WDS'09 Proceedings of Contributed Papers, Part III, 189–192, 2009.
ISBN 978-80-7378-103-3 © MATFYZPRESS
Elementar LIBS Analysis of Biological Samples
Z. Grolmusová, L. Mináriková, J. Rakovský, P. Čermák, and P. Veis
Department of Experimental Physics, Faculty of Mathematics, Physics and Informatics Comenius University,
Mlynská dolina F2, 842 48 Bratislava, Slovakia.
M. Kopáni, J. Jakubovský, and P. Babál
Department of Pathology, Faculty of Medicine Comenius University, Špitálska 24, 813 72 Bratislava, Slovakia.
M. Čaplovičová
Department of Geology of Mineral Deposits, Faculty of Natural Sciences, Comenius University,
Mlynská Dolina, 842 15 Bratislava, Slovakia.
Abstract. Trace element analysis of biological samples can be useful for diagnostics of
diseases caused by accumulation of some elements in the organism. The aim of this work
was to evaluate the performance of Laser induced breakdown spectroscopy (LIBS) for the
determination of elements present in human tissues. We have investigated samples of
human liver and human spleen using pulsed Nd:YAG laser operating at 532 nm coupled
with the Mechelle ME 5000 spectrometer covering range from 200 to 975 nm. Laser pulse
caused ablation of samples followed by plasma generation. In the emitted spectra we could
clearly detect Ca, Si, Mg, C, K, Fe, Cu, Na, Ti and Al spectral lines. In this work we
present the comparison of intensity of iron spectral lines from spleens with diagnosed
hereditary spherocytosis and hemosiderosis. This comparison indicates up to 4-times
higher intensity of iron lines of spleen with hemosiderosis and 8-times higher intensity of
iron lines of spleen with hereditary spherocytosis in comparison with the iron lines of
reference spleen. LIBS showed to be a promising analytical method for these applications.
Introduction
Laser induced breakdown spectroscopy (LIBS) is a promising analytical method which has many
advantages: simplicity, no sample preparation, rapid analysis, ability to sample gases, liquids and
solids equally well. LIBS has manifold utilizations e.g. detection of versatile hazardous materials
(Gottfried, 2007), chemical weapons (DeLucia, 2005), lead in paints (Marquardt, 1996) etc. Lately it is
investigated in biological field too: hair tissue analysis (Corsi, 2003), elementar analysis of teeth
(Samek, 2001), elementar composition of malignant and healthy animal tissues (Kumar, 2004) etc.
Our experiments dealt with laser induced plasma on soft human tissues (liver, spleen). Our
investigation studied the availability of LIBS technique to detect some interesting elements like iron,
copper, aluminium, titanium in mentioned tissues. It could be useful for investigating human tissues
with some pathologies characterised by cumulation of some elements. We have investigated iron
contents in human spleens with diagnosed hereditary spherocytosis and hemosiderosis in comparison
with healthy human spleen.
Principle and experimental setup
Principle of LIBS is based on the atomisation of investigated sample using high power laser pulse.
Laser beem is focused on the sample surface and the breakdown is caused by high power of laser.
Plasma is created above the surface of the sample and it causes dissociation and excitation of atoms
and molecules from the sample. Emission spectrum is collected by fiber optics into the spectrometer.
Recognition of atomic emission lines of output spectra provides species identification. For the
identification of spectral lines we have used the national NIST database (Ralchenko, 2007).
In our experiment we have used a frequency-doubled Nd3+:YAG laser (QUANTEL – Brilliant
EaZy, maximal energy 165 mJ, maximal power 1,65 W, pulse duration 4 ns, wavelength 532 nm).
Output beam was deviated in right by optical prism (Thorlabs, BK7) and focused using lens (Thorlabs,
focal length 4 cm) on the sample surface. Emitted light of the ignited spark was collected with lens
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GROLMUSOVÁ ET AL.: ELEMENTAR LIBS ANALYSIS OF BIOLOGICAL SAMPLES
(Thorlabs, focal length 8 cm) into the optic fiber connected to the Andor Mechelle spectrometer
system (Mechelle 5000 with an intensified CCD iStar (DH734i-18F-03)). This spectrometer allowed a
wide spectral range coverage (200-950 nm) with a relatively high resolution (Dl/l=5000) and a short
focal length (178 mm). The mentioned experimental setup is shown in Figure 1.
Figure 1. Experimental setup of the LIBS system.
Biological samples
We have used samples of human liver and human spleen. Some were dried as bulk tissues and
some were prepared on glass slides according to standard histological techniques (Foltinová, 2003).
We have investigated human spleen with diagnosed hereditary spherocytosis and hemosiderosis.
Hereditary spherocytosis is a heritable disease characterized by hemolysis of the pathological shaped
erythrocytes (named spherocytes) in the spleen, which can lead to higher contents of iron. In the case
of hemosiderosis serum iron is accumulated in the human body.
ResultsAblated area
After the laser shots there is a small amount of sample ablated. We can`t see it clearly with naked
eye, but here we show images taken by scanning electron microscope (Figures 2 and 3). These pictures
show thin liver sample layed on the glass slide after the laser ablation. In the center of the ablated area
is a crater created in the glass slide caused by high laser power in the middle of the laser beam. From
these pictures we have determined the average diameter of craters (460,4 ± 33,3 μm). From this value
we have calculated the ablated area (0,1665 ± 0,0009 mm2) of the sample. In the case of samples
coated on the glass slide we can clearly see that by laser shot we have ablated the glass slide too. It`s
the explanation for why we have also seen the elements of the glass in the spectra of samples. So we
have had to compare the spectra of the glass slide with the spectra of the sample fixed on the glass
slide.
Example of spectra
On spectra of human tissues supposed to be healthy we can clearly recognize spectral lines of Ca,
Si, Mg, C, K, Fe, Cu, Na, Ti, Al and (the spectral lines) of N, O which originate from the ambient air.
In the obtained spectra some elements not natural in the body are shown in Figures 4 and 5.
Pathological spleens
We have investigated two samples of pathological spleens using LIBS. One was with diagnosed
hemosiderosis and the other with diagnosed hereditary spherocytosis. Figure 6 shows high intensity of
iron lines in the case of spleen with hereditary spherocytosis. These spectra were normalized to the
spectral line of carbon (C I: 247.856 nm) since the content of carbon should be the same in each
spleen. Then we compared the ratio of the iron line (Fe I: 302.107 nm) and the carbon line (C I:
247.856 nm) from each spleen. In the case of the spleen with hemosiderosis this ratio is 3.67-times
higher than in the reference spleen. The mentioned ratio is 8.25-times higher for spleen with hereditary
spherocytosis than in the reference spleen.
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GROLMUSOVÁ ET AL.: ELEMENTAR LIBS ANALYSIS OF BIOLOGICAL SAMPLES
Figure 2. Thin liver sample coated on the glass
slide after the laser ablation. This photography is
made by scannig electron microscope JXA 840 A
(JEOL, Japan) at accelerating voltage 10 kV and
magnification 35-times.
Figure 3. Thin liver sample coated on the glass
slide after the laser ablation. This photography is
made by scannig electron microscope JXA 840 A
(JEOL, Japan) at accelerating voltage 10 kV and
magnification 150-times.
human spleen
glass slide
dried human liver
40000
70000
Cu I
Cu I
60000
Intensity
Intensity
30000
Ti II
20000
50000
40000
Ca II
30000
10000
Al I
20000
10000
0
322
323
324
325
326
327
392
328
394
395
396
397
398
wavelength [nm]
wavelength [nm]
Figure 4. The comparison of the spectra of
human spleen and spectra of the glass slide.
Arrows markt the copper lines of the spleen and
the titanium lines come from the glass slide.
(Cu I: 324.754 and 327.396 nm; Ti II: 323.452;
323.657; 323.904; 324.199 nm)
Figure 5. Spectrum of dried human liver. Arrows
mark the calcium and aluminium lines.
(Ca II: 393.366 and 396.847 nm; Al I: 394.401;
396.152 nm)
18
Intensity
393
glass slide
spleen - hemosiderosis
spleen - reference
spleen - hereditary
spherocytosis
9
0
300
w avelenght [nm]
304
Figure 6. Comparison of spectra of pathological spleen and the reference spleen. Numerous arrows
mark the iron lines.
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GROLMUSOVÁ ET AL.: ELEMENTAR LIBS ANALYSIS OF BIOLOGICAL SAMPLES
Conclusion
We have demonstrated suitability of LIBS for biological samples investigation, mostly for human
liver and spleen as we have shown above. Elementar analysis of human tissues can be used for
diagnosis of diseases characterized by abnormal accumulation of some elements. The presence of
some elements in human tissues may indicate poisoning or some noxious environmetal aspects.
Acknowledgments. We gratefully acknowledge financial support under contract numbers: VEGA
1/0609/08, 1/4015/07, 1/0851/09, 1/0337/08, APVV-0544-07, APVV-0267-06, SK-AT-0025-08, G-09-234-00
and G-09-265-00.
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