Abstract

Multispectral imaging has significantly enhanced the analysis of fixed specimens in pathology and cytogenetics. However, application of this technology to in vivo studies has been limited. This is due in part to the increased temporal resolution required to analyze changes in cellular function. Here we present a non-scanning instrument that simultaneously acquires full spectral information (460 nm to 740 nm) from every pixel within its 2-D field of view (200 µm×200 µm) during a single integration time (typically, 2 seconds). The current spatial and spectral sampling intervals of the spectrometer are 0.985 µm and 5 nm, respectively. These properties allow for the analysis of physiological responses within living biological specimens.

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References

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  1. W. T. Mason, ed. Fluorescent and Luminescent Probes for Biological Activity: A Practical Guide for Quantitative Real-Time Analysis, (Academic Press, 1999).
  2. R. Haugland, Handbook of fluorescent probes and research chemicals. Eighth Edition, (Molecular Probes, Inc., 2001).
  3. R. M. Lynch, K. D. Nullmeyer, B. K. Ford, L. S. Tompkins, V. L. Sutherland and M. R. Descour, "Multiparametric analysis of cellular and subcellular function by spectral imaging," in Molecular Imaging: Reporters, Dyes, Markers and Instrumentation, D. J. Burnhop and K. Licha, eds., Proc. SPIE 3924, 79-87 (2000).
  4. K. N. Richmond, S. Burnite and R. M. Lynch, "Oxygen sensitivity of mitochondrial metabolic state in isolated skeletal and cardiac myocytes," Am. J. Physiol. 273 (Cell 42), C1613- C1622 (1997).
  5. R. Martinez-Zaguilan, M. Gurule and R. M. Lynch, "Simultaneous easurement of pH and Ca2+ in single insulin secreting cells by microscopic spectral imaging," Am. J. Physiol. 270 (Cell 40), C1438-1446 (1996).
  6. R. Martinez-Zaguilan, L. S. Tompkins, R. J. Gillies and R. M. Lynch, "Simultaneous measurements of calcium and pH in cell populations," in Calcium Signaling Protocols, Meth. Molec. Biol. Series, Vol. 114, D.G. Lambert, ed. (Humana Press, 1999), Chap. 20.
  7. E. Schr�ck, S. du Manoir, T. Veldman, B. Schoell, J. Wienberg, M. A. Ferguson-Smith, Y. Ning, D. H. Ledbetter, I. Am-Bar, D. Soenksen, Y. Garini, T. Ried, "Multicolor spectral karyotyping of human chromosomes," Science 273, 494-497 (1996).
    [CrossRef] [PubMed]
  8. E. S. Wachman, W. Niu and D. L. Farkas, "AOTF microscope for imaging with increased speed and versatility," Biopys J. 73, 1215-1222 (1997).
    [CrossRef]
  9. H. R. Morris, Hoyt C. C., Treado P. J, "Imaging spectrometers for fluorescence and Raman microscopy- acoustooptic and liquid-crystal tunable filters," Appl. Spectrosc. 48:857-866
  10. N. M. Haralampus-Grynaviski, M. J. Stimson, and J. D. Simon, "Design and Applications of Rapid-Scan Spectrally Resolved Fluorescence Microscopy," Appl. Spectrosc. 54, 1727-1733 (2000).
    [CrossRef]
  11. M.E. Dickinson, "Spectral imaging with multiphoton excitation microscopy," in Imaging Life: From cells to whole animals. Microscopy and Microanalysis Pre-Meeting Congress, Long Beach California (2001).
  12. C. E. Volin, B. K. Ford, M. R. Descour, J. P. Garcia, P. D. Maker, G. H. Bearman, "High- speed spectral imager for imaging transient fluorescence phenomena," Appl. Opt. 37, 8112 -8119 (1998).
    [CrossRef]
  13. B. K. Ford, S. M. Murphy, C. E. Volin, R. M. Lynch, and M. R. Descour, "Computed-Tomography based video-rate spectral imaging system for fluorescence microscopy," Biophys. J. 80, 986-993 (2001).
    [CrossRef] [PubMed]
  14. S. A. Clark, B. L. Burnham, andW. L. Chick, "Modulation of glucose-induced insulin secretion from a rat clonal �-cell line," Endocrinology, 127(6), 2779-2788 (1990).
    [CrossRef]
  15. B. K. Ford, C. E. Volin, A. R. Rouse, R. M. Lynch, A. F. Gmitro, G. H. Bearman and M. R. Descour, "Video-rate spectral imaging system for fluorescence microscopy," in Systems and Technologies for Clinical Diagnostics and Drug Discovery II, G. E. Cohn, ed., Proc. SPIE 3603, 3603-3629 (1999).
  16. Olympus America, Inc. Melville NY, 11747, http://www.olympus.com.
  17. DALSA Tucson. Tucson, AZ 85713, http://www.dalsa.com.
  18. Jet Propulsion Laboratory. Pasadena, CA 91109.
  19. Volin, C. E, Portable snapshot infrared imaging spectrometer, Ph.D. Dissertation, University of Arizona. (2001).
  20. M. R. Descour, C. E. Volin, T. M. Gleeson, E. L. Dereniak, M. F. Hopkins, D. W. Wilson and P. D. Maker, "Demonstration of a Computed-Tomography Imaging Spectrometer using a computer-generated hologram disperser," Appl. Opt. 36, 3694-98 (1997).
    [CrossRef] [PubMed]
  21. M. R. Descour and E. Dereniak, "Computed-tomography imaging spectrometer: Experimental calibration and reconstruction results," Appl. Opt. 34, 4817-4826 (1995).
    [CrossRef] [PubMed]
  22. M. R. Descour, C. E. Volin, E. L. Dereniak, K. J. Thome, A. B. Schumacher, D. W. Wilson and P. D. Maker, "Demonstration of a High Speed Non-scanning Imaging Spectrometer," Opt. Lett. 22, 1271-1273 (1997).
    [CrossRef] [PubMed]
  23. J. D. Gaskill, Linear Systems, Fourier Transforms, and Optics (JohnWiley and Sons, Inc, 1978) Chap. 5.
  24. A. Lent, "A convergent algorithm for maximum entropy image restoration," in Image Analysis and Evaluation, Rodney Shaw, ed. SPSE Proceedings, 249-257 (1976).
  25. Ocean Optics, Inc. Dunedin, FL 34698, http://www.oceanoptics.com/homepage.asp.
  26. R. M. Lynch, K. E. Fogarty and F. S. Fay, "Analysis of hexokinase association with mitochondria by quantitative confocal microscopy," J. Cell Biol. 112, 385-395 (1991).
    [CrossRef] [PubMed]
  27. M. P Bruchez Jr., M. Moronne, P. Gin, S. Weiss, A. P. Alivosatos, "Semiconductor nanocrystals as fluorescent biological labels, Science 281, 2013-2016 (1998).
    [CrossRef] [PubMed]

Other (27)

W. T. Mason, ed. Fluorescent and Luminescent Probes for Biological Activity: A Practical Guide for Quantitative Real-Time Analysis, (Academic Press, 1999).

R. Haugland, Handbook of fluorescent probes and research chemicals. Eighth Edition, (Molecular Probes, Inc., 2001).

R. M. Lynch, K. D. Nullmeyer, B. K. Ford, L. S. Tompkins, V. L. Sutherland and M. R. Descour, "Multiparametric analysis of cellular and subcellular function by spectral imaging," in Molecular Imaging: Reporters, Dyes, Markers and Instrumentation, D. J. Burnhop and K. Licha, eds., Proc. SPIE 3924, 79-87 (2000).

K. N. Richmond, S. Burnite and R. M. Lynch, "Oxygen sensitivity of mitochondrial metabolic state in isolated skeletal and cardiac myocytes," Am. J. Physiol. 273 (Cell 42), C1613- C1622 (1997).

R. Martinez-Zaguilan, M. Gurule and R. M. Lynch, "Simultaneous easurement of pH and Ca2+ in single insulin secreting cells by microscopic spectral imaging," Am. J. Physiol. 270 (Cell 40), C1438-1446 (1996).

R. Martinez-Zaguilan, L. S. Tompkins, R. J. Gillies and R. M. Lynch, "Simultaneous measurements of calcium and pH in cell populations," in Calcium Signaling Protocols, Meth. Molec. Biol. Series, Vol. 114, D.G. Lambert, ed. (Humana Press, 1999), Chap. 20.

E. Schr�ck, S. du Manoir, T. Veldman, B. Schoell, J. Wienberg, M. A. Ferguson-Smith, Y. Ning, D. H. Ledbetter, I. Am-Bar, D. Soenksen, Y. Garini, T. Ried, "Multicolor spectral karyotyping of human chromosomes," Science 273, 494-497 (1996).
[CrossRef] [PubMed]

E. S. Wachman, W. Niu and D. L. Farkas, "AOTF microscope for imaging with increased speed and versatility," Biopys J. 73, 1215-1222 (1997).
[CrossRef]

H. R. Morris, Hoyt C. C., Treado P. J, "Imaging spectrometers for fluorescence and Raman microscopy- acoustooptic and liquid-crystal tunable filters," Appl. Spectrosc. 48:857-866

N. M. Haralampus-Grynaviski, M. J. Stimson, and J. D. Simon, "Design and Applications of Rapid-Scan Spectrally Resolved Fluorescence Microscopy," Appl. Spectrosc. 54, 1727-1733 (2000).
[CrossRef]

M.E. Dickinson, "Spectral imaging with multiphoton excitation microscopy," in Imaging Life: From cells to whole animals. Microscopy and Microanalysis Pre-Meeting Congress, Long Beach California (2001).

C. E. Volin, B. K. Ford, M. R. Descour, J. P. Garcia, P. D. Maker, G. H. Bearman, "High- speed spectral imager for imaging transient fluorescence phenomena," Appl. Opt. 37, 8112 -8119 (1998).
[CrossRef]

B. K. Ford, S. M. Murphy, C. E. Volin, R. M. Lynch, and M. R. Descour, "Computed-Tomography based video-rate spectral imaging system for fluorescence microscopy," Biophys. J. 80, 986-993 (2001).
[CrossRef] [PubMed]

S. A. Clark, B. L. Burnham, andW. L. Chick, "Modulation of glucose-induced insulin secretion from a rat clonal �-cell line," Endocrinology, 127(6), 2779-2788 (1990).
[CrossRef]

B. K. Ford, C. E. Volin, A. R. Rouse, R. M. Lynch, A. F. Gmitro, G. H. Bearman and M. R. Descour, "Video-rate spectral imaging system for fluorescence microscopy," in Systems and Technologies for Clinical Diagnostics and Drug Discovery II, G. E. Cohn, ed., Proc. SPIE 3603, 3603-3629 (1999).

Olympus America, Inc. Melville NY, 11747, http://www.olympus.com.

DALSA Tucson. Tucson, AZ 85713, http://www.dalsa.com.

Jet Propulsion Laboratory. Pasadena, CA 91109.

Volin, C. E, Portable snapshot infrared imaging spectrometer, Ph.D. Dissertation, University of Arizona. (2001).

M. R. Descour, C. E. Volin, T. M. Gleeson, E. L. Dereniak, M. F. Hopkins, D. W. Wilson and P. D. Maker, "Demonstration of a Computed-Tomography Imaging Spectrometer using a computer-generated hologram disperser," Appl. Opt. 36, 3694-98 (1997).
[CrossRef] [PubMed]

M. R. Descour and E. Dereniak, "Computed-tomography imaging spectrometer: Experimental calibration and reconstruction results," Appl. Opt. 34, 4817-4826 (1995).
[CrossRef] [PubMed]

M. R. Descour, C. E. Volin, E. L. Dereniak, K. J. Thome, A. B. Schumacher, D. W. Wilson and P. D. Maker, "Demonstration of a High Speed Non-scanning Imaging Spectrometer," Opt. Lett. 22, 1271-1273 (1997).
[CrossRef] [PubMed]

J. D. Gaskill, Linear Systems, Fourier Transforms, and Optics (JohnWiley and Sons, Inc, 1978) Chap. 5.

A. Lent, "A convergent algorithm for maximum entropy image restoration," in Image Analysis and Evaluation, Rodney Shaw, ed. SPSE Proceedings, 249-257 (1976).

Ocean Optics, Inc. Dunedin, FL 34698, http://www.oceanoptics.com/homepage.asp.

R. M. Lynch, K. E. Fogarty and F. S. Fay, "Analysis of hexokinase association with mitochondria by quantitative confocal microscopy," J. Cell Biol. 112, 385-395 (1991).
[CrossRef] [PubMed]

M. P Bruchez Jr., M. Moronne, P. Gin, S. Weiss, A. P. Alivosatos, "Semiconductor nanocrystals as fluorescent biological labels, Science 281, 2013-2016 (1998).
[CrossRef] [PubMed]

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Figures (9)

Fig. 1.
Fig. 1.

Digital image of the large-format CTIS aligned to the side photo port of an Olympus IX70 inverted fluorescence microscope.

Fig. 2.
Fig. 2.

Optical microscope image of a segment of the CGH disperser (Courtesy of D. W. Wilson and P. D. Maker, Jet Propulsion Laboratory).

Fig. 3.
Fig. 3.

Mapping of the signal from individual voxels to the imaging array. The signal on the imaging array is distributed in a diffraction pattern that depends on a voxel’s (x, y, λ) coordinates in the object cube. Calibration images are shown for two different voxels that occupy the same spatial position but different spectral bands. Part (a) corresponds to a voxel with a center wavelength of 470 nm while Part (b) corresponds to a center wavelength of 740 nm.

Fig. 4.
Fig. 4.

(821 KB) Movie demonstrating how the CTIS maps a 3-D (x,y,λ) object cube onto a 2-D image plane. The sequence corresponds to simulated raw images of the same specimen viewed through a filter of increasing spectral bandwidth. The movie begins with a narrow-band image (500 nm–510 nm). The bandwidth increases in steps of 10 nm to a maximum of 240 nm (500 nm–740 nm). The simulated object cube consists of eight spheres with four different emission maxima (530 nm, 515 nm, 560 nm, 615 nm).

Fig. 5.
Fig. 5.

Representative raw image of embryonic rat cerebellum glia. The cells were probed with primary antibodies to glial fibrillary acidic proteins (GFAP) and subsequently labeled with Alexa Fluor 488 goat anti-rabbit IgG (H+L) conjugate secondary antibody and the DNA/RNA probe, Ethidium Bromide. The image was taken using a 40x, NA=1.35 microscope objective. Object cubes were reconstructed with 55 spectral bands (470nm–740 nm, spectral sampling distance of 5 nm) and 203×203 spatial resolution elements. The spatial sampling distance between adjacent pixels is 0.985 µm for this application

Fig. 6.
Fig. 6.

Reconstructed image of a mixture of microspheres with different emission spectra. Color image is generated by converting the reconstructed spectra into sets of RGB values.

Fig. 7.
Fig. 7.

Reconstructed spectra corresponding to three different locations within the microsphere sample. See Fig. 6 for locations (A), (B) and (C). Crosses denote comparison spectra measured with a radiometrically calibrated non-imaging spectrometer.

Fig. 8.
Fig. 8.

Color image reconstructed from the raw image shown in Figure 5. Green fluorescence indicates the distribution of GFAP while orange-red indicates nucleic acid distribution.

Fig. 9.
Fig. 9.

Reconstructed spectra of two pixel locations within the reconstructed image of Figure 8. The locations (A) and (B) are indicated in Figure 8.

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