FCS Calibration of Burst Analysis Spectroscopy Microscope

Abstract

Fluorescence microscopy techniques are powerful tools for measuring protein kinematics in biological systems and for understanding protein aggregation pathways. In this paper, we discuss the theoretical and mathematical foundations behind several microscopy techniques including fluorescence correlation spectroscopy (FCS), photon counting histograms (PCH) and burst analysis spectroscopy (BAS). Next, we sought to calibrate a custom built confocal fluorescence microscope for future BAS experiments. We estimated the confocal volume using FCS measurements of fluorescent nanobeads and fluorescein dye. While nanobead measurements provided a reasonable preliminary estimate, subsequent experiments with fluorescein produced unexpected results, showing diffusion times similar to those of the nanobeads—an outcome inconsistent with the known molecular properties. We therefore believe there to be a mistake in out experimental setup. Despite extensive troubleshooting, we were unable to determine the source of the error. Further study is needed to determine the error and properly calibrate the microscope before it can be used for future experiments.