Abstract

We introduce an in vivo imaging flow cytometer, which provides fluorescence images simultaneously with quantitative information on the cell population of interest in a live animal. As fluorescent cells pass through the slit of light focused across a blood vessel, the excited fluorescence is confocally detected. This cell signal triggers a strobe beam and a high sensitivity CCD camera that captures a snapshot image of the cell as it moves down-stream from the slit. We demonstrate that the majority of signal peaks detected in the in vivo flow cytometer arise form individual cells. The instrument’s capability to image circulating T cells and measure their speed in the blood vessel in real time in vivo is demonstrated. The cell signal irradiance variation, clustering percentage, and potential applications in biology and medicine are discussed.

© 2006 Optical Society of America

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  1. J. Novak, I. Georgakoudi, X. Wei, A. Prossin, and C. P. Lin, "In vivo flow cytometer for real-time detection and quantification of circulating cells," Opt. Lett. 29, 77-79 (2004).
    [CrossRef] [PubMed]
  2. I. Georgakoudi, N. Solban, J. Novak, W. L. Rice, X. Wei, T. Hasan, and C. P. Lin, "In vivo flow cytometry: a new method for enumerating circulating cancer cells," Cancer Res. 64, 5044-5047 (2004).
    [CrossRef] [PubMed]
  3. X. Wei, D. A. Sipkins, C. M. Pitsillides, J. Novak, I. Georgakoudi, and C. P. Lin, "Real-time detection of circulating apoptotic cells by in vivo flow cytometry," Mol. Imaging. 4, 415-416 (2005).
    [PubMed]
  4. D. A. Sipkins, X. Wei, J. W. Wu, J. M. Runnels, D. Côté, T. K. Means, A. D. Luster, D. T. Scadden, and C. P. Lin, "In vivo imaging of specialized bone marrow endothelial microdomains for tumour engraftment," Nature 435, 969-973 (2005).
    [CrossRef] [PubMed]
  5. V. P. Zharov, E. I. Galanzha, and V. V. Tuchin, "In vivo photothermal flow cytometry: imaging and detection of individual cells in blood and lymph flow," J. Cell. Biochem. 97, 916-932 (2006).
    [CrossRef] [PubMed]
  6. A. Hafezi-Moghadam, K. L. Thomas, and C. Cornelssen, "A novel mouse-driven ex vivo flow chamber for the study of leukocyte and platelet function," Am. J. Physiol. Cell Physiol. 286, C876-C892 (2004).
    [CrossRef]
  7. M. Shin, K. Matsuda, O. Ishii, H. Terai, M. Kaazempur-Mofrad, J. Borenstein, M. Detmar, and J. P. Vacanti, "Endothelialized networks with a vascular geometry in microfabricated poly(dimethyl siloxane)," Biomed. Microdevices 6, 269-278 (2004).
    [CrossRef] [PubMed]

2006

V. P. Zharov, E. I. Galanzha, and V. V. Tuchin, "In vivo photothermal flow cytometry: imaging and detection of individual cells in blood and lymph flow," J. Cell. Biochem. 97, 916-932 (2006).
[CrossRef] [PubMed]

2005

X. Wei, D. A. Sipkins, C. M. Pitsillides, J. Novak, I. Georgakoudi, and C. P. Lin, "Real-time detection of circulating apoptotic cells by in vivo flow cytometry," Mol. Imaging. 4, 415-416 (2005).
[PubMed]

D. A. Sipkins, X. Wei, J. W. Wu, J. M. Runnels, D. Côté, T. K. Means, A. D. Luster, D. T. Scadden, and C. P. Lin, "In vivo imaging of specialized bone marrow endothelial microdomains for tumour engraftment," Nature 435, 969-973 (2005).
[CrossRef] [PubMed]

2004

I. Georgakoudi, N. Solban, J. Novak, W. L. Rice, X. Wei, T. Hasan, and C. P. Lin, "In vivo flow cytometry: a new method for enumerating circulating cancer cells," Cancer Res. 64, 5044-5047 (2004).
[CrossRef] [PubMed]

A. Hafezi-Moghadam, K. L. Thomas, and C. Cornelssen, "A novel mouse-driven ex vivo flow chamber for the study of leukocyte and platelet function," Am. J. Physiol. Cell Physiol. 286, C876-C892 (2004).
[CrossRef]

M. Shin, K. Matsuda, O. Ishii, H. Terai, M. Kaazempur-Mofrad, J. Borenstein, M. Detmar, and J. P. Vacanti, "Endothelialized networks with a vascular geometry in microfabricated poly(dimethyl siloxane)," Biomed. Microdevices 6, 269-278 (2004).
[CrossRef] [PubMed]

J. Novak, I. Georgakoudi, X. Wei, A. Prossin, and C. P. Lin, "In vivo flow cytometer for real-time detection and quantification of circulating cells," Opt. Lett. 29, 77-79 (2004).
[CrossRef] [PubMed]

Borenstein, J.

M. Shin, K. Matsuda, O. Ishii, H. Terai, M. Kaazempur-Mofrad, J. Borenstein, M. Detmar, and J. P. Vacanti, "Endothelialized networks with a vascular geometry in microfabricated poly(dimethyl siloxane)," Biomed. Microdevices 6, 269-278 (2004).
[CrossRef] [PubMed]

Cornelssen, C.

A. Hafezi-Moghadam, K. L. Thomas, and C. Cornelssen, "A novel mouse-driven ex vivo flow chamber for the study of leukocyte and platelet function," Am. J. Physiol. Cell Physiol. 286, C876-C892 (2004).
[CrossRef]

Côté, D.

D. A. Sipkins, X. Wei, J. W. Wu, J. M. Runnels, D. Côté, T. K. Means, A. D. Luster, D. T. Scadden, and C. P. Lin, "In vivo imaging of specialized bone marrow endothelial microdomains for tumour engraftment," Nature 435, 969-973 (2005).
[CrossRef] [PubMed]

Detmar, M.

M. Shin, K. Matsuda, O. Ishii, H. Terai, M. Kaazempur-Mofrad, J. Borenstein, M. Detmar, and J. P. Vacanti, "Endothelialized networks with a vascular geometry in microfabricated poly(dimethyl siloxane)," Biomed. Microdevices 6, 269-278 (2004).
[CrossRef] [PubMed]

Galanzha, E. I.

V. P. Zharov, E. I. Galanzha, and V. V. Tuchin, "In vivo photothermal flow cytometry: imaging and detection of individual cells in blood and lymph flow," J. Cell. Biochem. 97, 916-932 (2006).
[CrossRef] [PubMed]

Georgakoudi, I.

X. Wei, D. A. Sipkins, C. M. Pitsillides, J. Novak, I. Georgakoudi, and C. P. Lin, "Real-time detection of circulating apoptotic cells by in vivo flow cytometry," Mol. Imaging. 4, 415-416 (2005).
[PubMed]

J. Novak, I. Georgakoudi, X. Wei, A. Prossin, and C. P. Lin, "In vivo flow cytometer for real-time detection and quantification of circulating cells," Opt. Lett. 29, 77-79 (2004).
[CrossRef] [PubMed]

I. Georgakoudi, N. Solban, J. Novak, W. L. Rice, X. Wei, T. Hasan, and C. P. Lin, "In vivo flow cytometry: a new method for enumerating circulating cancer cells," Cancer Res. 64, 5044-5047 (2004).
[CrossRef] [PubMed]

Hafezi-Moghadam, A.

A. Hafezi-Moghadam, K. L. Thomas, and C. Cornelssen, "A novel mouse-driven ex vivo flow chamber for the study of leukocyte and platelet function," Am. J. Physiol. Cell Physiol. 286, C876-C892 (2004).
[CrossRef]

Hasan, T.

I. Georgakoudi, N. Solban, J. Novak, W. L. Rice, X. Wei, T. Hasan, and C. P. Lin, "In vivo flow cytometry: a new method for enumerating circulating cancer cells," Cancer Res. 64, 5044-5047 (2004).
[CrossRef] [PubMed]

Ishii, O.

M. Shin, K. Matsuda, O. Ishii, H. Terai, M. Kaazempur-Mofrad, J. Borenstein, M. Detmar, and J. P. Vacanti, "Endothelialized networks with a vascular geometry in microfabricated poly(dimethyl siloxane)," Biomed. Microdevices 6, 269-278 (2004).
[CrossRef] [PubMed]

Kaazempur-Mofrad, M.

M. Shin, K. Matsuda, O. Ishii, H. Terai, M. Kaazempur-Mofrad, J. Borenstein, M. Detmar, and J. P. Vacanti, "Endothelialized networks with a vascular geometry in microfabricated poly(dimethyl siloxane)," Biomed. Microdevices 6, 269-278 (2004).
[CrossRef] [PubMed]

Lin, C. P.

D. A. Sipkins, X. Wei, J. W. Wu, J. M. Runnels, D. Côté, T. K. Means, A. D. Luster, D. T. Scadden, and C. P. Lin, "In vivo imaging of specialized bone marrow endothelial microdomains for tumour engraftment," Nature 435, 969-973 (2005).
[CrossRef] [PubMed]

X. Wei, D. A. Sipkins, C. M. Pitsillides, J. Novak, I. Georgakoudi, and C. P. Lin, "Real-time detection of circulating apoptotic cells by in vivo flow cytometry," Mol. Imaging. 4, 415-416 (2005).
[PubMed]

J. Novak, I. Georgakoudi, X. Wei, A. Prossin, and C. P. Lin, "In vivo flow cytometer for real-time detection and quantification of circulating cells," Opt. Lett. 29, 77-79 (2004).
[CrossRef] [PubMed]

I. Georgakoudi, N. Solban, J. Novak, W. L. Rice, X. Wei, T. Hasan, and C. P. Lin, "In vivo flow cytometry: a new method for enumerating circulating cancer cells," Cancer Res. 64, 5044-5047 (2004).
[CrossRef] [PubMed]

Luster, A. D.

D. A. Sipkins, X. Wei, J. W. Wu, J. M. Runnels, D. Côté, T. K. Means, A. D. Luster, D. T. Scadden, and C. P. Lin, "In vivo imaging of specialized bone marrow endothelial microdomains for tumour engraftment," Nature 435, 969-973 (2005).
[CrossRef] [PubMed]

Matsuda, K.

M. Shin, K. Matsuda, O. Ishii, H. Terai, M. Kaazempur-Mofrad, J. Borenstein, M. Detmar, and J. P. Vacanti, "Endothelialized networks with a vascular geometry in microfabricated poly(dimethyl siloxane)," Biomed. Microdevices 6, 269-278 (2004).
[CrossRef] [PubMed]

Means, T. K.

D. A. Sipkins, X. Wei, J. W. Wu, J. M. Runnels, D. Côté, T. K. Means, A. D. Luster, D. T. Scadden, and C. P. Lin, "In vivo imaging of specialized bone marrow endothelial microdomains for tumour engraftment," Nature 435, 969-973 (2005).
[CrossRef] [PubMed]

Novak, J.

X. Wei, D. A. Sipkins, C. M. Pitsillides, J. Novak, I. Georgakoudi, and C. P. Lin, "Real-time detection of circulating apoptotic cells by in vivo flow cytometry," Mol. Imaging. 4, 415-416 (2005).
[PubMed]

J. Novak, I. Georgakoudi, X. Wei, A. Prossin, and C. P. Lin, "In vivo flow cytometer for real-time detection and quantification of circulating cells," Opt. Lett. 29, 77-79 (2004).
[CrossRef] [PubMed]

I. Georgakoudi, N. Solban, J. Novak, W. L. Rice, X. Wei, T. Hasan, and C. P. Lin, "In vivo flow cytometry: a new method for enumerating circulating cancer cells," Cancer Res. 64, 5044-5047 (2004).
[CrossRef] [PubMed]

Pitsillides, C. M.

X. Wei, D. A. Sipkins, C. M. Pitsillides, J. Novak, I. Georgakoudi, and C. P. Lin, "Real-time detection of circulating apoptotic cells by in vivo flow cytometry," Mol. Imaging. 4, 415-416 (2005).
[PubMed]

Prossin, A.

Rice, W. L.

I. Georgakoudi, N. Solban, J. Novak, W. L. Rice, X. Wei, T. Hasan, and C. P. Lin, "In vivo flow cytometry: a new method for enumerating circulating cancer cells," Cancer Res. 64, 5044-5047 (2004).
[CrossRef] [PubMed]

Runnels, J. M.

D. A. Sipkins, X. Wei, J. W. Wu, J. M. Runnels, D. Côté, T. K. Means, A. D. Luster, D. T. Scadden, and C. P. Lin, "In vivo imaging of specialized bone marrow endothelial microdomains for tumour engraftment," Nature 435, 969-973 (2005).
[CrossRef] [PubMed]

Scadden, D. T.

D. A. Sipkins, X. Wei, J. W. Wu, J. M. Runnels, D. Côté, T. K. Means, A. D. Luster, D. T. Scadden, and C. P. Lin, "In vivo imaging of specialized bone marrow endothelial microdomains for tumour engraftment," Nature 435, 969-973 (2005).
[CrossRef] [PubMed]

Shin, M.

M. Shin, K. Matsuda, O. Ishii, H. Terai, M. Kaazempur-Mofrad, J. Borenstein, M. Detmar, and J. P. Vacanti, "Endothelialized networks with a vascular geometry in microfabricated poly(dimethyl siloxane)," Biomed. Microdevices 6, 269-278 (2004).
[CrossRef] [PubMed]

Sipkins, D. A.

D. A. Sipkins, X. Wei, J. W. Wu, J. M. Runnels, D. Côté, T. K. Means, A. D. Luster, D. T. Scadden, and C. P. Lin, "In vivo imaging of specialized bone marrow endothelial microdomains for tumour engraftment," Nature 435, 969-973 (2005).
[CrossRef] [PubMed]

X. Wei, D. A. Sipkins, C. M. Pitsillides, J. Novak, I. Georgakoudi, and C. P. Lin, "Real-time detection of circulating apoptotic cells by in vivo flow cytometry," Mol. Imaging. 4, 415-416 (2005).
[PubMed]

Solban, N.

I. Georgakoudi, N. Solban, J. Novak, W. L. Rice, X. Wei, T. Hasan, and C. P. Lin, "In vivo flow cytometry: a new method for enumerating circulating cancer cells," Cancer Res. 64, 5044-5047 (2004).
[CrossRef] [PubMed]

Terai, H.

M. Shin, K. Matsuda, O. Ishii, H. Terai, M. Kaazempur-Mofrad, J. Borenstein, M. Detmar, and J. P. Vacanti, "Endothelialized networks with a vascular geometry in microfabricated poly(dimethyl siloxane)," Biomed. Microdevices 6, 269-278 (2004).
[CrossRef] [PubMed]

Thomas, K. L.

A. Hafezi-Moghadam, K. L. Thomas, and C. Cornelssen, "A novel mouse-driven ex vivo flow chamber for the study of leukocyte and platelet function," Am. J. Physiol. Cell Physiol. 286, C876-C892 (2004).
[CrossRef]

Tuchin, V. V.

V. P. Zharov, E. I. Galanzha, and V. V. Tuchin, "In vivo photothermal flow cytometry: imaging and detection of individual cells in blood and lymph flow," J. Cell. Biochem. 97, 916-932 (2006).
[CrossRef] [PubMed]

Vacanti, J. P.

M. Shin, K. Matsuda, O. Ishii, H. Terai, M. Kaazempur-Mofrad, J. Borenstein, M. Detmar, and J. P. Vacanti, "Endothelialized networks with a vascular geometry in microfabricated poly(dimethyl siloxane)," Biomed. Microdevices 6, 269-278 (2004).
[CrossRef] [PubMed]

Wei, X.

D. A. Sipkins, X. Wei, J. W. Wu, J. M. Runnels, D. Côté, T. K. Means, A. D. Luster, D. T. Scadden, and C. P. Lin, "In vivo imaging of specialized bone marrow endothelial microdomains for tumour engraftment," Nature 435, 969-973 (2005).
[CrossRef] [PubMed]

X. Wei, D. A. Sipkins, C. M. Pitsillides, J. Novak, I. Georgakoudi, and C. P. Lin, "Real-time detection of circulating apoptotic cells by in vivo flow cytometry," Mol. Imaging. 4, 415-416 (2005).
[PubMed]

J. Novak, I. Georgakoudi, X. Wei, A. Prossin, and C. P. Lin, "In vivo flow cytometer for real-time detection and quantification of circulating cells," Opt. Lett. 29, 77-79 (2004).
[CrossRef] [PubMed]

I. Georgakoudi, N. Solban, J. Novak, W. L. Rice, X. Wei, T. Hasan, and C. P. Lin, "In vivo flow cytometry: a new method for enumerating circulating cancer cells," Cancer Res. 64, 5044-5047 (2004).
[CrossRef] [PubMed]

Wu, J. W.

D. A. Sipkins, X. Wei, J. W. Wu, J. M. Runnels, D. Côté, T. K. Means, A. D. Luster, D. T. Scadden, and C. P. Lin, "In vivo imaging of specialized bone marrow endothelial microdomains for tumour engraftment," Nature 435, 969-973 (2005).
[CrossRef] [PubMed]

Zharov, V. P.

V. P. Zharov, E. I. Galanzha, and V. V. Tuchin, "In vivo photothermal flow cytometry: imaging and detection of individual cells in blood and lymph flow," J. Cell. Biochem. 97, 916-932 (2006).
[CrossRef] [PubMed]

Am. J. Physiol. Cell Physiol.

A. Hafezi-Moghadam, K. L. Thomas, and C. Cornelssen, "A novel mouse-driven ex vivo flow chamber for the study of leukocyte and platelet function," Am. J. Physiol. Cell Physiol. 286, C876-C892 (2004).
[CrossRef]

Biomed. Microdevices

M. Shin, K. Matsuda, O. Ishii, H. Terai, M. Kaazempur-Mofrad, J. Borenstein, M. Detmar, and J. P. Vacanti, "Endothelialized networks with a vascular geometry in microfabricated poly(dimethyl siloxane)," Biomed. Microdevices 6, 269-278 (2004).
[CrossRef] [PubMed]

Cancer Res.

I. Georgakoudi, N. Solban, J. Novak, W. L. Rice, X. Wei, T. Hasan, and C. P. Lin, "In vivo flow cytometry: a new method for enumerating circulating cancer cells," Cancer Res. 64, 5044-5047 (2004).
[CrossRef] [PubMed]

J. Cell. Biochem.

V. P. Zharov, E. I. Galanzha, and V. V. Tuchin, "In vivo photothermal flow cytometry: imaging and detection of individual cells in blood and lymph flow," J. Cell. Biochem. 97, 916-932 (2006).
[CrossRef] [PubMed]

Mol. Imaging.

X. Wei, D. A. Sipkins, C. M. Pitsillides, J. Novak, I. Georgakoudi, and C. P. Lin, "Real-time detection of circulating apoptotic cells by in vivo flow cytometry," Mol. Imaging. 4, 415-416 (2005).
[PubMed]

Nature

D. A. Sipkins, X. Wei, J. W. Wu, J. M. Runnels, D. Côté, T. K. Means, A. D. Luster, D. T. Scadden, and C. P. Lin, "In vivo imaging of specialized bone marrow endothelial microdomains for tumour engraftment," Nature 435, 969-973 (2005).
[CrossRef] [PubMed]

Opt. Lett.

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Figures (8)

Fig. 1.
Fig. 1.

Schematic diagram of the in vivo imaging flow cytometer. M1-M3 : mirrors, AL1-AL4: achromats, BS1-BS2: dichroic beam splitters , F1-F2: bandpass filters, CL : cylindrical lens, OL: microscope objective lens. Red lines indicate optical path. Dashed black arrows indicate electrical connections.

Fig. 2.
Fig. 2.

A typical result of in vitro flow cytometry of a DiD-labeled cell population. The X-axis in the scatter plot (a) represents forward scattering, which is correlated to cell size (increasing with cell diameter). The Y-axis represents the intensity of fluorescence labeling. About 99% of the labeled cells are the size of T cell, appearing in the upper left quadrant of the fluorescence and scattering plot. However, about 1% of population (upper right quadrant) is comprised of cells bigger than T cells or potential cell aggregates. The histogram (b) demonstrates that fluorescence intensity of DiD-labeled cells is well separated from unlabeled control cells.

Fig. 3.
Fig. 3.

Ex vivo images of DiD-labeled T cells. (a) image taken with a commercial fluorescence microscope and (b) images taken with the imaging flow cytometer

Fig. 4.
Fig. 4.

In vivo images and corresponding PMT traces of a single T cell ((a), (b)), a cluster of two T cells ((c), (d)), and two cells traveling in close proximity to each other ((e), (f)).

Fig. 5.
Fig. 5.

(a) Ex vivo image and (b) in vivo image of non T cell. These cells can be distinguished from a T cell due to its bigger size. Furthermore, their surface structure appears to be irregular and distinctly different from the usual appearance of T cells. However, it is not possible to clearly identify them with DiD labeling alone. They are most likely not dead cells, as apoptotic or necrotic cells are cleared from circulation within minutes [3].

Fig. 6.
Fig. 6.

In vivo images of DiD-labeled T cells and corresponding PMT traces. (a) brightest, (b) medium brightness, (c) darkest cell, and (d) the total photon count against PMT voltage. The first bell-shaped pulse is induced by a cell passing through the slit. The second short pulse is induced by the imaging pulse. Since the gain for the PMT and the intensified CCD camera were constant for these measurements, the difference in brightness is intrinsic to the cell labeling.

Fig. 7.
Fig. 7.

In vivo image of cells in circulation with different speed and corresponding PMT traces. The speed was estimated a straight path from the slit to the captured position. (a) a fast cell with a speed of 3.8 mm/s and (b) a slow cell with a speed of 2.1 mm/s.

Fig. 8.
Fig. 8.

In vivo image of a cell with two consecutive imaging pulses and corresponding PMT trace. Two pulses are temporally separated by 20 ms and the image pulse duration is 400 μs. The measured traveling speed of cell is 0.67 mm/s.

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