General
Information
GENERAL:
Microspectrophotometric data provide essential
information in the planning and in the interpretation of X-ray crystallographic
experiments. This is particularly true for studies on redox proteins, where the
oxidation state of redox centres may be altered by electrons liberated by X-rays
during data collection (for more details see Berglund, G.I., Carlsson, G.H.,
Smith, A.T., Szoke, H., Henriksen, A. & Hajdu, J. (2002) The catalytic
pathway of horseradish peroxidase at high resolution. Nature 417, 463-468. and
the supplementary material with this paper. Those papers can be found in the
bottom of the page).
(1)
DETECTORS AND MONOCHROMATORS:
We buy the detectors, spectrograph and the
software from a company in Belfast (Andor).
CCD
DETECTOR SYSTEM WITH USB2.0
INTERFACE AND SHAMROCK 163 MONOCHROMATOR
The read out speed of the CCD is very high, the
quantum efficiency is also high, the dark current is low, and illumination with
a low intensity light is sufficient. This is important if you have
photosensitive material in the beam.
(2)
LIGHT SOURCES
We have two different light sources: A very
powerful and very little HALOGEN LAMP is supplied as standard with the
instrument. This halogen lamp can be used for spectral measurements in the
350-800 nm range. We can also supply a Zeiss CLX 500 XENON LAMP in addition to
the halogen lamp with an output between ~180 - ~1000 nm, but this is extra.
(1)
THE HALOGEN LAMP that comes with the basic instrument is
specifically designed to inject a single optical fibre. And it does that very
effectively. The lamp has a tiny bulb with 5 W power only, yet the light output
at the other end of the fibre is many times higher than the output when one
uses, say, a 1000 W projectorlamp to inject the fibre. This is due to the source
size and the divergence just like with synchrotrons and anodes. This lamp is
used for spectral illumination AND also for producing light for the observation
microscope during sample manipulation.
(2)
The Zeiss CLX 500 XENON LAMP works between ~190 - ~1100
nm (for some reason Zeiss claims a different regime on their web site). It is
suitable for UV-VIS-NIR absorption measurements and also for fluorescence
applications. It is one of the best and simplest xenon lamps on the market. The
model is equipped with a slow electronic shutter, has a shutter cable (2 pins),
and SMA single fiber connector. For specs see http://www.zeiss.de/de/home_e.nsf
then click on search and enter clx 500 (note the SPACE between clx and 500).
THE
USEFUL SPECTRAL RANGE:
The mirror lenses focus the spectrum onto a
single spot without significant chromatic aberration. They work well in the
wavelength range of about 150 - 10,000 nm (far UV to far infrared) which is
quite spectacular. But..., we use optical fibers to link the mirror lenses to
the lamp and to the monochromator and detector. These fibers limit the
accessible spectral range. For application in protein crystallography, we use
very high grade quartz single fibres with excellent light transmission in the
230-800 nm regime (you can probably push this a bit further with averaging but
you are counting single photons then). If one would like to do infrared
measurements, one could use special IR fibres, monochromators and detectors. We
don't supply these presently but we have done FTIR measurements on thin crystals
with these mirror lenses.
This depends on the diameter of the illuminating
fibre. With a 50 micron fibre (the standard stuff), the spot size in 4DXSystems'
microspectrophotometer is between 20-25 micron. The size of this spot can be
increased or reduced (to about 10 micron) if necessary. One should always make
sure that the measurement spot is smaller than the sample.
This is done with a holmium filter built in the
halogen lamp that is supplied as standard with the instrument. When we do
experiments, we always calibrate the wavelength after switching on the
instrument. Safety first - there are hundreds of people who are obsessed with
turning micrometer screws and there is one on the monochromator.
REFERENCE
SPECTRA FOR SPECTROPHOTOMETRIC MEASUREMENTS:
We usually use air as a reference. You can also
pick up a reference spectrum from the mother liquor near the crystal. Have a
look at Wilmot, C.M., Sjogren, T., Carlsson, G.H., Berglund, G.I. & Hajdu,
J. (2002). Defining redox state of X-ray crystal structures by single-crystal
ultraviolet-visible microspectrophotometry. Methods in Enzymology, vol 353,
"Redox Cell Biology and Genetics, part B", pp 301-318, Ed. C. K. Sen
& L. Packer, Academic Press (USA).The most reliable measurements are
difference measurements following the trigger of a reaction. It is important to
keep the crystal in the same orientation throughout. In contrast to
SHAPE
OF THE CRYSTALS:
Crystals with parallel faces offer reproducible
measurements when the light beam falls normal to one of the crystal faces.
NON-FLAT
CRYSTALS:
Try a non-flat cuvette in spectrophotometry. The
effect is exactly the same as with a non flat crystal in microspectrophotometry.
If you change the orientation of the non-flat cuvette (you can call this a
prism), the apparent spectrum will change. You can however make accurate
difference measurements relative to a fixed orientation, e.g. as a function of
time, temperature, pH, etc.
MEASUREMENTS
ON CRYSTALS MOUNTED IN CAPILLARIES:
A
capillary filled with liquid is a cylindrical lens. In order to obtain good
spectra from such samples, it is important to center the capillary carefully and
have its optical axis perpendicular and intersecting with the optical axis of
the microspectrophotometer. Relative difference measurements can however be
performed accurately even if the sample and the capillary is in some random but
unchanging orientation.
FLUORESCENCE
CAPABILITY:
Two measurement modes on single crystals are
possible:(i) Orthogonal 3 axis arrangement with the excitation source
perpendicular to the emission probe and both axes perpendicular to the spindle
axis of the goniometer in the microspectrophotometer. The observation microscope
can either face the emission probe or the excitation probe in this system.