Digital Holographic Microscopy
Quantification of Drug-induced Alterations in Confluent Cell Layers
- Fig. 1: Sketch of a typical inverted off-axis digital holographic microscope in transmission configuration for label-free quantitative phase imaging of living cell cultures (modified from ).
- Fig. 2: Quantitative DHM phase images of confluent control cells (a) and after treatment with 50 µM capsaicin (b); (c), (d): False color-coded pseudo 3D representation of the images in (a), (b). (e): Histograms of the phase values Δϕ in images (a) and (b). The insert in (e) illustrates cell layer morphology changes after capsaicin treatment (adapted from , ).
- Fig. 3: Impact of free capsaicin and capsaicin filled chitosan nanocapsules on confluent MDCK-C7 cell layers. (a): Representative DHM quantitative phase images of MDCK-C7 cell monolayers at the beginning (t=0) and at the end of the experimental period. Open cell-cell contacts (dark gaps) are indicated by arrows. (b): Histograms of the quantitative phase images at distinct time points for treated cell layers and control cells. (c): Average phase contrast ΔϕAVG plotted vs. observation time in comparison with a linear fit. (d): Slopes and Y-intercepts retrieved from the plots in (c). Data are mean values ± SD for N = 3 independent experiments (adapted from , ).
Strategies to quantify changes in confluent cell layers were explored by quantitative phase imaging (QPI) for the example of digital holographic microscopy (DHM). Utilizing histogram based-evaluation the impact of free capsaicin and capaicin filled chitosan nanocapsules was analyzed in MDCK cells. The results demonstrate that histogram-based evaluation of quantitative phase images allows a reliable label-free quantification of global morphology changes in confluent cell layers.
Quantitative phase imaging (QPI) is based on the detection of specimen induced optical path length changes against the surrounding environment . As the technique is typically utilized in transmission only low light intensities for object illumination are required. Thus, the interaction with the sample is minimized which makes the technology in particular suitable for minimally-invasive long-term quantitative imaging of living cell cultures [1-3]. During the past years QPI techniques have been continuously improved for high resolution label-free quantitative live cell imaging . However, the analysis of confluent cell layers is challenging, as the retrieved phase images lack of specimen-free reference phase data, which hinders a further quantitative evaluation, e.g. by image segmentation for retrieval of absolute growth and morphology parameters like the cellular dry mass or the cell thickness. Strategies for the analysis of confluent cell layers utilizing histogram based-evaluation of quantitative phase images were explored . For the example of digital holographic microscopy (DHM) , a variant of QPI, the applicability of the proposed numerical procedures is illustrated by the quantification of the impact of free capsaicin and capsaicin filled chitosan nanocapsules on Madin Darby canine kidney (MDCK) epithelial cell layers.
Quantitative Phase Imaging with Digital Holographic Microscopy
Figure 1 shows a sketch of a typical off-axis setup for DHM that also can be integrated into commercial research microscopes . An inverted transmission arrangement allows investigations on mainly transparent samples in a liquid, e.g., living cell cultures in a Petri dish filled with cell culture medium.
Coherent light of a laser is divided into an object illumination wave O and a reference wave R. While O passes the sample, the undisturbed wave R is guided directly but slightly tiled to the digital image recording device, e.g., a charge-coupled device (CCD) sensor. From the recorded interference patterns (digital off-axis holograms), quantitative phase images can be numerically reconstructed which quantify the optical path length changes induced by the morphology of the investigated specimen [4,6,7].
Detection of Global Cell Layer Morphology Changes by Histogram-based Evaluation of Quantitative DHM Phase Contrast Images
Figure 2 illustrates the concept for determination of global morphology changes in confluent layers from quantitative DHM phase contrast images for the example of MDCK-C7 cells . Figures 2a and 2b depict representative QPI images of untreated control cells and cells after treatment with capsaicin. Figures 2c and 2d present corresponding false color-coded pseudo 3D representations of the quantitative phase images which illustrate the change of the cell layer surface roughness. Without capsaicin cells grow thin with tight cell-cell contacts that are almost not visible (fig. 2a and 2c). In contrast, capsaicin induces an opening of cell-cell contacts (fig. 2b and 2d). Figure 2e shows representative histograms of quantitative phase contrast images of capsaicin treated MDCK cells and control cells. The parameter Δφ quantifies the optical path length delay that is proportional to thickness changes of individual cells in the confluent mono layers  and thus detects alterations of the cell layer surface roughness after capsaicin treatment. For the control cells, the histogram is narrow which indicates a low cell layer surface roughness. The treatment with capsaicin causes a shift of the histogram towards higher phase values and a broadening is observed. Both changes are indicative for an increased cell layer surface roughness compared to the control cells (see schematic illustration in the insert of fig. 2e).
Impact of Free Capsaicin and Capsaicin Filled Chitosan Nano Capsules on MDCK-C7 Cells
Chitosan, known for its various interactions with biological barriers, was used as a model for lipophilic drugs and as a coating to generate capsaicin-loaded nanocapsules . Capsaicin is a natural compound occurring in hot chili peppers that is well-known for its pungency and was chosen due to its various biological activities and its widespread usage in traditional and scientific medicine . For QPI observations with DHM, MDCK-C7 cells were grown confluently in Petri dishes with glass lids and incubated either with a concentration of 50 μM capsaicin or capsaicin loaded nanocapsules .
Figure 3a shows quantitative phase images that illustrate the impact of free capsaicin and capsaicin filled chitosan nano capsules on confluent MDCK-C7 cell layers in comparison to untreated control cells for the beginning (t=0) and at the end of representative experiments. Capsaicin and capsaicin loaded nanocapsules induce opening of cell-cell contacts which is in indicated by dark gaps (see arrows in fig. 3a). The corresponding histograms of the quantitative phase images at different time points are plotted in figure 3b. For control cells, the histograms remain highly similar during the whole experimental period. Treatment with capsaicin and nanocapsules causes broadening and shift of the histograms towards higher phase values. Figure 3c shows the average phase values corresponding to the histogram data in figure 3b. For all treatments, the trends are linear. Figure 3d depicts the slopes and Y-axis intercepts extracted from linear fits to the temporal development of the average phase contrast in figure 3b. For free capsaicin, the slope is almost similar to the control while the treatment with capsaicin loaded nanocapsules results in a significant increase of the average phase contrast. The corresponding abscissa values in figure 3d show a similar ascending trend but also indicate that initial cell layer surface roughness changes are already induced within the range of several minutes after the addition of the different substances to the MDCK cells. All findings are in good agreement with the with the visible morphological appearance of cell layers in figure 3a as well as with findings from complementary studies on cell motility, calcium flux and fluorescence microscopy of selected tight junction and cytoskeleton proteins .
Our results show that histogram-based evaluation of quantitative phase images allows a reliable detection of global morphology changes in confluent cell layers. Moreover, for the example of epithelial cell treatment with capsaicin and nanocapsules it is demonstrated that the approach can be used to quantify the influence of drugs. In summary, our approach represents a versatile tool for simplified evaluation of quantitative phase images with the potential to address novel application areas of QPI.
Björn Kemper1, Mathias Kaiser2, Francisco M. Goycoolea3, Luisa Pohl1, Steffi Ketelhut1
1Biomedical Technology Center, Medical Faculty University of Münster, Germany
2Max Delbrück Center for Molecular Medicine, Germany
3University of Leeds, School of Food Science and Nutrition, Leeds, UK
Dr. Björn Kemper
Biomedical Technology Center
Medical Faculty University of Münster
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 Dominik Bettenworth, Arne Bokemeier, Christopher Poremba, Nik S. Ding, Steffi Ketelhut, Phillip Lenz, Björn Kemper: Quantitative phase microscopy for evaluation of intestinal inflammation and wound healing utilizing label-free biophysical markers, Histology and Histopathology, 33(5), 417-32 (2018) doi: 10.14670/HH-11-937
 Mathias Kaiser, Luisa Pohl, Steffi Ketelhut, Lena Kastl, Jürgen Schnekenburger, Martin Götte, Christian Gorzelanny, Francisco. M. Goycoolea, Björn Kemper: Nano encapsulated capsaicin influences cell migration behavior and morphology of madin darby canine kidney epithelial cell monolayers, PLoS ONE 12(11), e0187497 (2017) doi: 10.1371/journal.pone.0187497
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 Junwei Min, Baoli Yao, Steffi Ketelhut, Christian Engwer, Burkhard Greve, Björn Kemper: Simple and fast spectral domain algrorithm for quantitative phase imaging of living cells with digital holographic microscopy, Optics Letters 42(2), 227-30 (2017) doi: 10.1364/OL.42.000227
 Mathias Kaiser, Susana Pereira, Luisa Pohl, Steffi Ketelhut, Björen Kemper, Christian Gorzelanny, Hans-Joachim Galla, Bruno M. Moerschbacher, Francisco M. Goycoolea: Chitosan encapsulation modulates the effect of capsaicin on tight junctions on MDCK cells", Scientific Reports, 5, 10048 (2015) doi: 10.1038/srep10048
 Björn Kemper, Luisa Pohl, Mathias Kaiser, Eva Döpker, Jürgen Schnekenburger, Steffi Ketelhut: Label-free detection of global morphology changes in confluent cell layers utilizing quantitative phase imaging with digital holographic microscopy, Proceedings of SPIE, 11076, 110760T (2019) doi: 10.1117/12.2527189