Impedance-based tracking of the loss of intracellular components in microalgae cells

Highlights

• Impact analysis of intracellular density and distribution on the impedance signals.

• Characterization of cellular morphology by the low-frequency impedance detection.

• Intracellular phenotyping via the magnitude and shape of high-frequency impedance pulses.

Abstract

Cell staining is a typical procedure for assessing the distribution and density of intracellular components. In this work, a label-free alternative is developed and verified using impedance cytometry to characterize the loss of intracellular components in Euglena gracilis (E. gracilis) cells. By inhibiting chloroplast synthesis, the number of chloroplasts in single cells are reduced gradually, as is the density of intracellular components. As a result, low-frequency impedance signals (0.5 MHz) are shown to assess the cell morphology. With increasing voltage frequency (i.e., > 1 MHz), the resistance of the cell membrane lowers, and the cell membrane eventually becomes transparent to impedance detection. The magnitude and morphology of impedance signals (6 MHz) get the dependance on the density and distribution of intracellular components, respectively. Additionally, impedance-based cell phenotyping reveals that the shrinking of intracellular components and cell volume can cause two distinct declines in the high- and low-frequency electrical diameter of single cells, respectively. This conclusion is confirmed by simulation results and the time course of the changes in electrical diameters and electrical opacity. To sum up, our findings indicate that impedance cytometry and our analysis method can be further refined to serve as a powerful and non-invasive tool for assessing intracellular components at the single cell level.

Introduction

For a living cell, organelles, biomolecules, and cytoplasm all work together to control the cellular states and functionalities [1], [2], [3]. Within such a highly compacted system, the changes in intracellular densities are significant, as its effect on biophysical properties of cells such as macromolecular crowding, diffusion, mechanical stiffness, and phase transitions [4], [5], [6], [7], [8]. Although numerous approaches, such as live-cell imaging [6], [7], Raman flow cytometry [8], [9], [10], and chemical probes [11], [12], have shown the capability of detecting the changes in intracellular components, they are time-consuming, labor-intensive, and incapable of real-time analysis [13], [14]. In this study, we employed impedance cytometry [15], [16], [17], [18] to evaluate the changes in intracellular distribution and density of single cells, which is a simple and convenient method.

Impedance cytometry has the potential to characterize intracellular components of single cells in a label-free manner, which has been demonstrated by Xu et al. theoretically [19] using single-shell model. As the impedance of cell membrane is frequency-dependent, the cell shows high resistance to low-frequency electric field (typically < 1 MHz), resulting in the cellular morphology dependency of the low-frequency impedance signals [20], [21], [22]. Based on our past research [20], at low detection frequencies, impedance pulses show a tilt tendency for objects with asymmetric shapes, and the level of tilt is directly related to the level of asymmetry. By contrast, the impedance detection at high detection frequencies can characterize intracellular components [23]. The high-frequency electric field (typically >1 MHz) has the ability to pass through the membrane [24], [25]. The change in intracellular components would theoretically cause the change in conductivity at high detection frequencies. To date, although there are numerous applications of impedance cytometry for cell/particle detection (i.e., ~10–30 µm), the effects of intracellular components on impedance components are still rarely investigated [26], [27], [28], [29], [30].

In this paper, we established a new high-throughput single-cell characterization method that delivers information related to the distribution and density of intracellular components of cells at rates of up to 1500 cells per second. The impedance detection is carried out via a field programmable gate array (FPGA) board [31] with two detection frequencies, including 6 MHz high frequency ($\left|Z_{\mathit{HF}}\right|$) and 500 kHz low frequency ($\left|Z_{\mathit{LF}}\right|$). Through numerical simulation, we first found that the density and distribution of intracellular components have direct impacts on the high frequency impedance signals, which can be quantified through electrical diameter (${\left|Z_{\mathit{HF}}\right|}^{1/3}$) and tilt index ($T_{\mathit{HF}}^{\mathit{Left}}/T_{\mathit{HF}}^{\mathit{Right}}-1$). Besides, cell volume and shape can be quantified by low-frequency impedance signals based on low-frequency electrical diameter (${\left|Z_{\mathit{LF}}\right|}^{1/3}$) and tilt index ($T_{\mathit{LF}}^{\mathit{Left}}/T_{\mathit{LF}}^{\mathit{Right}}-1$). In experiments, Euglena gracilis (E. gracilis) cells were employed to verify the capability of impedance cytometry. Without the light stimulus, chloroplast synthesis was suppressed, resulting in the changes in the intracellular densities. Experimental results confirmed that the free space caused chloroplast loss can be reflected by the decrease in the high-frequency impedance signals and opacity (ratio of $\left|Z_{\mathit{HF}}\right|/\left|Z_{\mathit{LF}}\right|$) of single cells. Together, the simplicity of the measurement suggests that our method is suitable for a new generation of quick assays for the density and distribution of intracellular components at the single cell level.