Abstract

Focal Modulation Microscopy (FMM) is a single-photon excitation fluorescence microscopy technique which effectively rejects the out-of-focus fluorescence background that arises when imaging deep inside biological tissues. Here, we report on the implementation of FMM in which laser intensity modulation at the focal plane is achieved using acousto-optic modulators (AOM). The modulation speed is greatly enhanced to the MHz range and thus enables real-time image acquisition. The capability of FMM is demonstrated by imaging fluorescence labeled vasculatures in mouse brain as well as self-made tissue phantom.

© 2010 OSA

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S. J. Lunt, C. Gray, C. C. Reyes-Aldasoro, S. J. Matcher, and G. M. Tozer, “Application of intravital microscopy in studies of tumor microcirculation,” J. Biomed. Opt. 15(1), 011113 (2010).
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[CrossRef] [PubMed]

2009

C. H. Wong, S. P. Chong, C. J. R. Sheppard, and N. Chen, “Simple spatial phase modulator for focal modulation microscopy,” Appl. Opt. 48(17), 3237–3242 (2009).
[CrossRef] [PubMed]

D. Razansky, M. Distel, C. Vinegoni, R. Ma, N. Perrimon, R. W. Köster, and V. Ntziachristos, “Multispectral opto-acoustic tomography of deep-seated fluorescent proteins in vivo,” Nat. Photonics 3(7), 412–417 (2009).
[CrossRef]

2008

2007

2006

P. Theer and W. Denk, “On the fundamental imaging-depth limit in two-photon microscopy,” J. Opt. Soc. Am. A 23(12), 3139–3149 (2006).
[CrossRef] [PubMed]

H. F. Zhang, K. Maslov, G. Stoica, and L. V. Wang, “Functional photoacoustic microscopy for high-resolution and noninvasive in vivo imaging,” Nat. Biotechnol. 24(7), 848–851 (2006).
[CrossRef] [PubMed]

K. Svoboda and R. Yasuda, “Principles of two-photon excitation microscopy and its applications to neuroscience,” Neuron 50(6), 823–839 (2006).
[CrossRef] [PubMed]

2005

J. A. Conchello and J. W. Lichtman, “Optical sectioning microscopy,” Nat. Methods 2(12), 920–931 (2005).
[CrossRef] [PubMed]

F. Helmchen and W. Denk, “Deep tissue two-photon microscopy,” Nat. Methods 2(12), 932–940 (2005).
[CrossRef] [PubMed]

2003

2000

G. H. Patterson and D. W. Piston, “Photobleaching in two-photon excitation microscopy,” Biophys. J. 78(4), 2159–2162 (2000).
[CrossRef] [PubMed]

1997

1995

M. Firbank, M. Oda, and D. T. Delpy, “An improved design for a stable and reproducible phantom material for use in near-infrared spectroscopy and imaging,” Phys. Med. Biol. 40(5), 955–961 (1995).
[CrossRef] [PubMed]

1994

1990

W. Denk, J. H. Strickler, and W. W. Webb, “Two-photon laser scanning fluorescence microscopy,” Science 248(4951), 73–76 (1990).
[CrossRef] [PubMed]

Chen, N.

Chong, S. P.

Conchello, J. A.

J. A. Conchello and J. W. Lichtman, “Optical sectioning microscopy,” Nat. Methods 2(12), 920–931 (2005).
[CrossRef] [PubMed]

Delpy, D. T.

M. Firbank, M. Oda, and D. T. Delpy, “An improved design for a stable and reproducible phantom material for use in near-infrared spectroscopy and imaging,” Phys. Med. Biol. 40(5), 955–961 (1995).
[CrossRef] [PubMed]

Deng, X.

Denk, W.

P. Theer and W. Denk, “On the fundamental imaging-depth limit in two-photon microscopy,” J. Opt. Soc. Am. A 23(12), 3139–3149 (2006).
[CrossRef] [PubMed]

F. Helmchen and W. Denk, “Deep tissue two-photon microscopy,” Nat. Methods 2(12), 932–940 (2005).
[CrossRef] [PubMed]

W. Denk, J. H. Strickler, and W. W. Webb, “Two-photon laser scanning fluorescence microscopy,” Science 248(4951), 73–76 (1990).
[CrossRef] [PubMed]

Distel, M.

D. Razansky, M. Distel, C. Vinegoni, R. Ma, N. Perrimon, R. W. Köster, and V. Ntziachristos, “Multispectral opto-acoustic tomography of deep-seated fluorescent proteins in vivo,” Nat. Photonics 3(7), 412–417 (2009).
[CrossRef]

Firbank, M.

M. Firbank, M. Oda, and D. T. Delpy, “An improved design for a stable and reproducible phantom material for use in near-infrared spectroscopy and imaging,” Phys. Med. Biol. 40(5), 955–961 (1995).
[CrossRef] [PubMed]

Gaidosh, G.

Y. Li, Y. Song, L. Zhao, G. Gaidosh, A. M. Laties, and R. Wen, “Direct labeling and visualization of blood vessels with lipophilic carbocyanine dye DiI,” Nat. Protoc. 3(11), 1703–1708 (2008).
[CrossRef] [PubMed]

Gray, C.

S. J. Lunt, C. Gray, C. C. Reyes-Aldasoro, S. J. Matcher, and G. M. Tozer, “Application of intravital microscopy in studies of tumor microcirculation,” J. Biomed. Opt. 15(1), 011113 (2010).
[CrossRef] [PubMed]

Gu, M.

Heintzmann, R.

Hell, S. W.

Helmchen, F.

F. Helmchen and W. Denk, “Deep tissue two-photon microscopy,” Nat. Methods 2(12), 932–940 (2005).
[CrossRef] [PubMed]

Juskaitis, R.

Knüttel, A.

Köster, R. W.

D. Razansky, M. Distel, C. Vinegoni, R. Ma, N. Perrimon, R. W. Köster, and V. Ntziachristos, “Multispectral opto-acoustic tomography of deep-seated fluorescent proteins in vivo,” Nat. Photonics 3(7), 412–417 (2009).
[CrossRef]

Laties, A. M.

Y. Li, Y. Song, L. Zhao, G. Gaidosh, A. M. Laties, and R. Wen, “Direct labeling and visualization of blood vessels with lipophilic carbocyanine dye DiI,” Nat. Protoc. 3(11), 1703–1708 (2008).
[CrossRef] [PubMed]

Li, Y.

Y. Li, Y. Song, L. Zhao, G. Gaidosh, A. M. Laties, and R. Wen, “Direct labeling and visualization of blood vessels with lipophilic carbocyanine dye DiI,” Nat. Protoc. 3(11), 1703–1708 (2008).
[CrossRef] [PubMed]

Lichtman, J. W.

J. A. Conchello and J. W. Lichtman, “Optical sectioning microscopy,” Nat. Methods 2(12), 920–931 (2005).
[CrossRef] [PubMed]

Lunt, S. J.

S. J. Lunt, C. Gray, C. C. Reyes-Aldasoro, S. J. Matcher, and G. M. Tozer, “Application of intravital microscopy in studies of tumor microcirculation,” J. Biomed. Opt. 15(1), 011113 (2010).
[CrossRef] [PubMed]

Ma, R.

D. Razansky, M. Distel, C. Vinegoni, R. Ma, N. Perrimon, R. W. Köster, and V. Ntziachristos, “Multispectral opto-acoustic tomography of deep-seated fluorescent proteins in vivo,” Nat. Photonics 3(7), 412–417 (2009).
[CrossRef]

Maslov, K.

H. F. Zhang, K. Maslov, G. Stoica, and L. V. Wang, “Functional photoacoustic microscopy for high-resolution and noninvasive in vivo imaging,” Nat. Biotechnol. 24(7), 848–851 (2006).
[CrossRef] [PubMed]

Matcher, S. J.

S. J. Lunt, C. Gray, C. C. Reyes-Aldasoro, S. J. Matcher, and G. M. Tozer, “Application of intravital microscopy in studies of tumor microcirculation,” J. Biomed. Opt. 15(1), 011113 (2010).
[CrossRef] [PubMed]

Mertz, J.

Mo, W.

Neil, M. A. A.

Ntziachristos, V.

D. Razansky, M. Distel, C. Vinegoni, R. Ma, N. Perrimon, R. W. Köster, and V. Ntziachristos, “Multispectral opto-acoustic tomography of deep-seated fluorescent proteins in vivo,” Nat. Photonics 3(7), 412–417 (2009).
[CrossRef]

Oda, M.

M. Firbank, M. Oda, and D. T. Delpy, “An improved design for a stable and reproducible phantom material for use in near-infrared spectroscopy and imaging,” Phys. Med. Biol. 40(5), 955–961 (1995).
[CrossRef] [PubMed]

Patterson, G. H.

G. H. Patterson and D. W. Piston, “Photobleaching in two-photon excitation microscopy,” Biophys. J. 78(4), 2159–2162 (2000).
[CrossRef] [PubMed]

Perrimon, N.

D. Razansky, M. Distel, C. Vinegoni, R. Ma, N. Perrimon, R. W. Köster, and V. Ntziachristos, “Multispectral opto-acoustic tomography of deep-seated fluorescent proteins in vivo,” Nat. Photonics 3(7), 412–417 (2009).
[CrossRef]

Piston, D. W.

G. H. Patterson and D. W. Piston, “Photobleaching in two-photon excitation microscopy,” Biophys. J. 78(4), 2159–2162 (2000).
[CrossRef] [PubMed]

Razansky, D.

D. Razansky, M. Distel, C. Vinegoni, R. Ma, N. Perrimon, R. W. Köster, and V. Ntziachristos, “Multispectral opto-acoustic tomography of deep-seated fluorescent proteins in vivo,” Nat. Photonics 3(7), 412–417 (2009).
[CrossRef]

Reyes-Aldasoro, C. C.

S. J. Lunt, C. Gray, C. C. Reyes-Aldasoro, S. J. Matcher, and G. M. Tozer, “Application of intravital microscopy in studies of tumor microcirculation,” J. Biomed. Opt. 15(1), 011113 (2010).
[CrossRef] [PubMed]

Schmitt, J. M.

Sheppard, C. J.

Sheppard, C. J. R.

Song, Y.

Y. Li, Y. Song, L. Zhao, G. Gaidosh, A. M. Laties, and R. Wen, “Direct labeling and visualization of blood vessels with lipophilic carbocyanine dye DiI,” Nat. Protoc. 3(11), 1703–1708 (2008).
[CrossRef] [PubMed]

Stoica, G.

H. F. Zhang, K. Maslov, G. Stoica, and L. V. Wang, “Functional photoacoustic microscopy for high-resolution and noninvasive in vivo imaging,” Nat. Biotechnol. 24(7), 848–851 (2006).
[CrossRef] [PubMed]

Strickler, J. H.

W. Denk, J. H. Strickler, and W. W. Webb, “Two-photon laser scanning fluorescence microscopy,” Science 248(4951), 73–76 (1990).
[CrossRef] [PubMed]

Svoboda, K.

K. Svoboda and R. Yasuda, “Principles of two-photon excitation microscopy and its applications to neuroscience,” Neuron 50(6), 823–839 (2006).
[CrossRef] [PubMed]

Theer, P.

Tozer, G. M.

S. J. Lunt, C. Gray, C. C. Reyes-Aldasoro, S. J. Matcher, and G. M. Tozer, “Application of intravital microscopy in studies of tumor microcirculation,” J. Biomed. Opt. 15(1), 011113 (2010).
[CrossRef] [PubMed]

Ventalon, C.

Vinegoni, C.

D. Razansky, M. Distel, C. Vinegoni, R. Ma, N. Perrimon, R. W. Köster, and V. Ntziachristos, “Multispectral opto-acoustic tomography of deep-seated fluorescent proteins in vivo,” Nat. Photonics 3(7), 412–417 (2009).
[CrossRef]

Wang, L. V.

H. F. Zhang, K. Maslov, G. Stoica, and L. V. Wang, “Functional photoacoustic microscopy for high-resolution and noninvasive in vivo imaging,” Nat. Biotechnol. 24(7), 848–851 (2006).
[CrossRef] [PubMed]

Webb, W. W.

W. Denk, J. H. Strickler, and W. W. Webb, “Two-photon laser scanning fluorescence microscopy,” Science 248(4951), 73–76 (1990).
[CrossRef] [PubMed]

Wen, R.

Y. Li, Y. Song, L. Zhao, G. Gaidosh, A. M. Laties, and R. Wen, “Direct labeling and visualization of blood vessels with lipophilic carbocyanine dye DiI,” Nat. Protoc. 3(11), 1703–1708 (2008).
[CrossRef] [PubMed]

Wichmann, J.

Wilson, T.

Wong, C. H.

Yadlowsky, M.

Yasuda, R.

K. Svoboda and R. Yasuda, “Principles of two-photon excitation microscopy and its applications to neuroscience,” Neuron 50(6), 823–839 (2006).
[CrossRef] [PubMed]

Zhang, H. F.

H. F. Zhang, K. Maslov, G. Stoica, and L. V. Wang, “Functional photoacoustic microscopy for high-resolution and noninvasive in vivo imaging,” Nat. Biotechnol. 24(7), 848–851 (2006).
[CrossRef] [PubMed]

Zhao, L.

Y. Li, Y. Song, L. Zhao, G. Gaidosh, A. M. Laties, and R. Wen, “Direct labeling and visualization of blood vessels with lipophilic carbocyanine dye DiI,” Nat. Protoc. 3(11), 1703–1708 (2008).
[CrossRef] [PubMed]

Appl. Opt.

Biophys. J.

G. H. Patterson and D. W. Piston, “Photobleaching in two-photon excitation microscopy,” Biophys. J. 78(4), 2159–2162 (2000).
[CrossRef] [PubMed]

J. Biomed. Opt.

S. J. Lunt, C. Gray, C. C. Reyes-Aldasoro, S. J. Matcher, and G. M. Tozer, “Application of intravital microscopy in studies of tumor microcirculation,” J. Biomed. Opt. 15(1), 011113 (2010).
[CrossRef] [PubMed]

J. Opt. Soc. Am. A

Nat. Biotechnol.

H. F. Zhang, K. Maslov, G. Stoica, and L. V. Wang, “Functional photoacoustic microscopy for high-resolution and noninvasive in vivo imaging,” Nat. Biotechnol. 24(7), 848–851 (2006).
[CrossRef] [PubMed]

Nat. Methods

J. A. Conchello and J. W. Lichtman, “Optical sectioning microscopy,” Nat. Methods 2(12), 920–931 (2005).
[CrossRef] [PubMed]

F. Helmchen and W. Denk, “Deep tissue two-photon microscopy,” Nat. Methods 2(12), 932–940 (2005).
[CrossRef] [PubMed]

Nat. Photonics

D. Razansky, M. Distel, C. Vinegoni, R. Ma, N. Perrimon, R. W. Köster, and V. Ntziachristos, “Multispectral opto-acoustic tomography of deep-seated fluorescent proteins in vivo,” Nat. Photonics 3(7), 412–417 (2009).
[CrossRef]

Nat. Protoc.

Y. Li, Y. Song, L. Zhao, G. Gaidosh, A. M. Laties, and R. Wen, “Direct labeling and visualization of blood vessels with lipophilic carbocyanine dye DiI,” Nat. Protoc. 3(11), 1703–1708 (2008).
[CrossRef] [PubMed]

Neuron

K. Svoboda and R. Yasuda, “Principles of two-photon excitation microscopy and its applications to neuroscience,” Neuron 50(6), 823–839 (2006).
[CrossRef] [PubMed]

Opt. Express

Opt. Lett.

Phys. Med. Biol.

M. Firbank, M. Oda, and D. T. Delpy, “An improved design for a stable and reproducible phantom material for use in near-infrared spectroscopy and imaging,” Phys. Med. Biol. 40(5), 955–961 (1995).
[CrossRef] [PubMed]

Science

W. Denk, J. H. Strickler, and W. W. Webb, “Two-photon laser scanning fluorescence microscopy,” Science 248(4951), 73–76 (1990).
[CrossRef] [PubMed]

Supplementary Material (1)

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Figures (5)

Fig. 1
Fig. 1

Setup for a FMM. Laser beam is split by a beam splitter (BS) in which light beam in each arm undergoing frequency shifting by two acoustic-optical modulators (AOM) with different resonance frequencies. Phase-shifted beams are reflected back to the BS by retroreflectors (R1 and R2). They are aligned in parallel non-overlapping manner when being split second times at BS in which one combined beam will be used to generate a reference signal while another combined beam is directed to the scanning unit of Olympus FV300 to excite the samples through the objective lens (OBJ). PD is a photodetector and M is a mirror.

Fig. 2
Fig. 2

Flow diagrams showing the signal processing pathway.

Fig. 3
Fig. 3

Demonstration of FMM in retaining high-resolution features by rejecting out-of-focus fluorescence background. A blood vessel bifurcation image acquired with Olympus LUCPLANFLN 20x/0.45 NA using (a) CM and (b) FMM shows much finer features can be revealed by FMM even at penetration depth up 200um. Figure 3 (c) and (d) shows the digital magnification near the bifurcation of the main blood vessel which reveals much richer features on the image captured by FMM. The plot at the bottom of the images shows the intensity profiles of the white lines (top-down) in the images.

Fig. 4
Fig. 4

(Media 1) Image of cluster of TiO2 embedded 320um inside tissue phantom captured with Olympus LUCPLANFLN 20x/0.45 NA using (a) CM and (b) FMM. Better image quality is achieved using FMM, with less blurring and structures are more clearly distinguishable due to sharp boundaries. The plot at the bottom of the images shows the intensity profile of the white lines in the images. Peaks are more distinct with better contrast can be observed.

Fig. 5
Fig. 5

Normalized spatial spectra (with respect to dc values) of the region of interests labeled by square boxes in Fig. 4 (a) and (b) showing the spatial frequency components of CM and FMM respectively.

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