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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2003 (8)

T. Misteli (guest editor), “In vivo imaging,” Methods 29, 1–122 (2003).
[Crossref] [PubMed]

F. Iborra, P. R. Cook, and D. A. Jackson, “Applying microscopy to the analysis of nuclear structure and function,” Methods 29, 131–141 (2003).
[Crossref] [PubMed]

S.-W. Chuet al., “In vivo developmental biology study using noninvasive multi-harmonic generation microscopy,” Opt. Express 11, 3093–3099 (2003), http://www.opticsexpress.org/abstract.cfm?URI=OPEX-11-23-3093.
[Crossref] [PubMed]

D. Yelinet al., “Multiphoton plasmon-resonance microscopy,” Opt. Express 11, 1385–1391 (2003), http://www.opticsexpress.org/abstract.cfm?URI=OPEX-11-12-1385.
[Crossref] [PubMed]

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

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

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

A. B. Verkhovskyet al., “Orientational order of the lamellipodial actin network as demonstrated in living motile cells,” Mol. Biol. Cell 14, 4667–4675 (2003).
[Crossref] [PubMed]

2002 (1)

T. Haraguchi (preface to a series of reviews), “Live cell imaging: Approaches for studying protein dynamics in living cells,” Cell Struct. Funct. 27, 333–334 (2002).
[Crossref] [PubMed]

2001 (3)

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]

F. S. Wouters, P. J. Verveer, and P. I. H. Bastiaens, “Imaging biochemistry inside cells,” Trends Cell Biol. 11, 203–211 (2001).
[Crossref] [PubMed]

G. S. Harmset al., “Autofluorescent proteins in single-molecule research: Applications to live cell imaging microscopy,” Biophys. J. 80, 2396–2408 (2001).
[Crossref] [PubMed]

2000 (2)

D. Margineantu, R. A. Capaldi, and A. H. Marcus, “Dynamics of the mitochondrial reticulum in live cells using Fourier imaging correlation spectroscopy and digital video microscopy,” Biophys. J. 79, 1833–1849 (2000).
[Crossref] [PubMed]

M. A. Leveret al., “Rapid exchange of histone H1.1 on chromatin in living human cells,” Nature 408, 873–876 (2000).
[Crossref] [PubMed]

1999 (1)

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]

1998 (2)

Alberts, B.

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

Bastiaens, P. I. H.

F. S. Wouters, P. J. Verveer, and P. I. H. Bastiaens, “Imaging biochemistry inside cells,” Trends Cell Biol. 11, 203–211 (2001).
[Crossref] [PubMed]

Capaldi, R. A.

D. Margineantu, R. A. Capaldi, and A. H. Marcus, “Dynamics of the mitochondrial reticulum in live cells using Fourier imaging correlation spectroscopy and digital video microscopy,” Biophys. J. 79, 1833–1849 (2000).
[Crossref] [PubMed]

Chu, S.-W.

Cook, P. R.

F. Iborra, P. R. Cook, and D. A. Jackson, “Applying microscopy to the analysis of nuclear structure and function,” Methods 29, 131–141 (2003).
[Crossref] [PubMed]

Devès, G.

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]

Ellenberg, J.

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

Gerlich, D.

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

Guo, C.-L.

Haraguchi, T.

T. Haraguchi (preface to a series of reviews), “Live cell imaging: Approaches for studying protein dynamics in living cells,” Cell Struct. Funct. 27, 333–334 (2002).
[Crossref] [PubMed]

Harms, G. S.

G. S. Harmset al., “Autofluorescent proteins in single-molecule research: Applications to live cell imaging microscopy,” Biophys. J. 80, 2396–2408 (2001).
[Crossref] [PubMed]

Iborra, F.

F. Iborra, P. R. Cook, and D. A. Jackson, “Applying microscopy to the analysis of nuclear structure and function,” Methods 29, 131–141 (2003).
[Crossref] [PubMed]

Jackson, D. A.

F. Iborra, P. R. Cook, and D. A. Jackson, “Applying microscopy to the analysis of nuclear structure and function,” Methods 29, 131–141 (2003).
[Crossref] [PubMed]

Kogure, T.

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

Lee, C.-H.

Lever, M. A.

M. A. Leveret al., “Rapid exchange of histone H1.1 on chromatin in living human cells,” Nature 408, 873–876 (2000).
[Crossref] [PubMed]

Marcus, A. H.

D. Margineantu, R. A. Capaldi, and A. H. Marcus, “Dynamics of the mitochondrial reticulum in live cells using Fourier imaging correlation spectroscopy and digital video microscopy,” Biophys. J. 79, 1833–1849 (2000).
[Crossref] [PubMed]

Margineantu, D.

D. Margineantu, R. A. Capaldi, and A. H. Marcus, “Dynamics of the mitochondrial reticulum in live cells using Fourier imaging correlation spectroscopy and digital video microscopy,” Biophys. J. 79, 1833–1849 (2000).
[Crossref] [PubMed]

Miyawaki, A.

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

Mohler, W. A.

Moretto, P.

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]

Ortega, R.

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]

Prasad, P. B. V.

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]

Sako, Y.

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

Sambasiva Rao, G.

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]

Sawano, A.

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

Shashikanth, P. B.

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]

Verkhovsky, A. B.

A. B. Verkhovskyet al., “Orientational order of the lamellipodial actin network as demonstrated in living motile cells,” Mol. Biol. Cell 14, 4667–4675 (2003).
[Crossref] [PubMed]

Verveer, P. J.

F. S. Wouters, P. J. Verveer, and P. I. H. Bastiaens, “Imaging biochemistry inside cells,” Trends Cell Biol. 11, 203–211 (2001).
[Crossref] [PubMed]

Wang, J.

White, J. G.

Wouters, F. S.

F. S. Wouters, P. J. Verveer, and P. I. H. Bastiaens, “Imaging biochemistry inside cells,” Trends Cell Biol. 11, 203–211 (2001).
[Crossref] [PubMed]

Yanagida, T.

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

Yelin, D.

Biophys. J. (2)

D. Margineantu, R. A. Capaldi, and A. H. Marcus, “Dynamics of the mitochondrial reticulum in live cells using Fourier imaging correlation spectroscopy and digital video microscopy,” Biophys. J. 79, 1833–1849 (2000).
[Crossref] [PubMed]

G. S. Harmset al., “Autofluorescent proteins in single-molecule research: Applications to live cell imaging microscopy,” Biophys. J. 80, 2396–2408 (2001).
[Crossref] [PubMed]

Cell Struct. Funct. (1)

T. Haraguchi (preface to a series of reviews), “Live cell imaging: Approaches for studying protein dynamics in living cells,” Cell Struct. Funct. 27, 333–334 (2002).
[Crossref] [PubMed]

Cryst. Res. Technol. (1)

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]

Methods (2)

T. Misteli (guest editor), “In vivo imaging,” Methods 29, 1–122 (2003).
[Crossref] [PubMed]

F. Iborra, P. R. Cook, and D. A. Jackson, “Applying microscopy to the analysis of nuclear structure and function,” Methods 29, 131–141 (2003).
[Crossref] [PubMed]

Mol. Biol. Cell (1)

A. B. Verkhovskyet 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. (2)

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. (1)

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

Nature (1)

M. A. Leveret al., “Rapid exchange of histone H1.1 on chromatin in living human cells,” Nature 408, 873–876 (2000).
[Crossref] [PubMed]

Nucl. Instrum. Meth. B (1)

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 (3)

Opt. Lett. (1)

Trends Cell Biol. (1)

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 (2)

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

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

Supplementary Material (7)

» Media 1: MOV (2567 KB)     
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» Media 4: MOV (2348 KB)     
» Media 5: MOV (2554 KB)     
» Media 6: MOV (1939 KB)     
» Media 7: MOV (2485 KB)     

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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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