Abstract

A snapshot Image Mapping Spectrometer (IMS) with high sampling density is developed for hyperspectral microscopy, measuring a datacube of dimensions 285 × 285 × 60 (x, y, λ). The spatial resolution is ~0.45 µm with a FOV of 100 × 100 µm2. The measured spectrum is from 450 nm to 650 nm and is sampled by 60 spectral channels with average sampling interval ~3.3 nm. The channel’s spectral resolution is ~8nm. The spectral imaging results demonstrate the potential of the IMS for real-time cellular fluorescence imaging.

© 2010 OSA

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2010 (5)

2009 (3)

L. Gao, R. T. Kester, and T. S. Tkaczyk, “Compact Image Slicing Spectrometer (ISS) for hyperspectral fluorescence microscopy,” Opt. Express 17(15), 12293–12308 (2009), http://www.opticsinfobase.org/abstract.cfm?URI=oe-17-15-12293 .
[CrossRef] [PubMed]

R. A. Schwarz, W. Gao, C. Redden Weber, C. Kurachi, J. J. Lee, A. K. El-Naggar, R. Richards-Kortum, and A. M. Gillenwater, “Noninvasive evaluation of oral lesions using depth-sensitive optical spectroscopy,” Cancer 115(8), 1669–1679 (2009).
[CrossRef] [PubMed]

C. A. Fernandez, A. Wagadarikar, D. J. Brady, S. C. McCain, and T. Oliver, “Fluorescence microscopy with a coded aperture snapshot spectral imager,” Proc. SPIE 7184, 71840Z (2009).
[CrossRef]

2007 (3)

J. Y. Ye, C. J. Divin, J. R. Baker, and T. B. Norris, “Whole spectrum fluorescence detection with ultrafast white light excitation,” Opt. Express 15(16), 10439–10445 (2007), http://www.opticsinfobase.org/oe/abstract.cfm?URI=oe-15-16-10439 .
[CrossRef] [PubMed]

T. Vo-Dinh, “A hyperspectral imaging system for in vivo optical diagnostics,” IEEE Eng. Med. Biol. Mag. 23, 40–49 (2007).

W. R. Johnson, D. W. Wilson, W. Fink, M. Humayun, and G. Bearman, “Snapshot hyperspectral imaging in ophthalmology,” J. Biomed. Opt. 12(1), 014036 (2007).
[CrossRef] [PubMed]

2005 (2)

V. Ntziachristos, J. Ripoll, L. V. Wang, and R. Weissleder, “Looking and listening to light: the evolution of whole-body photonic imaging,” Nat. Biotechnol. 23(3), 313–320 (2005).
[CrossRef] [PubMed]

K. Ritchie, X. Y. Shan, J. Kondo, K. Iwasawa, T. Fujiwara, and A. Kusumi, “Detection of non-Brownian diffusion in the cell membrane in single molecule tracking,” Biophys. J. 88(3), 2266–2277 (2005).
[CrossRef]

2004 (1)

R. H. Berg, “Evaluation of spectral imaging for plant cell analysis,” J. Microsc. 214(2), 174–181 (2004).
[CrossRef] [PubMed]

2003 (1)

T. Zimmermann, J. Rietdorf, and R. Pepperkok, “Spectral imaging and its applications in live cell microscopy,” FEBS Lett. 546(1), 87–92 (2003).
[CrossRef] [PubMed]

2002 (2)

Y. Hiraoka, T. Shimi, and T. Haraguchi, “Multispectral imaging fluorescence microscopy for living cells,” Cell Struct. Funct. 27(5), 367–374 (2002).
[CrossRef] [PubMed]

K. J. Zuzak, M. D. Schaeberle, E. N. Lewis, and I. W. Levin, “Visible reflectance hyperspectral imaging: characterization of a noninvasive, in vivo system for determining tissue perfusion,” Anal. Chem. 74(9), 2021–2028 (2002).
[CrossRef] [PubMed]

2001 (3)

B. K. Ford, C. E. Volin, S. M. Murphy, R. M. Lynch, and M. R. Descour, “Computed tomography-based spectral imaging for fluorescence microscopy,” Biophys. J. 80(2), 986–993 (2001).
[CrossRef] [PubMed]

B. Ford, M. Descour, and R. Lynch, “Large-image-format computed tomography imaging spectrometer for fluorescence microscopy,” Opt. Express 9(9), 444–453 (2001).
[CrossRef] [PubMed]

R. Lansford, G. Bearman, and S. E. Fraser, “Resolution of multiple green fluorescent protein color variants and dyes using two-photon microscopy and imaging spectroscopy,” J. Biomed. Opt. 6(3), 311–318 (2001).
[CrossRef] [PubMed]

2000 (1)

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

1999 (1)

K. N. Richmond, R. D. Shonat, R. M. Lynch, and P. C. Johnson, “The critical oxygen tension of skeletal muscle in vivo,” Am. J. Physiol. 277(5 Pt 2), H1831–H1840 (1999).
[PubMed]

Baker, J. R.

Bearman, G.

W. R. Johnson, D. W. Wilson, W. Fink, M. Humayun, and G. Bearman, “Snapshot hyperspectral imaging in ophthalmology,” J. Biomed. Opt. 12(1), 014036 (2007).
[CrossRef] [PubMed]

R. Lansford, G. Bearman, and S. E. Fraser, “Resolution of multiple green fluorescent protein color variants and dyes using two-photon microscopy and imaging spectroscopy,” J. Biomed. Opt. 6(3), 311–318 (2001).
[CrossRef] [PubMed]

Bedard, N.

R. T. Kester, L. Gao, N. Bedard, and T. S. Tkaczyk, “Real-time hyperspectral endoscope for early cancer diagnostics,” Proc. SPIE 7555, 75550A (2010).
[CrossRef]

Berg, R. H.

R. H. Berg, “Evaluation of spectral imaging for plant cell analysis,” J. Microsc. 214(2), 174–181 (2004).
[CrossRef] [PubMed]

Booth, M. J.

M. J. Booth, A. Jesacher, R. Juå Kaitis, and T. Wilson, “Full spectrum filterless fluorescence microscopy,” J. Microsc. 237(1), 103–109 (2010).
[CrossRef] [PubMed]

Brady, D. J.

C. F. Cull, K. Choi, D. J. Brady, and T. Oliver, “Identification of fluorescent beads using a coded aperture snapshot spectral imager,” Appl. Opt. 49(10), B59–B70 (2010), http://www.opticsinfobase.org/abstract.cfm?URI=ao-49-10-B59 .
[CrossRef] [PubMed]

C. A. Fernandez, A. Wagadarikar, D. J. Brady, S. C. McCain, and T. Oliver, “Fluorescence microscopy with a coded aperture snapshot spectral imager,” Proc. SPIE 7184, 71840Z (2009).
[CrossRef]

Choi, K.

Cull, C. F.

Descour, M.

Descour, M. R.

B. K. Ford, C. E. Volin, S. M. Murphy, R. M. Lynch, and M. R. Descour, “Computed tomography-based spectral imaging for fluorescence microscopy,” Biophys. J. 80(2), 986–993 (2001).
[CrossRef] [PubMed]

Divin, C. J.

El-Naggar, A. K.

R. A. Schwarz, W. Gao, C. Redden Weber, C. Kurachi, J. J. Lee, A. K. El-Naggar, R. Richards-Kortum, and A. M. Gillenwater, “Noninvasive evaluation of oral lesions using depth-sensitive optical spectroscopy,” Cancer 115(8), 1669–1679 (2009).
[CrossRef] [PubMed]

Fernandez, C. A.

C. A. Fernandez, A. Wagadarikar, D. J. Brady, S. C. McCain, and T. Oliver, “Fluorescence microscopy with a coded aperture snapshot spectral imager,” Proc. SPIE 7184, 71840Z (2009).
[CrossRef]

Fink, W.

W. R. Johnson, D. W. Wilson, W. Fink, M. Humayun, and G. Bearman, “Snapshot hyperspectral imaging in ophthalmology,” J. Biomed. Opt. 12(1), 014036 (2007).
[CrossRef] [PubMed]

Fletcher-Holmes, D. W.

Ford, B.

Ford, B. K.

B. K. Ford, C. E. Volin, S. M. Murphy, R. M. Lynch, and M. R. Descour, “Computed tomography-based spectral imaging for fluorescence microscopy,” Biophys. J. 80(2), 986–993 (2001).
[CrossRef] [PubMed]

Fraser, S. E.

R. Lansford, G. Bearman, and S. E. Fraser, “Resolution of multiple green fluorescent protein color variants and dyes using two-photon microscopy and imaging spectroscopy,” J. Biomed. Opt. 6(3), 311–318 (2001).
[CrossRef] [PubMed]

Fujiwara, T.

K. Ritchie, X. Y. Shan, J. Kondo, K. Iwasawa, T. Fujiwara, and A. Kusumi, “Detection of non-Brownian diffusion in the cell membrane in single molecule tracking,” Biophys. J. 88(3), 2266–2277 (2005).
[CrossRef]

Gao, L.

Gao, W.

R. A. Schwarz, W. Gao, C. Redden Weber, C. Kurachi, J. J. Lee, A. K. El-Naggar, R. Richards-Kortum, and A. M. Gillenwater, “Noninvasive evaluation of oral lesions using depth-sensitive optical spectroscopy,” Cancer 115(8), 1669–1679 (2009).
[CrossRef] [PubMed]

Gillenwater, A. M.

R. A. Schwarz, W. Gao, C. Redden Weber, C. Kurachi, J. J. Lee, A. K. El-Naggar, R. Richards-Kortum, and A. M. Gillenwater, “Noninvasive evaluation of oral lesions using depth-sensitive optical spectroscopy,” Cancer 115(8), 1669–1679 (2009).
[CrossRef] [PubMed]

Gorman, A.

Haraguchi, T.

Y. Hiraoka, T. Shimi, and T. Haraguchi, “Multispectral imaging fluorescence microscopy for living cells,” Cell Struct. Funct. 27(5), 367–374 (2002).
[CrossRef] [PubMed]

Harvey, A. R.

Hiraoka, Y.

Y. Hiraoka, T. Shimi, and T. Haraguchi, “Multispectral imaging fluorescence microscopy for living cells,” Cell Struct. Funct. 27(5), 367–374 (2002).
[CrossRef] [PubMed]

Humayun, M.

W. R. Johnson, D. W. Wilson, W. Fink, M. Humayun, and G. Bearman, “Snapshot hyperspectral imaging in ophthalmology,” J. Biomed. Opt. 12(1), 014036 (2007).
[CrossRef] [PubMed]

Iwasawa, K.

K. Ritchie, X. Y. Shan, J. Kondo, K. Iwasawa, T. Fujiwara, and A. Kusumi, “Detection of non-Brownian diffusion in the cell membrane in single molecule tracking,” Biophys. J. 88(3), 2266–2277 (2005).
[CrossRef]

Jesacher, A.

M. J. Booth, A. Jesacher, R. Juå Kaitis, and T. Wilson, “Full spectrum filterless fluorescence microscopy,” J. Microsc. 237(1), 103–109 (2010).
[CrossRef] [PubMed]

Johnson, P. C.

K. N. Richmond, R. D. Shonat, R. M. Lynch, and P. C. Johnson, “The critical oxygen tension of skeletal muscle in vivo,” Am. J. Physiol. 277(5 Pt 2), H1831–H1840 (1999).
[PubMed]

Johnson, W. R.

W. R. Johnson, D. W. Wilson, W. Fink, M. Humayun, and G. Bearman, “Snapshot hyperspectral imaging in ophthalmology,” J. Biomed. Opt. 12(1), 014036 (2007).
[CrossRef] [PubMed]

Juå Kaitis, R.

M. J. Booth, A. Jesacher, R. Juå Kaitis, and T. Wilson, “Full spectrum filterless fluorescence microscopy,” J. Microsc. 237(1), 103–109 (2010).
[CrossRef] [PubMed]

Kester, R. T.

Kondo, J.

K. Ritchie, X. Y. Shan, J. Kondo, K. Iwasawa, T. Fujiwara, and A. Kusumi, “Detection of non-Brownian diffusion in the cell membrane in single molecule tracking,” Biophys. J. 88(3), 2266–2277 (2005).
[CrossRef]

Kurachi, C.

R. A. Schwarz, W. Gao, C. Redden Weber, C. Kurachi, J. J. Lee, A. K. El-Naggar, R. Richards-Kortum, and A. M. Gillenwater, “Noninvasive evaluation of oral lesions using depth-sensitive optical spectroscopy,” Cancer 115(8), 1669–1679 (2009).
[CrossRef] [PubMed]

Kusumi, A.

K. Ritchie, X. Y. Shan, J. Kondo, K. Iwasawa, T. Fujiwara, and A. Kusumi, “Detection of non-Brownian diffusion in the cell membrane in single molecule tracking,” Biophys. J. 88(3), 2266–2277 (2005).
[CrossRef]

Lansford, R.

R. Lansford, G. Bearman, and S. E. Fraser, “Resolution of multiple green fluorescent protein color variants and dyes using two-photon microscopy and imaging spectroscopy,” J. Biomed. Opt. 6(3), 311–318 (2001).
[CrossRef] [PubMed]

Lee, J. J.

R. A. Schwarz, W. Gao, C. Redden Weber, C. Kurachi, J. J. Lee, A. K. El-Naggar, R. Richards-Kortum, and A. M. Gillenwater, “Noninvasive evaluation of oral lesions using depth-sensitive optical spectroscopy,” Cancer 115(8), 1669–1679 (2009).
[CrossRef] [PubMed]

Levin, I. W.

K. J. Zuzak, M. D. Schaeberle, E. N. Lewis, and I. W. Levin, “Visible reflectance hyperspectral imaging: characterization of a noninvasive, in vivo system for determining tissue perfusion,” Anal. Chem. 74(9), 2021–2028 (2002).
[CrossRef] [PubMed]

Lewis, E. N.

K. J. Zuzak, M. D. Schaeberle, E. N. Lewis, and I. W. Levin, “Visible reflectance hyperspectral imaging: characterization of a noninvasive, in vivo system for determining tissue perfusion,” Anal. Chem. 74(9), 2021–2028 (2002).
[CrossRef] [PubMed]

Lynch, R.

Lynch, R. M.

B. K. Ford, C. E. Volin, S. M. Murphy, R. M. Lynch, and M. R. Descour, “Computed tomography-based spectral imaging for fluorescence microscopy,” Biophys. J. 80(2), 986–993 (2001).
[CrossRef] [PubMed]

K. N. Richmond, R. D. Shonat, R. M. Lynch, and P. C. Johnson, “The critical oxygen tension of skeletal muscle in vivo,” Am. J. Physiol. 277(5 Pt 2), H1831–H1840 (1999).
[PubMed]

McCain, S. C.

C. A. Fernandez, A. Wagadarikar, D. J. Brady, S. C. McCain, and T. Oliver, “Fluorescence microscopy with a coded aperture snapshot spectral imager,” Proc. SPIE 7184, 71840Z (2009).
[CrossRef]

Murphy, S. M.

B. K. Ford, C. E. Volin, S. M. Murphy, R. M. Lynch, and M. R. Descour, “Computed tomography-based spectral imaging for fluorescence microscopy,” Biophys. J. 80(2), 986–993 (2001).
[CrossRef] [PubMed]

Norris, T. B.

Ntziachristos, V.

V. Ntziachristos, J. Ripoll, L. V. Wang, and R. Weissleder, “Looking and listening to light: the evolution of whole-body photonic imaging,” Nat. Biotechnol. 23(3), 313–320 (2005).
[CrossRef] [PubMed]

Oliver, T.

C. F. Cull, K. Choi, D. J. Brady, and T. Oliver, “Identification of fluorescent beads using a coded aperture snapshot spectral imager,” Appl. Opt. 49(10), B59–B70 (2010), http://www.opticsinfobase.org/abstract.cfm?URI=ao-49-10-B59 .
[CrossRef] [PubMed]

C. A. Fernandez, A. Wagadarikar, D. J. Brady, S. C. McCain, and T. Oliver, “Fluorescence microscopy with a coded aperture snapshot spectral imager,” Proc. SPIE 7184, 71840Z (2009).
[CrossRef]

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]

Pepperkok, R.

T. Zimmermann, J. Rietdorf, and R. Pepperkok, “Spectral imaging and its applications in live cell microscopy,” FEBS Lett. 546(1), 87–92 (2003).
[CrossRef] [PubMed]

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]

Redden Weber, C.

R. A. Schwarz, W. Gao, C. Redden Weber, C. Kurachi, J. J. Lee, A. K. El-Naggar, R. Richards-Kortum, and A. M. Gillenwater, “Noninvasive evaluation of oral lesions using depth-sensitive optical spectroscopy,” Cancer 115(8), 1669–1679 (2009).
[CrossRef] [PubMed]

Richards-Kortum, R.

R. A. Schwarz, W. Gao, C. Redden Weber, C. Kurachi, J. J. Lee, A. K. El-Naggar, R. Richards-Kortum, and A. M. Gillenwater, “Noninvasive evaluation of oral lesions using depth-sensitive optical spectroscopy,” Cancer 115(8), 1669–1679 (2009).
[CrossRef] [PubMed]

Richmond, K. N.

K. N. Richmond, R. D. Shonat, R. M. Lynch, and P. C. Johnson, “The critical oxygen tension of skeletal muscle in vivo,” Am. J. Physiol. 277(5 Pt 2), H1831–H1840 (1999).
[PubMed]

Rietdorf, J.

T. Zimmermann, J. Rietdorf, and R. Pepperkok, “Spectral imaging and its applications in live cell microscopy,” FEBS Lett. 546(1), 87–92 (2003).
[CrossRef] [PubMed]

Ripoll, J.

V. Ntziachristos, J. Ripoll, L. V. Wang, and R. Weissleder, “Looking and listening to light: the evolution of whole-body photonic imaging,” Nat. Biotechnol. 23(3), 313–320 (2005).
[CrossRef] [PubMed]

Ritchie, K.

K. Ritchie, X. Y. Shan, J. Kondo, K. Iwasawa, T. Fujiwara, and A. Kusumi, “Detection of non-Brownian diffusion in the cell membrane in single molecule tracking,” Biophys. J. 88(3), 2266–2277 (2005).
[CrossRef]

Schaeberle, M. D.

K. J. Zuzak, M. D. Schaeberle, E. N. Lewis, and I. W. Levin, “Visible reflectance hyperspectral imaging: characterization of a noninvasive, in vivo system for determining tissue perfusion,” Anal. Chem. 74(9), 2021–2028 (2002).
[CrossRef] [PubMed]

Schwarz, R. A.

R. A. Schwarz, W. Gao, C. Redden Weber, C. Kurachi, J. J. Lee, A. K. El-Naggar, R. Richards-Kortum, and A. M. Gillenwater, “Noninvasive evaluation of oral lesions using depth-sensitive optical spectroscopy,” Cancer 115(8), 1669–1679 (2009).
[CrossRef] [PubMed]

Shan, X. Y.

K. Ritchie, X. Y. Shan, J. Kondo, K. Iwasawa, T. Fujiwara, and A. Kusumi, “Detection of non-Brownian diffusion in the cell membrane in single molecule tracking,” Biophys. J. 88(3), 2266–2277 (2005).
[CrossRef]

Shimi, T.

Y. Hiraoka, T. Shimi, and T. Haraguchi, “Multispectral imaging fluorescence microscopy for living cells,” Cell Struct. Funct. 27(5), 367–374 (2002).
[CrossRef] [PubMed]

Shonat, R. D.

K. N. Richmond, R. D. Shonat, R. M. Lynch, and P. C. Johnson, “The critical oxygen tension of skeletal muscle in vivo,” Am. J. Physiol. 277(5 Pt 2), H1831–H1840 (1999).
[PubMed]

Tkaczyk, T. S.

Vo-Dinh, T.

T. Vo-Dinh, “A hyperspectral imaging system for in vivo optical diagnostics,” IEEE Eng. Med. Biol. Mag. 23, 40–49 (2007).

Volin, C. E.

B. K. Ford, C. E. Volin, S. M. Murphy, R. M. Lynch, and M. R. Descour, “Computed tomography-based spectral imaging for fluorescence microscopy,” Biophys. J. 80(2), 986–993 (2001).
[CrossRef] [PubMed]

Wagadarikar, A.

C. A. Fernandez, A. Wagadarikar, D. J. Brady, S. C. McCain, and T. Oliver, “Fluorescence microscopy with a coded aperture snapshot spectral imager,” Proc. SPIE 7184, 71840Z (2009).
[CrossRef]

Wang, L. V.

V. Ntziachristos, J. Ripoll, L. V. Wang, and R. Weissleder, “Looking and listening to light: the evolution of whole-body photonic imaging,” Nat. Biotechnol. 23(3), 313–320 (2005).
[CrossRef] [PubMed]

Weissleder, R.

V. Ntziachristos, J. Ripoll, L. V. Wang, and R. Weissleder, “Looking and listening to light: the evolution of whole-body photonic imaging,” Nat. Biotechnol. 23(3), 313–320 (2005).
[CrossRef] [PubMed]

Wilson, D. W.

W. R. Johnson, D. W. Wilson, W. Fink, M. Humayun, and G. Bearman, “Snapshot hyperspectral imaging in ophthalmology,” J. Biomed. Opt. 12(1), 014036 (2007).
[CrossRef] [PubMed]

Wilson, T.

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Supplementary Material (2)

» Media 1: MOV (337 KB)     
» Media 2: MOV (833 KB)     

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Figures (12)

Fig. 1
Fig. 1

High-sampling IMS optical layout and system configuration

Fig. 2
Fig. 2

Diffraction-limited re-imaging lens design

Fig. 3
Fig. 3

Pupil array configuration at the back focal plane of the collecting lens. Chief rays are drawn from a periodic block on the image mapper.

Fig. 4
Fig. 4

Image mapping principle in IMS. Two coordinate systems (x, y, k) and (x’, y’) are set up on the datacube and CCD camera respectively.

Fig. 5
Fig. 5

Location of an image line in spectral spread direction under “elemental object” illumination. Each dot in the image represents a sweeping wavelength.

Fig. 6
Fig. 6

“Elemental image” and assembly of full datacube

Fig. 7
Fig. 7

Image line distortion and correction

Fig. 8
Fig. 8

LSF measurement of image lines on the IMS raw image

Fig. 9
Fig. 9

MTF measurement at the image plane. The solid line is the measured MTF. The dashed line is the diffraction limit.

Fig. 10
Fig. 10

BPAE cells; (a) direct imaging image and (b) IMS raw image. The 11-bit IMS raw image was captured with an integration time of 1 second.

Fig. 11
Fig. 11

(Media 1) IMS spectral channel images. Cell Nuclei labeled with DAPI are visible in the blue spectral channels (460-472 nm); Filamentous actin labeled with Alexa Fluor 488 phalloidin are visible in the green spectral channels (520-549 nm); Mitochondria labeled with MitoTracker Red CMXRos are visible in the red spectral channels (596-649 nm).

Fig. 12
Fig. 12

(Media 2) 3D datacube 285 × 285 × 60 (x, y, λ) acquired by IMS. The displayed values result from assigning each voxel an opacity value in addition to its irradiance, and summing (with attenuation) all voxels along each ray defined by the lines of sight. The color bar is in arbitrary units proportional to photons/s/band.

Equations (4)

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Δ S = f × 2 Δ α = 90 m m × 2 × 0.01 = 1.8 m m D = f × 2 N . A . r e f l e c t = 90 m m × 2 × 0.004 = 0.72 m m
I ( x , y , k ) I ( x ' , y ' )
I ( x , y , k ) = M { I ( x ' , y ' ) / S ( x ' , y ' ) }
N = Length of spectral spread region FWHM of LSF = 60 pixels 2.49 pixels = 24.1

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