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Fluorescence Lifetime Imaging of Molecular Rotors in Living Cells
Published on: February 9, 2012
Probing Red Blood Cell Membrane Microviscosity Using Fluorescence Anisotropy Decay Curves of the Lipophilic Dye PKH26
Alexey N Semenov1, Daniil A Gvozdev1, Anastasia M Moysenovich1
1Faculty of Biology, M.V. Lomonosov Moscow State University, 1-12 Leninskie Gory Str., 119991 Moscow, Russia.
Abstract:
Red blood cell (RBC) aggregation and deformation are governed by the molecular processes occurring on the membrane. Since several social important diseases are accompanied by alterations in RBC aggregation and deformability, it is important to develop a diagnostic parameter of RBC membrane structural integrity and stability. In this work, we propose membrane microviscosity assessed by time-resolved fluorescence anisotropy of the lipophilic PKH26 fluorescent probe as a diagnostic parameter. We measured the fluorescence decay curves of the PKH26 probe in the RBC membrane to establish the optimal parameters of the developed fluorescence assay. We observed a complex biphasic profile of the fluorescence anisotropy decay characterized by two correlation times corresponding to the rotational diffusion of free PKH26, and membrane-bounded molecules of the probe. The developed assay allowed us to estimate membrane microviscosity ηm in the range of 100-500 cP depending on the temperature, which paves the way for assessing RBC membrane properties in clinical applications as predictors of blood microrheological abnormalities.
Insights
Researchers developed a new diagnostic parameter using membrane microviscosity to assess red blood cell (RBC) membrane integrity. This method uses a fluorescent probe to measure RBC membrane stability, aiding in predicting blood microrheological abnormalities.
Area of Science:
- Biophysics
- Biochemistry
- Hematology
Background:
- Red blood cell (RBC) aggregation and deformation are crucial for blood flow.
- Alterations in RBC properties are linked to various diseases.
- Assessing RBC membrane integrity is vital for clinical diagnostics.
Purpose of the Study:
- To develop a diagnostic parameter for RBC membrane structural integrity and stability.
- To utilize membrane microviscosity as a novel diagnostic marker.
- To establish a fluorescence-based assay for clinical applications.
Main Methods:
- Time-resolved fluorescence anisotropy of the lipophilic PKH26 fluorescent probe was employed.
- Fluorescence decay curves of PKH26 in the RBC membrane were measured.
- Membrane microviscosity (ηm) was estimated based on probe rotational diffusion.
Main Results:
- A complex biphasic fluorescence anisotropy decay profile was observed.
- Two correlation times indicated distinct probe rotational behaviors.
- The assay successfully estimated membrane microviscosity (ηm) between 100-500 cP.
Conclusions:
- Membrane microviscosity assessed by PKH26 fluorescence anisotropy is a viable diagnostic parameter.
- This method can predict blood microrheological abnormalities.
- The developed assay holds promise for clinical assessment of RBC membrane properties.
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