Abstract
Scientists studying biological molecules often use single-molecule fluorescence techniques to observe individual molecules in real time. This approach allows researchers to track the movements and interactions of molecules as they behave naturally. To prepare samples for fluorescence measurements, scientists typically immobilize molecules on a cover slip using various techniques. These include purification, labeling the molecules with fluorescent tags, and attaching the molecules to the cover slip. Once the sample is ready, researchers use fluorescence spectrometers to excite the fluorescent tags and detect the emitted light, revealing the individual molecules' locations and behaviors. Software is then used to analyze the fluorescence data and interpret the results. By compiling the positions and behaviors of molecules over time into trajectory data, researchers can gain insights into the structures, functions, and interactions of biological molecules. This project explores the processes involved in studying single molecules using fluorescence spectroscopy. We discuss preparing cover slips and samples for fluorescence measurements in a scanning confocal microscope. We also cover the microscope’s operation, collecting fluorescence data from single molecules over time. The resulting trajectory data is then analyzed using software like MASH-FRET to interpret the behaviors and interactions of the molecules. Additionally, we tweaked existing MATLAB code used in the science department to optimize the data analysis process. By applying these experimental and analysis techniques, we collected and interpreted trajectory data for a variety of samples. Overall, this project offers an in-depth look at the methods used to study single molecules via their fluorescence.
Included in
Single-Molecule Dynamics: Fluorescent Tracking and Analysis Techniques
Scientists studying biological molecules often use single-molecule fluorescence techniques to observe individual molecules in real time. This approach allows researchers to track the movements and interactions of molecules as they behave naturally. To prepare samples for fluorescence measurements, scientists typically immobilize molecules on a cover slip using various techniques. These include purification, labeling the molecules with fluorescent tags, and attaching the molecules to the cover slip. Once the sample is ready, researchers use fluorescence spectrometers to excite the fluorescent tags and detect the emitted light, revealing the individual molecules' locations and behaviors. Software is then used to analyze the fluorescence data and interpret the results. By compiling the positions and behaviors of molecules over time into trajectory data, researchers can gain insights into the structures, functions, and interactions of biological molecules. This project explores the processes involved in studying single molecules using fluorescence spectroscopy. We discuss preparing cover slips and samples for fluorescence measurements in a scanning confocal microscope. We also cover the microscope’s operation, collecting fluorescence data from single molecules over time. The resulting trajectory data is then analyzed using software like MASH-FRET to interpret the behaviors and interactions of the molecules. Additionally, we tweaked existing MATLAB code used in the science department to optimize the data analysis process. By applying these experimental and analysis techniques, we collected and interpreted trajectory data for a variety of samples. Overall, this project offers an in-depth look at the methods used to study single molecules via their fluorescence.