Fluorescence Correlation Spectroscopy
University of Florida, June 27-29, 2018
Fluorescence correlation spectroscopy (FCS) is an optical technique that uses the statistics of photon detection to measure diffusion, reactions, and interactions of molecules in solution at very low concentrations. Following its invention in the early 1970’s FCS has become a valuable tool of biological physics, allowing direct, non-destructive and non-invasive study of biomolecules such as DNA and protein, including the kinetics and dynamics of their synthesis, diffusion, conformational changes, and interactions. The essence of the FCS method is a correlation analysis of random, temporal fluctuations in the fluorescence signal from small numbers of microscopic particles as they undergo random motion through the focal volume of a microscope. Consequently FCS teaches principles of laser optics, microscopy, Brownian motion, photon counting, and correlation analysis, as well as chemical kinetics.
(One set-up) CLOSED
This Immersion will present a benchtop FCS apparatus constructed from Thorlabs components mounted on a vibration-isolation table. A 532 nm DPSS laser beam is focused through a microscope objective, exciting the fluorescence of a solution of fluorescently-labeled particles at ~pM – nM concentration within a diffraction limited volume. Information about the molecular dynamics is contained in the temporal correlations in the fluctuations of the detected fluorescence. To capture this signal, fluorescence from the excitation volume is collected by the objective and projected at high magnification onto the conjugate image plane where a 75 micron pinhole is positioned. The pinhole passes light only from the selected focal volume to a photomultiplier. Hardware counters time photon arrivals with 10~ns resolution and LabVIEW software performs an autocorrelation analysis on this data to extract (e.g.) the diffusion coefficient of the particles. Optionally, a 50/50 beam splitter can be inserted after the pinhole to randomly direct photons toward one of two photomultipliers and a cross-correlation analysis can be performed. The cross-correlation technique removes photomultiplier dead time and after-pulsing artifacts so that molecular dynamics on sub-microsecond time scales can be observed.
This workshop will cover the construction and alignment of the FCS apparatus, the theoretical basis, software algorithms, and the calibration of the apparatus using diffusion standards such as fluorescent microspheres or dye molecules.
Host and Mentor:
Bob DeSerio received his B.Sc. from the Polytechnic Institute of Brooklyn and his Ph.D. from the University of Chicago in experimental atomic beam physics performed at Argonne National Lab and at the University of Notre Dame. After post-doctoral work at the University of Arizona involving molecular physics, he joined the faculty at the University of Tennessee with research performed at Oak Ridge National Lab. He is now at the University of Florida where he directs the instructional physics laboratories. Recently, he has designed introductory physics labs for online students that can be done at home using a wireless data acquisition devise. He also teaches in the advanced physics lab and has designed and written guides for experiments such as the chaotic pendulum, optical tweezers and fluorescence correlation (with Steve Hagen), muon physics (with Darin Acosta) and studies of a quartz crystal tuning fork in superfluid helium (with Yoonseok Lee).
Robert DeSerio, University of Florida, Department of Physics, PO Box 118440, Gainesville FL 32611-8440. Email: firstname.lastname@example.org. Telephone: (352) 392-1690.
Please note that the Jonathan F. Reichert Foundation has established a grant program to help purchase apparatus used in Laboratory Immersions. Limitations and exclusions apply, but generally speaking the foundation may support up to 40% of the cost of the required equipment.