Quantitative assessment of changes in cellular morphology at photodynamic treatment in vitro by means of digital holographic microscopy

Abstract

Temporal dependence of changes in the morphological characteristics of cells of two cultured lines of cancer origin, HeLa and A549, induced by photodynamic treatment with Radachlorin photosensitizer, have been monitored using digital holographic microscopy during first two hours after short-term irradiation. The observed post-treatment early dynamics of the phase shift in the transmitted wavefront indicated several distinct scenarios of cell behavior depending upon the irradiation dose. In particular the phase shift increased at low doses, which can be associated with apoptosis, while at high doses it decreased, which can be associated with necrosis. As shown, the two cell types responded differently to similar irradiation doses. Although the sequence of death scenarios with the increase of the irradiation dose was the same, each scenario was realized at substantially different doses. These findings suggest that the average phase shift of the transmitted wavefront can be used for quantitative non-invasive cell death characterization. The conclusions made were cofirmed by commonly used test assays using confocal fluorescent microscopy.

Fig. 1.

3D pseudocolored phase plots of HeLa cells obtained before photodynamic treatment (a, c) and in 60 minutes after irradiation at 22.1 ${\mathrm{mW/cm}}^2$ (b) and 93 ${\mathrm{mW/cm}}^2$ (d).

Fig. 2.

Average phase shift dynamics in HeLa (a) and A549 (b) cells at the indicated irradiation doses. (c) HeLa cells dry mass dynamics at indicated irradiation doses.(d) average phase shift dynamics in photosensitised but not irradiated HeLa cells during 6 hours of their continuous monitoring. (e) Schematics of average phase shift variation scenarios as function of fluence rate for the two cell lines. Colors in (e) correspond to those on the graphs in (a, b, c).

Fig. 3.

Typical 2D phase images of HeLa and A549 cells before and after PD treatment. Irradiation doses are indicated on the left of each pair of images.

Fig. 4.

Images of HeLa cells subjected to PD treatment at different irradiation doses. Images of cells before irradiation (a-c) and in 50 minutes after irradiation at the fluence rates of 15 ${\mathrm{W/cm}}^2$ (d-f) and 62 ${\mathrm{W/cm}}^2$ (g-i). Left column: AO(green)/EB(red) fluorescent images; middle column: Annexin-V(green)/PI(red) fluorescent images; right column: phase contrast images.