Image Contrast in Embedded Specimens
Using NEXAFS Spectroscopy
N. I. Khaleque and C. J. Buckley
Department of Physics, King’s College, London, WC2R 2LS, UK
Abstract. Absorption spectra across the carbon edge were taken frommethacrylate, spurrs and araldite and compared to that from protein. It
was found that at a wavelength of 44.425Å, therewas a significant difference in the absorption profiles providing contrast between biological
specimens and embedding media. To observethis difference in an imaging
mode, rabbit muscle fibres wereembedded in the three resins, sectioned
and imaged at this wavelength.It was found that methacrylate gave the
best contrast, followed by spurrs and araldite.
1
Introduction
Much of the motivation for the use of X-ray microscopy on biological specimens
is derived from the utilization of native specimen contrast. Recently, chemical
as well as elemental contrast has been obtained by Zhang [1] using the near
edge X-ray absorption fine structure (NEXAFS). Although the organelles studied by Zhang did not require embedding,many other specimens do; especially
when ultra-thin sections areneeded.This is the case in the study of undecalcified
bone and other mineralised tissues [2]. In recent work [3], the mass thicknesses
of mineral and protein content have been mapped using NEXAFS on 150 nm
thick sections of femoral neck from osteoporotic like mice. Because of the dense
nature of undemineralised bone tissue, it is important to use a resin which has
a low viscosity to avoid excessively long penetration times. Also, in order to
obtain good contrast, the resin needs to have a significantly different absorption
spectrum to that of protein. Further, the resin must also have little shrinkage,
uniform polymerization and ease of sectioning.
There is a number of commonly used protocols using resins such as spurrs,
araldite and methacrylate [4], each having different properties,e.g., viscosity,
hardness and degree of shrinkage. However, none of the embedding mediaso far
developed possess all the desired qualities, and some of theproperties may be
incompatible with others. For example, a mediumwhich polymerizes with little
shrinkage usually has a high initialviscosity. The epoxy resins are the most widely
used since they have mostof the properties required. The polyester resins have
similar excellent properties, but are less easily available and some of the componentsof the embedding medium are not stable during storage. The methacrylatespolymerize unevenly and with considerable shrinkage. Wolosewick [5] and
Kondo [6] have reported that reasonable success has been obtainedwhen imaging
biological tissue embedded in polyethylene glycol (PEG). Herethey remove the
PEG after sectioning which alleviates the problem ofobtaining contrast between
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N. I. Khaleque and C. J. Buckley
the embedding medium and biological tissue.However, the use of PEG did not
prove practical, due to its softness.
The differences in the NEXAFS features, between the pre- and post-carbon
K absorption edge, of three commercial resins and protein were used to select
a wavelength, λ, at which good contrast was expected. To asses the contrast
achievable in an imaging environment, unstained, regular protein structures (in
this case rabbit muscle fibres) were embedded in the three resins, sectioned to a
thickness of ≈150 nm and imaged using theselected wavelength (λ). Methacrylate gave the best contrast, as was expected by its spectra followed by spurrs
and araldite.
2
Materials and Methods
The resins studied were LR White Methacrylate (80% Polyhydroxyl substituted
Bis-phenol A Dimethacrylate with 20% C12 Methacrylate ester), spurrs, (TAAB
premix kit: SO32 medium) and araldite, (TAAB premix kit: CY212, medium
E029). The resinblocks were cured at 60◦ C for 18 hours and ≈150 nm thick
sections were cut and mounted on silicon nitride windows.
Fig. 1. A schematic diagram (not to scale) illustrating the X-ray microscope at the
NSLS.
Spectra were taken of each of the resin sections and Bovine Serum Albumin(a
standard protein), across the carbon K absorption edge, using an X-ray microscope (figure 1) at the National Synchrotron Light Source (NSLS) at Brookhaven
National Laboratories [7]. The spectra (figure 2)were processed using,
µρt = −ln
I
Io
(1)
where I is the transmitted intensity, Io is the incident intensity,µ is the mass
absorption coefficient, ρ is the density and t is the thickness of the material.
Muscle fibres were then embedded in the resins, sectioned and imaged at a
wavelength of 42.425Å, where good contrast was expected by comparisonof all
the spectra.
Image Contrast in Embedded Specimens
3
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Results and Conclusion
The spectra in figure 2 show the NEXAFS features of the three resins comparedto
that of protein. The histogram which compares the contrast obtained betweenthe
protein and each of the embedding media is calculated using,
Contrast =
Iprotein − Iresin
Iprotein + Iresin
(2)
where Iprotein is the intensity through an area of protein only, e.g.,striations
and Iresin is the intensity through an area of resin only.
Comaprison of NEXAFS Spectra
2.0
Contrast Between Protein and Resin
0.35
Protein
Methacrylate
Spurrs
Araldite
0.3
0.25
Contrast
Optical density
2.5
1.5
0.2
0.15
1.0
0.1
0.5
0.05
0.0
42.0 42.1 42.2 42.3 42.4 42.5 42.6 42.7 42.8 42.9 43.0
Wavelength (A)
0.0
Methacrylate
Spurrs
Araldite
Resin
Fig. 2. The spectra shows a comparison of methacrylate, spurrs,araldite and BSA
NEXAFS features at the carbon K absorption edge. Thehistogram shows the contrast,
between muscle fibres and resin, measuredfrom the images at a wavelength of 42.425Å
and the image is of anunstained muscle fibre embedded in methacrylate (scale bar =
100 µm).
The spectra in figure 2 show that, at a wavelength of 42.425Å,there is considerable difference in the optical density (OD) ofall three resins when compared
to that from protein. The spectra also show that methacrylate should give the
greatest contrast followed by spurrs and araldite. The increased peak height of
araldite at the wavelengths of42.425 and 42.75Å is due to the thickness effect
[8]; it wasimportant to use thin sections to avoid this problem. To observe these
differences in absorption in an imaging mode rabbitmuscle fibres were embedded
in the resins and imaged at 42.425Å.
The results from the histogram shows that methacrylate gives the best contrastat 42.425Å, followed by spurrs and araldite, as expected from the difference in their OD at that wavelength. The spectra alsoindicates that spurrs and
araldite should give similar contrast in imaging mode, but the contrast measured
from the images show that spurrs givesa much bigger difference in contrast than
araldite. This is due tothe thickness effect, which tends to distort the NEXAFS
peaks from their true value.
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N. I. Khaleque and C. J. Buckley
Lastly, the image in figure 2 is of an unstained rabbit muscle fibre embedded
in LR White methacrylate and sectioned to ≈150 nm in thickness. Due to the
natural contrast available, the striations of the fibre can be seen clearly without
the need for stains.
4
Discussion
It has been shown that natural contrast is achievable between biological material
and embedding medium without the need for stains when using theNEXAFS
features. Out of the three resins studied, methacrylate gavethe best contrast
followed by spurrs and then araldite.
The study of obtaining natural contrast between biological materialand embedding medium will be extended by using different amounts of the components
in each of the resins described earlier, by using a number of other resins such as
epon and also studying the contrast achievable by using other biological material.
Acknowledgements
The authors would like to thank Dr. P. Bennett for providing the rabbit muscle
fibres, the Stony Brook X-ray group for the use of their microscope at the X1a
beam line of the NSLS and Mr. Copper of TAAB Laboratories for supplying the
information on the structure of LR White Methacrylate.
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Zhang, X. et al., J. of Structural Biology 116, 335–344 (1996).
Buckley, C.J., Rev. Sci. Instrum. 66, 1318–1321 (1995).
Buckley, C.J., these Proceedings.
Glauert, A.M., Hall, C., Microscopy and Analysis, 15–20 (September 1991).
Wolosewick, J.J., The Journal of Cell Biology 86, 675–681 (1980).
Kondo, H., Ushiki, T., J. of Electron Microscopy 2, 457–462 (1985).
Jacobsen, C. et al., Optics Communications 86, 351–364 (1991).
Buckley, C.J. et al., Rev. Sci. Instrum. 6, 1322–1324 (1995).
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