Long-Term Low-Intensity Live Cell Imaging of Therapeutically Treated Cells in Cultures

Abstract

Phase contrast, fluorescence and oblique incidence reflection imaging techniques have been used for the purpose of monitoring long-term cellular activity of therapeutically treated cancerous cells in cultures. Morphological changes in living cells that were induced by either irradiation or chemical agent were monitored over several days. Oblique incidence reflection (OIR) microscopy was used to obtain information on cell dynamics with minimal photodamage and sample degradation.Time-lapse imaging and cell culturing techniques used in this study were as described in [1]. Both cell lines for which data are presented were monolayer-adherent type cultures of human origin. With the exception of a few complementary fluorescence frames, the OIR imaging method was used to gather time-lapse information from a targeted cell group.Radiation is commonly and successfully used in the treatment of certain cancers, the dose being delivered in fractions or a sequence of incremental amounts. A preliminary assessment of the effects of irradiation was performed using cancer cells known to be responsive to radiation treatment, and a single dose fraction. A dose of 600cGy was delivered to histone H1-GFP labeled breast cancer cells (MCF-7) using the 6MV therapeutic x-ray beam from a Siemens ONCOR linear accelerator. Phase contrast images of randomly selected cell groups were taken each day for a period of 3 weeks postirradiation. Figure 1 (top) shows representative images of both untreated control (a) and treated (b through d) cells at one-week intervals. Typically, major morphological changes were visible after 4 days; treated cells were many times larger than normal and contained multiple nuclei. Morphologically normal cell colonies began to appear after 1 week and eventually took over the culture after 3 weeks of incubation (untreated control became confluent after 1 week). Figure 1 e) through g) illustrate examples of still frames selected from a time-lapse imaging session performed 6 days post-irradiation. Sequences of oblique images provided clear visualisation of macromolecular streaming, structural movement, nucleoli dynamics, and cell death.Induction of cellular differentiation is a possible treatment of cancers, and clinical trials are underway on several such inducers. Neuroblastoma cell lines have been used as a model to study cell differentiation [2,3]. To induce morphological changes typically seen in differentiation, untagged SK-N-SH neuroblastoma cells were incubated with 5mM sodium butyrate (a histone deacetylase inhibitor known to initiate differentiation in numerous cell lines). Time-lapse OIR imaging experiments of untreated control and treated cells were performed over several days.

Oblique incidence reflection imaging of live cells: improving contrast and image quality with an objective mask

“…Preventing cell damage caused by the illumination source while monitoring cellular activity is crucial for all imaging methods. Time‐lapse OIR microscopy has been demonstrated to be an ideal tool for the long‐term monitoring of untagged living cells with little photodamage and sample degradation (Landry et al ., 2004, 2006). However, for the results reported thus far by this group, images were acquired from samples mounted in shallow imaging chambers (i.e.…”