Journal of Nature and Science (JNSCI), Vol.1, No.4, e61, 2015

Biological Sciences

 

Imaging the Electromagnetic Field of Plants (Vigna radiata) Using Iron Particles: Qualitative and quantitative correlates

 

&Benjamin J. Scherlag, §Bing Huang, #Ling Zhang, ##Kaustuv Sahoo, ƮRheal Towner, ƮNatalya Smith, ***Abraham A. Embi, &Sunny S. Po

 

&Heart Rhythm Institute, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma, USA. §Department of Cardiology, Renmin Hospital of Wuhan University, Wuhan, China. #Arrhythmia Research Lab, First Affiliated Hospital of Xinjiang Medical University, Urumqi, China. ## School of Pharmacy, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma, USA. ƮOklahoma Medical Research Foundation, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma, USA. ***13442 SW 102 Lane, Miami, FL 33186, USA


A recent study utilized a sensitive atomic magnetometer to measure the electromagnetic field (EMF) of a plant. We aimed to use a simplified method for recording direct and indirect images of EMFs from the leaves of the plant, Vigna radiata. Protocol 1. Consisted of Iron particle solutions of 200 or 2000 nanometers (nm) and Prussian Blue stain (PBS) which were applied to leaves in a 2 glass slide “sandwich”. When the liquid had dried, the leaves were microphotographed. Protocol 2. Leaves prepared as in Protocol 1 were then covered by a second pair of slides within which was a solution of iron particles and PBS. Protocol 3. Six pairs of leaves treated with solutions of iron particles and PBS or deionized water for 24 hours were analyzed by magnetic resonance imaging (MRI). Leaves with direct and indirect contact with the iron particle/PBS solutions showed aggregation of iron particles outlining the leaf edges and leaf hairs (trichomes, Protocol 1) or EMF images of leaf edges, trichomes as well as interior veins were seen in slides containing iron particles and PBS not directly in contact with the leaves (Protocol 2). Protocol 3: MRI studies (n=12) allowed a quantitative comparison of leaves treated with iron particles compared to those exposed to deionized water. In the former group, there was a significant loss of T2 values indicative of the replacement of water by iron particles adherent to the treated leaves, p≤0.0001 compared to the group in deionized water. Nanometer sized paramagnetic iron particles can be used to demonstrate the EMF of the leaves of the plant Vigna radiata. Journal of Nature and Science, 1(4):e61, 2015.

 

Electromagnetic fields | Vigna radiata | Plant leaf | Magnetic resonance imaging

 

A metabolic process common to plants (photosynthesis) and animals (cellular respiration) involves the electron transport chain. This process consists of electrons transferred along a series of electron donor and receptor compounds coupled to a proton, H+, gradient across cell membranes. The ensuing charge differential or voltage is used to drive energy production in the form of adenosine triphosphate (ATP). As inferred by Faraday’s law, electron movement within cells will induce an electromagnetic field (EMF) emanating from both plant and animal cells.

In this regard, a recent report from Corsini et al. [1] stated, “To our knowledge, no one has yet detected the electromagnetic field from a plant.” These investigators made measurements with a sensitive atomic magnetometer on the flowering plant, Titan arum (Amorphophallus titanium). The EMF at the surface of the plant was approximated to be as high as 0.6 µG. In the first report of EMF measurements made from the human heart, Baule and McFee [2] used two large coils placed over the chest, to cancel ambient magnetic interference. Better resolution and less noise was achieved by Cohen and his associates who recorded EMFs from the brain [3] and the heart [4, 5] using a superconducting quantum interference device (SQUID).

In the present study we hypothesized that a solution containing paramagnetic iron (Fe3+) oxide particles (mean particle size, 2000 nm or mean particle size, 200 nm) could be applied to plants or parts of plants as a means of imaging their EMFs.

 

Methods

Preparation of Iron containing Solution

A fine iron particle solution was prepared by mixing several grams of powdered iron filings (Edmond Scientific, Co., Tonawanda, NY) in 200 cc of deionized water (resistivity, 18.2 MΩ.cm). After standing for several hours aliquots of the supernatant were subjected to homogenization (Brinkmann Instruments Co., Sybron Corporation Westbury, NY) for 10 min. These samples were  centrifuged  for  20-25 min  and the  upper third of the supernatant was carefully decanted for sizing of the iron nanoparticles.

The particle size and distribution of the nanoparticles from the supernatant of the initial and from the centrifuged solution were determined using dynamic light scattering (DLS) and the zeta potential using phase analysis light scattering by a Zeta potential analyzer (ZetaPALS,  Brookhaven Instruments Corp, Holtsville, NY). For sizing, 1.5 ml of the solution in de-ionized water was scanned at 25 °C and the values obtained in nanometers (nm). A similar aliquot of the fine iron particle solution was scanned for 25 runs at 25 °C. for determining zeta potentials. Zeta potential values were displayed as millivolts (mV).

 

Imaging Procedures

Protocol 1. Direct Application of Iron Containing Solutions to Leaves

Seeds of the Genus and species, Vigna radiata were germinated in tap water for 2 weeks. Oklahoma City treated water contains 0.3 ppm [6]. Mature green leaves were carefully cut from the plants. To avoid any metal contact with the leaves wooden applicator sticks were used to transfer the leaves to clean glass slides. Using a transfer pipette, several drops of a solution containing aliquots of the iron (mean particle size, 200 or 2000 nM) and a solution of Prussian Blue stain (PBS,2.5%  potassium ferrocyanide, 2.5% hydrochloric acid) was added to the two leaves on the slide. One leaf was placed with the upper surface in contact with the slide and the other leaf with the underside of the leaf in contact with slide surface. A second glass slide was applied to hold the leaves in place.

Similarly, two leaves were prepared with deionized water (resistivity, 18.2 megohms) as the applied solution (no iron or PBS) between slides which served as a control set.

These slides were allowed to stand until the liquid between the slides had essentially dried (at least 48 hours). The specimens were then examined under a microscope and microphotographs were made of examples from each group.

 

Protocol 2. Separation of Leaves and Iron Containing Solution

Mature green leaves were cut from the plants and transferred to glass slides, as above. A second glass slide was added to hold the leaves in place. Using a transfer pipette, several drops of a solution containing aliquots of the iron solution (mean particle size, 200 nM) and a solution of Prussian Blue stain (2.5% potassium ferrocyanide,

2.5% hydrochloric acid) was “sandwiched” between two thin glass coverslips. This sandwich of the glass slides was then added to cover the slides enclosing the leaves.

Two leaves were prepared similarly but with deionized water as the substitute for the iron particle solution applied between the coverslips that were placed over the glass slides enclosing the leaves.

These slides were allowed to stand until the liquid between the slides had essentially dried (≥48 hours). The specimens were then examined under a microscope and microphotographs were made of examples from each group.

 

Figure 1. Microphotographs of leaf images. Panel A. An example of the formation of aggregated iron particles aligned with the leaf edge as a uniform wavefront and surrounding the larger trichomes (arrows) Magnification 40X. Panel B shows a dense cloud of particles at the leaf edge, after treatment with iron particles (2000 nm) and Prussian blue staining solutions magnification 40X. Panel C. A leaf treated with deionized water shows a clear field at the leaf edge. Magnification 40X

 

Protocol 3. Magnetic Resonance Imaging

Mature green leaves (n=6) were cut from the plants and placed in a solution of iron nanoparticles (200 nm) for 24 hours. A similar number of leaves were placed in deionized water for 24 hours, as well. The leaves were transferred to a specially constructed plexiglass holder that was machined to be inserted into a small animal Magnetic Resonance Imaging (MRI) unit. Each of the leaves was covered with a glass slide and a plexiglass plate to hold them in place during the imaging procedure.

 

Statistical Analysis

Data was represented as the mean ± the standard deviation (STD). An unpaired student T-test was used to compare specific categories determined in the MRI experiments. For example T-2 values of the leaves upper surface was compared between the experimental and control groups. A p-value of ≤0.05 was considered significant.

 

 

Figure 2. Microphotograph of interior leaf veins before (Panel A) and after treatment with iron particles and Prussian Blue staining solutions (Panel B). A. The leaf vein shows the cellular structure of xylem and phloem vessels as well as adjacent leaf parenchyma (green color) Magnification 40X. B. After iron particle treatment, lignin striations comprising the cellular “braces” maintaining the integrity of xylem cells in the leaf vein are readily visualized (between arrows). Also note the darkened adjacent atria tissue. See text for discussion. Magnification 40X.

 

 

Figure 3. Images made of leaf parts with solution of iron particles and Prussian Blue stain not in direct contact with leaves treated with these same solutions. Panel A shows outlines of the leaf edge and trichomes in the form of aggregated iron particles. Magnification 40X. Panel B shows the leaf interior with outlines of the network of veins and an area of atria tissue (arrow). See text for further discussion. Magnification 40X.

 

 

Results

Protocol 1:

Figure 1 illustrates the micrographs of images obtained from a leaf exposed to the iron particle solution (2000 nm)  with added Prussian blue staining solution 24-48 hours after initially prepared as described in Protocol 1, above. Panel A shows a relatively uniform wave of aggregated iron particle closely associated with the leaf edge and leaf hairs called trichomes (arrows) extending from the leaf edge. Panel B illustrates the aggregated iron particles as a cloud surrounding the trichomes and leaf edge.

Panel C. is a representative portion of a leaf that was treated with deionized water and viewed at a similar time interval after preparation. No particle cloud was observed at the leaf edge or surrounding the trichomes.

Figure 2. Panel A. shows the interior or the untreated leaf, specifically the large vein with the well delineated cellular structure of the xylem cells. Also note the trichomes extending from the vein indicating that the leaf hairs are present in the interior of the leaf as well as on the leaf edges. Panel B. In the leaves treated with the iron particle solution, there was a characteristic display of closely spaced striations running perpendicularly to the longitudinal arrangement of the xylem cells (between arrows). Note, the difference in the green color of the plant tissues adjacent to the vein in the untreated specimen, (panel A) compared to the dark color of the tissues adjacent to the treated leaf (panel B). The striations and the color difference provide biomarkers for the presence of the iron particles in the treated leaves as discussed below.

 

Protocol 2:

In this group, the leaves were not in contact with the bathing solution since the solution containing iron particles and the Prussian blue staining solution were enclosed in a slide “sandwich” placed onto and in close proximity to the glass slides containing the leaves. Figure 3A shows the leaf edge and trichomes as imaged by the surrounding aggregated iron particles. In figure 3B, images of the veins in the leaf interior are clearly delineated. Occasional patches of leaf parenchmyma were also imaged (arrow).

 

Protocol 3:

Table 1 is a compilation of the data (n=6) obtained by the MRI studies of the leaves treated with iron nanoparticle solution compared to those immersed in deionized water for the same 24 hour period. The absolute T2 data (Table 1) was normalized (Table 2) using the average T2 value for pure water (2000 ms) that was determined in our experiments. In all categories, upper leaf surface, lower leave surface and upper surface central vein and lower surface vein, there was a significant difference (p≤0.0001) in the loss of T-2 values in the experimental group (iron particle displacement of water) showing approximately half the T-2 values as the control group (pure water content).

 

 

Figure 4. Figure 4 A illustrates the image obtained from layering 2 leaves which were treated. Two separate linear images were observed (arrows) outlining the leaf edge and some of the trichomes. Magnification 10X. Figure 4 B shows the result of layering 4 leaves. The arrows indicate 4 separate linear aggregates of iron particles associated with each of the 4 layered leaves. Magnification 10X.

 

Ancillary Experiments

Another series of experiments (n= 6) were performed to provide further supportive evidence for our imaging studies. We layered either 2 or 4 leaves each onto 3 clean slides and treated  each separately with a mixture of Prussian blue stain (2 parts) and iron particles (2000 nm, 3 parts). Figure 4 A illustrates the image obtained from layering 2 leaves which were treated. The leaf section shown was close to the leaf base. Two separate linear images were observed (arrows) delineating the leaf edge and some of the trichomes. Figure 4 B shows the result of layering 4 leaves. The arrows indicate 4 separate linear aggregates of iron particles presumably associated with each of the layered leaves.

 

 

Table 1. A comparison of the absolute values of T2 (ms) between the control group (leaves in deionized water) and the experimental group (leaves treated with iron nanoparticles)

Control Group (Absolute Values)

NO.

upper surface

lower surface

vein-upper surface

vein-lower surface

1

1124

1314

765

1403

2

1047

1182

819

1089

3

1003

1089

759

1031

4

854

1169

686

883

5

963

1635

758

720

6

1119

1630

860

1013

7

933

1328

702

895

8

1080

1186

654

720

9

1065

1755

759

927

10

1254

1106

850

1089

AVERAGE

1044

1339

761

977

STD

113

245

68

200

 

Experimental Group (Absolute Values)

NO.

upper surface

lower surface

vein-upper surface

vein-lower surface

1

521

630

340

456

2

517

531

317

401

3

570

655

336

464

4

452

681

425

427

5

395

880

378

339

6

364

580

351

483

7

357

566

322

442

8

373

505

343

458

9

391

617

284

455

10

366

578

408

430

AVERAGE

431

622

350

436

STD

79

105

43

41

p-value

<0.0001

<0.0001

<0.0001

<0.0001

 

 

Discussion

We expected at the outset that the EMF from a plant or plant part would be extremely small [1]. Using very small iron particles in solution we hypothesized that they would be attracted to a magnetic source and would aggregate as these paramagnetic particles themselves became little magnets. This hypothesis was confirmed with the imaging experiments (Figure 1 A, B) and the quantitative data from the MRI experiment. In the latter, the data suggested that the leaves treated with iron particles displaced water resulting in a significant T2 loss (p≤0.001) compared to leaves treated with deionized water (Table 1 and 2). Further support for this finding was the marked darkening of the leaf parenchyma and the appearance of the lignin striations on the xylem vessels. Compare Figure 2B, iron treated and Figure 2A, in which the leaf was untreated.

It could be argued that the attraction of the iron particles was due to the action of surface tension, particularly as the solution around the leaves dried. Also it has been shown that iron has an affinity to plant fiber, specifically to the lignin found in plant cell walls [7-10]. Indeed, the treatment of the leaves with iron particles revealed the lignin ‘braces’ known to maintain the integrity of xylem cells.  Xylem tubes, called tracheids have spiral bands which became lignified as plants evolved [11] to contend with higher levels of water pressure required to move water from the roots to the leaves. This iron affinity explanation notwithstanding, figures 3 A, B showed that the EMF images of the leaf edges and trichomes as well as interior structures, i.e., veins and leaf parenchyma, were transmitted to an adjacent “sandwich” of iron particles plus stain even though these paramagnetic iron particles were not in direct contact with the leaves. Furthermore, the character of these images were granular similar to the aggregated images of iron particles ingressing toward the leaf as seen in Figure 1 A, B.

 

 

Table 2. A comparison of the normalized values of T2 (ms) between the control group (leaves in deionized water) and the experimental group (leaves treated with iron nanoparticles)

Control Group (Normalized Values)

NO.

upper surface

lower surface

vein-upper surface

vein-lower surface

1

0.56

0.66

0.38

0.70

2

0.52

0.59

0.41

0.54

3

0.50

0.54

0.38

0.52

4

0.43

0.58

0.34

0.44

5

0.48

0.82

0.38

0.36

6

0.56

0.82

0.43

0.51

7

0.47

0.66

0.35

0.45

8

0.54

0.59

0.33

0.36

9

0.53

0.88

0.38

0.46

10

0.63

0.55

0.43

0.54

AVERAGE

0.52

0.67

0.38

0.49

STD

0.06

0.13

0.03

0.10

 

Experimental Group (Normalized Values)

NO.

upper surface

lower surface

vein-upper surface

vein-lower surface

1

0.26

0.31

0.17

0.23

2

0.26

0.27

0.16

0.20

3

0.29

0.33

0.17

0.23

4

0.23

0.34

0.21

0.21

5

0.20

0.44

0.19

0.17

6

0.18

0.29

0.18

0.24

7

0.18

0.28

0.16

0.22

8

0.19

0.25

0.17

0.23

9

0.20

0.31

0.14

0.23

10

0.18

0.29

0.20

0.21

AVERAGE

0.22

0.31

0.18

0.22

STD

0.04

0.05

0.02

0.02

p-value

<0.0001

<0.0001

<0.0001

<0.0001

 

Implications and Future Studies

The findings of the present studies using iron particles to image the EMF of leaves of plants provides impetus for the study of EMF properties of other plant parts as well as whole plants. Furthermore, the use of iron nanoparticle solutions (mean diameter, ≤100nm) may provide additional information on internal EMFs field within plants. A recent article by Boutilier et al. [12] demonstrated that xylem cells filter out particles at a cutoff point of 100 nm. A logical extension of these studies is the application of iron particle treatment to animals on the organism, organ, tissue and cellular levels. 

 

Limitations

One of the consistent observations during these studies was the variability of the images in regard to the sites along the plant margins at which images were found. In general, more images were detected at the leaf base where the major vein entered the leaf with fewer images found proceeding toward the leaf tip. It is interesting to note that early studies of leaf photosynthesis noted a non-uniform pattern of this essential function. Tereshima in 1992, [13] noted that non-uniform stomatal behavior was a critical factor underlying the mechanism for non-uniform photosynthesis across the leaf surface. This finding might explain the variability we found in the EMF images which were related to the level of electron transfer activity inducing the EMF associated with photosynthesis.

 

Conclusions

In the present study we demonstrated EMF images induced by direct and indirect treatment of live plant leaves of Vigna radiata with very small iron particles. Moreover, MRI studies allowed quantitative analysis of leaves treated with iron particles compared to those exposed to deionized water. There was a significant loss of T2 values indicative of the replacement of water by iron particles adherent to the treated leaves. Finally, layering of treated leaves provided evidence of EMF images associated with each of the layered leaves. On the basis of these findings, further studies in plants and animals with this technique seem appropriate.

 

Acknowledgements

We thank Andrea Moseley for help in the preparation of this manuscript and Jack Scherlag for his help with growing our plants.

 

Contribution of the Authors:

BJS conceived the experimental concept, performed the plant experiments and wrote the majority of the manuscript; LZ and BH prepared the solutions and performed the analysis of the magnetic resonance imaging (MRI) data; KS advised and performed the sizing of the iron particles; RT and NS provided tutorials and advice for the use of the MRI equipment and reviewed the analysis of the MRI data. AAE provided the studies of the indirect effects of EMFs. SSP edited the manuscript.


 

 


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__________

Conflict of interest: No conflicts declared.

Corresponding Author: Benjamin J. Scherlag, PhD. Heart Rhythm Institute, Oklahoma University Health Sciences Center, 1200 Everett Drive (6E103), Oklahoma City, OK 73104, USA.

Phone: 405-271-9696, ext. 37501; Fax: 405-271-7455

E-mail: benjamin-scherlag@ouhsc.edu

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