Expanding the multiplexing capability of HIDE probes via fluorescence lifetime imaging microscopy.

Justin H Kwon, Alanna Schepartz

Abstract read

In one paragraph

Article in Methods in enzymology, 2026. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Not yet cited in PubMed.

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0citing papers in PubMed

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1 · What the graph read from it

What it found

Each row is one number read from the abstract, on the scale the paper reported it, with its interval. Left of the dashed line favours the treatment, right favours the comparator. Under each row is the sentence it came from. New to these charts? A ten-minute tutorial.

The abstract states no effect estimate the extractor could read, or names no intervention and outcome on the map, so this paper lights no cell and moves no belief. It is still indexed, cited and linked below.

2 · The registry

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3 · Its place in the literature

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0 citing papers in PubMed.

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4 · The record

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5 · Who and what money

Authors and funding

2 authors.

Justin H KwonDepartment of Chemistry, University of California, Berkeley, CA, United States.

Alanna SchepartzDepartment of Chemistry, University of California, Berkeley, CA, United States; Department of Molecular and Cellular Biology, University of California, Berkeley, CA, United States; California Institute for Quantitative Biosciences, University of California, Berkeley, CA, United States; Chan Zuckerberg Biohub, San Francisco, CA, United States; ARC Institute, Palo Alto, CA, United States. Electronic address: schepartz@berkeley.edu.

Funding

Fluorescence tools that illuminate biology and inspire translationR35GM134963 · NIGMS · UNIVERSITY OF CALIFORNIA BERKELEY · PI Alanna Schepartz · 2020 to 2026

$4.7M

NIGMS NIH HHS R35 GM134963

6 · The paper itself

Abstract

Fluorescence lifetime imaging microscopy (FLIM) measures the average time that a fluorophore spends in the excited state. FLIM is exceptionally useful as an imaging tool because the lifetime of a fluorophore is independent of its excitation and emission profiles. As such, even spectrally identical fluorophores can often be distinguished on the basis of their fluorescence lifetimes. Furthermore, given the dependence of fluorescence lifetime on the relative magnitude of radiative and nonradiative decay rates, it is possible to both predict and modulate the lifetime of a single fluorophore for applications in FLIM multiplexing. Herein, we provide an overview of the protocols necessary for transfection-free FLIM multiplexing in live cells. Protocols are provided for the in vitro reactions required to initially screen bioorthogonal handles and dyes, incubation of the small molecule probes required for FLIM multiplexing with live mammalian cells, and the setup of FLIM experiments. We also provide a discussion of methods available to analyze FLIM data to extract lifetime data.

Indexed as

Fluorescent DyesAnimalsHumansMicroscopy, FluorescenceFluorescent DyesClick chemistryFluorogenicityLive cell imagingOrganelle-organelle interactionsSmall molecule probes

Identifiers

PMID41720524

DOI10.1016/bs.mie.2025.11.011

PMCPMC13182769

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