C146-E317

TechnicalReport

Development of In Situ ImidaclopridVisualization Technique for Tomatoesusing iMScope

Saki Sumikura1, Atsushi Okazawa2, Hisashi Nishiwaki3, Eiichiro Fukusaki1, Shuichi Shimma1

Abstract:Neonicotinoid pesticide is assumed to be one of the causes of large-scale loss of honey bees and their abnormal behavior in recent years. Although neonicotinoid pesticides are known to be systemic in nature, which implies that they are highly penetrative and can be transported easily through organisms, there are almost no case studies examining penetration pathways of pesticides. This report describes a technique for determining penetration pathways of pesticides using imaging mass spectrometry with neonicotinoid imidacloprid used as the sample.

Keywords: iMScope TRIO, imaging mass spectrometry, pesticide analysis, imidacloprid

1. Introduction1. IntroductionNeonicotinoid pesticide sprayed on plants is assumed to be the sus-pected cause of the large-scale loss of honey bees and their abnormal behavior in recent years. Many studies suggest a link between neo-nicotinoid pesticide use and the loss of honeybees and their abnormal behavior. Although neonicotinoid pesticides are known to be system-ic in nature, characterized by their high penetrability and mobility in organisms, there have been almost no reported case studies regard-ing penetration pathways of pesticides. The transfer mechanism of sprayed pesticides in plants is almost unknown.

The dynamics of a chemical in an organism is normally analyzed by la-beling it with a radioisotope. The problems associated with this method are high cost, labor involved in marker synthesis, and inability to distinguish between the original chemical and its metabolites, when distributed throughout an organism.

In light of the above, imaging mass spectrometry (IMS) has been gain-ing attention in recent years. The IMS procedure involves observing a sample with a microscope, performing mass spectrometry without positional information loss of molecules after sample pre-treatment, and presenting the obtained spectra. It also provides information on multiple and diverse molecules in a single analysis and allows for visu-alization of those molecules without labeling, differentiation be-tween the original compound and its metabolites using mass spectra, and acquisition of highly specific data using MS/MS. The IMS tech-nique has been applied not only in the fields of medicine and biology, but also in fields related to plants and food.

This report describes a technique that uses iMScope TRIO to deter-mine the penetration pathways of imidacloprid, which belongs to the neonicotinoid pesticide family, in a nightshade plant.

2. Experimental Method2. Experimental MethodA Micro-Tom tomato plant (Solanum lycopersicum) of the nightshade family of plants, which was cultivated hydroponically, was used for analysis.

After plant culture, the leaves, flowers, stems, and fruits that had been exposed to imidacloprid were harvested and used for IMS analysis.

Ice crystals are formed when plant tissues with high water content are frozen. The presence of ice crystals can cause cell damages, rendering the samples inappropriate for IMS analysis. Therefore, plant tissues must be rapidly frozen in liquid nitrogen so that thin slices of the tis-sues can be obtained without destroying their structures (Fig. 1).

Leaf and flower samples were prepared without freezing and by im-mobilizing them on indium tin oxide (ITO)-coated glass using conduc-tive double-sided adhesive tape. Because of the high water content of the fruits, they were frozen in liquid nitrogen, like the stems, and then, 40-µm-thick sections were prepared.

200 µm

Frozen using a −80 °C freezer

200 µm

Frozen using liquid nitrogen

Fig. 1 Sections Prepared Using Different Freezing Methods

(20-µm-Thick Section)

1 Division of Advanced Science and Biotechnology, Graduate School of Engineering, Osaka University2 Applied Life Sciences, Graduate School of Life and Environmental Studies, Osaka Prefecture University3 Department of Bioscience, Graduate School of Agriculture, Ehime University 1

3. Matrix Investigation and Selection of Matrix Application Method3. Matrix Investigation and Selection of Matrix Application MethodFor sample ionization, the matrix materials used were alpha-cyano- 4-hydroxycinnamic acid (α-CHCA) and 2,5-dihydroxybenzoic acid (2,5-DHB). Two mixures — imidacloprid standard material and α-CHCA, and imidacloprid standard material and 2,5-DHB — were dripped onto the ITO-coated glasses. Precursor ions m/z 255.66 were used to create fragment ions, analyzed using MS/MS, and the resulting peak intensities were compared (Fig. 2). For both matrix materials, the two highest peaks were observed at m/z 209.06 and m/z 175.10. These peaks may correspond to protonated ion fragments of imidacloprid, where cleavage of the molecular structure occurs in the positions shown by the arrows in Fig. 2. Higher peaks are observed for the mix-ture associated with α-CHCA than for that associated with 2,5-DHB.

Two matrix application methods were compared: a dry coating method with vapor deposition of the matrix material onto the tissue surface and a two-step method, which involved the dry coating followed by the appli-cation of matrix solution as a spray (α-CHCA concentration: 10 mg/mL, solvent: mixture of 30% acetonitrile, 10% isopropanol, and 0.1% formic acid). iMLayer was used for matrix vapor deposition. When comparing the matrix application methods for stem sections exposed to imidacloprid, the vapor-deposition-only method resulted in stronger peaks, whereas the two-step method caused imidacloprid to bleed outside the tissue, as a result of which, its distribution could not be determined accurately (Fig. 3). These results indicate that the vapor-deposition-only method is more appropriate for application of the matrix material to plant tissue.

α-CHCA

175.10

209.06

Inte

nsity 4 106

3.5 106

3 106

2.5 106

2 106

1.5 106

1 106

5 105

0160 180 200 220 240 m/z

2,5-DHB

175.10 209.06

Inte

nsity 4 106

3.5 106

3 106

2.5 106

2 106

1.5 106

1 106

5 105

0160 180 200 220 240 m/z

Fig. 2 Comparison of Peak Intensities with Different Matrices

(a) (b)209.06

100

66

33

0

(c)209.06

100

66

33

0

(d)209.06

100

66

33

0

(e)209.06

100

66

33

0

Fig. 3 Comparison of α-CHCA

Application Methods

(a) Microscopic image of stem section(b) Imidacloprid distribution obtained by vapor deposition only(c) Superposition of microscopic image and imidacloprid

distribution obtained by vapor deposition only(d) Imidacloprid distribution obtained by two-step matrix

application(e) Superposition of microscopic image and imidacloprid

distribution obtained by two-step matrix application

Table 1 Analytical Conditions

Sample : Micro-Tom tomato plant exposed to 100 ppm imidacloprid for 1 dayMatrix : α-CHCAMeasurement mode : Positive ion mode

Precursor ion : m/z 255.66Laser irradiation diameter parameter : 2Measurement pitch : 50 µm

2

4. Tissue Imaging4. Tissue ImagingFig. 4 shows the results of using the iMScope TRIO to analyze the root close to the stem, leaf, fruit, and flower of the tomato plant. The ana-lytical conditions were identical to those in Table 1.

Fig. 5 shows the results of high-resolution (10 µm pitch) analysis of a stem section. The corresponding analytical conditions are shown in Table 2.

(1)

Xylem

Phloem

Endodermis andpericycle

Cortex

209.06

100

66

33

0

(2)209.06

100

66

33

0

(3)209.06

100

66

33

0

(4)209.06

100

66

33

0

Fig. 4 Tissue Imaging

(1) Root close to stem (2) Stem (3) Leaf (4) Flower

3

209.06100

66

33

0

Fig. 5 High-Resolution Stem Imaging

Table 2 Analytical Conditions

Sample : Micro-Tom tomato plant exposed to 100 ppm imidacloprid for 1 dayMatrix : α-CHCAMeasurement mode : Positive ion mode

Precursor ion : m/z 255.66Laser irradiation diameter parameter : 1Measurement pitch : 10 µm

These results show that imidacloprid penetrates the entire plant. Fig. 4-(1) shows no accumulation of imidacloprid in the root phloem, but show imidacloprid accumulation in the root cortex, endodermis, peri-cycle, and xylem. Fig. 4-(2) shows imidacloprid penetration in the stem endodermis, epidermis, cortex, xylem, and pith, with higher quantity of imidacloprid distributed in the interior of the stem than in its exte-

rior. Fig. 4-(3) shows imidacloprid distributed along leaf veins, and Fig. 4-(4) shows that imidacloprid distributed throughout the flower.

The results show that it is possible to visualize the distribution of pesti-cides in plants using a section preparation method, immobilization onto ITO-coated glass, and the matrix application methods, as de-scribed in sections 2 and 3.

Section preparation

Cryostat

Placement in holder Optical image acquisitioniMScope TRIO

Matrix application

iMLayer

Imaging data acquisition

Fig. 6 Experimental Instruments and Work�ow

iMScope TRIO was commercialized based on the results obtained from the Development of Systems and Technology for Advanced Measurement and Analysis Program of the Japan Science and Technology Agency (JST).

First Edition: December, 2016

© Shimadzu Corporation, 2016Printed in Japan 3655-06607-500NS