Accepted Manuscript Analytical Methods Visualization of calcium and zinc ions in Saccharomyces cerevisiae cells treated with PEF (pulse electric fields) by laser confocal microscopy Pankiewicz Urszula, Jamroz Jerzy, Monika Sujka, Radosław Kowalski PII: DOI: Reference:

S0308-8146(15)00678-0 http://dx.doi.org/10.1016/j.foodchem.2015.04.121 FOCH 17522

To appear in:

Food Chemistry

Received Date: Revised Date: Accepted Date:

17 February 2015 20 April 2015 25 April 2015

Please cite this article as: Urszula, P., Jerzy, J., Sujka, M., Kowalski, R., Visualization of calcium and zinc ions in Saccharomyces cerevisiae cells treated with PEF (pulse electric fields) by laser confocal microscopy, Food Chemistry (2015), doi: http://dx.doi.org/10.1016/j.foodchem.2015.04.121

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Visualization of calcium and zinc ions in Saccharomyces cerevisiae cells treated

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with PEF (pulse electric fields) by laser confocal microscopy

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Pankiewicz Urszula*, Jamroz Jerzy, Monika Sujka, Radosław Kowalski

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Department of Analysis and Evaluation of Food Quality

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Faculty of Food Science and Biotechnology

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University of Life Sciences

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Skromna 8; 20-704 Lublin, Poland

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Running title: visualization of Ca, Zn in cells treated with PEF by microscopy

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*Corresponding author: Urszula Pankiewicz

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e-mail address:[email protected];

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Postal address: University of Life Sciences, Skromna Street 8; 20-704 Lublin,

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Poland, Phone: +48814623333; Fax: +48814623376

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ABSTRACT

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The aim of the present work was to visualize the areas of increased

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concentration of calcium and zinc ions inside Saccharomyces cerevisiae cells with

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the use of confocal microscopy and to make an attempt to asses semi-quantitatively

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their concentration within the limits of the cells. Semi-quantitave analysis revealed

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that fluorescence inside cells from control samples was three-times lower than that

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observed for cells from the sample enriched with calcium. Differences in distribution

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of fluorescence intensity between cells originated from the samples enriched with

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zinc and control samples were also observed.

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On the basis of the optical sections, the 3D reconstructions of ion-rich areas

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distribution in the cell were made. The obtained results showed that confocal

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microscopy is an useful technique for visualization of the areas in Saccharomyces

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cerevisiae cells which contain higher amount of calcium and zinc and it may be also

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used for semi-quantitative analysis.

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.

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Keywords: zinc, calcium, Saccharomyces cerevisiae, pulsed electric fields, laser

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confocal microscopy

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1. Introduction

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Zinc and calcium ions are very interesting because they have a positive effect on

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the respiratory activity and the growth rate of Saccharomyces cerevisiae. Zinc is

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required as cofactor and structural component in nearly 300 enzymes, including

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alcohol dehydrogenase (ADH), the terminal enzyme of the fermentation pathway (De

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Nicola & Walker, 2009). In the yeast Saccharomyces cerevisiae, zinc is estimated to

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be required for the function of nearly 3% of the proteome (Rebar & Miller, 2004).

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Zinc compromises yeast cell metabolism impairing protein synthesis and affects

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phospholipids composition of membranes (De Nicola et al., 2007; Iwanyshyn, Han,

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& Carman, 2004). Calcium ions are not necessary for the yeast growth, but they

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participate in the synthesis of cell structures and control protein-protein interactions.

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Calcium, being actively excluded from the yeast cell, acts mainly extracellularly, e.

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g. it is essential for α-amylase activity and phosphate precipitation, therefore playing

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a role in wort pH control (Pasternakiewicz & Tuszyński, 1997; Rees & Steward,

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1997). Accumulated metals, most often, are stored in the cell wall, cytoplasm and in

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inner structures of the cell. The majority of microorganisms absorbs metals by

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passive accumulation on the surface, binding in the cell wall (carboxyl, hydroxyl,

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amine, hydrogen sulphate, phosphate groups), and then in the next phase, active

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transport of the ion through the cell membrane to the cytoplasm (Avery & Tobin,

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1993; Vinopal, Ruml, & Kotrba, 2007). Metal ions adsorbed on the cells surface may

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next be a subject of intracellular bioaccumulation. This way yeasts produce metal-

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protein complexes called metaloproteins or more generally – bioplexes.

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Macroelements (e.g. zinc, calcium) adsorbed to proteins are available by human and

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animal organisms far better than their inorganic or organic salts. In the present work

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electroporation was applied for enrichment of yeast with zinc and calcium 3

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(Pankiewicz & Jamroz, 2011, 2013). Electroporation is one of the relatively easy and

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at the same time nontoxic and inexpensive methods for the introduction of specific

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macromolecules to the cytoplasm by PEF (pulse electric field) processing (Arronson,

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Rőnner, & Borch, 2005; Korolczuk, Mc Keag, Fernandez, Baron, Grosset, & Jeantet,

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2006; Pankiewicz & Jamroz, 2010).

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florescent complex with zinc (Zn) and is thus used to localize Zn in yeast cells.

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Several methods can be used to assess the uptake and accumulation of metal ions in

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plant cells. Bioaccumulation involved in the metal resistance of these protozoa has

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been made evident using transmission electron and fluorescence microscopies

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(Calderon-Miranda, Barbosa-Canovas, & Swanson, 1999; De Nicola & Walker,

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2009; Martin-Gonzalez, Diaz, Borniquel, Gallego, & Gutierrez, 2006; Suh, Kim,

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Yun, & Song, 1998; Suh, Yun, & Kim, 1999; Vinopal, Ruml, & Kotrba, 2007).

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However, the low sensitivity and poor spatial resolution of these staining techniques

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did not allow them to be used as tools for studying the cellular distribution of metal

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ions. Fluorophores such as morin and calcium orange are highly sensitive and can

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detect very low concentrations of Zn and Ca (Eggert, 1970; Eticha, Staβ, & Horst,

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2005; Kataoka, Likura, & Nakanishi, 1997). Morin is a pentaprotic acid that forms a

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highly fluorescent complex with Zn. The Zn-morin complex has excitation and

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emission wavelengths of 488 nm and 512 nm, respectively. Its florescence detection

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limit is as low as 2x10-9 M (Lian et al., 2003b) and thus morin is used along with

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florescence microscopy to sensitively localize Zn in plant cells. Calcium orange is

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one of the fluorescent calcium indicators. Its fluorescence excitation maximum is

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detected at 551 nm, whereas maximum emission is observed at 574 nm. Thanks to

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this property calcium orange can be used for determination of Ca2+ with the use of

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confocal microscopy.

Morin is a fluorochrome which forms a

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The aim of the present work was to visualize the areas of increased concentration of

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calcium and zinc ions inside Saccharomyces cerevisiae cells with the use of confocal

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microscopy and to make an attempt to asses semi-quantitatively their concentration

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within the limits of cells.

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2. Materials and methods

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2.1. Culture maintenance and inoculum preparation

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Saccharomyces cerevisiae 11 B1 (industrial strain) from the Yeast Plant (Lublin,

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Poland), was used. Medium for agar slants and inoculum growth (g/L): sucrose (20);

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NH4Cl (3.2); KH2PO4 (2.5); Na2SO4 (2.0); MgCl2 · 6H2O (1.5) (POCH, Gliwice,

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Poland); yeast extract (YE) (5.0); agar (15) (DIFCO, Detroit, MI, USA); and

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unhoped wort (40.0 mL) (Lublin Breweries S.A., Lublin, Poland) had

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Experimental medium for S. cerevisiae contained (g/L): peptone (10) (Sigma-Aldrich

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CO, St. Louis, MI, USA); YE (5); glucose (10) (POCH, Gliwice, Poland) (Blackwell,

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Tobin, & Avery, 1997).

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2.2. PEF treatment and enrichment with calcium or zinc

pH 5.

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The cultures were treated with PEF (ECM 830 electroporator, BTX Harvard

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Apparatus, USA) at optimum parameters for the maximum accumulation of calcium

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and zinc itself.

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PEF treatment chamber consists of four parallel plexiglas plates which have

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stainless steel electrodes of area equal 4 cm2, facing each other with a gap of 5 mm.

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The culture was agitated in a chamber during PEF treatment with a magnetic stirrer.

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The electrical conductivity measured for the treated samples was between 3.9 mS/cm

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- 7.36 mS/cm, temperature 25°C, and field frequency 1Hz. Electroporation of cell

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membranes conducted at optimized parameters: electric field strength (5 kV/cm), 5

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exposure time (20 min), the pulse width (20 µs) and the point of treatment in course

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of the growth of cultures (20 h), at the optimum 100 µg/ml calcium concentration in

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the medium, provided the highest level of the calcium accumulation in

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Saccharomyces cerevisiae (sample W). In the case of zinc, optimized parameters of

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electroporation for the highest level of accumulation were as follows: electric field

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strength (3.0 kV/cm), exposure time (15 min), the pulse width (10 µs) and the point

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of treatment in course of the growth of cultures (20 h), the optimum zinc

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concentration in the medium 100 µg/ml (sample Z). Samples not treated with PEF

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and without calcium or zinc in the medium (K1) or with 100 µg /mL calcium or zinc

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(K2) served as controls.

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2.3. Determination of the calcium or zinc concentration

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Calcium or zinc were determined using the method of flame atomic absorption

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spectrometry (FAAS, VARIAN AA 280 FS). Samples were conducted as follows:

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250 mg samples of lyophilized calcium- or zinc-enriched yeast were weighed into

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glass thimbles, and 3 ml of HNO3 was added. Determination by the FAAS method

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was carried out according to the Norm PN-EN 14082:2004. A standard curve was

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plotted with the following concentrations: for Zn 0.5, 1, 2, 5, 10, 15 and 20 mg/l; and

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Ca 5, 10, 20, 40, 60, 80, 100 mg/l (determination coefficient was equal 1). Samples

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were digested in a MARS5 (CEM Corporation) microwave oven at 200°C for 30

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min. After cooling, solutions were transferred to 50 ml measuring flasks and topped

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up with deionized water.(Pankiewicz & Jamroz, 2011; Pankiewicz & Jamroz, 2013).

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2.4. Data analysis

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Significant differences between particular groups were found involving the Student

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t-test applied to compare independent samples in pairs, and variance analysis 6

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(ANOVA) was used for more than two groups. Statistical processing of results was

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performed using Statistica 6.0 software.

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2.5. Yeast staining with calcium orange

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Stock solution of the indicator (2 mM) was prepared according to the producer’s

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recommendations by dissolving calcium orange in anhydrous DMSO. 0.5 ml of yeast

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cells was transferred to an eppendorf tube and then 0.5 ml of PBS (phosphate –

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buffered saline) was added. Homogenous suspension of cells was obtained by

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vortexing of a sample for a few seconds. 498 µl of the suspension was combined

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with 2 µl of 2 mM solution of the indicator resulting in a concentration of 8 µM.

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During staining of yeast cells, which was carried out for 30 min. at 20ºC, the sample

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was vortexed three times.

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2.6. Yeast staining with morin

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30 mg of morin hydrate was dissolved in 1 ml of methanol to obtain 0.1 M

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solution. The final concentration of morin (200 µM) was obtained by adding

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respective amount of the solution to the yeast suspension. Because there is a lack of

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information concerning a precise concentration of morin, two-fold higher dose was

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applied than in the case of staining procedure for stem’s growing point. Yeasts from

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the culture were transferred to 1 ml PBS at 20ºC and mixed. 20 µl of morin solution

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was added to yeast suspension. Cells were stained for 20 min. at 20ºC in darkness.

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2.7. Sample preparation for microscopy

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About 100 µl of yeast suspension stained with calcium orange or morin was placed

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on a cover slip and covered with another one. Typical arrangement: microscope slide

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– cover slip were not used to avoid undesirable phenomena connected with laser light

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scattering by a thick slide.

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2.8. Microscopic observations and microphotograph documentation

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Confocal laser scanning micrographs displaying distribution of calcium orange and

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morin in yeast cells were acquired on an Olympus FV1000 microscope (Olympus

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America Inc., Melville, New York) at magnification 1000x. calcium orange is a dye

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which fluoresces in the presence of Ca2+ ions with the maximum at 576 nm after

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excitation with wavelength of 549 nm, whereas zinc-morin complex shows green

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fluorescence after excitation with 488 nm laser light. Documentation was prepared

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using adjustments of excitation source and filters according to the specification for

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FITC. The techniques of two- and three-dimensional laser scanning were applied.

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The 2D scans were taken based on fluorescence as well as transmitted light. The 3D

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image reconstructions of cell areas rich of ions were made on the basis of optical

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sections.

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3.

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3.1. Accumulation of zinc and calcium by Sacchromyces cerevisiae

Results and discussion

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Treatment of the cells with pulsed electric field (PEF) is called electroporation or

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electroperforation and is based on an action of alternating current on the cells. In the

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cell treated with PEF, the induced transmembrane potential causes pore formation in

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the membrane and leads to an increase of its permeability. According to the model

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proposed by Zimmermann (1986) charges of opposite sign are induced by the

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electric field on the outer and inner surface of the cell membrane. Once the

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transmembrane potential reaches the critical value, bilateral attraction of charges

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leads to the formation of the large number of pores. Permeability of the cell 8

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membrane can increase to the level that allows such molecules as DNA or metal ions

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to enter the cell. When an action of the electric field stops, pores are sealed and the

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cells retain introduced molecules or ions.

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The cell membrane loses its continuity when the membrane potential exceeds

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0,5-1,0 V. Electroporation can be a reversible process when pores are sealed again. It

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depends on intensity and exposition time of the external field. Process is reversible

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when electric impulses of the field intensity in the range 1 - 20 kV/cm last from

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micro- to milliseconds. Pores open during a few microseconds and close after various

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time, depending on temperature, from a few seconds at 37°C to several minutes at

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4°C (Torrgerosa, Esteve, Frigola, & Cortes, 2006). When the electric field intensity

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exceeds considerably the critical value, pore formation can be irreversible and leads

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to destruction of a cell.

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Our previous studies showed that optimization of particular PEF parameters and ions

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concentrations in the medium caused 2-fold increase in accumulation of magnesium

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(Pankiewicz & Jamroz, 2010) and zinc (Pankiewicz & Jamroz, 2011) ions and 3.5-

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fold higher accumulation of calcium ions (Pankiewicz & Jamroz, 2013) in the cells.

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In the case of ion couple, accumulation of magnesium and zinc was, respectively,

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1.5-fold and 2-fold higher in comparison to the control cultures (Pankiewicz, Sujka,

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Włodarczyk-Stasiak, Mazurek, & Jamroz, 2014).

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Optimized electric field strength (5 kV/cm), exposure time (20 min), the pulse

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width (20 µs) and the point of treatment in course of the growth of cultures (20 h), at

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the optimum concentration of 100 µg Ca2+/mL medium provided the highest level of

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the calcium accumulation in Saccharomyces cerevisiae - 3 mg/g d.m. The control

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culture K1 without calcium supplementation and PEF treatment accumulated calcium

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on the level of 0.16 mg/g d.m. The supplementation with calcium without the PEF

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treatment (control K2) provided an increase in accumulated calcium up to 0.5 mg/g

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d.m. Under optimized conditions (15 min exposure of the 20 h grown culture to PEF

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of the 1500 V and 10 µs pulse width) accumulation of zinc in the yeast biomass

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reached maximum 15.57 mg/g d.m. Under optimum zinc concentration (100 µg/mL

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nutrient medium), its accumulation in the cells was higher by 63% in comparison to

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the control (without PEF). In the control K1 which was not supplemented with zinc

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and not treated with PEF, zinc accumulation was on the level 0.5 mg/g d.m.

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Statistically significant increase of zinc accumulation was observed in the case of the

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control K2 which was supplemented with the total dose of zinc but was not treated

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with PEF. Simultaneously, the level of accumulation (8.16 mg/g s.s.) was lower than

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in the case of the media treated with PEF.

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3.2. Microscopic analysis of Sacchromyces cerevisiae cells enriched with calcium.

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Yeasts from the sample K1 (Fig.1. A and B; Fig. 2 A) were cultured in the medium

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not containing calcium and not treated with PEF. Nevertheless fluorescence caused

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by the presence of calcium orange inside cells was observed. A fluorochrome

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calcium orange is characterized by a small difference in fluorescence intensity

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between the free form and the form connected with calcium ions. Ratio FCa2+|Ffree is

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only 3:1 so it is possible to observe fluorescence also in the case of the control

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sample. Semi-quantitave analysis revealed that fluorescence inside cells from K1 was

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three-times lower than that observed for cells from the sample W, which is consistent

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with information given by the producer.

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At magnification 1000x numerous granules filled with a dye can be observed.

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The 3D reconstruction revealed that intensity of fluorescence between granules was 10

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distinctly lower. The areas of higher concentration of a dye were mainly localized at

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the edge of cells. Quantitative analysis of fluorescence intensity performed for yeast

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cells from this culture showed the lowest results among all investigated cultures.

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Yeasts from the sample K2 (Fig.1. C, D and Fig. 2. B) were cultured in the

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medium containing increased concentration of calcium. Granules in the cytoplasm

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were less visible when compared with the control culture. Fluorescence intensity

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distribution in the cytoplasm was more regular and granules were larger than these

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observed in the cells from K1. Their number was also significantly lower (a few per

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a cell). In some cells a dark area, probably a vacuole, began to appear. Result of

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quantitative analysis of fluorescence intensity was similar to this obtained for the

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sample K1.

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Yeasts from the sample W (Fig.1. E, F and Fig. 2. C) showed higher intensity

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of fluorescence in the cytoplasm in comparison to cells from the samples K1 and K2.

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Luminosity was very uniform, granules were very large and rare (0-2 per a cell). A

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dark area (probably a vacuole) was not visible in the majority of cells which suggests

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permeating of calcium ions through the cell membrane and tonoplast.

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3.3. Comparison of fluorescence distribution in yeast cells from the samples K1, K2

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and W

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When higher number of cells is available it is possible to analyze fluorescence

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intensity inside them. Such analysis was carried out for 173 cells from K1 (control),

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108 cells from K2 and 109 cells from W. Based on the observations, four modes of

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fluorescence intensity distribution in a cell were identified (Fig.3.).

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In the first case (Fig. 3aA) fluorescence is visible in a whole cell and any granules or

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dark vacuoles are not observed. As it is seen in the second image (Fig. 3aB),

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fluorescence intensity can also be distributed in the cytoplasm with distinctly visible

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dark area of vacuole and without granules. The third mode (Fig 3aC) is characterized

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by intensity of fluorescence regularly distributed in entire cell, brightly shining

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granules and lack of vacuoles. And finally, this phenomenon can be observed in the

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cytoplasm and shiny granules as well as a dark vacuole are well visible (Fig. 3aD).

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Cellular granules can be considered as mitochondria, peroxisomes or transport

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vesicles. The highest percentage of cells with regularly distributed intensity of

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florescence was observed in the sample W. It could be related to the increase of

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permeability of the tonoplast and cell membrane by an electrical impulse.

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Consequently calcium ions were absorbed and distributed uniformly all over a cell.

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The largest percentage of cells with uniform localization of fluorescence intensity

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was observed in the sample treated with PEF (W) (Fig. 3b).

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3.4. Microscopic analysis of Sacchromyces cerevisiae cells enriched with zinc

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Microphotographs presented in Fig. 4 show differences in distribution of

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fluorescence intensity between cells originated from the samples Z and K2. Intensity

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of fluorescence detected for the cell wall was very low, but higher than background

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(Fig. 4H). Such fluorescence can be observed in the images of the samples Z and K2

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within the limits of cells not showing fluorescence in the cytoplasm (pointed out with

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arrows).

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Fig. 5 presents a single optical section of a yeast cell. Non-uniform distribution of

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fluorescence intensity in yeast cells noticeable in the images A and D resulted from

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different localization of fluorescent areas in the cytoplasm and was not related to the

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cell wall staining. Dark areas in the images of some cells may be considered as

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vacuoles.

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3.5.

An attempt to asses distribution of zinc ions in cells

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The aim of the analysis of fluorescence intensity profiles (results presented in

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Fig. 6), which was performed for a single cell originated from each of the samples,

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was only to show potentiality of this method in determination of zinc distribution in

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yeast cells.

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In our opinion, confocal laser scanning microscopy can be suitable for visualization

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of the areas of increased calcium and zinc concentration inside yeast cells. After

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taking into consideration some remarks, this technique also can be used as a one of

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the methods of semi-quantitative analysis of ions concentration inside a cell. In

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general, the characterization of biosorbents by scanning electron microscopy (SEM)

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offers topographical and elemental information of the solids with a virtually large

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depth of field, allowing different specimen parts to stay in focus at a time. SEM also

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has high resolution, making higher magnification possible for closely spaced

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materials (Gupta & Rastogi, 2008). The ability of transmission electron microscopy

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(TEM) to provide information on crystalline structures as well as density maps that

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reach subatomic resolution is of great interest and widely applicable for the

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characterization of miscellaneous biomass (Han, Wong, & Tam, 2006; Srivastava &

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Thakur, 2007)

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De Nicola and Walker (2009) show, using fluorescent probes, that zinc localized

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predominantly in the yeast vacuole. Therefore, higher zinc cell content accumulated

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by larger (mother) cells may be related to larger vacuolar size in such cells. The 13

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change morphology of S. cerevisiae during the process of Pb2+ accumulation was

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observed by transmission electron microscope (TEM) (Suh, Kim, Yun, & Song,

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1998; Suh, Yun, & Kim, 1999). Prior to accumulation there was essentially no Pb2+

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on the cell surface, cell membrane, and in the cytoplasm. After about 30 min, Pb2+

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appeared on the cell wall and membrane but not in the cytoplasm. As the Pb2+

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accumulation proceeded, Pb2+ penetrated into the inner cellular parts, and

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consequently plasmolysis of the cell was often observed after 24 h. The black spots

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were proved as Pb2+ precipitates using an energy dispersive X-ray analysis (EDX).

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Martin-Gonzalez et al. (2006) attempted to confirm the existence of heavy metal

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bioaccumulation in the ciliate cytoplasm using two types of methods, fluorescence

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and transmission electron microscopy. In all ciliates exposed to Zn, several

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fluorescent cytoplasm granules (putative metal accumulations) were detected. For

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instance, U. nigricans BQ2 treated with 50 mg Zn/L for 24 h showed quite high

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putative cytoplasmic metal deposits with regard to the control.

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Several authors used morin to study the cellular distribution of Al. However, results

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are conflicting with regard to the major cellular site of Al accumulation. Ahn et al.

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(2002) observed Al-morin fluorescence in the cell wall of squash root apices after 3 h

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of Al treatment, whereas Vitorello and Haug (1996) did not see any fluorescence in

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the cell wall of cultured tobacco cells. They observed Al-morin fluorescence in the

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cytoplasm in a discrete zone of the cell periphery. Similarly, Tice, Parker and

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DeMason (1992) observed Al-morin fluorescence, particularly in the cytoplasm and

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the nucleus and less in the cell wall of wheat root tips. The objective of Eticha, Staβ

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and Horst (2005) work was to investigate whether Al localization with morin

328

staining can show the proper cellular distribution of Al. Fresh root cross-sections

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were made from root apices of maize (cv.Lixis) treated with 25 µM Al for 6 h and 14

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stained with morin. Fluorescence microscopic investigation shoved Al-morin

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fluorescence in the cytosol, but not in the cell wall. This is in contrast to the growing

332

evidence which shows that Al mainly accumulates in the cell wall, especially bound

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to the pectin matrix. Results from fluorescence microscopy suggest that this

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methodology is only useful for locating cytoplamic metallic deposits when cells are

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exposed to rather high heavy metal concentrations and metallic accumulations inside

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ciliates are rather large.

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4. Conclusion

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The obtained results showed that confocal microscopy is an useful technique for

339

visualization of the areas in Saccharomyces cerevisiae cells which contain higher

340

amount of calcium and zinc. It may be also used for semi-quantitative analysis of

341

their concentration in a cell. Our studies revealed that fluorescence inside cells from

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the control samples was lower than that observed for cells from the sample enriched

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with calcium or zinc.

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Acknowledgments This study was supported by a grant N N312 689840 awarded for the years 2011-2014 from the Polish Ministry of Science and Higher Education.

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Figure captions:

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Fig. 1. Images of cells presenting differences in fluorescence intensity and

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fluorescence localization in yeast cells from the investigated samples. A and B -

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yeast cells from the sample K1 (without calcium and PEF treatment), C and D - yeast

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cells from the sample K2 (with calcium, not treated with PEF), E and F: yeast cells

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from the sample W (with calcium and PEF treatment).

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Fig. 2. Comparison of fluorescence localization in yeast cells from all samples.

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Images: A - yeast cells from the sample K1 (without calcium and PEF treatment); B -

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yeast cells from the sample K2 (with calcium, not treated with PEF); C - yeast cells

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from the sample W (with calcium and PEF treatment).

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Fig. 3. a) Different modes of fluorescence intensity distribution in a cell. b)

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Percentage of cells with various localization of fluorescence intensity.

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Fig. 4. Microphotographs of yeast samples obtained in the range of green light. A, B,

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C – sample Z (with zinc and PEF treatment).; D, E, F – sample K2 (with zinc, not

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treated with PEF) and G, H, I - sample K1 (without zinc and PEF treatment). Images

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C, F and I present combined microphotographs of the same area taken in green light

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range and with the use of Nomarski contrast.

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Fig. 5. 3D images of a single optical section (thickness 1,1 µm) of yeast cells

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obtained in the range of green light emitted by zinc-morin complex. A and B: sample

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Z (with zinc and PEF treatment); C and D: sample K2 (with zinc, not treated with 20

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PEF); E and F: sample K1 (without zinc and PEF treatment). Images were analyzed

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using IMARIS (Bitplane, Inc.) software.

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Fig. 6. Fluorescence intensity profiles in a single cell taken along the yellow line.

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Gray value means a value of fluorescence intensity in 8 bit scale. Yeas cell originated

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from the culture: enriched with zinc and treated with PEF (A), enriched with zinc and

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no treated with PEF (B), without zinc addition and PEF treatment (C).

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Figure(s)

●Method of supplementation of yeast with Ca and Zn to obtain bioplexes is proposed ●Ions concentration and their mutual interactions affect accumulation in cells ●Optimized PEF parameters provide the highest level of the Ca2+ and Zn2+ in yeast ●Parameters of PEF and concentration of Ca and Zn affect biomass and cell viability

Visualization of calcium and zinc ions in Saccharomyces cerevisiae cells treated with PEFs (pulse electric fields) by laser confocal microscopy.

The aim of the present work was to visualize the areas of increased concentration of calcium and zinc ions inside Saccharomyces cerevisiae cells with ...
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