Quantitating Fluorescence Intensity From Fluorophore: Assignment of MESF Values.

Area of Science:

• Analytical Chemistry
• Biophysics

Background:

• Accurate quantitation of fluorophores is crucial in various scientific disciplines.
• Existing methods for comparing fluorescence signals can be limited by instrument variations and sample properties.

Purpose of the Study:

• To develop a standardized procedure for comparing fluorescence yields (FY) irrespective of measurement conditions.
• To establish a robust method for quantitation using molecules of equivalent soluble fluorophore (MESF).

Main Methods:

• Revised the measurement model relating fluorescence signals to fluorescence yields.
• Utilized a calibration curve generated from serial dilutions of fluorescein solutions.
• Measured fluorescence signals from microspheres immobilized with fluorescein isothiocyanate (FITC).

Main Results:

• Demonstrated that equality of fluorescence yields allows for direct comparison of fluorophore concentrations.
• Successfully quantified soluble fluorophore concentration equivalent to microsphere suspensions.
• Established a method to determine the number of soluble fluorescein molecules equivalent to a single microsphere.

Conclusions:

• The presented procedure accurately converts measured fluorescence signals into comparable fluorescence yields.
• The method provides a reliable basis for quantitation in terms of molecules of equivalent soluble fluorophore (MESF).
• This approach enhances the accuracy and comparability of fluorescence-based measurements in solutions and suspensions.