The Confocal Microscopy Facility of the Department of Biochemistry and Molecular Biophysics is open to Washington University researchers. The facility has a Zeiss LSM 510 laser-scanning microscope equipped with an argon ion laser and two Helium/Neon lasers which allow excitation over the blue-red visible spectrum. Two fluorescence detectors and a transmitted light detector are supplemented by a META detector head which provides 32 additional wavelength-selective fluorescence channels. The linear unmixing capability of the Zeiss software can discriminate emission spectra separated by only a few nanometers. Fluorescence recovery after photobleaching (FRAP) can be easily implemented to measure the kinetics of molecular motion in cells or cell membranes.
Additionally, the microscope has a ConfoCor 2 head for fluorescence correlation spectroscopy (FCS). FCS measures the dynamics of labeled molecules at sub-nanomolar concentrations inside living cells, on the cell membrane or in free solution. Diffusion coefficients, rates of directed transport, rates of chemical reactions or conformational changes and the number of fluorescent molecules in the observation volume (~femtoliter) can be determined. With appropriate labeling, cross-correlation can measure the rates and extents of molecular associations.
$25/hr; nights (6:00PM-8:00AM) and weekends are half price. Training costs are $50/hour and will vary according to the needs of the researcher - the more you already know, the less you pay for training, but there is a half-hour minimum training/set-up/check-out charge. Technical assistance and consultation (beyond quick-and-easy fixes) are also $50/hour.
To sign up for a time slot, go to the sign-up calendar. Contact Tony Pryse for a username/password if you don't already have one. If you cancel at least 24 hours before the start of your time, no charges will be levied. Within 24 hours, the time will be charged at ½ price unless it can be filled by someone else.
Steering Committee: Professors Elliot Elson and Carl Frieden
Management: Tony Pryse (362-3345, email)
LSM 510 Manual (Manuals in pdf, will open in new
Chapters 1-4 of the LSM manual, The only two chapters of interest to most people are: 3. Introduction to Laser Scanning Microscopy. "Here you will find an introduction to Laser Scanning Microscopy, with an explanation of the principles of confocal imaging. The section also outlines the ways to present LSM image series in three dimensions, and introduces you to the performance features of your LSM 510." and 4. Quickstart
Chapter 5 of the LSM manual, Long chapter, but it's all in here. 5 Operation in Expert Mode "In the Operation section you will find the most important steps and procedures of the LSM menu structure. The step-by-step description how to get an image will be shown by typical application examples."
Chapters 6-10 of the LSM manual Most relevant chapters: 7. Routine Mode and Tools "This section contains a description of the Routine Mode for scanning images using the LSM scanning module and the use of the tools for setting the microscope." 8. 3D for LSM 510 "This section contains a description of the LSM 3D software package (basic program and addons). At the same time, all functions and settings are presented in a systematic form and in the order in which they can be reached from the basic menu via sub-menus and dialog boxes."
In the begining ...
More recent work:
Not enough?? Here are the titles
and abstracts of all the 2007 and 2006 FCS papers (from SciFinder Scholar.)
Want some older ones? No problem: 2004
A good FCS primer by Petra Schwille and Elke Haustein: FCS: An Introduction to its Concepts and Applications
Confocal microscopy sites