New Preparation Method of Bone. Samples for Raman. Microspectrometry. G. PENEL,* G. LEROY, and E. BRES. U 279 INSERM 1 rue du Prof. Calmette, 59019 ...
New Preparation Method of Bone Samples for Raman Microspectrometry G. PENEL,* G. LEROY, and E. BRES U 279 INSERM 1 rue du Prof. Calmette, 59019 Lille Cedex, France (G.P., G.L.); Laboratoire d’ Anatomie bucco-dentaire, UFR de Chirurgie-Dentaire, place de Verdun 59000 Lille, France (G.P.); and Laboratoire de MeÂtallurgie Physique, LSPES URA CNRS 234 USTL BaÃt. C6 59655, Villeneuve d’ Ascq, France (E.B.)
Index Headings: Raman microspectrom etry; Bone; Hydrogen peroxyde; Acetone.
INTRODUCTION Raman microspectrometry is increasingly becoming recognized as a signi® cant method for mineralized tissues investigations. For bone studies, conventional Raman spectra are disturbed by ¯ uorescence problems.1,2 A hydrazine treatment of bone has been used ef® ciently to solve this problem,3 but this method is destructive in terms of the organic part of this tissue and reduces some of the phosphate band intensities on the Raman spectra.4 Since we know that some biological apatites (dentine and bone for example) exhibit a very similar spectrum with respect to mineral components, the nondestruction of the organic part appears to be an important condition for spectral investigations. The aim of this work is to ® nd a simple method to reduce the bone ¯ uorescence that allows the Raman spectral attribution of a bone sample without destruction of the organic part of the tissue.
FIG. 1. MicroRaman spectra of bone samples processed with NaCl (NaCl) and acetone/hydrogen peroxyde (H2 O2 ).
MATERIALS AND METHODS
obtained in a range of 200± 3600 cm2 1 . A 1003 microscope objective lens was used in a confocal con® guration. The power on the samples was 7 mW. For the NaCl sample, the signal was saturated for an integration time longer than 10 s. Thus the spectro-
Preparation of Bone Samples. Bone samples were obtained from rabbit femur, cleaned of soft tissue and periosteum. The diaphysis were broken in the middle to facilitate the cleaning of bone marrow. Then they were immersed in a NaCl solution (100 g/L) for 24 h in order to reduce the ¯ uorescence (NaCl sample). The spectroscopic assay provided some good results, but we still had to deal with ¯ uorescence artifacts (Fig. 1). Secondarily, in order to further decrease ¯ uorescence, a second method was used. The samples were immersed successively for 2 h in a 30 volume hydrogen peroxyde solution, in acetone, and in hydrogen peroxyde again (H2 O2 sample). The samples were attached to a microscope slide for the spectral acquisition. Raman Microspectrometry. The Raman microspectrometer was a LABRAM type from DILOR (Lille); argon-ion laser (514.5 nm) excitation was used. The overall spectral resolution was 2 cm2 1 . Spectra were Received 3 January 1997; accepted 27 August 1997. * Author to whom correspondence should be sent.
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TABLE I. Raman band position s and assignments of the bone tissue prepared with the acetone and hydrogen peroxyde method. Band assignments
n
2
PO342
n
4
PO342
P± OH stretch 32 1 PO4 n 3 HPO242 n 3 PO342
n
B type n 1 CO232 A type n 1 CO232 Amide IIIa C± H bendinga Amide Ia C± H stretching a
a
Band positions (cm2 1) 432 452 584 590 611 924 961 1005 1032 1044 1071 1071 1107 1243 1262 1449 1662 2882 2946 2986
From Ref. 5.
0003-7028 / 98 / 5202-0312$2.00 / 0 q 1998 Society for Applied Spectroscopy
APPLIED SPECTROSCOPY
scopic conditions used were an integration time of 10 s for 250 accumulations. On the H2 O2 sample it was possible to use a 40 s integration time for 20 accumulations. It should be noted that the total laser beam exposure was 2500 s for the NaCl sample and 800 s for the H2 O2 sample. To validate the ef® ciency of the different treatments on the background noise reduction, we carried out spectral acquisition for the 850± 1150 cm2 1 region. For each sample, the same spectroscopic conditions were used: 10 s integration time and 10 accumulations. RESULTS For the 10 s integration time and 10 accumulations assays, the background noise intensity values were 5800 and 900 for samples prepared with NaCl and H2 O2 , respectively. The acetone and hydrogen peroxyde processing we used for bone treatment complements the results previously published.5 The signi® cant reduction of the ¯ uorescence (Fig. 1) allows a complete Raman study of the tissue including both mineral and organic parts. The band positions and assignments are listed in Table I. We think that ¯ uorescence encountered without or with insuf® cient bone preparation comes from blood chromophores. Hydrogen peroxyde eliminates them, and
the acetone facilitates this procedure by reduction of the fat tissue contents. CONCLUSION The method described in this presentation allows a full Raman spectral attribution of bone samples. Fluorescence and background noise are signi® cantly reduced. As opposed to spectra obtained from samples processed with the hydrazine method, the organic part of the bone is preserved. In the future, extensive investigations and comparisons with other biological apatites will be carried out with the aim of detecting a possible relationship between the organic and mineral parts of calci® ed tissues. In contrast to the histological methods, which identi® ed only the organic part, the Raman micro-spectrometry method allows a strict characterization of the biological apatites. 1. G. R. Sauer, W. B. Zunic, J. R. Durig, and R. E. Wuthier, Calcif. Tissue. Int. 54, 414 (1994). 2. M. A. Walters, Y. C. Leung, N. C. Blumenthal, R. Z. Le Gros, and K. A. Konsker, J. Inorg. Biochem. 39, 193 (1990). 3. J. D. Termine, E. D. Eanes, D. J. Green® eld, M. U. Nylen, and R. A. Harperr, Calcif. Tissue Res. 12, 73 (1973). 4. I. Rehman, R. Smith, L. L. Hench, and W. Bon® eld, J. Biomed. Mat. Res. 29, 1287 (1995). 5. S. Nie, K. L. Bergbauer, J. J. Ho, J. Kuck and N. Yu, Spectrosc. Int. 3, 20 (1990).
APPLIED SPECTROSCOPY
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