
==== Front
bioRxiv
BIORXIV
bioRxiv
2692-8205
Cold Spring Harbor Laboratory

10.1101/2024.08.17.605585
preprint
1
Article
Ultraplex microscopy: versatile highly-multiplexed molecular labeling and imaging across scale and resolution
Perez-Garza Janeth http://orcid.org/0000-0002-3023-7741

Orea Jairo
Deane Zachary http://orcid.org/0009-0009-0223-3290

Raimondi Gianna http://orcid.org/0000-0002-2664-1362

Tripp Rebecca
Charles Imani
Ostroff Linnaea http://orcid.org/0000-0002-3348-342X

19 8 2024
2024.08.17.605585https://creativecommons.org/licenses/by-nc-nd/4.0/ This work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License, which allows reusers to copy and distribute the material in any medium or format in unadapted form only, for noncommercial purposes only, and only so long as attribution is given to the creator.
http://biorxiv.org/lookup/doi/10.1101/2024.08.17.605585
nihpp-2024.08.17.605585.pdf
The molecular organization of cells and tissue is challenging to study due to the inefficiency of multiplexed molecular labeling methods and the limited options for combining microscopy modalities in a single specimen, especially when high spatial resolution is needed. Here we describe ultraplex microscopy, which combines serial multiplexing, ultrathin sectioning, and reversible embedding to circumvent incompatibilities between labeling and imaging techniques, enhance resolution, and expand multiplexing capacity within and across modalities. Samples can be labeled with antibodies, RNA probes, and tissue stains for imaging by brightfield, epifluorescence, super-resolution, and electron microscopy without specialized reagents or materials. We demonstrate applications in brain tissue including molecular profiling of single cells and axonal boutons, high-resolution molecular colocalization, and correlative imaging of fluorescent proteins with confocal and ultraplex microscopy. The power and versatility of ultraplex microscopy will be valuable in addressing currently intractable experimental questions in many systems and contexts.
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