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Y Goltsev (2018)968
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Profiling molecular expression in situ allows the integration of biomolecular and cellular features, enabling an in‑ depth understanding of biological systems. Multiplexed immunofluorescence methods can visualize tens to hun‑ dreds of proteins from individual tissue samples, but their application is usually limited to thin tissue sections. Multi‑ plexed immunofluorescence of thick tissues or intact organs will enable high‑throughput profiling of cellular protein expression within 3D tissue architectures (e.g., blood vessels, neural projections, tumors), opening a new dimension in diverse biological research and medical applications. We will review current multiplexed immunofluorescence meth‑ ods and discuss possible approaches and challenges to achieve 3D multiplexed immunofluorescence. Keywords Immunofluorescence, Multiplexed immunofluorescence, 3D immunostaining Here we will review current multiplexed IF methods Main text and 3D immunostaining methods. Then we will discuss Immunofluorescence (IF) can visualize proteins in tissues possible approaches and challenges to realize 3D multi- using antibodies and fluorophores . The number of plexed IF. proteins IF can visualize from individual tissue is limited to 4–5 due to the spectral overlapping of fluorophores [1, Current multiplexed IF methods 2]. Current methods can be divided into four categories: flu - Multiplexed IF methods can visualize tens to hundreds orescence inactivation, antibody stripping, oligonucleo- of proteins from each tissue [1, 3–8], enabling in-depth tide conjugation, and spectral unmixing methods (Fig. 1). analysis of tissues in diverse fundamental and clinical Among those, fluorophore inactivation and antibody research. However, multiplexed IF methods are currently stripping methods can be collectively termed “Cyclic limited to thin tissue section [1, 2, 7]. 3D multiplexed IF” as those involve multiple cycles of immunostaining, IF, multiplexed IF of millimeter-thick tissues or intact imaging, removal of fluorescence signals, and re-immu - organs, can be achieved by developing a multiplexed nostaining with another set of antibodies . IF method applicable to 3D tissues and by visualizing A fluorophore inactivation method removes fluo - immunolabeled 3D tissues using tissue clearing and 3D rescence signals by quenching fluorophores via pho - microscopy techniques. tobleaching, fluorophore oxidation, or fluorophore unconjugation [1, 3]. This method allows removing fluo - rescence signals with mild processes compared to anti- Wonjin Cho and Sehun Kim contributed equally to this work. body stripping methods [1, 3]. However, this method can *Correspondence: suffer from low signal quality caused by steric hindrance Young‑Gyun Park among antibodies because residual bound antibodies firstname.lastname@example.org from the previous rounds can interfere with antibody Department of Bio and Brain Engineering, Korea Advanced Institute of Science and Technology, Daejeon, Republic of Korea binding of the next rounds . © The Author(s) 2023. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/. The Creative Commons Public Domain Dedication waiver (http:// creat iveco mmons. org/ publi cdoma in/ zero/1. 0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. to 3D tissues Cho et al. Molecular Brain (2023) 16:37 Page 2 of 4 Fluorescence inactivation Antibody stripping Oligonucleotide conjugation Spectral unmixing (e.g. SWITCH, Opal-IHC, 4i) (e.g. CODEX, Immuno-SABER, DEI) (e.g. LUMos, PICASSO, Histo-cytometry) (e.g. MxIF, MELC, IBEX, t-CyCIF, SAFE) Immunostaining: multiple rounds Immunostaining: multiple rounds Immunostaining: one round Immunostaining: one round Multiplexed Sample mounting and imaging: multiple rounds Sample mounting and imaging: multiple rounds Sample mounting and imaging: multiple rounds Sample mounting and imaging: one round IF method - A 3D immunostaining technique - A 3D immunostaining technique - A 3D immunostaining technique - A 3D immunostaining technique Things necessary for - A way to efficiently transport fluorescent - A spectral unmixing algorithm applicable - A way to efficiently transport quenching - A way to efficiently transport antibody- 3D multiplexed chemicals into thick tissues stripping chemicals into thick tissues imager DNAs into thick tissues IF - A 3D image co-registration algorithm - A 3D image co-registration algorithm - A 3D image co-registration algorithm - Low signals due to steric hindrance - The need of robust tissue - Low signals due to steric hindrance - Low signals due to steric hindrance Challenges of - The hardship of 3D image co-registration - The hardship of 3D image co-registration - The hardship of 3D image co-registration - The hardship of 3D image spectral 3D multiplexed unmixing IF - Relatively long procedure - Relatively long procedure - Relatively long procedure Profiling cells and subcellular processes of neurons (axon, synapse) Potential Profiling cells and tissue microenvironment of intact tumors for based on their protein co-expression patterns within 3D landscape of applications in-depth cancer diagnosis. thick brain tissues or intact brains. Addressable questions in neuroscience How many cell types exist in the brain based on the How many subtypes of neuronal What are the protein profiles of cells and processes of 3D brain combinatorial expressions of cell type markers? subcellular processes exist in the brain? pathology (e.g., plaques, glioblastoma) and its surrounding? Fig. 1 Potential approaches and challenges to achieve 3D multiplexed IF with its impacts An antibody stripping method removes fluorescence highly depends on the accuracy of the spectral unmixing signals by detaching antibodies from tissues [4, 5]. Unlike algorithm that is sensitive to tissue properties, imaging all the other methods, this method is free from the steric conditions, and microscope setup. This method also suf - hindrance problem. However, the antibody stripping pro- fers from the steric hindrance happening during immu- cess can deform tissue and damage tissue antigenicity nostaining with tens of antibodies . , which can be ameliorated by using tissue protection techniques [5, 10]. Current 3D immunostaining methods An oligonucleotide conjugation method utilizes DNA- 3D multiplexed IF requires uniformly immunostain 3D barcoded antibodies [1, 6]. Tissue is simultaneously tissues. Current 3D immunostaining methods can be immunolabeled with tens of different antibodies conju - categorized based on the strategies facilitating antibody gated with unique DNA oligomers of which subset can transport into tissues: increasing antibody diffusion rate, be visualized by using fluorescent imager DNAs. This decreasing antibody reaction rate, or the combination of method needs just a single round of immunostaining, both. although requires multiple rounds of labeling with differ - Increasing antibody diffusion rate has been achieved ent sets of imager DNAs . Moreover, steric hindrance by increasing tissue pore sizes using chemicals , or can happen among tens of different antibodies compet - by utilizing forces that facilitate diffusion. For example, ing for binding during immunostaining . ACT-PRESTO utilizes centrifugal force , and sto- A spectral unmixing method involves imaging tens of chastic electrotransport utilizes an electric field . antibodies with overlapping fluorescence spectra fol - Another approach for 3D immunostaining involves lowed by unmixing the spectra using a computational decreasing antibody reaction rate, which in turn increases algorithm. This method is simple because it requires the distances that antibodies travel into a tissue before only one round of immunostaining and sample mount- binding to target proteins, facilitating antibody transport ing for imaging [7, 8], which exempts this method from into tissue . For example, CUBIC-HistoVIsion utilizes the need for image co-registration (registering images quadrol and urea to attenuate antibody binding . generated from iterative rounds of sample mounting and There is a method combining both approaches. imaging of the same tissue) that is necessary for all the eFLASH increases antibody diffusion rate by using sto - other methods. However, a spectral unmixing method chastic electrophoresis while reducing antibody reaction Cho et al. Molecular Brain (2023) 16:37 Page 3 of 4 rate using pH and a detergent . Then the gradual be shorter than other methods since it needs only one change of pH and the concentration of the detergent round of immunostaining. allows distributed antibodies to bind to nearby target proteins, achieving uniform immunostaining of 3D tis- Outlook and prospects sues within 1–2 days . Many challenges exist on realizing 3D multiplexed IF in addition to the general hardship of tissue clearing, 3D tis- Potential approaches and challenges to achieve 3D sue imaging, 3D immunostaining, and 3D image analysis. multiplexed IF However, once realized, 3D multiplexed IF will answer 3D multiplexed IF requires 3D immunostaining com- fundamental questions of neuroscience and will enable bined with a strategy for multiplexing as well as novel translational applications (Fig. 1). techniques for visualization of labeled 3D tissues. Mul- tiplexing strategies of the fluorescence inactivation and Abbreviations antibody stripping methods can be scalable to 3D tissues 3D Three‑dimensional by engendering ways to transfer quenching and anti- DNA Deoxyribonucleic acid body-stripping chemicals into 3D tissues, respectively. Acknowledgements The multiplexing strategy of the oligonucleotide conju - Not applicable. gation method is applicable to 3D tissues by devising a Author contributions way to efficiently transport imager DNAs into thick tis - WC, SK, and YP wrote the manuscript. All authors read and approved the final sues. A spectral unmixing algorithm that is applicable manuscript. to 3D tissue images can enable spectral unmixing-based Funding 3D multiplexed IF. However, several challenges exist on This research was supported by the National Research Foundation achieving 3D multiplexed IF. of Korea (NRF) grant funded by the Korean government (MSIT ) (No. Steric hindrance problem the steric hindrance problem 2021R1C1C1011567), the Bio & Medical Technology Development Program of the NRF funded by the Korean government (MSIT ) (NRF‑2021M3A9E4080780), can lower signal quality , although high signal quality and the KAIST UP Program. is crucial for 3D IF because high levels of light scattering and absorption of thick tissue imaging will reduce detect- Availability of data and materials Not applicable. No data was generated during the current study. ability of fluorescence signals. This problem can be over - come by adapting signal amplification methods, utilizing Declarations tissue clearing method that preserves proteins inside tis- sues, or using microscopy that minimizes photobleach- Ethics approval and consent to participate ing during imaging such as two-photon or light-sheet Not applicable. microscopy. The antibody stripping-based 3D multi - Consent for publication plexed IF will be free from this problem . All authors have agreed to publish this manuscript. The hardship of 3D image co-registration Except for the Competing interests spectral unmixing method, the other three multiplexing The authors declare no competing interest. methods need an image co-registration algorithm to inte- grate results from multiple images acquired from a single Received: 22 February 2023 Accepted: 14 April 2023 tissue, indicating the need of 3D image co-registration for 3D multiplexed IF. 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Molecular Brain – Springer Journals
Published: May 2, 2023
Keywords: Immunofluorescence; Multiplexed immunofluorescence; 3D immunostaining
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