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Chemiluminescence imaging offers a low background and high sensitivity approach to imaging analytes in living cells and animals. Intensity-based measurements have been developed, but require careful consideration of kinetics, probe localization, and fluctuations in quantum yield, all of which complicate quantification. Here, we report a ratiometric strategy for quantitative chemiluminescence imaging of pH. The strategy relies on an energy transfer cascade of chemiluminescence emission from a spiroadamantane 1,2-dioxetane to a ratiometric pH indicator via fluorescent dyes in Enhancer solutions. Monitoring the pH-dependent changes in chemiluminescence emission at multiple wavelengths enables ratiometric imaging and quantification of pH independent from variations due to kinetics and probe concentration.
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Developing 1,4-Diethyl-1,2,3,4-tetrahydroquinoxalin-substituted Fluorogens Based on GFP Chromophore for Endoplasmic Reticulum and Lysosome Staining
In the present study, we demonstrated that the introduction of a 1,4-diethyl-1,2,3,4-tetrahydroquinoxalin moiety into the arylidene part of GFP chromophore-derived compounds results in the formation of environment-sensitive fluorogens. The rationally designed and synthesized compounds exhibit remarkable solvent- and pH-dependence in fluorescence intensity. The solvent-dependent variation in fluorescence quantum yield makes it possible to use some of the proposed compounds as polarity sensors suitable for selective endoplasmic reticulum fluorescent labeling in living cells. Moreover, the pH-dependent emission intensity variation of other fluorogens makes them selective fluorescent labels for the lysosomes in living cells.
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- Award ID(s):
- 2003550
- PAR ID:
- 10545133
- Publisher / Repository:
- MDPI
- Date Published:
- Journal Name:
- International Journal of Molecular Sciences
- Volume:
- 25
- Issue:
- 19
- ISSN:
- 1422-0067
- Page Range / eLocation ID:
- 10448
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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- Free Publicly Accessible Full Text
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Accepted Manuscript1.0
- Journal Article:
- https://doi.org/10.3390/ijms251910448
