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.
The Details
- Microscope:
Zeiss Axiovert 200M, inverted design, infinity-corrected optics.
- Objectives:
- C-Apochromat
40X, 1.2 NA, correction collar, water immersion;
- Plan-Apochromat
20X, .75 NA, dry;
- Plan-Neofluar
10X, .30 NA, dry.
- Fluorescence
excitation:
- Argon
laser lines: 458, 477, 488, 514 nm;
- HeNe
laser: 543 nm;
- HeNe
laser: 633 nm;
- 50-watt
mercury lamp.
- Other
imaging modes: transmission brightfield
(non-confocal) and DIC.
- Temperature
control: A temperature-controlled stage
can keep cells at 37°C.
- Stage
control: The microscope stage is
motor-controlled, and a piezo stage can be used for finer x-y control.
- Dichroics and filters
Costs
$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.
Sign-up policy
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.
Administration
Steering Committee: Professors Elliot Elson and Carl Frieden
Management: Tony Pryse (362-3345, email)
Useful Links
LSM 510 Manual (Manuals in pdf, will open in new
window)
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."
FCS/ConfoCor Manuals
Chapters
1-4, Chapter 5, and Chapters 6-10.
FCS papers
In the begining ...
- Magde, D.,
Elson, E.L. & Webb, W.W. Thermodynamic Fluctuations in a Reacting
System — Measurement by Fluorescence Correlation Spectroscopy(1972) Phys.Rev.Lett.29,705-708.
- Elson, E.
L. & Magde, D. (1974) Biopolymers 13, 1-27, and following paper.
More recent work:
- Chattopadhyay
K., Elson E.L., Frieden C. The kinetics of conformational fluctuations
in an unfolded protein measured by fluorescence methods. Proc Natl Acad Sci
U S A. 2005 Feb 15;102(7):2385-9
- Qian H.,
Elson E.L. Fluorescence correlation spectroscopy with high-order and
dual-color correlation to probe nonequilibrium steady states. Proc Natl Acad Sci
U S A. 2004 Mar 2;101(9):2828-33
- Christian
Eggeling, Peet Kask, Dirk Winkler and Stefan Jäger, Rapid Analysis of
Förster Resonance Energy Transfer by Two-Color Global Fluorescence
Correlation Spectroscopy: Trypsin Proteinase Reaction Biophysical
Journal 89:605-618(2005)
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
and 2005
A good FCS primer by Petra Schwille and Elke Haustein:
FCS: An Introduction
to its Concepts and Applications
Confocal microscopy sites
- Molecular
Expressions at Florida State is one of the most complete and informative
microscopy sites around: Basic Concepts in
Optical Microscopy
- This
Olympus site also has a lot of information, some of which links to the
Florida State site above: Theory of Confocal
Microscopy
- One of the
Java tutorials on the Olympus site above is very useful for checking
bleed-through of two labels: Choosing
Fluorophore Combinations for Confocal Microscopy
- A very
useful collection of ImageJ plug-ins, including the LSM image reader and
ImageJ itself, can be downloaded and installed together from here: Wright Cell Imaging
Facility.
- This Nikon
site has some good stuff: Microscopy
U.
- The ImageJ site itself, with the most
recent version.
- Zeiss
site Lots of technical data on Zeiss products scattered around.