Tonicity induced changes in volume and refractive index of suspended cells quantified with digital holographic microscopy

The analysis of changes in the volume and intracellular solute concentrations of biological cells represents an important topic in cytometry. We present results from a comparative study in which the cell volume and the refractive index of living rat inner medullary collecting duct cells and human pancreatic tumor cells was determined in media with different tonicity by using quantitative digital holographic phase microscopy. The results show that the inverse cell volume correlates linearly with the integral cellular refractive index which is directly related to the concentration of intracellular solutes. The volume of biological cells depends mainly on the intracellular concentration of osmotic active compounds because the outer plasma membrane is highly permeable to water compared to solutes like Na + , K + , or Cl . A decrease of the extracellular tonicity leads to an increase of the cellular volume. An increase of the extracellular tonicity is followed by a decrease of the cell volume. Factors and underlying mechanisms for the regulation of the cell volume maintenance are summarized e.g. by Hoffmann et al. [1]. Beside other factors the activation of several transport proteins is involved in the cell volume regulation. These include Cl channels, K + channels or proteins participating in the osmolyte release pathway. Prominent transporters belong to the family of Na + /H + exchangers (SLC9), Na + -K + -2Cl – co-transporters (SLC12) or to transporters involved in the uptake of organic osmolytes [1]. To reveal the function of such transporters in cell volume regulation the cellular volume and the density of intracellular solutes has to be quantified. However, simultaneous measurement of these parameters is a challenging and a variety of experimental approaches have been proposed. For example, cell volume changes were detected utilizing fluorescent probes [2], laser scanning microscopy [3] and electronic sizing with a flow cytometer [4]. In contrast to [2-4], digital holographic microscopy (DHM) [5-8] enables minimally invasive live cell analysis by quantitative multi-focus phase contrast imaging without the need of labeling or complex sample * E-mail: bkemper@uni-muenster.de preparation. Furthermore, DHM provides not only access to the cell volume but also information about the refractive index of adherent [8-10] and suspended single cells [11-13], which is directly related to the intracellular solute concentration [14], as well as capabilities for refractive index tomography [15-18]. Thus, in this study we used DHM to analyze the absolute cell volume and the mean refractive index of primary cultured inner medullary collecting duct (IMCD) cells [19], which are capable to tolerate large changes in extracellular osmolalities. We also compared the obtained data to the results obtained from pancreatic tumor cells (PaTu 8988 T) [8]. IMCD cells were initially cultivated at 600 mOsmol/kg as described in [19]. The PaTu 8988 T cells were cultivated under typical cell culture conditions at 320 mOsmol/kg (for details see the description in [8]). For measurements both cell types were trypsinized and suspended to Petri dishes with a HEPES buffered medium. IMCD cells were investigated in MEM (Modified Eagle Medium) while PaTu 8988 T cells were observed in DMEM (Dulbecco’s Modified Eagle Medium). In order to stimulate the IMCD cells, starting from the initial medium of 600 mOsmol/kg equimolar amounts of NaCl and urea were added or the medium was diluted with deionized water in order to increase or decrease the osmolality. For hypotonic stimulation of PaTu 8988 T cells the initial medium of 320 mOsmol/kg was diluted by deionized water while investigations with hypertonic medium were performed with a solution generated by diluting a concentrated version of DMEM (10x). For quantitative DHM phase contrast imaging of the IMCD cells an inverted microscope (iMIC, Till Photonics, Gräfelfing, Germany) was used with an attached digital holographic add-on module as described in [120]. The PaTu 8988 T cells were analyzed with a self-interference DHM setup based on a Zeiss Axio Observer A1 microscope (Carl Zeiss MicroImaging GmbH, Germany) [21]. In both experimental setups the coherent light source for the recording of digital holograms was a frequency doubled Tonicity induced changes in volume and refractive index of suspended cells quantified with digital holographic microscopy B. Kemper, *1 J. Klokkers, 2 S. Przibilla, 1 A. Vollmer, 1 S. Ketelhut, 1 G. von Bally, 1 E. Schlatter, 2 H. Pavenstädt, 2 and B. Edemir 2 1 Center for Biomedical Optics and Photonics, University of Muenster, Robert-Koch-Str. 45, D-48149 Muenster,Germany 2 Department of Internal Medicine D, University of Muenster, Albert-Schweitzer-Campus 1, A 14, D-48149 Muenster, Germany Received May 25, 2012; accepted June 23, 2012; published June 30, 2012 doi: 10.4302/plp.2012. 2.02 PHOTONICS LETTERS OF POLAND, VOL. 4 (2), 45-47 (2012) http://www.photonics. pl/PLP © 2012 Photonics Society of Poland 46 Nd:YAG laser (Compass 315M-100, Coherent, Lübeck, Germany, λ = 532nm). Prior to each measurement with digital holographic microscopy cells were equilibrated for 5 min in the respective medium. Then digital off-axis holograms of cells with mainly spherical appearance were recorded. For each osmolality N=86-164 cells were recorded at room temperature (T = 22°C) during the following 20 min. A numerical reconstruction of digitally captured holograms was performed by spatial phase shifting reconstruction as described in [5, 8] combined with digital holographic autofocusing [22]. From the resulting quantitative phase images an average cell radius R and an average integral cellular refractive index ncell were obtained for each cell by two dimensional fitting of the sphere model as described in detail in [13]. Assuming a mainly spherical shape of the suspended cells an average cell volume was calculated from obtained cell radii R while a simultaneously obtained parameter ncell was used to characterize intracellular solute concentration.