Abstract

We have performed multimodal imaging of live fibroblast cells infected by murine cytomegalovirus (mCMV). The infection process was monitored by imaging the two-photon fluorescence signal from a GFP-expressing strain of mCMV, whilst changes to lipid droplet configuration were observed by CARS imaging. This allowed us to identify three visually distinct stages of infection. Quantitative analysis of lipid droplet number and size distributions were obtained from live cells, which showed significant perturbations across the different stages of infection. The CARS and two-photon images were acquired simultaneously and the experimental design allowed incorporation of an environmental control chamber to maintain cell viability. Photodamage to the live cell population was also assessed.

© 2011 OSA

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2010 (5)

D. M. Jones and J. McLauchlan, “Hepatitis C virus: assembly and release of virus particles,” J. Biol. Chem. 285, 22733–22739 (2010).
[CrossRef] [PubMed]

R. K. Lyn, D. C Kennedy, A. Stolow, A. Ridsdale, and J. P. Pezacki, “Dynamics of lipid droplets induced by the hepatitis C virus core protein,” Biochem. Biophys. Res. Commun. 399, 518–524 (2010).
[CrossRef] [PubMed]

E. Herker, C. Harris, C. Hernandez, A. Carpentier, K. Kaehlcke, A. R. Rosenberg, R. V. Farese, and M. Ott, “Efficient hepatitis C virus particle formation requires diacylglycerol acyltransferase-1,” Nat. Med. 16, 1295–1298 (2010).
[CrossRef] [PubMed]

R. Bartenschlager, F. Penn, V. Lohmann, and P. Andre, “Assembly of infectious hepatitis C virus particles,” Trends Microbiol. 19, 95–103 (2010).
[CrossRef] [PubMed]

I. Robinson, M. A. Ochsenkühn, C. J. Campbell, G. Giraud, W. J. Hossack, J. Arlt, and J. Crain, “Intracellular imaging of host-pathogen interactions using combined CARS and two-photon fluorescence microscopies,” J. Biophoton. 3, 138–146 (2010).
[CrossRef]

2009 (1)

Y. Guo, K. R. Cordes, R.V. Farese, and T.C. Walther, “Lipid droplets at a glance,” J. Cell Sci. 122, 749–752 (2009).
[CrossRef] [PubMed]

2008 (2)

C. L. Evans and X. S. Xie, “Coherent anti-Stokes Raman scattering microscopy: chemical imaging for biology and medicine,” Annu. Rev. Anal. Chem. 1, 883–909 (2008).
[CrossRef]

D. J. Stevenson, D. J. Carnegie, B. Agate, F. Gunn-Moore, and K. Dholakia, “Long-term cell culture on a microscope stage: the carrel flask revisited.” Microsc. Anal. 22, 9–11 (2008).

2007 (1)

2005 (1)

A. Yu, X. Ye, D. Ionascu, W. Cao, and P.M. Champion, “Two-colour pump-probe laser spectroscopy instrument with picosecond time-resolved electronic delay and extended scan range,” Rev. Sci. Instrum. 76114301 (2005).
[CrossRef]

2003 (2)

X. Nan, J. X. Cheng, and X. S. Xie, “Vibrational imaging of lipid droplets in live fibroblast cells with coherent anti-Stokes Raman scattering microscopy,” J. Lipid. Res. 44, 2202–2208 (2003).
[CrossRef] [PubMed]

H. Fried and U. Kutay, “Nucleocytoplasmic transport: taking an inventory,” Cell. Mol. Life Sci. 60, 1659–1688 (2003).
[CrossRef] [PubMed]

2002 (1)

W. Muranyi, J. Haas, M. Wagner, G. Krohne, and U. H. Koszinowski, “Cytomegalovirus recruitment of cellular kinases to dissolve the nuclear lamina,” Science 297, 854–857 (2002).
[CrossRef] [PubMed]

2000 (2)

A. Angulo, P. Ghazal, and M. Messerle, “The major immediate-early gene ie3 of mouse cytomegalovirus is essential for viral growth,” J. Virol. 74, 11129–11136 (2000).
[CrossRef] [PubMed]

N. E. Bishop, “Hepatitis A virus replication: an intermediate in the uncoating process,” Intervirology 43, 36–47 (2000).
[CrossRef] [PubMed]

1998 (1)

J. Bewersdorf and S. W. Hell, “Picosecond pulsed two-photon imaging with repetition rates of 200 and 400 MHz,” J. Microsc. 191, 28–38 (1998).
[CrossRef]

Agate, B.

D. J. Stevenson, D. J. Carnegie, B. Agate, F. Gunn-Moore, and K. Dholakia, “Long-term cell culture on a microscope stage: the carrel flask revisited.” Microsc. Anal. 22, 9–11 (2008).

Andre, P.

R. Bartenschlager, F. Penn, V. Lohmann, and P. Andre, “Assembly of infectious hepatitis C virus particles,” Trends Microbiol. 19, 95–103 (2010).
[CrossRef] [PubMed]

Angulo, A.

A. Angulo, P. Ghazal, and M. Messerle, “The major immediate-early gene ie3 of mouse cytomegalovirus is essential for viral growth,” J. Virol. 74, 11129–11136 (2000).
[CrossRef] [PubMed]

Arlt, J.

I. Robinson, M. A. Ochsenkühn, C. J. Campbell, G. Giraud, W. J. Hossack, J. Arlt, and J. Crain, “Intracellular imaging of host-pathogen interactions using combined CARS and two-photon fluorescence microscopies,” J. Biophoton. 3, 138–146 (2010).
[CrossRef]

Bartenschlager, R.

R. Bartenschlager, F. Penn, V. Lohmann, and P. Andre, “Assembly of infectious hepatitis C virus particles,” Trends Microbiol. 19, 95–103 (2010).
[CrossRef] [PubMed]

Bewersdorf, J.

J. Bewersdorf and S. W. Hell, “Picosecond pulsed two-photon imaging with repetition rates of 200 and 400 MHz,” J. Microsc. 191, 28–38 (1998).
[CrossRef]

Bishop, N. E.

N. E. Bishop, “Hepatitis A virus replication: an intermediate in the uncoating process,” Intervirology 43, 36–47 (2000).
[CrossRef] [PubMed]

Campbell, C. J.

I. Robinson, M. A. Ochsenkühn, C. J. Campbell, G. Giraud, W. J. Hossack, J. Arlt, and J. Crain, “Intracellular imaging of host-pathogen interactions using combined CARS and two-photon fluorescence microscopies,” J. Biophoton. 3, 138–146 (2010).
[CrossRef]

Cao, W.

A. Yu, X. Ye, D. Ionascu, W. Cao, and P.M. Champion, “Two-colour pump-probe laser spectroscopy instrument with picosecond time-resolved electronic delay and extended scan range,” Rev. Sci. Instrum. 76114301 (2005).
[CrossRef]

Carnegie, D. J.

D. J. Stevenson, D. J. Carnegie, B. Agate, F. Gunn-Moore, and K. Dholakia, “Long-term cell culture on a microscope stage: the carrel flask revisited.” Microsc. Anal. 22, 9–11 (2008).

Carpentier, A.

E. Herker, C. Harris, C. Hernandez, A. Carpentier, K. Kaehlcke, A. R. Rosenberg, R. V. Farese, and M. Ott, “Efficient hepatitis C virus particle formation requires diacylglycerol acyltransferase-1,” Nat. Med. 16, 1295–1298 (2010).
[CrossRef] [PubMed]

Champion, P.M.

A. Yu, X. Ye, D. Ionascu, W. Cao, and P.M. Champion, “Two-colour pump-probe laser spectroscopy instrument with picosecond time-resolved electronic delay and extended scan range,” Rev. Sci. Instrum. 76114301 (2005).
[CrossRef]

Cheng, J.

Cheng, J. X.

X. Nan, J. X. Cheng, and X. S. Xie, “Vibrational imaging of lipid droplets in live fibroblast cells with coherent anti-Stokes Raman scattering microscopy,” J. Lipid. Res. 44, 2202–2208 (2003).
[CrossRef] [PubMed]

Cordes, K. R.

Y. Guo, K. R. Cordes, R.V. Farese, and T.C. Walther, “Lipid droplets at a glance,” J. Cell Sci. 122, 749–752 (2009).
[CrossRef] [PubMed]

Crain, J.

I. Robinson, M. A. Ochsenkühn, C. J. Campbell, G. Giraud, W. J. Hossack, J. Arlt, and J. Crain, “Intracellular imaging of host-pathogen interactions using combined CARS and two-photon fluorescence microscopies,” J. Biophoton. 3, 138–146 (2010).
[CrossRef]

Denk, W.

W. Denk, D. W. Piston, and W. W. Webb, “Multi-photon molecular excitation in laser scanning microscopy,” in Handbook of Biological Confocal Microscopy J. B. Pawley, (Springer, 2006) pp. 535–549.
[CrossRef]

Dholakia, K.

D. J. Stevenson, D. J. Carnegie, B. Agate, F. Gunn-Moore, and K. Dholakia, “Long-term cell culture on a microscope stage: the carrel flask revisited.” Microsc. Anal. 22, 9–11 (2008).

Evans, C. L.

C. L. Evans and X. S. Xie, “Coherent anti-Stokes Raman scattering microscopy: chemical imaging for biology and medicine,” Annu. Rev. Anal. Chem. 1, 883–909 (2008).
[CrossRef]

Farese, R. V.

E. Herker, C. Harris, C. Hernandez, A. Carpentier, K. Kaehlcke, A. R. Rosenberg, R. V. Farese, and M. Ott, “Efficient hepatitis C virus particle formation requires diacylglycerol acyltransferase-1,” Nat. Med. 16, 1295–1298 (2010).
[CrossRef] [PubMed]

Farese, R.V.

Y. Guo, K. R. Cordes, R.V. Farese, and T.C. Walther, “Lipid droplets at a glance,” J. Cell Sci. 122, 749–752 (2009).
[CrossRef] [PubMed]

Fried, H.

H. Fried and U. Kutay, “Nucleocytoplasmic transport: taking an inventory,” Cell. Mol. Life Sci. 60, 1659–1688 (2003).
[CrossRef] [PubMed]

Ghazal, P.

A. Angulo, P. Ghazal, and M. Messerle, “The major immediate-early gene ie3 of mouse cytomegalovirus is essential for viral growth,” J. Virol. 74, 11129–11136 (2000).
[CrossRef] [PubMed]

Giraud, G.

I. Robinson, M. A. Ochsenkühn, C. J. Campbell, G. Giraud, W. J. Hossack, J. Arlt, and J. Crain, “Intracellular imaging of host-pathogen interactions using combined CARS and two-photon fluorescence microscopies,” J. Biophoton. 3, 138–146 (2010).
[CrossRef]

Gunn-Moore, F.

D. J. Stevenson, D. J. Carnegie, B. Agate, F. Gunn-Moore, and K. Dholakia, “Long-term cell culture on a microscope stage: the carrel flask revisited.” Microsc. Anal. 22, 9–11 (2008).

Guo, Y.

Y. Guo, K. R. Cordes, R.V. Farese, and T.C. Walther, “Lipid droplets at a glance,” J. Cell Sci. 122, 749–752 (2009).
[CrossRef] [PubMed]

Haas, J.

W. Muranyi, J. Haas, M. Wagner, G. Krohne, and U. H. Koszinowski, “Cytomegalovirus recruitment of cellular kinases to dissolve the nuclear lamina,” Science 297, 854–857 (2002).
[CrossRef] [PubMed]

Harris, C.

E. Herker, C. Harris, C. Hernandez, A. Carpentier, K. Kaehlcke, A. R. Rosenberg, R. V. Farese, and M. Ott, “Efficient hepatitis C virus particle formation requires diacylglycerol acyltransferase-1,” Nat. Med. 16, 1295–1298 (2010).
[CrossRef] [PubMed]

Hayat, M. A.

M. A. Hayat, “Chemical fixation,” in Principles and Techniques of Electron Microscopy Biological Applications (Cambridge University Press, 2000), pp. 43–44.

Hell, S. W.

J. Bewersdorf and S. W. Hell, “Picosecond pulsed two-photon imaging with repetition rates of 200 and 400 MHz,” J. Microsc. 191, 28–38 (1998).
[CrossRef]

Herker, E.

E. Herker, C. Harris, C. Hernandez, A. Carpentier, K. Kaehlcke, A. R. Rosenberg, R. V. Farese, and M. Ott, “Efficient hepatitis C virus particle formation requires diacylglycerol acyltransferase-1,” Nat. Med. 16, 1295–1298 (2010).
[CrossRef] [PubMed]

Hernandez, C.

E. Herker, C. Harris, C. Hernandez, A. Carpentier, K. Kaehlcke, A. R. Rosenberg, R. V. Farese, and M. Ott, “Efficient hepatitis C virus particle formation requires diacylglycerol acyltransferase-1,” Nat. Med. 16, 1295–1298 (2010).
[CrossRef] [PubMed]

Hinchcliffe, E. H.

G. Sluder, J. J. Nordberg, F. J. Miller, and E. H. Hinchcliffe, “A sealed preparation for long-term observations of cultured cells,” in Live Cell Imaging: A Laboratory Manual , R. D. Goldman and D. L. Spector eds. (Cold Spring Harbor Laboratory Press, 2005), pp. 345–349.

Hossack, W. J.

I. Robinson, M. A. Ochsenkühn, C. J. Campbell, G. Giraud, W. J. Hossack, J. Arlt, and J. Crain, “Intracellular imaging of host-pathogen interactions using combined CARS and two-photon fluorescence microscopies,” J. Biophoton. 3, 138–146 (2010).
[CrossRef]

Ionascu, D.

A. Yu, X. Ye, D. Ionascu, W. Cao, and P.M. Champion, “Two-colour pump-probe laser spectroscopy instrument with picosecond time-resolved electronic delay and extended scan range,” Rev. Sci. Instrum. 76114301 (2005).
[CrossRef]

Jones, D. M.

D. M. Jones and J. McLauchlan, “Hepatitis C virus: assembly and release of virus particles,” J. Biol. Chem. 285, 22733–22739 (2010).
[CrossRef] [PubMed]

Kaehlcke, K.

E. Herker, C. Harris, C. Hernandez, A. Carpentier, K. Kaehlcke, A. R. Rosenberg, R. V. Farese, and M. Ott, “Efficient hepatitis C virus particle formation requires diacylglycerol acyltransferase-1,” Nat. Med. 16, 1295–1298 (2010).
[CrossRef] [PubMed]

Kennedy, D. C

R. K. Lyn, D. C Kennedy, A. Stolow, A. Ridsdale, and J. P. Pezacki, “Dynamics of lipid droplets induced by the hepatitis C virus core protein,” Biochem. Biophys. Res. Commun. 399, 518–524 (2010).
[CrossRef] [PubMed]

Koszinowski, U. H.

W. Muranyi, J. Haas, M. Wagner, G. Krohne, and U. H. Koszinowski, “Cytomegalovirus recruitment of cellular kinases to dissolve the nuclear lamina,” Science 297, 854–857 (2002).
[CrossRef] [PubMed]

Krohne, G.

W. Muranyi, J. Haas, M. Wagner, G. Krohne, and U. H. Koszinowski, “Cytomegalovirus recruitment of cellular kinases to dissolve the nuclear lamina,” Science 297, 854–857 (2002).
[CrossRef] [PubMed]

Kutay, U.

H. Fried and U. Kutay, “Nucleocytoplasmic transport: taking an inventory,” Cell. Mol. Life Sci. 60, 1659–1688 (2003).
[CrossRef] [PubMed]

Lohmann, V.

R. Bartenschlager, F. Penn, V. Lohmann, and P. Andre, “Assembly of infectious hepatitis C virus particles,” Trends Microbiol. 19, 95–103 (2010).
[CrossRef] [PubMed]

Lyn, R. K.

R. K. Lyn, D. C Kennedy, A. Stolow, A. Ridsdale, and J. P. Pezacki, “Dynamics of lipid droplets induced by the hepatitis C virus core protein,” Biochem. Biophys. Res. Commun. 399, 518–524 (2010).
[CrossRef] [PubMed]

McLauchlan, J.

D. M. Jones and J. McLauchlan, “Hepatitis C virus: assembly and release of virus particles,” J. Biol. Chem. 285, 22733–22739 (2010).
[CrossRef] [PubMed]

Messerle, M.

A. Angulo, P. Ghazal, and M. Messerle, “The major immediate-early gene ie3 of mouse cytomegalovirus is essential for viral growth,” J. Virol. 74, 11129–11136 (2000).
[CrossRef] [PubMed]

Miller, F. J.

G. Sluder, J. J. Nordberg, F. J. Miller, and E. H. Hinchcliffe, “A sealed preparation for long-term observations of cultured cells,” in Live Cell Imaging: A Laboratory Manual , R. D. Goldman and D. L. Spector eds. (Cold Spring Harbor Laboratory Press, 2005), pp. 345–349.

Mocarski, E. S.

E. S. Mocarski, T. Shenk, and R. F. Pass, “Cytomegaloviruses,” in Fields Virology , D. M. Knipe and P. M. Howley eds. (Lippincott Williams and Wilkins, 2006) pp. 2702–2772.

Muranyi, W.

W. Muranyi, J. Haas, M. Wagner, G. Krohne, and U. H. Koszinowski, “Cytomegalovirus recruitment of cellular kinases to dissolve the nuclear lamina,” Science 297, 854–857 (2002).
[CrossRef] [PubMed]

Nan, X.

X. Nan, J. X. Cheng, and X. S. Xie, “Vibrational imaging of lipid droplets in live fibroblast cells with coherent anti-Stokes Raman scattering microscopy,” J. Lipid. Res. 44, 2202–2208 (2003).
[CrossRef] [PubMed]

Nordberg, J. J.

G. Sluder, J. J. Nordberg, F. J. Miller, and E. H. Hinchcliffe, “A sealed preparation for long-term observations of cultured cells,” in Live Cell Imaging: A Laboratory Manual , R. D. Goldman and D. L. Spector eds. (Cold Spring Harbor Laboratory Press, 2005), pp. 345–349.

Ochsenkühn, M. A.

I. Robinson, M. A. Ochsenkühn, C. J. Campbell, G. Giraud, W. J. Hossack, J. Arlt, and J. Crain, “Intracellular imaging of host-pathogen interactions using combined CARS and two-photon fluorescence microscopies,” J. Biophoton. 3, 138–146 (2010).
[CrossRef]

Ott, M.

E. Herker, C. Harris, C. Hernandez, A. Carpentier, K. Kaehlcke, A. R. Rosenberg, R. V. Farese, and M. Ott, “Efficient hepatitis C virus particle formation requires diacylglycerol acyltransferase-1,” Nat. Med. 16, 1295–1298 (2010).
[CrossRef] [PubMed]

Pass, R. F.

E. S. Mocarski, T. Shenk, and R. F. Pass, “Cytomegaloviruses,” in Fields Virology , D. M. Knipe and P. M. Howley eds. (Lippincott Williams and Wilkins, 2006) pp. 2702–2772.

Penn, F.

R. Bartenschlager, F. Penn, V. Lohmann, and P. Andre, “Assembly of infectious hepatitis C virus particles,” Trends Microbiol. 19, 95–103 (2010).
[CrossRef] [PubMed]

Pezacki, J. P.

R. K. Lyn, D. C Kennedy, A. Stolow, A. Ridsdale, and J. P. Pezacki, “Dynamics of lipid droplets induced by the hepatitis C virus core protein,” Biochem. Biophys. Res. Commun. 399, 518–524 (2010).
[CrossRef] [PubMed]

Piston, D. W.

W. Denk, D. W. Piston, and W. W. Webb, “Multi-photon molecular excitation in laser scanning microscopy,” in Handbook of Biological Confocal Microscopy J. B. Pawley, (Springer, 2006) pp. 535–549.
[CrossRef]

Ridsdale, A.

R. K. Lyn, D. C Kennedy, A. Stolow, A. Ridsdale, and J. P. Pezacki, “Dynamics of lipid droplets induced by the hepatitis C virus core protein,” Biochem. Biophys. Res. Commun. 399, 518–524 (2010).
[CrossRef] [PubMed]

Robinson, I.

I. Robinson, M. A. Ochsenkühn, C. J. Campbell, G. Giraud, W. J. Hossack, J. Arlt, and J. Crain, “Intracellular imaging of host-pathogen interactions using combined CARS and two-photon fluorescence microscopies,” J. Biophoton. 3, 138–146 (2010).
[CrossRef]

Rosenberg, A. R.

E. Herker, C. Harris, C. Hernandez, A. Carpentier, K. Kaehlcke, A. R. Rosenberg, R. V. Farese, and M. Ott, “Efficient hepatitis C virus particle formation requires diacylglycerol acyltransferase-1,” Nat. Med. 16, 1295–1298 (2010).
[CrossRef] [PubMed]

Shenk, T.

E. S. Mocarski, T. Shenk, and R. F. Pass, “Cytomegaloviruses,” in Fields Virology , D. M. Knipe and P. M. Howley eds. (Lippincott Williams and Wilkins, 2006) pp. 2702–2772.

Sluder, G.

G. Sluder, J. J. Nordberg, F. J. Miller, and E. H. Hinchcliffe, “A sealed preparation for long-term observations of cultured cells,” in Live Cell Imaging: A Laboratory Manual , R. D. Goldman and D. L. Spector eds. (Cold Spring Harbor Laboratory Press, 2005), pp. 345–349.

Stevenson, D. J.

D. J. Stevenson, D. J. Carnegie, B. Agate, F. Gunn-Moore, and K. Dholakia, “Long-term cell culture on a microscope stage: the carrel flask revisited.” Microsc. Anal. 22, 9–11 (2008).

Stolow, A.

R. K. Lyn, D. C Kennedy, A. Stolow, A. Ridsdale, and J. P. Pezacki, “Dynamics of lipid droplets induced by the hepatitis C virus core protein,” Biochem. Biophys. Res. Commun. 399, 518–524 (2010).
[CrossRef] [PubMed]

Wagner, M.

W. Muranyi, J. Haas, M. Wagner, G. Krohne, and U. H. Koszinowski, “Cytomegalovirus recruitment of cellular kinases to dissolve the nuclear lamina,” Science 297, 854–857 (2002).
[CrossRef] [PubMed]

Walther, T.C.

Y. Guo, K. R. Cordes, R.V. Farese, and T.C. Walther, “Lipid droplets at a glance,” J. Cell Sci. 122, 749–752 (2009).
[CrossRef] [PubMed]

Webb, W. W.

W. Denk, D. W. Piston, and W. W. Webb, “Multi-photon molecular excitation in laser scanning microscopy,” in Handbook of Biological Confocal Microscopy J. B. Pawley, (Springer, 2006) pp. 535–549.
[CrossRef]

Xie, X. S.

C. L. Evans and X. S. Xie, “Coherent anti-Stokes Raman scattering microscopy: chemical imaging for biology and medicine,” Annu. Rev. Anal. Chem. 1, 883–909 (2008).
[CrossRef]

X. Nan, J. X. Cheng, and X. S. Xie, “Vibrational imaging of lipid droplets in live fibroblast cells with coherent anti-Stokes Raman scattering microscopy,” J. Lipid. Res. 44, 2202–2208 (2003).
[CrossRef] [PubMed]

Ye, X.

A. Yu, X. Ye, D. Ionascu, W. Cao, and P.M. Champion, “Two-colour pump-probe laser spectroscopy instrument with picosecond time-resolved electronic delay and extended scan range,” Rev. Sci. Instrum. 76114301 (2005).
[CrossRef]

Yu, A.

A. Yu, X. Ye, D. Ionascu, W. Cao, and P.M. Champion, “Two-colour pump-probe laser spectroscopy instrument with picosecond time-resolved electronic delay and extended scan range,” Rev. Sci. Instrum. 76114301 (2005).
[CrossRef]

Annu. Rev. Anal. Chem. (1)

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Figures (15)

Fig. 1
Fig. 1

Portable cell chamber for live cell imaging. A glass-bottom Petri dish containing the cell sample is sealed into the chamber in a 5 % CO2 environment. The aluminium chamber body is fitted with heating elements and connected to a temperature controller which maintains a constant temperature of 37.0 °C. The chamber attaches directly to the stage of the (custom-built) microscope.

Fig. 2
Fig. 2

Multimodal CARS and TPF microscopy system. The Stokes laser is tuned to 896 nm and the pump to 714 nm, to target a Raman shift of 2845 cm−1. The two lasers are mode-locked and the pulses are synchronized. An xy pair of galvanometer mirrors is used to scan the combined beams across the sample. The CARS signal is detected by a photomultiplier tube (PMT) in the forward direction (F-CARS) and TPF is detected, also by a PMT, in the backward direction. The set-up can also perform bright field imaging with a halogen lamp and camera.

Fig. 3
Fig. 3

Images of fibroblast cells infected with a genetically modified mCMV which causes cellular expression of GFP. The CARS image (A) shows the distribution of cellular lipid droplets. The TPF image (B), acquired simultaneously with the CARS, shows the sites of GFP, indicating that the cell is infected. A second TPF image (C) was recorded after the CARS image (sequentially) using only the Stokes laser beam; the pump beam was blocked.

Fig. 4
Fig. 4

A stack of CARS images through a single cell. Slices were taken at 0.25 μm increments in the z-direction. The laser power at the sample was approximately 35 mW. The bright spots visible in the images were identified as lipid droplets.

Fig. 5
Fig. 5

Brightfield images recorded before (A) and after (B) the acquisition of the stack shown in Fig. 4. Visible photodamage is apparent after laser scanning.

Fig. 6
Fig. 6

CARS (A) and brightfield (B) images of the same cell shown in Fig. 4 and Fig. 5 recorded 2 hours after acquisition of the stack.

Fig. 7
Fig. 7

CARS (A) and TPF (B) images of cells fixed at the early stage of infection. Lipid droplets of regular size are uniformly distributed in the cytosol. The low level of fluorescence provides limited contrast in the TPF image.

Fig. 8
Fig. 8

CARS (A) and TPF (B) images of cells in the intermediate stage of infection. The distribution of lipid droplets is less uniform than in the early stage (Fig. 7) with the emergence of some clustering. The TPF image shows an increased amount of GFP expression.

Fig. 9
Fig. 9

CARS (A) and TPF (B) images of a sample of cells fixed in the late infection stage. (C) and (D) are CARS and TPF images of a different area of the same sample. The contrast in the two CARS images (A,C) is very limited due to the absence of lipid droplets. The TPF images (B,D) show a high level of GFP expression as well as fragmentation of cell nuclei suggesting apoptosis.

Fig. 10
Fig. 10

Graph showing the average intensities for TPF images acquired pre-infection; and during the early, intermediate and late stages. Error bars indicate 1 standard deviation. To account for small changes in laser power between images the two-photon intensities have been normalized by dividing by the square of the power.

Fig. 13
Fig. 13

CARS (A), TPF (B) and overlaid (C) images of a group of live cells in the late infection stage. (D–F) show a different group of cells. The pump and Stokes laser power at the sample was approximately 43 mW and 47 mW respectively. The CARS images (A,D) show a small number of lipid clusters. The high intensity in the corresponding TPF images (B,E) confirm the late stage of infection. Inhomogeneous infection is evident in image (B), where most cells are in the late infection stage with the exception of the two cells marked X which are in the intermediate stage, identified using fixed cell data as a reference.

Fig. 11
Fig. 11

CARS (A) and bright field (B) images of a group of live cells during the early stage of infection. The CARS image shows lipid droplets of regular size uniformly distributed throughout the cytosol. The pump and Stokes laser powers at the sample were approximately 25 mW and 30 mW respectively. The bright field image shows the cell outlines only.

Fig. 12
Fig. 12

CARS (A) and TPF (B) images of two cells in the intermediate stage of infection. The images are overlaid in (C). The pump and Stokes laser powers at the sample were approximately 28 mW and 32 mW respectively. The CARS image shows a non-uniform distribution of lipid droplets within the cytosol, with the appearance of clustering, specifically towards the neighbouring cell. The TPF image confirms that the cells are infected, and also shows that some GFP has been imported into the nucleus.

Fig. 14
Fig. 14

Histogram showing the probability distribution of the number of lipid droplets in a cell for the early, intermediate and late infection stages. The separate counts for each infection stage group were binned and are plotted in the histogram. An empty bin for a particular infection stage indicates that there were no cells whose lipid droplet count was within the bin range.

Fig. 15
Fig. 15

Histogram showing the probability distribution of lipid droplet area for the early, intermediate and late infection stages. Inset: histogram (of the same data) showing the probability distribution of the lipid droplets with area greater than 2 μm2.

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