Abstract

The relative performances of fluorescence, oblique incidence reflection and phase contrast imaging techniques have been studied for the purpose of monitoring long-term cellular activity and cell viability of several types of normal and cancerous cells in cultures. Time-lapse movies of live cell imaging of untagged and green fluorescent protein (GFP) tagged cell lines are presented. Oblique incidence reflection microscopy is the simplest and least expensive method to implement, appears to be the least phototoxic to cells, and is recommended for use in long-term optical monitoring of cell viability.

© 2004 Optical Society of America

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References

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P. B. Shashikanth, P. B. V. Prasad and G. Sambasiva Rao, �??Oblique incidence reflection microscopy (OIRM) study of hydrocarbon films,�?? Cryst. Res. Technol. 34, 1287-1292 (1999).
[CrossRef]

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[CrossRef] [PubMed]

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[CrossRef] [PubMed]

Mol. Biol. Cell

A. B. Verkhovsky et al., �??Orientational order of the lamellipodial actin network as demonstrated in living motile cells,�?? Mol. Biol. Cell 14, 4667-4675 (2003).
[CrossRef] [PubMed]

Nat. Cell Biol.

D. Gerlich and J. Ellenberg, �??4D imaging to assay complex dynamics in live specimens,�?? Nat. Cell Biol. 5, S14-S19 (2003).

A. Miyawaki, A. Sawano and T. Kogure, �??Lighting up cells: labelling proteins with fluorophores,�?? Nat. Cell Biol. 5, S1-S7 (2003).

Nat. Rev. Mol. Cell Bio.

Y. Sako and T. Yanagida, �??Single-molecule visualization in cell biology,�?? Nat. Rev. Mol. Cell Bio. 4, SS1-SS5 (2003).

Nature

M. A. Lever et al., �??Rapid exchange of histone H1.1 on chromatin in living human cells,�?? Nature 408, 873-876 (2000).
[CrossRef] [PubMed]

Nucl. Instrum. Meth. B

R. Ortega, G. Devès and P. Moretto, �??In-air scanning transmission ion microscopy of cultured cancer cells,�?? Nucl. Instrum. Meth. B 181, 475-479 (2001).
[CrossRef]

Opt. Express

Opt. Lett.

Trends Cell Biol.

F. S. Wouters, P. J. Verveer and P. I. H. Bastiaens, �??Imaging biochemistry inside cells,�?? Trends Cell Biol. 11, 203-211 (2001).
[CrossRef] [PubMed]

Other

W. T. Mason (editor), Fluorescent and luminescent probes for biological activity (Academic Press, 1999).

B. Alberts et al., Molecular biology of the cell (Taylor & Francis Group, New York, 2002).

Supplementary Material (7)

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Figures (7)

Fig. 1.
Fig. 1.

(a) Oblique incidence reflection imaging system. Two low-intensity incandescent lamps located over the microscope stage illuminate the sample. (b) Schematic ray diagram showing the useful subset of unfocussed lamp rays interacting with the sample.

Fig. 2.
Fig. 2.

Fluorescence: (a) (2.5 MB) Time-lapse sequence of Actin-GFP labeled HeLa cells showing plasma membrane ruffling (left), cell division (right) and overall cell motility. (b) (2.4 MB) Time-lapse sequence of histone H1-GFP labeled MCF-7 cells illustrating chromosome dynamics throughout mitosis. Scale=20µm.

Fig. 3.
Fig. 3.

Phase contrast: (2.2MB) Time-lapse sequence of unlabeled HeLa cells showing lamellipodium growth (left), cell roll-up followed by a partial division (right) and overall streaming of organelles and nuclear bodies. Scale=20µm.

Fig. 4.
Fig. 4.

Oblique incidence reflection: Time-lapse sequences of untagged cell lines clearly showing organelle and macromolecular streaming as well as structural movement. (a) (2.3 MB) HeLa cells displaying a wide range of cellular activity including crawling, cell roll-up followed by division and reattachment. (b) (2.5 MB) HeLa cells, and (c) (1.9 MB) HSF-55 cells: these two sequences illustrate the remarkable crawling capability of epithelial and fibroblast cells (peak velocity ~1µm/min). Scale=20µm.

Fig. 5.
Fig. 5.

Oblique incidence reflection: Untagged HeLa cell still frames 4(a) and 4(b) processed with a non-linear function in order to compress the dynamic range of each image so that the faintest and brightest components of the cells are visible. Scale=20µm.

Fig. 6.
Fig. 6.

(a) Fluorescence, (b) phase contrast and (c) oblique incidence reflection images of the same histone H1-GFP labeled breast cancer (MCF-7) cell group. Scale=20µm.

Fig. 7.
Fig. 7.

Fluorescence and oblique incidence reflection imaging: (2.4 MB) Time-lapse series of actin-GFP labeled HeLa cells illustrating concurrent, sequential and separate illuminations. Examples of still frames taken from the sequence: (a) combined fluorescence and oblique, (b) oblique only, and (c) fluorescence only. Scale=20µm.

Tables (2)

Tables Icon

Table 1. Cell lines for which time-lapse movies are presented.

Tables Icon

Table 2. Time-lapse movies: a summary.

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