu et al., 2016Liu A.K. Lai H.M. Chang R.C. Gentleman S.M. Free-of-acrylamide sodium doceyl sulphate (SDS)-based Tissue Clearing (FASTClear): a novel protocol of tissue clearing for three-dimensional visualisation of human brain tissues.Neuropathol. Appl. Neurobiol. 2016; https://doi.org/10.1111/nan.12361Crossref Scopus (11) Google Scholar). One way of dealing with sinks is to use a large amount of probe to saturate the sink sites and then supply additional probe in divided doses to avoid nonspecific staining (Liu et al., 2016Liu A.K. Lai H.M. Chang R.C. Gentleman S.M. Free-of-acrylamide sodium doceyl sulphate (SDS)-based Tissue Clearing (FASTClear): a novel protocol of tissue clearing for three-dimensional visualisation of human brain tissues.Neuropathol. Appl. Neurobiol. 2016; https://doi.org/10.1111/nan.12361Crossref Scopus (11) Google Scholar). In a different strategy, Murray et al., 2015Murray E. Cho J. Goodwin D. Ku T. Swaney J. Kim S. Choi H. Park Y. Park J. Hubbert A. et al.Simple, scalable proteomic imaging for high-dimensional profiling of intact systems.Cell. 2015; 163: 1500-1514Abstract Full Text Full Text PDF PubMed Google Scholar applied binding inhibitors in the staining solution to prevent probe from binding their targets, thus avoiding the sink effect to favor probe diffusion; this is followed by selective removal of binding inhibitors to achieve homogeneous staining at great depths. Physical methods, such as the use of an electric field (Li et al., 2015Li J. Czajkowsky D.M. Li X. Shao Z. Fast immune-labeling by electrophoretically driven infiltration for intact tissue imaging.Sci. Rep. 2015; 5: 10640Crossref PubMed Google Scholar, Kim et al., 2015Kim S. Cho J.H. Murray E. Bakh N. Choi H. Ohn K. Ruelas L. Hubbert A. McCue M. Vassallo S.L. et al.Stochastic electrotransport selectively enhances the transport of highly electromobile molecules.Proc. Natl. Acad. Sci. USA. 2015; 112: E6274-E6283Crossref PubMed Google Scholar) and pressure (Lee et al., 2016Lee E. Choi J. Jo Y. Kim J.Y. Jang Y.J. Lee H.M. Kim S.Y. Lee H. Cho K. Jung N. et al.ACT-PRESTO: rapid and consistent tissue clearing and labeling method for 3-dimensional (3D) imaging.Sci. Rep. 2016; 6: 18631Crossref PubMed Scopus (39) Google Scholar), may also increase the rate of probe diffusion and decrease the probability of being trapped by the sinks. Although we describe above some of the ways to work around the issue of limited probe penetration, we would like to point out that, unfortunately, a general solution to this problem remains elusive. When it comes to fluorescent labeling of neuronal tissues, an ideal probe should be specific for a desired structure or molecule, sensitive with a good signal-to-noise ratio, and robust in order to function in the highly variable sources of brain tissues. In addition, ideal chemical probes for brain visualization must be compatible with tissue clearing methods, resistant to bleaching, active in red or near-infrared (NIR) fluorescent spectra, adaptable to multiplexed tissue interrogation, and simple to use. The basic components of a small-molecule probe are a target-binding moiety and a fluorescence moiety, where the two can be contained within the same or separate molecular structures (Figure 2). The fluorescence moiety can also be replaced by different functional groups that can be indirectly detected by in situ bioconjugation methods or specific proteins to amplify the signal. The probe's target specificity is one of its most critical properties. In general, this is governed by the structure of the molecule and its properties under the conditions of the tissue clearing method applied. In designing a chemical probe for tissue clearing, targeting the primary structures or the protein states under a specific tissue clearing condition might be more realistic than using the native protein structure as the starting point for probe design. Given the fragility of the structure of proteins under many of the tissue clearing methods in use, designing chemical probes for other biomolecules such as carbohydrates and nucleic acids should be considered. To overcome autofluorescence of the thick human brain tissues, it is important for probes to produce a high signal-to-noise contrast. In this respect, the higher the quantum yield, the better the probe. Low signals can also be due to lack of target engagement, due to either low specificity or other off-target effects. One category of chemical probes developed to address this problem are the so-called smart probes which change their fluorescent spectra when bound to target. For example, thioflavin T and other amyloid probes fluoresce only when bound to amyloids, as we discuss in more detail below. Tissue clearing processes frequently disrupt the probe-target interactions, as the use of denaturants tends to decrease the binding affinity of the probes to their targets when compared with most in vitro situations. Thus, maximizing binding affinities between probes and targets needs to be one of the core goals of probe development. In cases where the affinity cannot be further enhanced, as is the case for lipophilic tracers, the addition of fixable aliphatic amine or hydrazine groups can allow post-labeling fixation before proceeding to harsh tissue clearing treatment, maximizing compatibility with various tissue clearing techniques (Johnson and Spence, 2010Johnson I. Spence M. The Molecular Probes Handbook. A Guide to Fluorescent Probes and Labeling Technologies.Eleventh Edition. Molecular Probes, 2010Google Scholar). The fluorescent properties of the probes need to be carefully optimized as well. As mentioned, the ideal fluorescent range is in the red or near-infrared (NIR) region. Interested readers may refer to several excellent reviews on the structural-fluorescence relationships and comprehensive summaries of recently developed NIR fluorescent (NIRF) probes (Yuan et al., 2013Yuan L. Lin W. Zheng K. He L. Huang W. Far-red to near infrared analyte-responsive fluorescent probes based on organic fluorophore platforms for fluorescence imaging.Chem. Soc. Rev. 2013; 42: 622-661Crossref PubMed Google Scholar, Umezawa et al., 2014Umezawa K. Citterio D. Suzuki K. New trends in near-infrared fluorophores for bioimaging.Anal. Sci. 2014; 30: 327-349Crossref PubMed Scopus (51) Google Scholar). In addition, brain imaging probes need to be applicable for multicolor imaging, and the best probes for those applications have narrow excitation/emission peaks to minimize crosstalk. The ability to multiplex probes depends on the available fluorescent spectral options in each probe series, and can be expanded when a probe contains a tunable fluorogenic scaffold, as illustrated by the carbocyanine dyes (Meyers et al., 1994Meyers F. Marder S.R. Pierce B.M. Bredas J.L. 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