OUTLINE OF THE LAB


    1. Align the excitation laser so that it passes through and is focused by a microscope objective.

    2. Align the fluorescence emission into the end of an optical fiber, which guides the light to an avalanche photodiode detector.

    3. Collect data of various dilutions of a fluorophore sample. Using statistical methods, demonstrate when fluorescence data comes predominantly from single fluorophores.

Equipment

Pictures





OTHER LAB MATERIALS

Sapphire 488-20 Laser Manual

Modeled Experiment

The FCS experiment was simulated in Matlab, and the resulting modeled data was saved in the same format as the data files collected by the LabView data acquisition software. Included here are (a) the five modeled datasets, (b) the Matlab program used to create the modeled data, and (c) a document describing both the creation of the simulated data and analysis of this data with the same Matlab software used to analyze real data. The document is also included as Appendix 2 in the protocols for this lab. (zipped folder)

Lab Software

These are the computer programs used to collect and analyze data. Included are three LabView programs and one Matlab m-file. The software is described in detail in the protocols. (zipped folder)



PAPERS RELATED TO THE LAB

Webb, Robert H., Rep. Prog. Phys. 59, 1996, 427-471.Confocal optical microscopy.
  • A thorough review of confocal microscopy.

    • Rieger, Robert, Carlheinz Rockwer, and G. Ulrich Nienhas, Am. J. Phys. 73(12), 2005, 1129-1134. Fluctuation correlation spectroscopy for the advanced physics laboratory.
  • The design of an FCS lab for a physics laboratory class. The goals of this lab were similar to the goals of the Chem 184 lab.

    • Moerner, W.E. and David P. Fromm, Rev. Sci. Instrum. 74(8), 2003, 3597-3619. Methods of single-molecule fluorescence spectroscopy and microscopy.
  • Review of single-molecule microscopy techniques, focusing on the technical challenges that must be overcome to implement single-molecule spectroscopy.

    • Krichevsky, Oleg and Gregoire Bonnet, Fromm, Rep. Prog. Phys. 65, 2002, 251-297. Fluorescence correlation spectroscopy: the technique and its applications .
  • Review of fluorescence correlation spectroscopy.

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