2.1.

Cell Culture and Sample Preparation

The investigated human pancreatic ductal adenocarcinoma cell line PaTu 8988T was obtained from the German Collection of Microorganisms and Cell Cultures (DSMZ, Braunschweig, Germany). It is one of two cell lines established from a liver metastasis of a primary pancreatic adenocarcinoma from a $64\text{-year}$ old woman in 1985, representing a poorly differentiated adenocarcinoma with high metastatic potential.³⁹ Pancreatic tumor cells were cultured in Dulbecco’s modified Eagle’s Medium (DMEM) supplemented with 5% fetal calf serum, 5% horse serum, and $2\text{-}\mathrm{mM}$ L-glutamine under standard cell culture conditions ( $37°\mathrm{C}$ and 5% $\mathrm{C}{\mathrm{O}}_2$ ). For digital holographic cell imaging, cells were trypsinized and $1\times {10}^4\text{cells}∕\mathrm{ml}$ were seeded on petri dishes ( $\mu$ -dishes Grid-500, ibidi GmbH, Munich, Germany). Cells were cultured for $24\mathrm{h}$ and rinsed twice with Dulbecco’s phosphate buffered saline (PBS) (DPBS, Lonza, Verviers, Belgium). Live cell imaging experiments were conducted using petri dishes filled with either PBS or a mixture of glycerol/PBS (50%, v/v) and a climate chamber (ibidi GmbH, Munich, Germany), allowing for temperature-stabilized analysis at $37°\mathrm{C}$ and normal atmospheric conditions. Chemically fixated cells ( $30\min$ at room temperature) using 4% neutral buffered formalin were analyzed after mounting with PBS and glycerol, respectively.

The refractive indices of PBS ( $n$ : 1.337), glycerol ( $n$ : 1.474), and the 50% (v/v) mixture of glycerol/PBS ( $n$ : 1.408) were measured with a digital Abbe refractometer (WYA-2S, Ningbo Hinotek Technology Company, Limited, Ningbo, China).

2.2.

Digital Holographic Imaging and Experimental Setup

Live cell imaging experiments as well as analysis of fixated cells were performed using an inverse fluorescence microscope Olympus IX81 (Olympus Deutschland GmbH, Hamburg, Germany) with attached DHM module based on a principle described in Ref. 40. The coherent light source is a frequency-doubled Nd:YAG laser (Compass 315M-100, Coherent, Luebeck, Germany, $\lambda =532\mathrm{nm}$ ). Figure 1 illustrates the general concept for the integration of DHM into the microscopy system. Microinjection was performed using an Eppendorf InjectMan NI 2 and FemtoJet system (both Eppendorf GmbH, Hamburg, Germany) and an injection needle of approximately $0.5\mu \mathrm{m}$ inner and $0.7\mu \mathrm{m}$ outer diameter, respectively. An overview of the experimental setup is shown in Fig. 1, whereas Fig. 1 presents the microscope condenser with DHM unit, the opened climate chamber, and the micromanipulator under operating conditions in detail.

Fig. 1

General setup of a digital holographic microscopy system with an inverse microscope arrangement and microinjection unit: (a) schematic setup, (b) inversed Olympus IX81 fluorescence microscope with DHM module in transmission mode, (c) condenser with the DHM unit for coherent illumination, opened climate chamber (ibidi GmbH, Munich, Germany) and micromanipulator InjectMan NI2 (Eppendorf GmbH, Hamburg, Germany).

3.1.

Cell Injection Process Monitoring

First, the microinjection process is analyzed to assure the applicability of DHM in combination with the injection unit. In general, the microinjection process of a liquid into a living cell has to be performed fast to minimize cell membrane injury. Also, the applied injection pressure should be as low as possible to avoid a high flow stream inside the cell. This results in special demands for the experimental conditions. Thus, in an initial experiment, we performed a cell free approach to optimize our setup in respect to the acquisition rate of holograms, bright field images, and fluorescence images, as well as in respect of the presentability of the injection process and data processing. Due to high viscosity and refractive index of the pure substance, glycerol has been diluted with PBS (50%, v/v), leading to a refractive index of $n$ : 1.408. Figure 2 shows bright field images (first row), the quantitative DHM phase contrast images (second row), and the corresponding false color plots (third row) that result from the injection process into pure PBS. Figures 2, 2, 2 depict the micrographs before the injection with the tip of the injection needle extending into the section. To avoid a buffer influx into the needle due to capillary effects, the compensation pressure was set to $15\mathrm{hPa}$ . The images in Figs. 2, 2, 2 indicate no efflux of liquid into the buffer solution at the same time. After triggering the efflux ( $500\mathrm{hPa}$ , $1\mathrm{s}$ ), the glycerol/PBS mixture was injected into the buffer [Figs. 2, 2, 2]. The effectively released amount of liquid depends on injection pressure, duration (time), viscosity of the injected substance, and the actual inner diameter of the needle. Thus, the injected volume cannot be absolutely controlled and quantified. However, the different gray levels (256) in the DHM phase contrast images [Figs. 2, 2, 2, 2] represent the local change refractive index distribution. High gray values correlate with a high refractive index, while the lowest values display a refractive index of the pure PBS ( $n$ : 1.337). Obviously, the injection of the glycerol/PBS dilution leads to a maximum concentration of the mixture at the tip of the needle, which is indicated by the brightest gray levels and highest refractive index, respectively [Figs. 2 and 2]. The false color-coded representation of the phase contrast images in Figs. 2 and 2 illustrate these findings. The gentle transition from the tip to distant areas demonstrate the fast mixing process of both liquids to a finally homogeneous mixture [Figs. 2 and 2] within three seconds.

Fig. 2

(a)-(l) Injection of a glycerol/PBS mixture 50%, v/v in pure PBS. The injection pressure $\left(p_i\right)$ was set to $500\mathrm{Pa}$ with an injection time $\left(t_i\right)$ of $1\mathrm{s}$ . (a)-(d) Bright field images, (e)-(h) DHM phase contrast images, and (i)-(l) corresponding false color plot; (m) and (n) microinjection of a PaTu 8988T cell ( $p_i=20\mathrm{hPa}$ , $t_i=1\mathrm{s}$ ) with $10\text{-}\mathrm{mM}$ DCF 2,7-dichlorofluorescein in a glycerol/PBS mixture (50% v/v). (m) Bright field image of a PaTu 8988T cell after microinjection. (n) Corresponding fluorescence image (exposure time $1000\mathrm{ms}$ ) after microinjection.

To analyze the characteristics of the injected material flow inside a cell, we injected the fluorescent dye DCF [2,7-dichlorofluorescein in glycerol/PBS (50%, v/v)] into PaTu 8988T cells ( $30\mathrm{hPa}$ , $1\mathrm{s}$ ). Figure 2 shows a bright field image of a single cell prior to the injection. The fluorescence micrograph immediately after the injection is depicted in Fig. 2. The results demonstrated that cell membrane integrity is not affected by the microinjection. Furthermore, the homogeneous distribution of the fluorescent emission throughout the cytoplasm supports the proposed concept that the entire cytosol can be passed through by the injected mixture.

3.2.

Impact of the Intracellular Injection of Glycerol/Phosphate Buffered Saline on the Quantitative Phase Contrast

In the next experimental step, the influence of the intracellular contrast on quantitative phase contrast images of a living cell was investigated by injection of a glycerol/PBS mixture. It is expected that the injection causes changes in the cytosolic composition, either directly by the injected liquid or indirectly by the triggered cellular reaction. For the experiment, digital holograms were recorded before, during, and after the injection process. In addition, micrographs in the bright field mode were captured as a reference. Due to the experimental setup, the minimum delay between image acquisition in the two different modes amounted in the range of $1\text{to}2\mathrm{s}$ . Following the settings of the cell free approach in Sec. 3.1, the compensation pressure was set to $15\mathrm{hPa}$ and the injection pressure was adjusted to $20\mathrm{hPa}$ for $1\mathrm{s}$ . Considering the results of Sec. 3.1, a very gentle influx into the cell can be expected. Figure 3 shows an exemplarily bright field image of a PaTu 8988T cell $24\mathrm{h}$ after seeding. Flat membrane extension and lamellipodia are typical for healthy cells under cell culture conditions. The corresponding DHM phase contrast image in Fig. 3 displays the cell in different gray levels $\left(8\text{-bit}\right)$ . The gray level map represents the optical path length changes that are affected by the cell in comparison to the surrounding medium due to its thickness and inner refractive index distribution. Thin membrane extensions are resolved and the cell borders can be identified. However, due to a rather homogeneous intracellular refractive index, the phase contrast inside the cell is low. Only a few intracellular structures are visible, and a correlation to specific intracellular compartments is not possible. Figure 3 represents a DHM phase contrast image immediately after the injection of a glycerol/PBS mixture in the perinuclear region. Lamellipodia at the cell border are defined with almost similar details in comparison to the situation before the injection. However, the central part of the cell around the nucleus appears with enhanced phase contrast, and areas of brighter gray levels become visible. These structures are surrounded by an area with only smooth gray value changes and with a partly visible border to the ambient cell body [arrow, Fig. 3]. Comparisons with the bright field image [captured $1\mathrm{s}$ after the hologram, Fig. 3] allow the identification of these structures as the nucleus with nucleoli. Moreover, this image verifies the successful injection of the glycerol/PBS mixture, as a fluid-filled membrane bleb occurs in the right part of the cell. Additionally, small blebs at the distal parts of the membrane extensions are visible, revealing a direct connection to the main part of the cytosol. After one minute [Figs. 3 and 3], the cellular reaction appears increased and the membrane curled. Because of the rather low local differences of the refractive index inside the blebs and the cytosol, the injected liquid can be assumed to have immediately mixed with the intracellular compounds. Eight minutes after the microinjection, blebs have degenerated and the cell surface appears a little rougher in the bright field image [Fig. 3] compared to the unaffected cell in Fig. 3. Fine, long membrane extensions are still visible and indicate a living cell. In the corresponding DHM phase images [Fig. 3], the contrast of the cytosol to the surrounding buffer is low compared to the results of the prior measurements.

Fig. 3

Injection of a glycerol/PBS mixture (50%, v/v) into a PaTu 8988T cell. (a)-(d) Bright field images and (e)-(h) corresponding DHM phase contrast images showing a PaTu 8988T cell (a) and (e) before, (b) and (f) directly after, (c) and (g) $1\min$ after, and (d) and (h) $8\min$ after injection ( $p_i=20\mathrm{hPa}$ , $t_i=1\mathrm{s}$ ). (e) through (h) Black arrowheads indicate the positions of two cross sections to quantify the relative phase contrast of two different nucleoli (1 and 2) at each time step. The black arrow in (f) marks the nuclear envelope. (i) The plots for cross section 1 and (j) cross section 2 illustrate the alteration of the relative phase contrast $\Delta \Phi \left(\mathrm{rad}\right)$ per pixel, with the solid line representing the nucleolus prior to injection, the triangle line representing the nucleolus directly after injection, the circle line representing the nucleolus $1\min$ after injection, and the asterisk line representing the nucleolus $8\min$ after injection. The phase range of $6.6\mathrm{rad}$ is coded to 256 gray levels. Scale bar: $10\mu \mathrm{m}$ .

To quantify the enhanced intracellular contrast, the DHM phase contrast images were analyzed by cross sections through the nucleoli and surrounding parts of the nucleus [cross sections between the arrowheads in Figs. 3, 3, 3, 3]. Figures 3 and 3 depict the relative phase contrast differences [ $\Delta \Phi$ (rad)] for the cross sections before and after microinjection. The solid line in Fig. 3 represents the situation before the injection, and the relative phase contrast of nucleolus 1 is about $1.2\mathrm{rad}$ higher than the surrounding area. Directly after the injection of the glycerol/PBS mixture, $\Delta \Phi$ raises to $1.8\mathrm{rad}$ in maximum, one minute later to $2.0\mathrm{rad}$ , and eight minutes after the injection process to $2.3\mathrm{rad}$ . The results for the second analyzed nucleolus confirm this measurement. Here, before the injection, the relative phase contrast amounts to $1.2\mathrm{rad}$ in maximum, and eight minutes after the injection it shows an increase to $1.7\mathrm{rad}$ . These results of quantitative phase contrast analysis support the effect of contrast enhancement for the nucleoli inside the living cell after injection of a glycerol/PBS mixture.

Figure 4 represents another exemplarily pancreatic tumor cell before and after the injection process under the same experimental conditions described before and confirms the results. Similar to the results in Fig. 3, the cell body with its lamellipodia and membrane extensions is silhouetted against the environmental buffer, but on the contrary, the phase contrast of intracellular structures of the unaffected cell is low [Fig. 4]. After the injection of the glycerol/PBS mixture, two individual structures can be distinguished in the central part of the cell [Fig. 4]. One minute after the injection, a small bleb occurs in the lower cell extension [Fig. 4], but disappears within the next few minutes [Fig. 4]. The reconstructed phase images documented the cellular reactions as well, and in contrast to the first cell (Fig. 3), even thin membrane extensions at the cell borders stay visible. The differences of the phase contrast between the two nucleoli and the surrounding area were analyzed quantitatively and are depicted in Figs. 4 and 4. The findings are in good agreement with the results for the described first cell, and show an increasing difference in the phase contrast for the nucleoli and the environmental area after the injection of a glycerol/PBS mixture into the cell.

Fig. 4

Injection of a PaTu 8988T cell with a mixture of glycerol/PBS (50%, v/v). (a)-(d) Bright field images and (e)-(h) corresponding DHM phase contrast images showing a PaTu 8988T cell (a) and (e) before, (b) and (f) directly after, (c) and (g) $1\min$ after, and (d) and (h) $8\min$ after injection ( $p_i=20\mathrm{hPa}$ , $t_i=1\mathrm{s}$ ). Indication of the two cross sections [black arrowheads in (e)-(h)] plot lines [solid, triangle, circle, and asterisk in (i) and (j)] is conformed to Fig. 3. The phase range of $6.6\mathrm{rad}$ is coded to 256 gray levels. Scale bar: $10\mu \mathrm{m}$ .

3.3.

Influence of the Extracellular Buffer Composition on the Quantitative Phase Contrast

As shown in Sec. 3.2, the direct injection of a glycerol/PBS mixture into a living cell increased the intracellular contrast. In the next experimental step, the influence of an extracellularly offered mixture on the DHM phase contrast was investigated. Pancreatic tumor cells were first incubated in PBS, and bright field images as well as digital holograms were captured with a delay of $1\text{to}2\mathrm{s}$ . Figure 5 illustrates the results for an exemplarily selected PaTu 8988T cell. Flat lamellipodia and membrane extensions documented the healthy status of the cell. Two nucleoli can be identified in the central part of the nucleus [Fig. 5]. Particularly, the DHM phase contrast image allows the identification of these structures despite a weak contrast [Fig. 5]. After the initial bright field and DHM imaging, the complete buffer medium was changed from PBS to a glycerol/PBS mixture (50%, v/v). The cellular reaction was rapid (within seconds) and resulted in a distinct shrinkage of the cell body [Fig. 5]. In the bright field micrograph, lamellipodia could still be identified, but especially in the central part of the cell a correlation to specific cellular compartments was not possible. In the corresponding DHM phase contrast image [Fig. 5], cell boundaries and wide parts of lamellipodia are not displayed properly. The central part of the cell was silhouetted against the background, but a homogeneous refractive index inhibited the identification of subcellular structures.

Fig. 5

Influence of the surrounding buffer conditions on the intracellular contrast. (a) and (b) Bright field images and (c) and (d) corresponding DHM phase contrast images showing (a) and (c) a PaTu 8988T cell in PBS, and (b) and (d) after change to a mixture (50%, v/v) of glycerol/PBS as the surrounding media. (c) and (d) Black arrowheads indicate the positions of two cross sections to quantify the relative phase contrast of two different nucleoli (1 and 2) before and after buffer exchange. (e) The plots for cross section 1 and (f) cross section 2 illustrate the alteration of the relative phase contrast $\Delta \Phi \left(\mathrm{rad}\right)$ per pixel, with the solid line representing the nucleolus before and the triangle line representing the nucleolus after change of buffer. The phase range of $6.6\mathrm{rad}$ is coded to 256 gray levels. Scale bar: $10\mu \mathrm{m}$ .

The decreased intracellular contrast was analyzed quantitatively following the same protocol as described in Sec. 3.2. The cross sections were selected to cut the nucleoli but also the ambient area (sections marked with arrowheads). Figures 5 and 5 show the relative phase contrast $\left(\Delta \Phi \right)$ for the two cross sections before and after the buffer exchange. In PBS, in both cases $\Delta \Phi$ was increased at the site of the nucleoli and can be defined to $1.5\mathrm{rad}$ and to $1.9\mathrm{rad}$ , respectively. After external buffer exchange, these differences of relative phase contrast decreased to a maximum of $0.6\mathrm{rad}$ and $0.8\mathrm{rad}$ . Although in general those differences should be sufficient to identify a cellular component, the irregular curve profile clarifies that the maximum differences are caused by local unspecific peaks and not by a solid nucleoli.

Figure 6 exemplifies another pancreatic tumor cell before and after the change of the surrounding medium, demonstrating the reproducibility of the experiment. Before external buffer change, the cell has pronounced flat membrane extensions and lamellipodia, which were visible in both bright field [Fig. 6] and DHM mode [Fig. 6]. The intracellular contrast allowed the identification of nucleoli, although the contrast was low. Again, the change of the extracellular buffer to the glycerol/PBS mixture started a rapid shrinking process of the cell, leading to a reduced cell size displayed in Fig. 6. Moreover, the central part of the cell appeared unstructured and irregular. In the corresponding phase contrast image, the contrast is quite low, the membrane extensions are displayed only partly in front of the background, and the nucleoli cannot be allocated [Fig. 6]. The analysis of the relative phase contrast by cross sections is depicted in Figs. 6 and 6. Solid lines show the status of the development of the relative phase contrast before buffer exchange. Although the contrast is low, the progression of the curve allows a correlation of the high values to nucleoli and the lower ones to ambient areas of the nucleus. After the cell shrinkage, however, the correlation of the relative phase contrast to visible cellular compartments is difficult.

Fig. 6

The surrounding buffer conditions influence the intracellular contrast. (a) and (b) Bright field images and (c) and (d) corresponding DHM phase contrast images showing (a) and (c) a PaTu 8988T cell in PBS, and (b) and (d) after change to a mixture (50%, v/v) of glycerol/PBS as the surrounding media. Indication of the two cross sections [black arrowheads in (c) and (d)] and plot lines in [solid and triangle in (e) and (f)] conform to Fig. 5. The phase range of $4.5\mathrm{rad}$ is coded to 256 gray levels. Scale bar: $10\mu \mathrm{m}$ .

The findings in Figs. 5 and 6 indicate that the change of the extracellular PBS buffer to a mixture of glycerol/PBS leads to cell shrinkage and to a decrease of the intracellular DHM phase contrast.

3.4.

Impact of the Mounting Medium on the Intracellular Quantitative Phase Contrast of Chemically Fixated Cells

In the last part of this study, the effect of the mounting reagent on the intracellular contrast of chemically fixated cells was analyzed. Therefore, PaTu 8988T cells were grown on a petri dish ( $\mu$ -dish, ibidi, Munich, Germany) and $24\mathrm{h}$ after inoculation the cells were fixated by an unspecific chemical protein crosslink by formalin. This prevalently used method has been chosen to provide stable samples for histological investigations, and in contrast to the incubation with methanol (e.g.), the cell membranes are not completely eliminated.⁴² DHM phase contrast images of the same cells were recorded in PBS and in pure glycerol. In analogy to the previous sections, the visibility of the nucleoli was used as a marker for the intracellular contrast that was quantified by the difference $\Delta \Phi$ between the phase contrast of the nucleolus and the phase contrast of the surrounding area. Figure 7 shows the results for the mean relative phase contrast obtained from $n=32$ individual fixated cells that were subsequently embedded in PBS and glycerol. The values of the relative phase contrast for PBS $(0.7\pm 0.2\mathrm{rad})$ and for glycerol $(0.8\pm 0.3\mathrm{rad})$ both indicate that the nucleoli could be identified in the reconstructed images. Moreover, the data revealed that within the standard deviation of the measurement, there was no definite influence of the two different mounting media on the intracellular contrast in the case of fixated pancreatic tumor cells.

Fig. 7

Mean relative phase contrast of fixated cells. The column plot displays the measurement of the mean relative phase contrast of $n=32$ chemically fixated PaTu 8988T cells initially mounted with PBS (left column) and then with glycerol (right column).