Single molecule blinking and photobleaching separated by wide-field fluorescence microscopy

Area of Science:

• Physical Chemistry
• Spectroscopy
• Materials Science

Background:

• Single molecule fluorescence microscopy is crucial for understanding material properties at the nanoscale.
• Poly(vinyl alcohol) (PVA) is a versatile polymer with applications in various fields.
• Atto590 is a fluorescent dye commonly used in biological and materials science imaging.

Purpose of the Study:

• To investigate the single molecule fluorescence properties of Atto590 embedded in PVA.
• To analyze the time evolution of fluorescence signals and identify blinking and photobleaching characteristics.
• To develop a method for kinetically separating photobleaching from blinking phenomena.

Main Methods:

• Utilizing wide-field epifluorescence microscopy with CCD-camera detection for single molecule imaging.
• Acquiring image sequences to generate time traces of individual Atto590 molecules.
• Analyzing fluorescence intensity fluctuations over time to characterize molecular behavior.

Main Results:

• Observed distinct differences in fluorescence time evolution between molecules detected initially versus those detected throughout the sequence.
• Atto590 exhibited prolonged blinking times and photobleaching/photoblinking quadratically dependent on irradiation power density.
• Developed a method to kinetically separate photobleaching from blinking events.
• Demonstrated the ability to select and analyze different molecular ensembles, including initially dark or low-emitting molecules.

Conclusions:

• The study provides a detailed characterization of Atto590 photophysics in PVA at the single molecule level.
• The developed approach enables a more comprehensive understanding of ensemble behavior by including often overlooked molecules.
• This work contributes to the advancement of single molecule spectroscopy techniques for materials characterization.