DOI: 10.1007/s00340-003-1262-z Appl. Phys. B 77, 391–397 (2003) Lasers and Optics Applied Physics B a. assion 1 m. wollenhaupt 1 l. haag 1 f. mayorov 1 c. sarpe-tudoran 1 m. winter 1 u. kutschera 2 t. baumert 1, ✉ Femtosecond laser-induced-breakdown spectrometry for Ca 2+ analysis of biological samples with high spatial resolution 1 Fachbereich für Naturwissenschaften und Center of Interdisciplinary Nanostructure Science and Technology (CINSaT), Institut für Physik 1 , Institut für Biologie 2 , Universität Kassel, 34109 Kassel, Germany Received: 1 July 2003 Published online: 10 September 2003 • © Springer-Verlag 2003 ABSTRACT We describe an analytical method for element- specific in situ investigations of biological samples with high spatial resolution, using laser-induced-breakdown spectroscopy (LIBS). The prerequisites for spatially highly resolved LIBS are an appropriate analytical performance in combination with precise micro-ablation conditions, both laterally and axially. In order to identify the best suitable laser source, the analytical per- formance and the ablation process were studied for ns-LIBS and fs-LIBS. The analytical performance was studied on an aque- ous solution of CaCl 2 , where water served as a first substitute for biological material. The ablation process was investigated in the outer epidermal wall of a sunflower seedling stem. Besides direct measurements, the physiology of the sunflower seedling can be used in order to estimate the ablation depth. Our results show that analytical measurements with high spatial resolution can be performed on biological samples using fs-LIBS. This was demonstrated by in situ measurements of wall-associated calcium ion (Ca 2+ ) distributions within the peripheral cell wall of the sunflower seedling (Helianthus annuus L.) stem. In this biological sample an axial resolution of about 100 nm was achieved. PACS 52.50.Jm; 42.62.Be; 78.47.+p; 87.16.-b 1 Introduction The objective of this work is the development of a minimal invasive analytical method for spectrochemical in situ investigations of biological samples, with an axial resolution in the range of 100 nm, employing laser-induced- breakdown spectroscopy (LIBS). Several decades ago, LIBS was established as a spectro- chemical analysis method, where a laser-induced plasma is used as a spectrochemical source. The fundamental advan- tages of this approach can be summarized as follows: no sam- ple preparations are necessary, remote sensing measurements are possible and samples of any state of aggregation can be analyzed. The method is sensitive to the detection of different chemical elements. Moreover, multi-element analysis can be performed. Sensitivity limits in the ppm range can be achieved ✉ Fax: +49-561/804-4453, E-mail: baumert@physik.uni-kassel.de and spatial information can be obtained [1]. Mainly ns lasers have been used for LIBS and primarily analytical questions have been addressed. Only a few LIBS studies have investi- gated the spatial resolution from a basic point of view [2–5]. If spatial resolution becomes important, laser ablation pro- cesses have to be taken into account in addition. For trans- parent media and laser sources operating in the visible and infrared, ablation is initiated by the generation of a critical density of free electrons. Employing ultra-short laser pulses, multiphoton ionization is the dominating free electron for- mation path [6]. The non-linear dependence of multiphoton ionization on the laser intensity results in a sharply defined threshold for the optical breakdown. Additionally, during the interaction with the ultra-short laser pulse the energy ab- sorbed by the electrons is not transferred to the lattice. For ns-laser pulses, the electron plasma is generated by collisional (avalanche) ionization [7]. Statistical fluctuations of the num- ber of starting electrons for the avalanche ionization lead to strong fluctuations of the damage threshold. Therefore, in comparison to ns lasers, the optical breakdown threshold for fs lasers is a well-defined quantity. The breakdown threshold energy decreases with the fs-laser-pulse duration. Precise ab- lation, i.e. low ablation rates and minimal collateral damage, can be achieved, not only for technical [8–17] but also for biological samples [18–21]. Based on these considerations it is natural to assume that fs lasers are the ideal tool for minimal invasive LIBS with high spatial resolution. However, extensive experimental ex- perience on LIBS, demonstrating the high sensitivity of this method, exists mainly for ns-laser systems. With fs-laser sys- tems only a few experiments have been carried out, in which the plasma dynamics of fs-laser-generated plasmas of metallic samples was investigated [22–24]. From the discussion above, it is not obvious what kinds of laser sources enable LIBS with high sensitivity and high spa- tial resolution simultaneously, where sample volumes in the order of femtoliters and below have to be analyzed. Therefore, we investigated which laser source (fs laser or ns laser) is more suitable for LIBS with high spatial resolution. This paper is composed of four parts. In Sect. 2, the ex- perimental setup and the biological sample are described. Sec- tion 3 deals with basic considerations concerning the spatial resolution of LIBS. In Sect. 4, the analytical performances of ns-LIBS and fs-LIBS are discussed. In Sect. 5, a biological