Phluorin Imaging
Phluorin Imaging - Related Videos
09:41
Confocal Imaging of Neuropeptide Y-pHluorin: A Technique to Visualize Insulin Granule Exocytosis in Intact Murine and Human Islets
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Cited by 12 •
2017
We describe a protocol for visualization of insulin exocytosis in intact islets using pHluorin, a pH-sensitive green fluorescent protein. Isolated islets are infected with adenovirus encoding pHluorin coupled to the vesicle cargo neuropeptide Y. This allows for the detection of insulin granule fusion events by confocal microscopy.
12:58
Imaging pHluorin-tagged Receptor Insertion to the Plasma Membrane in Primary Cultured Mouse Neurons
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Cited by 8 •
2012
By tagging the extracellular domains of membrane receptors with superecliptic pHluorin, and by imaging these fusion receptors in cultured mouse neurons, we can directly visualize individual vesicular insertion events of the receptors to the plasma membrane. This technique will be instrumental in elucidating the molecular mechanisms governing receptor insertion to the plasma membrane.
09:59
Utilizing pHluorin-tagged Receptors to Monitor Subcellular Localization and Trafficking
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Cited by 3 •
2017
Labeling the extracellular domain of a membrane protein with a pH sensitive fluorophore, superecliptic pHluorin (SEP), allows subcellular localization, expression, and trafficking to be determined. Imaging SEP-labeled proteins with total internal reflection fluorescence microscopy (TIRFM) enables the quantification of protein levels in the peripheral ER and plasma membrane.
10:02
Applications of pHluorin for Quantitative, Kinetic and High-throughput Analysis of Endocytosis in Budding Yeast
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Cited by 18 •
2016
Accurate quantification of vesicular trafficking events often provides key insights into roles for specific proteins and the effects of mutations. This paper presents methods for using superecliptic pHluorin, a pH-sensitive GFP variant, as a tool for quantification of endocytic events in living cells using quantitative fluorescence microscopy and flow cytometry.
10:30
Use of pHluorin to Assess the Dynamics of Axon Guidance Receptors in Cell Culture and in the Chick Embryo
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Cited by 10 •
2014
We describe here the use of a pH-sensitive green fluorescent protein variant, pHluorin, to study the spatio-temporal dynamics of axon guidance receptors trafficking at the cell surface. The pHluorin-tagged receptor is expressed both in cell culture and in vivo, using electroporation of the chick embryo.
FAQs
The key signal comes from proton-sensitive fluorescence, not from membrane movement itself. When proton concentration changes, pHluorin brightness shifts, allowing optical tracking of local acidification or neutralization. This converts an otherwise invisible chemical change into a time-varying fluorescence signal that can be measured in intracellular compartments, organelles, or neuronal structures.
During exocytosis, a synaptic vesicle becomes continuous with the cell surface, exposing its pH-sensitive probe to the neutral extracellular environment. The resulting change in proton conditions increases fluorescence. This event links a visible optical change to vesicle fusion, enabling investigators to monitor neurotransmitter release-related membrane trafficking in living neurons.
After endocytosis, a retrieved vesicle is separated from the extracellular environment and undergoes reacidification. The associated decrease in pHluorin fluorescence marks the return toward its acidic internal state. Observing the increase during exposure and the subsequent decrease after retrieval helps distinguish successive phases of synaptic vesicle cycling.
Phluorin imaging can show how neuronal activity regulates presynaptic function by tracking changes in vesicle cycling over time. Because the optical signal reports release and retrieval dynamics, measurements can be used to examine activity-dependent differences in neurotransmitter release and membrane recovery rather than viewing presynaptic signaling as a single static event.
The approach requires a genetically encoded pH-sensitive fluorescent protein, such as pHluorin, positioned in the neuronal structure or compartment of interest. Fluorescence is then monitored in living neurons while proton conditions change. Depending on the experimental target, the same principle can be applied to intracellular regions, organelles, or synaptic vesicles.
This method is particularly useful when researchers need optical information about dynamic membrane trafficking in living neurons. It can support studies of synaptic vesicle cycling, neurotransmitter release, vesicle retrieval, and activity-dependent presynaptic regulation. Its value lies in connecting changing fluorescence with distinct trafficking-related pH states during neuronal signaling.