In our previous studies we developed a novel method for imaging the electromagnetic fields generated by plant and animal tissues^[1-4] using a solution containing fine iron particles and a Prussian blue stain (PBS) for iron. We have also shown that these electromagnetic fields can pass through glass^[5] (Figures 3 and Figure 5), as well as other types of barriers^[6] to be captured using the same imaging technology. In the present report we applied the PBS, fine iron particle methodology applied to samples taken from a culture of the single cell plant, Euglena gracilis.

Methods

Preparation of Solutions
   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 the supernatant was carefully decanted for sizing of the iron particles. The particle size and distribution of the particles from the supernatant was determined using dynamic light scattering and the zeta potential using phase analysis light scattering by a Zeta potential analyzer (ZetaPALS, Brook haven Instruments Corp, Holtsville, NY). For particle sizing, 1.5 ml of the solution in de-ionized water was scanned at 25°C and the median size of the particles was determined to be 2 microns. A similar aliquot of the fine iron particle solution was scanned for 25 runs at 25°C, to determine the zeta potential measured in millivolts (mV), the zeta potential for these particles was between +5 and -5 millivolt, in the hyper-aggregable range. A solution having diamagnetic properties was prepared by mixing aliquots of a 2.5% Potassium Ferrocyanide solution (K₄Fe(CN)₆ and a 2.5% hydrochloric acid solution (HCl). Also added were two parts of the fine iron particle solution. The composite solution is abbreviated throughout the manuscript as “PBS Fe2.”

  For ancillary studies a similar solution with paramagnetic properties was prepared by mixing aliquots of a 2.5 % Potassium Ferricyanide solution (K₃Fe(CN)₆ and a 2.5% solution of HCl. Also added were two parts of the fine iron particle solution. The composite paramagnetic solution is abbreviated throughout the manuscript as “PBS Fe3.”

Procedures
  Using a clean glass slide (25 mm x 75 mm x 1 mm width) as a base, a clean coverslip (24 mm x 50 mm x 0.017 width) was placed onto the base. Using a transfer tube, a drop of media containing Euglena gracilis, a one-celled mobile plant (Carolina Biological Supply, Co., Burlington, NC) was applied to the middle of the coverslip and a second cover slip applied to create a “sandwich” (SDW). Another coverslip SDW containing either the PBS Fe2 or PBS Fe3 solution was then applied offset to the first so that part was in direct contact with the underlying SDW (active zone) whereas a part of the second SDW was in the area of non-contact (control zone).

  Area measurements of the actual encysted cells in the evaporated deep well slides were compared to the areas of the encysted cells imaged through the glass barrier separating the two coverslips. We used the Image Pro-plus histomorphometry software program (Media Cybernetics, Inc., Rockville MD) to compare the area of several of the actual encysted cell randomly selected from the evaporated deep well slides of 4 different experiments (21 values) with those from the cells imaged though the glass barrier from 6 of the coverslip experiments (17 values).

Statistical Analysis
  Means of the areas of the actual encysted cells seen in the evaporated deep well slides were compared with those obtained through the glass barrier separating the paired coverslip using an unpaired T-test. A p value of 0.05 was considered significant.

Results

Figure 1: A microphotograph illustrated the encysted cells taken at 24 hours after the culture media had evaporated in the uncovered deep well slide. Most of the cells became rounded or elliptical in shape while still retaining their green coloration. Magnification 10X.

Figure 2: shows the images of the encysted cells in the second SDW containing the PBS Fe 2 or PBS Fe 3. Magnifications 20X.

Figure 3: In the PBS solution, the replicate images showed small crystals and/ or iron aggregates which clustered around the replicate cells. Magnification 40X.

Figure 4: In the PBS Fe3 solution the perimeter of the replicate images were directly contacted by different sizes and shapes of Fe3 crystals. Magnification 20X.

Figure 5:

Table 1: The comparison of the area of 21 actual encysted cells randomly selected from the evaporated deep well slides with 17 cells imaged though the glass barrier from the coverslip experiments. There was no significant difference between the two groups, p = 0.4.

Discussion

Major findings
   In the present study, living unicellular plant cells which undergo an encysted state in unfavorable environments, i.e., liquid culture evaporation, were enclosed in an SDW with a second SDW overlying the first. The second SDW contained either a diamagnetic, PBS Fe2^[7] or paramagnetic, PBS Fe3^[8,9] solution. We recorded replicate images that passed through a 0.034 mm glass barrier between the two SDWs. There was no significant difference in the areas encompassed by the replicate images of the encysted cells and those of the actual encysted cells observed in deep well slides after the culture media had evaporated.

Background
  The development of a novel and simplified method for the detection of electromagnetic signals^[1] emitted by electron transport during photosynthesis and cellular respiration of living plant an animal tissues has been described in several recent reports from our group^[2-6]. Furthermore we have previously reported that a living animal tissue, i.e., human hair follicle, interacts differently when exposed to a diamagnetic (Ferrocyanide) or a paramagnetic (Ferricyanide) solution. Specifically, electromagnetic energy emanating from hair follicles or actual magnet fragments caused surrounding ferricyanide crystals and incorporated iron aggregates of the evaporating solution to adhere to the follicle; whereas, the ferrocyanide crystals and incorporated iron aggregates were deposited at a distance from the follicle and shaft^[10]. In the present study the images that appeared through the glass barriers also displayed crystallization. This was more prominently seen in the replicate images developed in the paramagnetic, ferricyanide solution in which relatively large crystals were adherent to the cell images.

Implications
  If in fact, the crystallization evidence, albeit indirect, suggest that the replicate images are inherently electromagnetic, several questions arise:
1. Are these virtual images of the Euglena cells in a vegetative but still living state?
2. Could these images still be captured at some distance after passing through the glass barrier?
3. Can these replicates be revitalized in the same way as the encysted cells seen in the deep well slides after evaporation caused them to encyst. Further experiments may provide evidence to address some of these questions.

Limitations
  No direct measurement of electromagnetic fields emanating from these actual encysted or replicate plant cells has been performed. Others have used a highly sensitive atomic magnetometer to measure the electromagnetic field emanating from a giant plant^[11]. Their result was on the order of 0.6 Gauss. It would be expected that detection of the biomagnetic signal emitted by single cell plants, even in clusters would be immeasurably smaller.

Conclusion

  In the present study we were able to consistently demonstrate that replicate images of the single cell plant, Euglena gracilis, can be retrieved through a glass barrier. Evidence was presented suggesting that these replicate images had inherent electromagnetic properties.

Funding Source:
  An unrestricted grant from the Wilton and Helen Webster Research Foundation through the Oklahoma Research Foundation

Conflict of interest:
None declared.

References