Understanding the role of elemental analytes and metals within biological systems is a key area of research in disciplines ranging from clinical, medical, and pharmaceutical to food and environmental sciences. Researchers typically use various bioimaging and metallomics techniques to explore their samples of interest. Example techniques include liquid-chromatography (LC)-ICP-MS, single cell (sc)-ICP-MS, and laser ablation (LA)-ICP-MS, which have been used for protein quantification1, single-cell analysis2, and bioimaging, respectively. Bioimaging focuses on visualizing tissues, cells, and low molecular weight compounds, while metallomics examines the functions and roles of metals in biological structures.

As demonstrated in this article, two research groups based in Japan have used LA-ICP-MS to provide detailed information about the localization of elements on the surface of solid samples. Yukako Shintani-Domoto’s group at the Department of Diagnostic Pathology, Nippon Medical School Hospital in Tokyo investigated the distribution of metals on thin sections of human heart samples using their Agilent 8900 ICP-QQQ. Yuki Sugiura’s group at the Multiomics Platform, Center for Cancer Immunotherapy and Immunobiology, Kyoto University Graduate School of Medicine in Kyoto studied mouse kidney samples using their Agilent 7900 ICP-MS.

LA-ICP-MS instrumentation and software compatibility

In the separate studies, the 8900 ICP-QQQ and 7900 ICP-MS instruments were coupled with an ESL213 Laser Ablation System equipped with an argon (Ar) mass flow controller (Elemental Scientific Lasers, Bozeman, MT, USA). Ar was used as the carrier gas and makeup gas instead of helium (He) gas in response to the shortage of He supplies in some countries.

The LA system was controlled directly from the Agilent ICP-MS MassHunter software via the ESL-developed ActiveView2 (AV2) plug-in for ICP-MS MassHunter (Figure 1), enabling fully integrated sample analysis, data processing, and reporting.

Agilent ICP-MS MassHunter software user interface with ActiveView2 (AV2) integrated control of the Elemental Scientific Lasers ESL213 laser ablation system and Agilent ICP-MS instrument.

Figure 1. The Run Queue tab and status panel of the ESL213 LA system as displayed in Agilent ICP-MS MassHunter software via the ESL AV2 plug-in.

Analytical workflow

The LA-ICP-MS instrument operating conditions are given in Tables 1 and 2.

Table 1. Laser ablation system parameters.

Table of laser ablation operating parameters for LA-ICP-MS. A smaller spot size (20mm) and Scan speed (20mm/s) were used for the 8900 ICP-QQQ, with a repetition rate of 20 and an argon carrier gas flow rate of 0.95 l/min.

Table 2. ICP-MS system parameters.

Table of ICP-MS operating parameters for LA-ICP-MS. The 8900 ICP-QQQ used an RF Power of 1600W, a sampling depth of 8mm, a makeup gas flow rate of 1.15 l/min, and an oxygen cell gas flow rate of 25%.

Distribution of metals in heart samples

Myocardial infarction (MI) is induced by thrombosis or obstruction in the coronary arteries. In the chronic phase, referred to as old MI, the myocardium undergoes fibrosis, evident as blue areas in Azan staining—see Figure 2a. Also, concentrations of 31P, 44Ca, and 56Fe determined by LA-ICP-MS using the 8900 in O2 mass-shift mode decreased on the left-side area compared to other regions—see Figure 2 (b to d). The results from the two complementary techniques showed that metal distribution information obtained by LA-ICP-MS can serve as an indicator of cell viability in the tissue.

Azan staining of heart sample showing diseased area in blue. Distribution of phosphorus-31, calcium-44, and iron-56 obtained by LA-ICP-MS.

Figure 2. (a) Azan staining of human heart. (b to d) Distribution of analytes on thin sections of heart determined by LA-ICP-MS. Scale: 1000 μm.

Characteristic localization of metal proteins in mouse liver

A 10 μm thin section of C57BL/6 mouse liver was analyzed by LA-ICP-MS using the 7900 in H2 and He mode. The images in Figure 3 show the presence of 95Mo, 98Mo, and 56Fe. 95Mo and 98Mo show a similar pattern, demonstrating the reliability of the method. The molybdenum signals suggest that the isotopes accumulate in the liver and exist as molybdopterin cofactors. The 56Fe signals mainly surround a blood vessel at the center bottom of the sample, which aligns well with the observed red blood cells, suggesting the existence of heme iron (Figure 3c). The superimposition of Figures 3a and 3c reveals that 56Fe and 95Mo are detected in distinct regions (Figure 3d), suggesting a characteristic localization of metal proteins in tissues.

Distribution of magnesium-24, iron-56, copper-63, molybdenum-95, and molybdenum-98 in a section of mouse liver obtained by LA-ICP-MS. Overlay of the iron-56 and molybdenum-95 signals in distinct regions of the sample.

Figure 3. (a) to (e) Distribution of 24Mg, 56Fe, 63Cu, 95Mo, and 98Mo, respectively, determined by LA-ICP-MS. (f) Red Green Blue (RGB) mode image produced by iolite 4 software (ESL), which combined 56Fe (red) and 95Mo (green) data into one image. Scale: 500 μm.

Benefits of integrated LA-ICP-MS methods

The study demonstrates the high performance of the Agilent 8900 ICP-QQQ and Agilent 7900 ICP-MS equipped with an ESL LA system for bioimaging of thin sections. The data provided useful insights into metal accumulation resulting from tissue lesions, and characteristic localization of metal proteins and cofactors in tissues.

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