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Application note Digital holography advantage

Viability analysis directly in culture vessel without staining Background

Cell death is often divided into two separate categories, necrosis and apoptosis (Kerr at al., 1972), but are quite often mixed (Loos & Engelbrecht, 2009). Cell death is a process that is considered reversible up to a point-of-no-return. Exactly what molecular event that pushes the cell past this point is mostly unknown (Galluzzi et al., 2009). Apoptosis, or programmed cell death, is initiated through tightly regulated extra- or intracellular processes. Several morphological changes follow the induction of apoptosis. The cells detach, appear rounder and shrink. Membrane blebbing subsequently results in separate small apoptotic bodies that are phagocytosed by neighbouring cells and DNA and nuclei are fragmented. Necrosis on the other hand is uncontrolled cell death, which usually begins with cell swelling. The cells round up and the cell membrane bursts, leaving cell fragments that may cause damage to other cells.

End-points

Cell death can be measured in numerous ways. Trypan blue staining is a common method where the leaking membranes of dying and dead cells allow the dye to enter the cell. The resulting blue cells are easily identified in a phase contrast microscope. Flow cytometry is also commonly used to determine the amount of dead cells in sample. These methods cannot distinguish between apoptosis and necrosis. There are also a large number of molecular assays where the level of various intracellular protein or molecules such as caspases or ATP are measured. Using the molecular assays it is possible to distinguish between necrosis and apoptosis. The common denominator for all these assays is that the cells are destroyed in the process and cannot be used for further studies after the assay has been performed.

Application

Using the HoloMonitor™ M3 to measure cell death, samples are placed culture vessel without staining Digital holography advantage Viability analysis directly in on the objective table. The study can then be performed either as a time-lapse study, in which case the cells should be put on a heating stage or in a micro-incubator, or as separate time-points after treatment, in which case no special equipment is necessary. Images are captured automatically or manually, as set by the operator. Each picture is reconstructed as previously described (Mölder et al., 2008), into 3D representations of the sample. These images are segmented to provide morphological data on a cell-to-cell basis. The data available after segmentation shows general proliferation data such as confluence and cell number as well as morphological data, including area, thickness and elongation. Changes in viability can be correlated to changes in morphology. As the cell is dying the membrane starts leaking, thus causing changes in the refractive index of the cell. As the refractive index approaches that of the surrounding media, the cells appear to become thinner. This is highly visible in scatterplots showing the correlation between cell area and cell thickness. Control samples are used to set the base line for healthy, dying/ dead and fragmented cells. As the data from each time-point come

Control

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Table 1. Percentage of cells found in the areas a, b and g after different treatments.

Control Cells found in (%)

50 μM

100 μM

etoposide etoposide

Area α (healthy)

89

61

58

Area β (dead/dying)

10

36

38

Area γ (fragmented)

1

3

4

from all cells in the images captured at that time point, the analysis results are statistically significant. Every analyzed cell can be followed back to the original image to guarantee the accuracy of the region settings.

Results

The changes in cell morphology are highly visible in scatterplots showing the correlation between cell area and cell thickness. Control samples are used to set the base line for healthy, dying/ dead and fragmented cells. As the data from each time-point come from all cells in the images captured at that time point, the analysis results in a statistically significant dataset. Each and every cell contributing to the data can be followed back to the original image to guarantee the accuracy of the region settings. Figure 1A and B show typical samples with healthy and dying etoposide-treated Jurkat cells respectively. The majority of the cells are healthy and clustered in area α of the plot (Table 1, Fig. 2A). There is always some degree of dead/dying (area β) and fragmented (area γ) cells present even in healthy cell cultures. By treating the cells with 50 μM of the cytotoxic substance etopo-

Etoposide treated


Application note A

a

B

C

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a

b

b

b

g

g

g

Figure 2. Morphological parameters studied in A: untreated Jurkat cells one day after seeding, B: Jurkat cells treated with 50 μM of the cytotoxic substance etoposide one day after seeding and C: Jurkat cells treated with 100 μM etoposide one day after seeding. Healthy cells are clustered in area α of the plot. There is always some degree of dead/dying (area β) or fragmented (area γ) cells present even in healthy cell cultures. Significantly more cells can be found in area β in cultures treated with 50 or 100 μM etoposide. N=3

side, the amount of cells found in area α decreases (Fig. 2B), while the amount of cells found in areas β and γ increase, indicating that the morphology of the cells has changed. Cells treated with 100 μM etoposide showed substantial changes in morphology (Fig. 2C) connected to changes in the viability of the culture.

Dead cells

Discussion

Cell death changes the morphology of the cells in drastic ways. These changes can be detected without any staining or other manipulations of the cells when using HoloMonitor ™ M3 (Fig. 3). Image libraries for each time-point result in a dataset spanning thousands of cells. Over the course of the experiment this means that there may be individual morphological data for thousands of cells, giving users an enormous amount of data that is easily accessible in standard spreadsheet format for further quick analysis of the experiment. Using flow cytometry with propidium iodide staining to investigate cell death results in scatterplots much like the ones produced by HoloMonitor ™ M3. The flow cytometry studies show only the proportion of dead cells, not if they are apoptotic or necrotic. The flow cytometry results can not be traced back to the individual cells, and the investigation is destructive. When investigating annexin-labeled cells with the flow cytometer an apoptotic procedure can be detected at an earlier stage, but otherwise with the same disadvantages as for propidium iodide staining. We have analyzed cells with HoloMonitor™ M3 and compared with trypan blue staining and counting in a heamocytometer. The percentage of dead cells found by this standard method correlates

Living cells

Figure 3. L929 cell line with various stages of cell death. When using the HoloMonitor™ M3 for assesing viability calibration of parameters must be set for the cell line used.

with the percentage of dead cells as determined by the HoloMonitor™ M3 (Fig. 4). The HoloMonitor™ M3 is compatible with all standard cell culture vessels. As samples analyzed with HoloMonitor™ M3 are not affected by the analysis, they can be used for consecutive investigations with other methods. The HoloMonitor™ M3 will thus allow the user to extract more data from each experiment for a small amount of extra time and with no extra material required. The experimental procedure simply consists of capturing images. As the HoloMonitor ™ M3 is non-invasive and non-destructive there is no need to seed cultures for each timepoint investigated. The cell culture can be analyzed at any number of time-points as the samples will not be

A

B

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affected by the measurements. HoloMonitor™ M3 thus allows for simplified time and resource saving procedures with high accuracy and many possibilities to double check the results. References

Galluzzi L. et al, Guidelines for the use and interpretation of assays for monitoring cell death in higher eukaryotes. Cell Death and Differentiation 16, p. 1093-1107, 2009. Kerr, J.F.R., Wyllie, A.H., Currie, A.R. Apoptosis: A basic biological phenomenon with wide-ranging implications in tissue kinetics. British Journal of Cancer 26(4), p. 239-257, 1972 Loos, B., Engelbrecht, A-M. Cell death: a dynamic response concept. Autophagy 5(5), p. 590-603, 2009. Mölder, A., Sebesta, M., Gustafsson, M., Gisselson, L., Gjörloff-Wingren, A., Alm, K. Non-invasive, label-free cell counting and quantitative analysis of adherent cells using digital holography. Journal of Microscopy 232, 240-247, 2008

Figure 4. Correlation of cell death between phase contrast and holographic imaging. A. Holographic image of Jurcat cells. B. Same field of view in phase contrast with trypan blue staining.


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