Application of AtomTrace LIBS Element Analysis Technology in Rapid Mapping of Plant Metal Element Distribution
May 05, 2023
AtomTrace is the sole derivative of the European Engineering Technology Center (CEITEC), and its members are researchers in the Laser Spectroscopy and Chemical Analysis Laboratory at the University of Brno. The laboratory started in 1997 and has nearly 20 years of experience in the field of LIBS application technology research and development. The Sci-Trace LIBS elemental analysis system developed and produced by the laboratory won the Czech Republic 2016 Best Cooperation Award. Prior to this, the AtomTrace team won the first place in the European LIBS Elemental Analysis Competition!
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LIBS technology has its unique and irreplaceable advantages over other elemental analysis methods. § Compared to the XRF technology, which can not detect light elements, LIBS can detect all elements;
§ Compared with traditional methods such as ICP-MS, samples do not need to be pretreated, and solid, liquid and gaseous samples can be directly detected and analyzed in real time;
§ Measuring speed can be up to 20 times in 1 second, and all the elements in the periodic table can be qualitatively and semi-quantitatively detected in a single measurement; Different nutrients and mineral content greatly affect the response of plants to growth and metabolism. Elemental measurement has always used the traditional method of ICP-OES or AAS. The disadvantage is that the sample pretreatment is complicated, it is easy to introduce new impurities and cause measurement error. But the most important thing is that you can't get the spatial information of the element distribution. However, if the distribution patterns are different, even if the content is similar, the effects on the physiological state of plants will vary greatly. The LIBS technology can make up for this shortcoming in the living state of plants without pre-processing for element mapping scanning. LA-ICP-MS technology can also perform elemental distribution scanning, but there are still many problems to be solved: laser ablation samples are transported to the ICP by carrier gas, and there will be particulate residue in the transport tube; large particle gasification in ICP is incomplete Memory effect (the effect of the previous measurement on the next measurement); the effect of the dead angle in the spray chamber and the transport tube on the signal strength and duration; multiple measurements must be taken at the same location to obtain a sufficiently strong signal. Therefore, for the measurement of the distribution of elements in plants, LIBS is considered to be the most optimal and promising measurement technique.
The AtomTrace research team has long been concerned about the application of LIBS technology in plant science. In 2006, Dr. Jozef Kaiser (CEO of AtomTrace, Director of the Spectroscopy Laboratory of Brno University of Technology) published the "Femtosecond laser spectrochemical analysis of plant samples" in the European Physical Journal, applying libs technology to Fe in the whole leaf of Hawthorn. The Mn element was subjected to a distribution mapping study. At that time, in this experiment, the LOD (detection limit) of Fe has been 5 ppm.
The AtomTrace team has been using LIBS technology to explore the field of plant element analysis, optimizing algorithms, developing software, and optimizing instruments—for example, using vacuum reaction chambers, double excitation techniques, etc. to improve mapping resolution and developing AtomAnalyzer spectral data analysis software. The calculation speed was increased by 10 times, and the UV vacuum module was developed to detect the 0-300 nm ultraviolet region spectrum. The test object includes both living plants and dry samples; including plant roots, stems, leaves and other parts of the plant; plant species including xerophytes, including high-moisture aquatic plants; qualitative and quantitative measurement of elements involved in plant importance Affected Al, Ca, C, Mg, P, Si, Sr, Zn, B, Cu, Fe, Mn, Pb, K, S, Na, Cl, H, N, Ni, Ba, Ag, and the like. And published a high impact factor article in the field of plant LIBS analysis as follows: - Pavlína M, Karel N, Pavel P, Jakub K, Přemysl L, Helena Z. G, Kaiser J, Comparative investigation of toxicity and bioaccumulation of Cd-based quantum dots and Cd salt in freshwater plant Lemna minor L. [J], Ecotoxicology And Environmental Safety, 147 (2018) 334–341.
- Krajcarová L, Novotný K, Kummerová M, J. Dubová J, Gloser V, Kaiser J. Mapping of the spatial distribution of silver nanoparticles in root tissues of Vicia faba by laser-induced breakdown spectroscopy (LIBS) [J], Talanta 173 ( 2017) 28–35.
- Lucie K, Novotny K, Petr B, Ivo P, Petra K, Vojtech A, Madhavi Z. Rene K, Kaiser J, Copper Transport and Accumulation in Spruce Stems Revealed by Laser-Induced Breakdown Spectroscopy, [J]. Electrochemical Science, 8 (2013) 4485 – 4504.
- Zitka O, Krystofova O, Hynek D, et al . Metal Transporters in Plants [M]. Heavy Metal Stress in Plants . 2013: 19-41.
- Kaiser J, Novotny K, Martin MZ, et al . Trace elemental analysis by laser-induced breakdown spectroscopy—Biological applications [J]. Surface Science Reports , 2012, 67 (11–12): 233-243.
- Michaela G, Jozef K, Karel N, et al. Utilization of laser-assisted analytical methods for monitoring of lead and nutrition elements distribution in fresh and dried Capsicum annuum I. leaves [J]. Microscopy Research and Technique, 2011, 74 (9 ): 845-852.
- Diopan V, Shestivska V, Zitka O, et al . Determination of Plant Thiols by Liquid Chromatography Coupled with Coulometric and Amperometric Detection in Lettuce Treated by Lead (II) Ions [J]. Electroanalysis , 2010, 22 (11): 1248-1259 .
- Kaiser J, Galiova M, Novotny K, et al . Utilization of the Laser Induced Plasma Spectroscopy for monitoring of the metal accumulation in plant tissues with high spatial resolution [J]. Networking IEEE/ACM Transactions on , 2010, 20 (4): 1096-1111.
- Kaiser J, Galiova M, Novotny K, et al . Mapping of lead, magnesium and copper accumulation in plant tissues by laser-induced breakdown spectroscopy and laser-ablation inductively coupled plasma mass spectrometry [J]. Spectrochimica Acta Part B Atomic Spectroscopy , 2009 , 64 (1): 67-73.
- Krystofova O, Shestivska V, Galiova M, et al . Sunflower Plants as Bioindicators of Environmental Pollution with Lead (II) Ions [J]. Sensors , 2009, 9 (7): 5040-5058.
- Kaiser J, Galiova M, Novotny K, et al . Mapping of the heavy-metal pollutants in plant tissues by Laser-Induced Breakdown Spectroscopy [C] Spectrochimica Acta Part B 64 (2009) 67–73.
- Galiova M, Kaiser J, Novotny K, et al . Investigation of heavy-metal accumulation in selected plant samples using laser induced breakdown spectroscopy and laser ablation inductively coupled plasma mass spectrometry [J]. Applied Physics A , 2008, 93 (4): 917-922.
- Sona K, Pavel R, Olga K, et al . Multi-instrumental analysis of tissues of sunflower plants treated with silver(I) ions – plants as bioindicators of environmental pollution [J]. Sensors , 2008, 8 (1): 445- 463.
- Stejskal K, Mendelova Z, et al. , Study of effects of lead ions on sugar beet [J]. Listy Cukrovarnicke A Reparske , 2008, 124 (4): 116-119.
- Galiova M, Kaiser J, Novotny K, et al . Utilization of laser induced breakdown spectroscopy for investigation of the metal accumulation in vegetal tissues [J]. Spectrochimica Acta Part B Atomic Spectroscopy , 2007, 62 (12): 1597-1605.
- Kaiser J, Samek O, Reale L, et al . Monitoring of the heavy-metal hyperaccumulation in vegetal tissues by X-ray radiography and by femto-second laser induced breakdown spectroscopy [J]. Microscopy Research and Technique , 2007, 70 (70) ): 147-153.
AtomTrace team applied LIBS technology to study the distribution of metal elements in plants Research case : Lemna minor L. is an indicator species of environmental pollution of metal elements and a model organism often used for metal toxicity and enrichment research. In this case, the AtomTrace team applied LIBS technology to map the element distribution of duckweed, and compared the enrichment mode of Cd elements in Cd salt and QDs in duckweed; and applied traditional ICP-OES technology to float different Cd-containing compounds. The content and enrichment of Pingzhong were measured. At the same time, the TEM method was used to explore the enrichment position of QDs--the surface of duckweed, the interior of cells, or the tissue of plants.
Note: Cd ions ranked 7th in the list of 275 important hazardous substances in 2015. Cd-containing quantum dots (QDs) are usually composed of CdS, CdSe, PbSe, and CdTe having a diameter of 3-6 nm and some other metal elements, which are coated with an organic polymer. Because the dye effect is superior to other bio-dyes, it is discharged into the water and releases Cd ions in water. Therefore, it is important to study the toxic effects of Cd quantum dots on organisms.
Study on the distribution of Cd elements in duckweed leaflets using double-excitation LIBS technique
experimental method:
§ The duckweed leaves were treated with cadmium-containing compounds CdCl 2 , MPA-QDs and GSH-QDs (the three solution concentrations were set to three gradients: 0.1, 1 and 10 mg/L, respectively). Applying the leaflets to the slide to make a sample;
§ LIBS measurements use orthogonal double excitation. The excitation laser wavelength is 266 nm, the pulse energy is 10 mJ; the secondary excitation laser wavelength is 1064 nm, and the pulse energy is 1064 mJ; the length of the two excitation laser pulses is 5 nm. The blade is broken down for each measurement.
§ Scan measurement resolution: 200 μm;
§ Cd detection main line: 508.58 nm; 
Fig. 4 Cd quantum dot and Cd salt treatment under duckweed leaflet element mapping image
Experimental results:
§ CdCl 2 and Cd-QDs pollution, there is no difference in the influence of Cd elements on the surface distribution of duckweed leaves;
§ Different concentrations have no difference in the influence of Cd elements on the surface distribution of duckweed leaves;
§ The concentration of three cadmium-containing compounds (CdCl 2, MPA-QDs, GSH-QDs) increased, and the LIBS detection signal increased.
§ Cd element enrichment at stem junctions is significantly higher than that of other tissues: Pavlína, M., Karel, N., Pavel, P., J, K., P, L., H, ZG, Jozef, K. 2018. Comparative investigation of toxicity and bioaccumulation of Cd-based quantum dots and Cd salt in freshwater plant Lemna minor L . [J]. Ecotoxicology and Environmental Safety 147 (2018) 334–341
In the second case , the LIBS mapping analysis of the elements of the plant tissue can determine the element type by the position of the line, and the element concentration can be obtained by the line intensity, and the element mapping image can obtain the element distribution and the related position distribution information of several elements. By continuously measuring at the same position, 3D information of the element profile distribution can be obtained.
Fig . 5 shows the experimental study:
Fig. 5 A: Effect of Pb element on the distribution of Mg elements on lettuce leaves. Mg is a key metal for chlorophyll, while Pb has a stronger affinity for chlorophyll. Therefore, when the concentration of Pb in the leaves increases, the concentration of Mg decreases.
Fig. 5 B: Pb treatment increased the Pb concentration in corn leaves.
Fig. 5 C: Plants have different resistance to metal ion toxicity. In the sunflower leaves as shown in the figure, Pb treatment had no effect on the distribution of Mg elements. The experimental results are consistent with the results of the morphological analysis experiment.
Fig. 5 D: LIBS technology can also be applied to elemental analysis in other tissues of plants. The resulting 3D element profile is measured by double-excited LIBS of pine twigs as shown. 
Fig. 5
Quoted from: Jozef, K., Karel, N., et al., Trace elemental analysis by laser-induced breakdown spectroscopy—Biological applications. Surface Science Reports 67 (2012) 233–243
The study's three roots play an important role in plant nutrient supply and protection of plants from excessive metal ions, but root elemental analysis is much more difficult than stem tissue, including: roots are usually smaller than stems and shoots. Many; the dry matter content is much smaller, which is very inconvenient for sample cutting; usually the relative content of the elements to be analyzed is relatively low; and how the soft and juicy sample retains its structural shape to obtain the correct result of the element distribution is also a problem.
AtomTrace has successfully explored the above challenges in this case---Mapping analysis of nano-silver particles (21.7±2.3 nm in diameter) at the roots of broad bean seedlings using double-excited LIBS technology, aiming at plant tissues in natural state Perform element detection to achieve high mapping resolution while ensuring detection sensitivity. This is also the first attempt at the distribution of nanoparticles in plant roots throughout the LIBS field.
LIBS double excitation technology -- that is, each measurement signal collected is excited by two laser pulses. This can reduce the ablation disturbance and improve the mapping resolution; at the same time, the two excitations can enhance the signal to obtain a more repeatable and better LOD (detection limit) detection result.
Experimental parameters: The secondary excitation pulse energies were 5 MJ@266 nm and 100 MJ@1064 nm, respectively, with an interval of 500 ns; the measurement frequency was 1 time/second; the experiment was carried out at 1 atmosphere.
Experimental materials: Broad bean seedlings were treated in AgNP solution, Cu+ and Ag+ ion solution for 7 days, and 40 μm thick sections were taken for LIBS mapping measurement.
Experimental results: It can be seen from the following experimental results that the detection speed of LIBS technology is fast; even for young roots with a diameter of only 2 mm, the mapping of metal ions and metal nanoparticles in the cross-section can be performed, and the detection accuracy and image resolution The rate is sufficient to meet the needs of the experiment. Using double-excitation technology, the mapping resolution can reach 50μm, which is enough to distinguish the distribution characteristics of elements in the root epidermis, cortex and middle column.
In addition, 7 days of short-term treatment can test the results, indicating that for plants in the natural environment and natural nutrient conditions, LIBS element mapping is also an effective experimental method for element distribution detection, so it will be plant physiology and environmental toxicity. Effective application in the field of science. 
Fig. 6 The roots of broad bean seedlings were cross-cut and subjected to single-line measurement with a resolution of 50 μm.
Fig.7 Cu + solution treatment of broad bean seedling root cross-cutting under different resolutions Mapping results: 100μm, 75μm, 50μm
Fig.8 Different concentrations of Cu 2+ solution [a) 100 μmol l −1 Cu 2+ ; b) 50 μmol l −1 Cu 2+ ; c) 10 μmol l −1 Cu 2+ ; d) 0 μmol l −1 Cu 2+ 】 treatment of broad bean seedling root cross-cutting mapping results; e) sample area characteristic line; f) reduction of Cu 2+ concentration, the corresponding line intensity is also reduced 
Fig.8 Microscopic images and element mapping results of root cuts of broad bean seedlings after 7 days of treatment with Cu2+, Ag+ and AgNPs (from: Krajcarová L, Novotný K, Kummerová M, J. Dubová J, Gloser V, Kaiser J. Mapping of the spatial distribution of silver nanoparticles in root tissues of Vicia faba by laser-induced breakdown spectroscopy (LIBS) [J], Talanta 173 (2017) 28–35.)
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