Fluorescence Lifetime Imaging Probes for Cell-Based Measurements of Enzyme Activity

Research output: Chapter in Book/Report/Conference proceedingChapter

1 Scopus citations


Posttranslational modification (PTM) enzymes are important modulators of protein structure and function. They typically act by chemically modifying amino acids, often on side chain functional groups, to change the physiochemical landscape of the protein and thus its biophysical behavior. In particular, protein kinases are enzymes that transfer phosphate from ATP to serine, threonine, or tyrosine in protein substrates. They are key regulators of vital cellular pathways such as survival, proliferation, and apoptosis, and their dysregulation in the context of cancer has been widely investigated for the purpose of development of anticancer drugs. However, several critical questions pertaining to their physiology, such as heterogeneity of kinase signaling within and between cells, and other factors that may play into the mechanisms of drug resistance, remain unanswered. Many of the current strategies to measure kinase activity lack the scope, subcellular resolution, and real-time monitoring ability needed to obtain the type of information needed about their dynamics and localization in cells. While FRET-based biosensors are capable of dynamic single cell imaging, their applications can be limited by difficulties in multiplexing and the inherent inadequacies of steady state measurements. In this chapter, we describe our fluorescence lifetime imaging microscopy (FLIM) probe technology in which peptide kinase substrates, linked to cell-penetrating peptides and labeled with small molecule fluorophores, are used to report kinase activity through time-resolved fluorescence imaging to visualize and quantify changes to the probe’s fluorescence lifetime. These can be multiplexed for more than one kinase at a time, and interpretation is not affected by differences in local intensity due to probe uptake and distribution or photobleaching. With careful choice of peptide substrate(s), fluorophore label, and imaging set-up, high specificity and spatiotemporal resolution can be achieved. Due to the mechanism by which the lifetime change occurs, this approach is compatible with other PTMs (such as acetylation, methylation), and so the considerations for kinase FLIM probe design described in this chapter should be broadly applicable for other PTMs as well.

Original languageEnglish (US)
Title of host publicationMethods in Molecular Biology
PublisherHumana Press Inc.
Number of pages30
StatePublished - 2022

Publication series

NameMethods in molecular biology (Clifton, N.J.)
PublisherHumana Press
ISSN (Print)1064-3745

Bibliographical note

Publisher Copyright:
© 2022, The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.


  • Cell-penetrating peptides
  • Fluorescence lifetime
  • Fluorescent probes
  • Kinase activity
  • Kinase substrates

PubMed: MeSH publication types

  • Journal Article


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