Expansion microscopy Ha V. Vo, Rong Xu, Edward S. Boyden, Yongxin Zhao Nature Reviews Methods Primers, 2026
An Omni-Mesoscope for multiscale high-throughput quantitative phase imaging of cellular dynamics and high-content molecular characterization Hongqiang Ma, Maomao Chen, Jianquan Xu, Yaxin Yang, Yongxin Zhao, Yang Liu Science Advances, 2024 The mesoscope has emerged as a powerful imaging tool in biomedical research, yet its high cost and low resolution have limited its broader application. Here, we introduce the Omni-Mesoscope, a high–spatial-temporal and multimodal mesoscopic imaging platform built from cost-efficient off-the-shelf components. This system uniquely merges the capabilities of label-free quantitative phase microscopy to capture live-cell morphodynamics across thousands of cells with highly multiplexed fluorescence imaging for comprehensive molecular characterization. This Omni-Mesoscope offers a mesoscale field of view of ~5 square millimeters with a high spatial resolution down to 700 nanometers, enabling the capture of detailed subcellular features. We demonstrate its capability in delineating molecular characteristics underlying rare morphodynamic cellular phenomena, including cancer cell responses to chemotherapy and the emergence of polyploidy in drug-resistant cells. We also integrate expansion technique to enhance three-dimensional volumetric super-resolution imaging of thicker tissues, opening the avenues for biological exploration at unprecedented scales and resolutions.
Improved immunostaining of nanostructures and cells in human brain specimens through expansion-mediated protein decrowding Pablo A. Valdes, Chih-Chieh (Jay) Yu, Jenna Aronson, Debarati Ghosh, Yongxin Zhao, Bobae An, Joshua D. Bernstock, Deepak Bhere, Michelle M. Felicella, Mariano S. Viapiano, Khalid Shah, E. Antonio Chiocca, Edward S. Boyden Science Translational Medicine, 2024 Proteins are densely packed in cells and tissues, where they form complex nanostructures. Expansion microscopy (ExM) variants have been used to separate proteins from each other in preserved biospecimens, improving antibody access to epitopes. Here, we present an ExM variant, decrowding expansion pathology (dExPath), that can expand proteins away from each other in human brain pathology specimens, including formalin-fixed paraffin-embedded (FFPE) clinical specimens. Immunostaining of dExPath-expanded specimens reveals, with nanoscale precision, previously unobserved cellular structures, as well as more continuous patterns of staining. This enhanced molecular staining results in observation of previously invisible disease marker–positive cell populations in human glioma specimens, with potential implications for tumor aggressiveness. dExPath results in improved fluorescence signals even as it eliminates lipofuscin-associated autofluorescence. Thus, this form of expansion-mediated protein decrowding may, through improved epitope access for antibodies, render immunohistochemistry more powerful in clinical science and, perhaps, diagnosis.
MicroMagnify: A Multiplexed Expansion Microscopy Method for Pathogens and Infected Tissues Zhangyu Cheng, Caroline Stefani, Thomas Skillman, Aleksandra Klimas, Aramchan Lee, Emma F. DiBernardo, Karina Mueller Brown, Tatyana Milman, Yuhong Wang, Brendan R. Gallagher, Katherine Lagree, Bhanu P. Jena, Jose S. Pulido, Scott G. Filler, Aaron P. Mitchell, N. Luisa Hiller, Adam Lacy‐Hulbert, Yongxin Zhao Advanced Science, 2023 Super‐resolution optical imaging tools are crucial in microbiology to understand the complex structures and behavior of microorganisms such as bacteria, fungi, and viruses. However, the capabilities of these tools, particularly when it comes to imaging pathogens and infected tissues, remain limited. MicroMagnify (µMagnify) is developed, a nanoscale multiplexed imaging method for pathogens and infected tissues that are derived from an expansion microscopy technique with a universal biomolecular anchor. The combination of heat denaturation and enzyme cocktails essential is found for robust cell wall digestion and expansion of microbial cells and infected tissues without distortion. µMagnify efficiently retains biomolecules suitable for high‐plex fluorescence imaging with nanoscale precision. It demonstrates up to eightfold expansion with µMagnify on a broad range of pathogen‐containing specimens, including bacterial and fungal biofilms, infected culture cells, fungus‐infected mouse tone, and formalin‐fixed paraffin‐embedded human cornea infected by various pathogens. Additionally, an associated virtual reality tool is developed to facilitate the visualization and navigation of complex 3D images generated by this method in an immersive environment allowing collaborative exploration among researchers worldwide. µMagnify is a valuable imaging platform for studying how microbes interact with their host systems and enables the development of new diagnosis strategies against infectious diseases.
Universal Molecular Retention with 11-Fold Expansion Microscopy Brendan R. Gallagher, Aleksandra Klimas, Zhangyu Cheng, Yongxin Zhao Journal of Visualized Experiments, 2023 The nanoscale imaging of biological specimens can improve the understanding of disease pathogenesis. In recent years, expansion microscopy (ExM) has been demonstrated to be an effective and low-cost alternative to optical super-resolution microscopy. However, it has been limited by the need for specific and often custom anchoring agents to retain different biomolecule classes within the gel and by difficulties with expanding standard clinical sample formats, such as formalin-fixed paraffin-embedded tissue, especially if larger expansion factors or preserved protein epitopes are desired. Here, we describe Magnify, a new ExM method for robust expansion up to 11-fold in a wide array of tissue types. By using methacrolein as the chemical anchor between the tissue and gel, Magnify retains multiple biomolecules, such as proteins, lipids, and nucleic acids, within the gel, thus allowing the broad nanoscale imaging of tissues on conventional optical microscopes. This protocol describes best practices to ensure robust and crack-free tissue expansion, as well as tips for handling and imaging highly expanded gels.
Magnify is a universal molecular anchoring strategy for expansion microscopy Aleksandra Klimas, Brendan R. Gallagher, Piyumi Wijesekara, Sinda Fekir, Emma F. DiBernardo, Zhangyu Cheng, Donna B. Stolz, Franca Cambi, Simon C. Watkins, Steven L. Brody, Amjad Horani, Alison L. Barth, Christopher I. Moore, Xi Ren, Yongxin Zhao Nature Biotechnology, 2023 Expansion microscopy enables nanoimaging with conventional microscopes by physically and isotropically magnifying preserved biological specimens embedded in a crosslinked water-swellable hydrogel. Current expansion microscopy protocols require prior treatment with reactive anchoring chemicals to link specific labels and biomolecule classes to the gel. We describe a strategy called Magnify, which uses a mechanically sturdy gel that retains nucleic acids, proteins and lipids without the need for a separate anchoring step. Magnify expands biological specimens up to 11 times and facilitates imaging of cells and tissues with effectively around 25-nm resolution using a diffraction-limited objective lens of about 280 nm on conventional optical microscopes or with around 15 nm effective resolution if combined with super-resolution optical fluctuation imaging. We demonstrate Magnify on a broad range of biological specimens, providing insight into nanoscopic subcellular structures, including synaptic proteins from mouse brain, podocyte foot processes in formalin-fixed paraffin-embedded human kidney and defects in cilia and basal bodies in drug-treated human lung organoids.
MAGNIFY: molecule anchorable gel-enabled nanoscale in-situ fluorescence microscopy for nanoscale imaging of biomolecules Aleksandra Klimas, Brendan R. Gallagher, Emma DiBernardo, Zhangyu Cheng, Yongxin Zhao Progress in Biomedical Optics and Imaging Proceedings of SPIE, 2023 Expansion microscopy (ExM) is a powerful imaging strategy that offers a low-cost solution for interrogating biological systems at the nanoscale using conventional optical microscopes. It achieves this by physically and isotropically magnifying preserved biological specimens embedded in a cross-linked water-swellable hydrogel. However, most reported techniques are unable to preserve endogenous epitopes due to strong protease digestion used to expand samples. In addition, these protocols rely on mechanically fragile hydrogels that only expand by at most 4.5× linearly. We present a new ExM framework, Molecule Anchorable Gel-enabled Nanoscale In-situ Fluorescence MicroscopY (MAGNIFY), that exhibits a broad retention of nucleic acids, proteins, and lipids without the need for a separate anchoring step. By using a mechanically sturdy hydrogel, MAGNIFY is capable of expanding biological specimens up to 11×. This facilitates nanoscale imaging (~25-nm effective resolution) using an ∼280-nm diffraction-limited objective lens on a conventional optical microscope and can be furthered to ~15 nm effective resolution if combined with computational methods such as Super-resolution Optical Fluctuation Imaging (SOFI). Here, we demonstrate that MAGNIFY provides a generalized solution for imaging nanoscale subcellular features of a broad range of biological specimens. We also show that MAGNIFY provides a novel, accessible tool for improving the precision, utility, and generality of nanoscopy.
Three-dimensional nanofabrication via ultrafast laser patterning and kinetically regulated material assembly Fei Han, Songyun Gu, Aleks Klimas, Ni Zhao, Yongxin Zhao, Shih-Chi Chen Science, 2022 A major challenge in nanotechnology is the fabrication of complex three-dimensional (3D) structures with desired materials. We present a strategy for fabricating arbitrary 3D nanostructures with a library of materials including metals, metal alloys, 2D materials, oxides, diamond, upconversion materials, semiconductors, polymers, biomaterials, molecular crystals, and inks. Specifically, hydrogels patterned by femtosecond light sheets are used as templates that allow for direct assembly of materials to form designed nanostructures. By fine-tuning the exposure strategy and features of the patterned gel, 2D and 3D structures of 20- to 200-nm resolution are realized. We fabricated nanodevices, including encrypted optical storage and microelectrodes, to demonstrate their designed functionality and precision. These results show that our method provides a systematic solution for nanofabrication across different classes of materials and opens up further possibilities for the design of sophisticated nanodevices.
Super-Resolution Vibrational Imaging Using Expansion Stimulated Raman Scattering Microscopy Lixue Shi, Aleksandra Klimas, Brendan Gallagher, Zhangyu Cheng, Feifei Fu, Piyumi Wijesekara, Yupeng Miao, Xi Ren, Yongxin Zhao, Wei Min Advanced Science, 2022 Stimulated Raman scattering (SRS) microscopy is an emerging technology that provides high chemical specificity for endogenous biomolecules and can circumvent common constraints of fluorescence microscopy including limited capabilities to probe small biomolecules and difficulty resolving many colors simultaneously. However, the resolution of SRS microscopy remains governed by the diffraction limit. To overcome this, a new technique called molecule anchorable gel-enabled nanoscale Imaging of Fluorescence and stimulated Raman scattering microscopy (MAGNIFIERS) that integrates SRS microscopy with expansion microscopy (ExM) is described. MAGNIFIERS offers chemical-specific nanoscale imaging with sub-50 nm resolution and has scalable multiplexity when combined with multiplex Raman probes and fluorescent labels. MAGNIFIERS is used to visualize nanoscale features in a label-free manner with CH vibration of proteins, lipids, and DNA in a broad range of biological specimens, from mouse brain, liver, and kidney to human lung organoid. In addition, MAGNIFIERS is applied to track nanoscale features of protein synthesis in protein aggregates using metabolic labeling of small metabolites. Finally, MAGNIFIERS is used to demonstrate 8-color nanoscale imaging in an expanded mouse brain section. Overall, MAGNIFIERS is a valuable platform for super-resolution label-free chemical imaging, high-resolution metabolic imaging, and highly multiplexed nanoscale imaging, thus bringing SRS to nanoscopy.
Neurophotonic tools for microscopic measurements and manipulation: status report Ahmed Abdelfattah, Srinivasa Rao Allu, Robert E. Campbell, Xiaojun Cheng, Tomáš Cižmár, Irene Costantini, Valentina Emiliani, Natalie Fomin-Thunemann, Ariel Gilad, Tomás Fernández Alfonso, Christopher G. L. Ferri, Andrew Harris, Elizabeth M. C. Hillman, Matthew G. Holt, Kivilcim Kiliç, Evan W. Miller, Rickson C. Mesquita, K.M. Naga Srinivas Nadella, U. Valentin Nägerl, Citlali Perez Campos, Francesca Puppo, Shy Shoham, R. Angus Silver, Vivek J. Srinivasan, Martin Thunemann, Lei Tian, Sergei A. Vinogradov, Flavia Vitale, Hana Uhlirova, Chris Xu, Mu-Han Yang, Yongxin Zhao, Sapna Ahuja, Taner Akkin, Joshua Brake, David A. Boas, Erin M. Buckley, Anderson I. Chen, Massimo De Vittorio, Anna Devor, Patrick Doran, Mirna El Khatib, Yeshaiahu Fainman, Xue Han, Ute Hochgeschwender, Na Ji, Evelyn Lake, Lei Li, Tianqi Li, Philipp Machler, Yusuke Nasu, Axel Nimmerjahn, Petra Ondrácková, Francesco S. Pavone, Darcy Peterka, Filippo Pisano, Ferruccio Pisanello, Bernardo L. Sabatini, Sanaz Sadegh, Sava Sakadžic, Sanaya N. Shroff, Ruth R. Sims, Spencer LaVere Smith, Lin Tian, Thomas Troxler, Antoine Valera, Alipasha Vaziri, Lihong V. Wang, Changhuei Yang, Gary Yellen, Ofer Yizhar Neurophotonics, 2022
Voltage imaging and optogenetics reveal behaviour-dependent changes in hippocampal dynamics Yoav Adam, Jeong J. Kim, Shan Lou, Yongxin Zhao, Michael E. Xie, Daan Brinks, Hao Wu, Mohammed A. Mostajo-Radji, Simon Kheifets, Vicente Parot, Selmaan Chettih, Katherine J. Williams, Benjamin Gmeiner, Samouil L. Farhi, Linda Madisen, E. Kelly Buchanan, Ian Kinsella, Ding Zhou, Liam Paninski, Christopher D. Harvey, Hongkui Zeng, Paola Arlotta, Robert E. Campbell, Adam E. Cohen Nature, 2019
Cortical column and whole-brain imaging with molecular contrast and nanoscale resolution Ruixuan Gao, Shoh M. Asano, Srigokul Upadhyayula, Igor Pisarev, Daniel E. Milkie, Tsung-Li Liu, Ved Singh, Austin Graves, Grace H. Huynh, Yongxin Zhao, John Bogovic, Jennifer Colonell, Carolyn M. Ott, Christopher Zugates, Susan Tappan, Alfredo Rodriguez, Kishore R. Mosaliganti, Shu-Hsien Sheu, H. Amalia Pasolli, Song Pang, C. Shan Xu, Sean G. Megason, Harald Hess, Jennifer Lippincott-Schwartz, Adam Hantman, Gerald M. Rubin, Tom Kirchhausen, Stephan Saalfeld, Yoshinori Aso, Edward S. Boyden, Eric Betzig Science, 2019
All-optical electrophysiology in mammalian neurons using engineered microbial rhodopsins Daniel R Hochbaum, Yongxin Zhao, Samouil L Farhi, Nathan Klapoetke, Christopher A Werley, Vikrant Kapoor, Peng Zou, Joel M Kralj, Dougal Maclaurin, Niklas Smedemark-Margulies, Jessica L Saulnier, Gabriella L Boulting, Christoph Straub, Yong Ku Cho, Michael Melkonian, Gane Ka-Shu Wong, D Jed Harrison, Venkatesh N Murthy, Bernardo L Sabatini, Edward S Boyden, Robert E Campbell, Adam E Cohen Nature Methods, 2014
Microfluidic cell sorter aided directed evolution of an improved fluorescent protein-based calcium ion indicator 17th International Conference on Miniaturized Systems for Chemistry and Life Sciences Microtas 2013, 2013
Optogenetic reporters Spencer C. Alford, Jiahui Wu, Yongxin Zhao, Robert E. Campbell, Thomas Knöpfel Biology of the Cell, 2013
Microfluidic cell sorter aided live cell screening for improved fluorescent protein Proceedings of the 16th International Conference on Miniaturized Systems for Chemistry and Life Sciences Microtas 2012, 2012
An expanded palette of genetically encoded Ca2+ indicators Yongxin Zhao, Satoko Araki, Jiahui Wu, Takayuki Teramoto, Yu-Fen Chang, Masahiro Nakano, Ahmed S. Abdelfattah, Manabi Fujiwara, Takeshi Ishihara, Takeharu Nagai, Robert E. Campbell Science, 2011
