Abstract

We present a small, lightweight two-photon fiberscope and demonstrate its suitability for functional imaging in the intact brain. Our device consists of a hollow-core photonic crystal fiber for efficient delivery of near-IR femtosecond laser pulses, a spiral fiber-scanner for resonant beam steering, and a gradient-index lens system for fluorescence excitation, dichroic beam splitting, and signal collection. Fluorescence light is remotely detected using a standard photomultiplier tube. All optical components have 1 mm dimensions and the microscope’s headpiece weighs only 0.6 grams. The instrument achieves micrometer resolution at frame rates of typically 25 Hz with a field-of-view of up to 200 microns. We demonstrate functional imaging of calcium signals in Purkinje cell dendrites in the cerebellum of anesthetized rats. The microscope will be easily portable by a rat or mouse and thus should enable functional imaging in freely behaving animals.

© 2008 Optical Society of America

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    [CrossRef]
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    [CrossRef] [PubMed]

2007

E. J. Seibel, R. S. Johnston, C. M. Brown, J. A. Dominitz, and M. B. Kimmey, "Novel ultrathin scanning fiber endoscope for cholangioscopy and pancreatoscopy," Gastrointest. Endosc. 65, Ab125-Ab125 (2007).
[CrossRef]

L. Fu and M. Gu, "Fibre-optic nonlinear optical microscopy and endoscopy," J. Microsc. 226, 195-206 (2007).
[CrossRef] [PubMed]

M. Murayama, E. Perez-Garci, H. R. Luscher, and M. E. Larkum, "Fiberoptic system for recording dendritic calcium signals in layer 5 neocortical pyramidal cells in freely moving rats," J. Neurophysiol. 98, 1791-1805 (2007).
[CrossRef] [PubMed]

J. Sawinski and W. Denk, "Miniature random-access fiber scanner for in vivo multiphoton imaging," J. Appl. Phys. 102, (2007).
[CrossRef]

W. Göbel and F. Helmchen, "New angles on neuronal dendrites in vivo," J. Neurophysiol. 98, 3770-3779 (2007).
[CrossRef] [PubMed]

D. A. Dombeck, A. N. Khabbaz, F. Collman, T. L. Adelman, and D. W. Tank, "Imaging large-scale neural activity with cellular resolution in awake, mobile mice," Neuron 56, 43-57 (2007).
[CrossRef] [PubMed]

2006

W. Piyawattanametha, R. P. J. Barretto, T. H. Ko, B. A. Flusberg, E. D. Cocker, H. J. Ra, D. S. Lee, O. Solgaard, and M. J. Schnitzer, "Fast-scanning two-photon fluorescence imaging based on a microelectromechanical systems two-dimensional scanning mirror," Opt. Lett. 31, 2018-2020 (2006).
[CrossRef] [PubMed]

L. Fu, A. Jain, H. Xie, C. Cranfield, and M. Gu, "Nonlinear optical endoscopy based on a double-clad photonic crystal fiber and a MEMS mirror," Opt. Express 14, 1027-1032 (2006).
[CrossRef] [PubMed]

A. Monfared, N. H. Blevins, E. L. M. Cheung, J. C. Jung, G. Popelka, and M. J. Schnitzer, "In vivo Imaging of mammalian cochlear blood flow using fluorescence microendoscopy," Otology & Neurotology 27, 144-152 (2006).
[CrossRef] [PubMed]

P. Vincent, U. Maskos, I. Charvet, L. Bourgeais, L. Stoppini, N. Leresche, J. P. Changeux, R. Lambert, P. Meda, and D. Paupardin-Tritsch, "Live imaging of neural structure and function by fibred fluorescence microscopy," EMBO Rep 7, 1154-1161 (2006).
[CrossRef] [PubMed]

E. J. Seibel, R. S. Johnston, and C. D. Melville, "A full-color scanning fiber endoscope," Optical Fibers and Sensors for Medical Diagnostics and Treatment Applications VI.Proceedings of the SPIE. 6083, 9-16 (2006).

C. J. Engelbrecht and E. H. K. Stelzer, "Resolution enhancement in a light-sheet-based microscope (SPIM)," Opt. Lett. 31, 1477-1479 (2006).
[CrossRef] [PubMed]

M. T. Myaing, D. J. MacDonald, and X. D. Li, "Fiber-optic scanning two-photon fluorescence endoscope," Opt. Lett. 31, 1076-1078 (2006).
[CrossRef] [PubMed]

A. F. Low, G. J. Tearney, B. E. Bouma, and I. K. Jang, "Technology insight: optical coherence tomography - current status and future development," Nature Clinical Practice Cardiovascular Medicine 3, 154-162 (2006).
[CrossRef] [PubMed]

Z. Yaqoob, J. G. Wu, E. J. McDowell, X. Heng, and C. H. Yang, "Methods and application areas of endoscopic optical coherence tomography," J. Biomed. Opt. 11, 063001 (2006).
[CrossRef]

2005

F. Helmchen and W. Denk, "Deep tissue two-photon microscopy," Nature Methods 2, 932-940 (2005).
[CrossRef] [PubMed]

E. Seibel, T. Soper, R. Johnston, and R. Glenny, "Ultrathin laser scanning bronchoscope and guidance system for the peripheral lung," Lung Cancer 49, S162-S162 (2005).
[CrossRef]

B. A. Flusberg, E. D. Cocker, W. Piyawattanametha, J. C. Jung, E. L. M. Cheung, and M. J. Schnitzer, "Fiber-optic fluorescence imaging," Nature Methods 2, 941-950 (2005).
[CrossRef] [PubMed]

H. Adelsberger, O. Garaschuk, and A. Konnerth, "Cortical calcium waves in resting newborn mice," Nat. Neurosci. 8, 988-990 (2005).
[CrossRef] [PubMed]

L. Fu, X. Gan, and M. Gu, "Nonlinear optical microscopy based on double-clad photonic crystal fibers," Opt. Express 13, 5528-5534 (2005).
[CrossRef] [PubMed]

B. A. Flusberg, J. C. Lung, E. D. Cocker, E. P. Anderson, and M. J. Schnitzer, "In vivo brain imaging using a portable 3.9 gram two-photon fluorescence microendoscope," Opt. Lett. 30, 2272-2274 (2005).
[CrossRef] [PubMed]

M. R. Sullivan, A. Nimmerjahn, D. V. Sarkisov, F. Helmchen, and S. S. Wang, "In vivo calcium imaging of circuit activity in cerebellar cortex," J. Neurophysiol. 94, 1636-1644 (2005).
[CrossRef] [PubMed]

2004

W. Göbel, A. Nimmerjahn, and F. Helmchen, "Distortion-free delivery of nanojoule femtosecond pulses from a Ti : sapphire laser through a hollow-core photonic crystal fiber," Opt. Lett. 29, 1285-1287 (2004).
[CrossRef] [PubMed]

W. Göbel, J. N. D. Kerr, A. Nimmerjahn, and F. Helmchen, "Miniaturized two-photon microscope based on a flexible coherent fiber bundle and a gradient-index lens objective," Opt. Lett. 29, 2521-2523 (2004).
[CrossRef] [PubMed]

A. D. Mehta, J. C. Jung, B. A. Flusberg, and M. J. Schnitzer, "Fiber optic in vivo imaging in the mammalian nervous system," Curr. Opin. Neurobiol. 14, 617-628 (2004).
[CrossRef] [PubMed]

J. C. Jung, A. D. Mehta, E. Aksay, R. Stepnoski, and M. J. Schnitzer, "In vivo mammalian brain Imaging using one- and two-photon fluorescence microendoscopy," J. Neurophysiol. 92, 3121-3133 (2004).
[CrossRef] [PubMed]

M. J. Levene, D. A. Dombeck, K. A. Kasischke, R. P. Molloy, and W. W. Webb, "In vivo multiphoton microscopy of deep brain tissue," J. Neurophysiol. 91, 1908-1912 (2004).
[CrossRef]

2003

J. C. Jung and M. J. Schnitzer, "Multiphoton endoscopy," Opt. Lett. 28, 902-904 (2003).
[CrossRef] [PubMed]

C. Stosiek, O. Garaschuk, K. Holthoff, and A. Konnerth, "In vivo two-photon calcium imaging of neuronal networks," Proc. Natl. Acad. Sci. U S A 100, 7319-7324 (2003).
[CrossRef] [PubMed]

2002

2001

F. Helmchen, M. S. Fee, D. W. Tank, and W. Denk, "A miniature head-mounted two-photon microscope: High-resolution brain imaging in freely moving animals," Neuron 31, 903-912 (2001).
[CrossRef] [PubMed]

J. Knittel, L. Schnieder, G. Buess, B. Messerschmidt, and T. Possner, "Endoscope-compatible confocal microscope using a gradient index-lens system," Opt. Commun. 188, 267-273 (2001).
[CrossRef]

1999

1997

W. Denk and K. Svoboda, "Photon upmanship: why multiphoton imaging is more than a gimmick," Neuron 18, 351-357 (1997).
[CrossRef] [PubMed]

1990

W. Denk, J. H. Strickler, and W. W. Webb, "Two-Photon Laser Scanning Fluorescence Microscopy," Science 248, 73-76 (1990).
[CrossRef] [PubMed]

Appl. Opt.

Curr. Opin. Neurobiol.

A. D. Mehta, J. C. Jung, B. A. Flusberg, and M. J. Schnitzer, "Fiber optic in vivo imaging in the mammalian nervous system," Curr. Opin. Neurobiol. 14, 617-628 (2004).
[CrossRef] [PubMed]

EMBO Rep

P. Vincent, U. Maskos, I. Charvet, L. Bourgeais, L. Stoppini, N. Leresche, J. P. Changeux, R. Lambert, P. Meda, and D. Paupardin-Tritsch, "Live imaging of neural structure and function by fibred fluorescence microscopy," EMBO Rep 7, 1154-1161 (2006).
[CrossRef] [PubMed]

Exp. Physiol.

F. Helmchen, "Miniaturization of fluorescence microscopes using fibre optics," Exp. Physiol. 87, 737-745 (2002).
[CrossRef] [PubMed]

Gastrointest. Endosc.

E. J. Seibel, R. S. Johnston, C. M. Brown, J. A. Dominitz, and M. B. Kimmey, "Novel ultrathin scanning fiber endoscope for cholangioscopy and pancreatoscopy," Gastrointest. Endosc. 65, Ab125-Ab125 (2007).
[CrossRef]

J. Appl. Phys.

J. Sawinski and W. Denk, "Miniature random-access fiber scanner for in vivo multiphoton imaging," J. Appl. Phys. 102, (2007).
[CrossRef]

J. Biomed. Opt.

Z. Yaqoob, J. G. Wu, E. J. McDowell, X. Heng, and C. H. Yang, "Methods and application areas of endoscopic optical coherence tomography," J. Biomed. Opt. 11, 063001 (2006).
[CrossRef]

J. Microsc.

L. Fu and M. Gu, "Fibre-optic nonlinear optical microscopy and endoscopy," J. Microsc. 226, 195-206 (2007).
[CrossRef] [PubMed]

J. Neurophysiol.

J. C. Jung, A. D. Mehta, E. Aksay, R. Stepnoski, and M. J. Schnitzer, "In vivo mammalian brain Imaging using one- and two-photon fluorescence microendoscopy," J. Neurophysiol. 92, 3121-3133 (2004).
[CrossRef] [PubMed]

M. J. Levene, D. A. Dombeck, K. A. Kasischke, R. P. Molloy, and W. W. Webb, "In vivo multiphoton microscopy of deep brain tissue," J. Neurophysiol. 91, 1908-1912 (2004).
[CrossRef]

M. Murayama, E. Perez-Garci, H. R. Luscher, and M. E. Larkum, "Fiberoptic system for recording dendritic calcium signals in layer 5 neocortical pyramidal cells in freely moving rats," J. Neurophysiol. 98, 1791-1805 (2007).
[CrossRef] [PubMed]

M. R. Sullivan, A. Nimmerjahn, D. V. Sarkisov, F. Helmchen, and S. S. Wang, "In vivo calcium imaging of circuit activity in cerebellar cortex," J. Neurophysiol. 94, 1636-1644 (2005).
[CrossRef] [PubMed]

W. Göbel and F. Helmchen, "New angles on neuronal dendrites in vivo," J. Neurophysiol. 98, 3770-3779 (2007).
[CrossRef] [PubMed]

J. Opt. Soc. Am. A

Lasers Surg. Med.

E. J. Seibel and Q. Y. J. Smithwick, "Unique features of optical scanning, single fiber endoscopy," Lasers Surg. Med. 30, 177-183 (2002).
[CrossRef] [PubMed]

Lung Cancer

E. Seibel, T. Soper, R. Johnston, and R. Glenny, "Ultrathin laser scanning bronchoscope and guidance system for the peripheral lung," Lung Cancer 49, S162-S162 (2005).
[CrossRef]

Nat. Neurosci.

H. Adelsberger, O. Garaschuk, and A. Konnerth, "Cortical calcium waves in resting newborn mice," Nat. Neurosci. 8, 988-990 (2005).
[CrossRef] [PubMed]

Nature Clinical Practice Cardiovascular Medicine

A. F. Low, G. J. Tearney, B. E. Bouma, and I. K. Jang, "Technology insight: optical coherence tomography - current status and future development," Nature Clinical Practice Cardiovascular Medicine 3, 154-162 (2006).
[CrossRef] [PubMed]

Nature Methods

B. A. Flusberg, E. D. Cocker, W. Piyawattanametha, J. C. Jung, E. L. M. Cheung, and M. J. Schnitzer, "Fiber-optic fluorescence imaging," Nature Methods 2, 941-950 (2005).
[CrossRef] [PubMed]

F. Helmchen and W. Denk, "Deep tissue two-photon microscopy," Nature Methods 2, 932-940 (2005).
[CrossRef] [PubMed]

Neuron

F. Helmchen, M. S. Fee, D. W. Tank, and W. Denk, "A miniature head-mounted two-photon microscope: High-resolution brain imaging in freely moving animals," Neuron 31, 903-912 (2001).
[CrossRef] [PubMed]

W. Denk and K. Svoboda, "Photon upmanship: why multiphoton imaging is more than a gimmick," Neuron 18, 351-357 (1997).
[CrossRef] [PubMed]

D. A. Dombeck, A. N. Khabbaz, F. Collman, T. L. Adelman, and D. W. Tank, "Imaging large-scale neural activity with cellular resolution in awake, mobile mice," Neuron 56, 43-57 (2007).
[CrossRef] [PubMed]

Opt. Commun.

J. Knittel, L. Schnieder, G. Buess, B. Messerschmidt, and T. Possner, "Endoscope-compatible confocal microscope using a gradient index-lens system," Opt. Commun. 188, 267-273 (2001).
[CrossRef]

Opt. Express

Opt. Lett.

W. Piyawattanametha, R. P. J. Barretto, T. H. Ko, B. A. Flusberg, E. D. Cocker, H. J. Ra, D. S. Lee, O. Solgaard, and M. J. Schnitzer, "Fast-scanning two-photon fluorescence imaging based on a microelectromechanical systems two-dimensional scanning mirror," Opt. Lett. 31, 2018-2020 (2006).
[CrossRef] [PubMed]

C. J. Engelbrecht and E. H. K. Stelzer, "Resolution enhancement in a light-sheet-based microscope (SPIM)," Opt. Lett. 31, 1477-1479 (2006).
[CrossRef] [PubMed]

W. Göbel, A. Nimmerjahn, and F. Helmchen, "Distortion-free delivery of nanojoule femtosecond pulses from a Ti : sapphire laser through a hollow-core photonic crystal fiber," Opt. Lett. 29, 1285-1287 (2004).
[CrossRef] [PubMed]

B. A. Flusberg, J. C. Lung, E. D. Cocker, E. P. Anderson, and M. J. Schnitzer, "In vivo brain imaging using a portable 3.9 gram two-photon fluorescence microendoscope," Opt. Lett. 30, 2272-2274 (2005).
[CrossRef] [PubMed]

M. T. Myaing, D. J. MacDonald, and X. D. Li, "Fiber-optic scanning two-photon fluorescence endoscope," Opt. Lett. 31, 1076-1078 (2006).
[CrossRef] [PubMed]

J. C. Jung and M. J. Schnitzer, "Multiphoton endoscopy," Opt. Lett. 28, 902-904 (2003).
[CrossRef] [PubMed]

W. Göbel, J. N. D. Kerr, A. Nimmerjahn, and F. Helmchen, "Miniaturized two-photon microscope based on a flexible coherent fiber bundle and a gradient-index lens objective," Opt. Lett. 29, 2521-2523 (2004).
[CrossRef] [PubMed]

D. G. Ouzounov, K. D. Moll, M. A. Foster, W. R. Zipfel, W. W. Webb, and A. L. Gaeta, "Delivery of nanojoule femtosecond pulses through large-core microstructured fibers," Opt. Lett. 27, 1513-1515 (2002).
[CrossRef]

Otology & Neurotology

A. Monfared, N. H. Blevins, E. L. M. Cheung, J. C. Jung, G. Popelka, and M. J. Schnitzer, "In vivo Imaging of mammalian cochlear blood flow using fluorescence microendoscopy," Otology & Neurotology 27, 144-152 (2006).
[CrossRef] [PubMed]

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Supplementary Material (1)

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Figures (3)

Fig. 1.
Fig. 1.

Schematic (a) and perspective (b) representations of the ultra-compact fiber-optic two-photon microscope. A hollow-core PCF guides near-IR ultrafast laser pulses (red) to the microscope headpiece. The fiber cantilever is scanned in a spiral pattern by applying appropriate piezo drive signals (PDS) to a resonant fiber scanner (RFS). The motion of the fiber cantilever is imaged onto the sample (S) by a custom-designed combination of GRINlenses and micro-beamsplitter prisms (BSP). Fluorescence emission light (green) passes the GRIN objective lens and the BSP, is guided through a large core fiber (LCF) and detected by a photomultiplier tube. All optical components in the headpiece have millimeter dimensions. (c) Piezo drive signals for x- (blue trace) and y-direction (orange trace; 90° phase shifted). Amplitude modulation is used to create the spiral pattern. (d) ‘Ideal’ (left) and measured (right) scan trajectories, exemplified for a very small number of spiral lines. (e) Two-photon fiberscope images of 10-micron diameter fluorescent microspheres using an ideal (left) and a measured (right) scan trajectory for image reconstruction, respectively. Average laser power levels of 1 mW and 10x averaging were used. Scale bars are 20 µm.

Fig. 2.
Fig. 2.

Micrometer resolution of the two-photon fiberscope. (a) Two-photon image of 500-nm fluorescent microspheres. Scale bar 3 µm. Fluorescence intensity profiles of an example microsphere along the lateral (b) and the axial (c) dimension. Red traces are Gaussian-shaped curves fitted to the data points. (d) Calculation of the effective NA from the observed lateral (left, red) and axial (right, blue) FWHM. Horizontal lines represent mean ± S.D. The shaded areas correspond to the estimated error ranges of the calculated NAs in lateral and axial direction, respectively. Average laser power levels of 25 mW and 3x averaging were used.

Fig. 3.
Fig. 3.

(a) The cerebellar cortex has a well-defined anatomical organization with flat dendritic trees of Purkinje cells lying in parasagittal planes. In top view, dendritic excitation is evident as band-like calcium signals. Three snapshots from a ΔF/F movie of 33 s total duration are provided. (b and c) Examples of spontaneous ΔF/F-traces in ROIs comprising different dendrites. Note the different time scales. (d) Example of semi-automated ROI-definition by ICA. (e) Corresponding ΔF/F-traces color-coded according to the ROI selections in (d). All scale bars are 15 µm. [Media 1]

Equations (2)

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FWHM 1 p , lat = λ 0 n 3 2 cos ( α ) cos ( 2 α )
FWHM 1 p , ax = λ 0 n [ 1 cos ( α ) ]

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