Abstract

We describe fluorescence spectral imaging results with the microscope computed-tomography imaging spectrometer (μCTIS). This imaging spectrometer is capable of recording spatial and spectral data simultaneously. Consequently, μCTIS can be used to image dynamic phenomena. The results presented consist of proof-of-concept imaging results with static targets composed of 6-μm fluorescing microspheres. Image data were collected with integration times of 16 ms, comparable with video-frame-rate integration times. Conversion of raw data acquired by the μCTIS to spatial and spectral data requires postprocessing. The emission spectra were sampled at 10-nm intervals between 420 and 710 nm. The smallest spatial sampling interval presented is 1.7 μm.

© 1998 Optical Society of America

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References

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    [CrossRef]
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    [CrossRef]
  12. D. R. Sandison, R. M. Williams, K. S. Wells, J. Strickler, W. W. Webb, “Quantitative fluorescence confocal laser scanning microscopy (CLSM),” Handbook of Biological Confocal Microscopy (Plenum, New York, 1995), pp. 39–53.
    [CrossRef]
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    [CrossRef] [PubMed]
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  15. A. Lent, “A convergent algorithm for maximum entropy image restoration,” in Image Analysis and Evaluation, SPSE Conference Proceedings, R. Shaw, ed. (Society of Photographic Scientists and Engineers, n.p.), pp. 249–257.
  16. Fluoresbrite polychromatic microspheres (6 μm), Catalog no. 19111, Polysciences Inc., 400 Valley Road, Warrington, Pa. 18976.
  17. S2000 Fiber Spectrometer, Ocean Optics, Inc., 380 Main Street, Dunedin, Fla. 34698.
  18. Optronic Laboratories, 4632 36th Street, Orlando, Fla. 32811; 1000-W Spectral Irradiance Standard, courtesy of Stuart Biggar, University of Arizona Optical Sciences Center, Remote Sensing Group (1998).
  19. Nikon, USA, 1300 Walt Whitman Road, Melville, N.Y. 11747-3064.

1997 (4)

1996 (3)

E. S. Wachman, W. H. Niu, D. L. Farkas, “Imaging acousto-optic tunable filter with 0.35-micrometer spatial resolution,” Appl. Opt. 35, 5220–5226 (1996).
[CrossRef] [PubMed]

C. Hoyt, “Liquid crystal tunable filters clear the way for imaging multiprobe fluorescence,” Biophotonics International 4, 49–51 (1996).

R. Martínez-Zaguilán, M. W. Gurulé, R. M. Lynch, “Simultaneous measurement of intracellular pH and Ca2+ in insulin-secreting cells by spectral imaging microscopy,” Am. J. Physiol. Cell Physiol. 270C1438–C1446 (1996).

Bacskai, B. J.

R. Y. Tsien, B. J. Bacskai, “Video-rate confocal microscopy,” Handbook of Biological Confocal Microscopy (Plenum, New York, 1995), pp. 459–478.
[CrossRef]

Braichotte, D. R.

Brodzik, A. K.

Buckwald, R. A.

Y. Garini, N. Katzir, D. Cabib, R. A. Buckwald, D. G. Soenksen, S. Malik, “Spectral bio-imaging,” Fluorescence Imaging Spectrometry and Microscopy, X. F. Wang, B. Herman, eds. (Wiley, New York, 1996), pp. 87–124.

Cabib, D.

Y. Garini, N. Katzir, D. Cabib, R. A. Buckwald, D. G. Soenksen, S. Malik, “Spectral bio-imaging,” Fluorescence Imaging Spectrometry and Microscopy, X. F. Wang, B. Herman, eds. (Wiley, New York, 1996), pp. 87–124.

Châtelain, A.

Dereniak, E. L.

Descour, M. R.

Despeursinge, C.

Farkas, D. L.

Flannery, B.

W. H. Press, S. Teukolsky, W. Vetterling, B. Flannery, Numerical Recipes in C pp. 78–81 (Cambridge U Press, N.Y., 1992).

Garini, Y.

Y. Garini, N. Katzir, D. Cabib, R. A. Buckwald, D. G. Soenksen, S. Malik, “Spectral bio-imaging,” Fluorescence Imaging Spectrometry and Microscopy, X. F. Wang, B. Herman, eds. (Wiley, New York, 1996), pp. 87–124.

Gleeson, T. M.

Gurulé, M. W.

R. Martínez-Zaguilán, M. W. Gurulé, R. M. Lynch, “Simultaneous measurement of intracellular pH and Ca2+ in insulin-secreting cells by spectral imaging microscopy,” Am. J. Physiol. Cell Physiol. 270C1438–C1446 (1996).

Herman, B.

X. F. Wang, B. Herman, Fluorescence Imaging Spectrometry and Microscopy (Wiley, New York, 1996).

Hopkins, M. F.

Hoyt, C.

C. Hoyt, “Liquid crystal tunable filters clear the way for imaging multiprobe fluorescence,” Biophotonics International 4, 49–51 (1996).

Katzir, N.

Y. Garini, N. Katzir, D. Cabib, R. A. Buckwald, D. G. Soenksen, S. Malik, “Spectral bio-imaging,” Fluorescence Imaging Spectrometry and Microscopy, X. F. Wang, B. Herman, eds. (Wiley, New York, 1996), pp. 87–124.

Lent, A.

A. Lent, “A convergent algorithm for maximum entropy image restoration,” in Image Analysis and Evaluation, SPSE Conference Proceedings, R. Shaw, ed. (Society of Photographic Scientists and Engineers, n.p.), pp. 249–257.

Lynch, R. M.

R. Martínez-Zaguilán, M. W. Gurulé, R. M. Lynch, “Simultaneous measurement of intracellular pH and Ca2+ in insulin-secreting cells by spectral imaging microscopy,” Am. J. Physiol. Cell Physiol. 270C1438–C1446 (1996).

Maker, P. D.

Malik, S.

Y. Garini, N. Katzir, D. Cabib, R. A. Buckwald, D. G. Soenksen, S. Malik, “Spectral bio-imaging,” Fluorescence Imaging Spectrometry and Microscopy, X. F. Wang, B. Herman, eds. (Wiley, New York, 1996), pp. 87–124.

Martínez-Zaguilán, R.

R. Martínez-Zaguilán, M. W. Gurulé, R. M. Lynch, “Simultaneous measurement of intracellular pH and Ca2+ in insulin-secreting cells by spectral imaging microscopy,” Am. J. Physiol. Cell Physiol. 270C1438–C1446 (1996).

Monnier, P.

Mooney, J. M.

Myoung, A.

Newton, I.

I. Newton, “Prop. V, Theorem IV,” Opticks (Dover, New York, 1952), p. 1704.

Niu, W. H.

Press, W. H.

W. H. Press, S. Teukolsky, W. Vetterling, B. Flannery, Numerical Recipes in C pp. 78–81 (Cambridge U Press, N.Y., 1992).

Sandison, D. R.

D. R. Sandison, R. M. Williams, K. S. Wells, J. Strickler, W. W. Webb, “Quantitative fluorescence confocal laser scanning microscopy (CLSM),” Handbook of Biological Confocal Microscopy (Plenum, New York, 1995), pp. 39–53.
[CrossRef]

Schumacher, A. B.

Soenksen, D. G.

Y. Garini, N. Katzir, D. Cabib, R. A. Buckwald, D. G. Soenksen, S. Malik, “Spectral bio-imaging,” Fluorescence Imaging Spectrometry and Microscopy, X. F. Wang, B. Herman, eds. (Wiley, New York, 1996), pp. 87–124.

Strickler, J.

D. R. Sandison, R. M. Williams, K. S. Wells, J. Strickler, W. W. Webb, “Quantitative fluorescence confocal laser scanning microscopy (CLSM),” Handbook of Biological Confocal Microscopy (Plenum, New York, 1995), pp. 39–53.
[CrossRef]

Studzinski, A. P.

Teukolsky, S.

W. H. Press, S. Teukolsky, W. Vetterling, B. Flannery, Numerical Recipes in C pp. 78–81 (Cambridge U Press, N.Y., 1992).

Thome, K. J.

Tsien, R. Y.

R. Y. Tsien, A. Waggoner, “Fluorophores for confocal microscopy,” Handbook of Biological Confocal Microscopy (Plenum, New York, 1995) pp. 267–279.
[CrossRef]

R. Y. Tsien, B. J. Bacskai, “Video-rate confocal microscopy,” Handbook of Biological Confocal Microscopy (Plenum, New York, 1995), pp. 459–478.
[CrossRef]

van den Bergh, H. E.

Vetterling, W.

W. H. Press, S. Teukolsky, W. Vetterling, B. Flannery, Numerical Recipes in C pp. 78–81 (Cambridge U Press, N.Y., 1992).

Vickers, V. E.

Volin, C. E.

Wachman, E. S.

Waggoner, A.

R. Y. Tsien, A. Waggoner, “Fluorophores for confocal microscopy,” Handbook of Biological Confocal Microscopy (Plenum, New York, 1995) pp. 267–279.
[CrossRef]

Wagnières, G. A.

Wang, X. F.

X. F. Wang, B. Herman, Fluorescence Imaging Spectrometry and Microscopy (Wiley, New York, 1996).

Webb, W. W.

D. R. Sandison, R. M. Williams, K. S. Wells, J. Strickler, W. W. Webb, “Quantitative fluorescence confocal laser scanning microscopy (CLSM),” Handbook of Biological Confocal Microscopy (Plenum, New York, 1995), pp. 39–53.
[CrossRef]

Wells, K. S.

D. R. Sandison, R. M. Williams, K. S. Wells, J. Strickler, W. W. Webb, “Quantitative fluorescence confocal laser scanning microscopy (CLSM),” Handbook of Biological Confocal Microscopy (Plenum, New York, 1995), pp. 39–53.
[CrossRef]

Williams, R. M.

D. R. Sandison, R. M. Williams, K. S. Wells, J. Strickler, W. W. Webb, “Quantitative fluorescence confocal laser scanning microscopy (CLSM),” Handbook of Biological Confocal Microscopy (Plenum, New York, 1995), pp. 39–53.
[CrossRef]

Wilson, D. W.

Am. J. Physiol. Cell Physiol. (1)

R. Martínez-Zaguilán, M. W. Gurulé, R. M. Lynch, “Simultaneous measurement of intracellular pH and Ca2+ in insulin-secreting cells by spectral imaging microscopy,” Am. J. Physiol. Cell Physiol. 270C1438–C1446 (1996).

Appl. Opt. (3)

Biophotonics International (1)

C. Hoyt, “Liquid crystal tunable filters clear the way for imaging multiprobe fluorescence,” Biophotonics International 4, 49–51 (1996).

J. Opt. Soc. Am. A (1)

Opt. Lett. (1)

Other (12)

I. Newton, “Prop. V, Theorem IV,” Opticks (Dover, New York, 1952), p. 1704.

Y. Garini, N. Katzir, D. Cabib, R. A. Buckwald, D. G. Soenksen, S. Malik, “Spectral bio-imaging,” Fluorescence Imaging Spectrometry and Microscopy, X. F. Wang, B. Herman, eds. (Wiley, New York, 1996), pp. 87–124.

X. F. Wang, B. Herman, Fluorescence Imaging Spectrometry and Microscopy (Wiley, New York, 1996).

R. Y. Tsien, B. J. Bacskai, “Video-rate confocal microscopy,” Handbook of Biological Confocal Microscopy (Plenum, New York, 1995), pp. 459–478.
[CrossRef]

R. Y. Tsien, A. Waggoner, “Fluorophores for confocal microscopy,” Handbook of Biological Confocal Microscopy (Plenum, New York, 1995) pp. 267–279.
[CrossRef]

D. R. Sandison, R. M. Williams, K. S. Wells, J. Strickler, W. W. Webb, “Quantitative fluorescence confocal laser scanning microscopy (CLSM),” Handbook of Biological Confocal Microscopy (Plenum, New York, 1995), pp. 39–53.
[CrossRef]

W. H. Press, S. Teukolsky, W. Vetterling, B. Flannery, Numerical Recipes in C pp. 78–81 (Cambridge U Press, N.Y., 1992).

A. Lent, “A convergent algorithm for maximum entropy image restoration,” in Image Analysis and Evaluation, SPSE Conference Proceedings, R. Shaw, ed. (Society of Photographic Scientists and Engineers, n.p.), pp. 249–257.

Fluoresbrite polychromatic microspheres (6 μm), Catalog no. 19111, Polysciences Inc., 400 Valley Road, Warrington, Pa. 18976.

S2000 Fiber Spectrometer, Ocean Optics, Inc., 380 Main Street, Dunedin, Fla. 34698.

Optronic Laboratories, 4632 36th Street, Orlando, Fla. 32811; 1000-W Spectral Irradiance Standard, courtesy of Stuart Biggar, University of Arizona Optical Sciences Center, Remote Sensing Group (1998).

Nikon, USA, 1300 Walt Whitman Road, Melville, N.Y. 11747-3064.

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Figures (10)

Fig. 1
Fig. 1

Mapping of signal from voxel to imaging array. (a) Voxel at a center wavelength of 450 nm, and the distribution of that voxel’s signal on the imaging array. (b) Voxel at a center wavelength of 710 nm, and the distribution of that voxel’s signal on the imaging array.

Fig. 2
Fig. 2

Layout of the CTIS microscope. Distances are not to scale. See text for details.

Fig. 3
Fig. 3

Representative raw image collected by the μCTIS microscope. This image results from five fluorescing 6-μm-diameter microspheres.

Fig. 4
Fig. 4

Comparison of calibration images collected at the center and the corner of the field stop. Both images are superimposed to illustrate the spatial shift invariance of the μCTIS. The box indicates the extent of the field stop.

Fig. 5
Fig. 5

Reconstructed spectra from four different locations within the dense-array target shown in Fig. 6. Crosses (+) denote the comparison spectra measured with a nonimaging, radiometrically calibrated reference spectrometer.

Fig. 6
Fig. 6

Corresponding locations of the reconstructed spectra in Fig. 5.

Fig. 7
Fig. 7

Raster display of 31 spectral images of the dense-microsphere target. Wavelength increases in raster fashion from left to right, from bottom to top, and in steps of 10 nm. The numbers provided in this figure are in nanometers and are intended to help orient the reader.

Fig. 8
Fig. 8

Raster display of 31 spectral images of the isolated microspheres target. Wavelength increases in raster fashion from left to right, from bottom to top, and in steps of 10 nm. The numbers provided in this figure are in nanometers and are intended to help orient the reader.

Fig. 9
Fig. 9

Reconstructed spectra from four different locations within the isolated microspheres target shown in Fig. 10. (d) Spectrum corresponding to a dark part of the scene. Crosses (+) denote the comparison spectra measured with a reference spectrometer.

Fig. 10
Fig. 10

Corresponding locations of the reconstructed spectra in Fig. 9.

Equations (2)

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f ˆ k + 1 = f ˆ k H T g H T H f ˆ k
RSE = s μ CTIS - α s ref | α s ref ,

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