Abstract

Time-domain fluorescence lifetime imaging (FLIM) and hyper-spectral imaging (HSI) are two advanced microscopy techniques widely used in biological studies. Typically both FLIM and HSI are performed with either a whole-field or raster-scanning approach, which often prove to be technically complex and expensive, requiring the user to accept a compromise among precision, speed, and spatial resolution. We propose the use of a digital micromirror device (DMD) as a spatial illuminator for time-domain FLIM and HSI with a laser diode excitation source. The rather unique features of the DMD allow both random and parallel access to regions of interest (ROIs) on the sample, in a very rapid and repeatable fashion. As a consequence both spectral and lifetime images can be acquired with a precision normally associated with single-point systems but with a high degree of flexibility in their spatial construction. In addition, the DMD system offers a very efficient way of implementing a global analysis approach for FLIM, where average fluorescence decay parameters are first acquired for a ROI and then used as initial estimates in determining their spatial distribution within the ROI. Experimental results obtained on phantoms employing fluorescent dyes clearly show how the DMD method supports both spectral and temporal separation for target identification in HSI and FLIM, respectively.

© 2008 Optical Society of America

Full Article  |  PDF Article

References

  • View by:
  • |
  • |

  1. T. Zimmermann, “Spectral imaging and linear unmixing in light microscopy,” Adv. Biochem. Eng./Biotechnol. 95, 245-265 (2005).
  2. J. R. Lakowicz, Principles of Fluorescence Spectroscopy (Kluwer Academic, 1999).
  3. D. Elson, J. Requejo-Isidro, I. Munro, F. Reavell, J. Siegel, K. Suhling, P. Tadrous, R. Benninger, P. Lanigan, J. McGinty, C. Talbot, B. Treanor, S. Webb, A. Sandison, A. Wallace, D. Davis, J. Lever, M. Neil, D. Phillips, G. Stamp, and P. French, “Time domain fluorescence lifetime imaging applied to biological tissue,” Photochem. Photobiol. Sci. 3, 795-801 (2004).
    [CrossRef] [PubMed]
  4. N. Gat, “Imaging spectroscopy using tunable filters: a review,” Proc. SPIE 4056, 50-64 (2000).
    [CrossRef]
  5. P. R. Barber, B. Vojnovic, G. Atkin, F. M. Daley, S. A. Everett, G. D. Wilson, and J. D. Gilbey, “Applications of cost-effective spectral imaging microscopy in cancer research,” J. Phys. D 36, 1729-1738 (2003).
    [CrossRef]
  6. M. Bouhifd, M. P. Whelan, and M. Aprahamian, “Fluorescence imaging spectroscopy utilising acousto-optic tunable filters,” Proc. SPIE 5826, 185-193 (2005).
    [CrossRef]
  7. B. W. Pogue, S. L. Gibbs, B. Chen, and M. Savellano, “Fluorescence imaging in vivo: raster scanned point source imaging provides more accurate quantification than broad beam geometries,” Technol. Cancer Res. Treat. 3, 15-21 (2004).
    [PubMed]
  8. N. Ramanujam, J. X. Chen, K. Gossage, R. Richards-Kortum, and B. Chance, “Fast and noninvasive fluorescence imaging of biological tissues in vivo using a flying spot scanner,” IEEE Trans. Biomed. Eng. 48, 1034-1041 (2001).
    [CrossRef] [PubMed]
  9. J. Requejo-Isidro, J. McGinty, I. Munro, D. S. Elson, N. P. Galletly, M. J. Lever, M. A. A. Neil, G. W. H. Stamp, P. M. W. French, P. A. Kellett, J. D. Hares, and A. K. L. Dymoke-Bradshaw, “High-speed wide-field time-gated endoscopic fluorescence-lifetime imaging,” Opt. Lett. 29, 2249-2251(2004).
    [CrossRef] [PubMed]
  10. D. S. Elson, J. Siegel, S. E. D. Webb, S. Lévêque-Fort, M. J. Lever, P. M. W. French, K. Lauritsen, M. Wahl, and R. Erdmann, “Fluorescence lifetime system for microscopy and multiwell plate imaging with blue picosecond diode laser,” Opt. Lett. 27, 1409-1411 (2002).
    [CrossRef]
  11. Y. Zhang, S. A. Soper, L. R. Middendorf, J. A. Wurm, R. Erdmann, and M. Wahl, “Simple near-infrared time-correlated single photon counting instrument with a pulsed diode laser and avalanche photodiode for time-resolved measurements in scanning applications,” Appl. Spectrosc. 53, 497-504(1999).
    [CrossRef]
  12. M. Kress, T. Meier, R. Steiner, F. Dolp, R. Erdmann, U. Ortmann, and A. Rück, “Time-resolved microspectrofluorometry and fluorescence lifetime imaging of photosensitizers using picosecond pulsed diode lasers in laser scanning microscopes,” J Biomed. Opt. 8, 26-32 (2003).
    [CrossRef] [PubMed]
  13. D. Dudley, W. M. Duncan, and J. Slaughter, “Emerging digital micromirror device (DMD) applications,” Proc. SPIE 4985, 14-25 (2003).
    [CrossRef]
  14. Q. S. Hanley, O. J. Verveer, and T. M. Jovin, “Optical sectioning fluorescence spectroscopy in a programmable array microscope,” Appl. Spectrosc. 52, 783-789 (1998).
    [CrossRef]
  15. Q. S. Hanley, K. A. Lidke, R. Heintzmann, D. J. Arngt-Jovin, and T. M. Jovin, “Fluorescence lifetime imaging in an optically sectioning programmable array microscope (PAM),” Cytometry 67A, 112-118 (2005).
    [CrossRef]
  16. K. K. Sharman and A. Periasamy, “Error analysis of the rapid lifetime determination method for double-exponential decays and new windowing schemes,” Anal. Chem. 71, 947-952(1999).
    [CrossRef] [PubMed]
  17. S. P. Chan, Z. J. Fuller, J. N. Demas, and B. A. De Graff, “Optimized gating scheme for rapid lifetime determinations of single-exponential luminescence lifetimes,” Anal. Chem. 73, 4486-4490 (2001).
    [CrossRef] [PubMed]
  18. G. Nishimura and M. Tamura, “Artefacts in the analysis of temporal response functions measured by photon counting,” Phys. Med. Biol. 50, 1327-1342 (2005).
    [CrossRef] [PubMed]
  19. R. A. Hoebe, C. H. Van Oven, T. W. J. Gadella, P. B. Dhonukshe, C. J. F. Van Noorden, and E. M. M. Manders, “Controlled light-exposure microscopy reduces photobleaching and phototoxicity in fluorescence live-cell imaging,” Nat. Biotechnol. 25, 249-253 (2007).
    [CrossRef] [PubMed]
  20. R. Höfling and E. Ahl, “ALP: universal DMD controller for metrology and testing,” Proc. SPIE 5289B, 322-329 (2004).
    [CrossRef]
  21. S. Pelet, M. J. R. Previte, L. H. Laiho, and P. T. C. So, “A fast global algorithm for fluorescence lifetime imaging microscopy based on image segmentation,” Biophys. J. 87, 2807-2817(2004).
    [CrossRef] [PubMed]
  22. A. Bednarkiewicz and M. P. Whelan, “Global analysis of microscopic fluorescence lifetime images using spectral segmentation and a digital micromirror spatial illuminator,” J. Biomed. Opt. (to be published).

2007 (1)

R. A. Hoebe, C. H. Van Oven, T. W. J. Gadella, P. B. Dhonukshe, C. J. F. Van Noorden, and E. M. M. Manders, “Controlled light-exposure microscopy reduces photobleaching and phototoxicity in fluorescence live-cell imaging,” Nat. Biotechnol. 25, 249-253 (2007).
[CrossRef] [PubMed]

2005 (4)

G. Nishimura and M. Tamura, “Artefacts in the analysis of temporal response functions measured by photon counting,” Phys. Med. Biol. 50, 1327-1342 (2005).
[CrossRef] [PubMed]

Q. S. Hanley, K. A. Lidke, R. Heintzmann, D. J. Arngt-Jovin, and T. M. Jovin, “Fluorescence lifetime imaging in an optically sectioning programmable array microscope (PAM),” Cytometry 67A, 112-118 (2005).
[CrossRef]

T. Zimmermann, “Spectral imaging and linear unmixing in light microscopy,” Adv. Biochem. Eng./Biotechnol. 95, 245-265 (2005).

M. Bouhifd, M. P. Whelan, and M. Aprahamian, “Fluorescence imaging spectroscopy utilising acousto-optic tunable filters,” Proc. SPIE 5826, 185-193 (2005).
[CrossRef]

2004 (5)

B. W. Pogue, S. L. Gibbs, B. Chen, and M. Savellano, “Fluorescence imaging in vivo: raster scanned point source imaging provides more accurate quantification than broad beam geometries,” Technol. Cancer Res. Treat. 3, 15-21 (2004).
[PubMed]

D. Elson, J. Requejo-Isidro, I. Munro, F. Reavell, J. Siegel, K. Suhling, P. Tadrous, R. Benninger, P. Lanigan, J. McGinty, C. Talbot, B. Treanor, S. Webb, A. Sandison, A. Wallace, D. Davis, J. Lever, M. Neil, D. Phillips, G. Stamp, and P. French, “Time domain fluorescence lifetime imaging applied to biological tissue,” Photochem. Photobiol. Sci. 3, 795-801 (2004).
[CrossRef] [PubMed]

J. Requejo-Isidro, J. McGinty, I. Munro, D. S. Elson, N. P. Galletly, M. J. Lever, M. A. A. Neil, G. W. H. Stamp, P. M. W. French, P. A. Kellett, J. D. Hares, and A. K. L. Dymoke-Bradshaw, “High-speed wide-field time-gated endoscopic fluorescence-lifetime imaging,” Opt. Lett. 29, 2249-2251(2004).
[CrossRef] [PubMed]

R. Höfling and E. Ahl, “ALP: universal DMD controller for metrology and testing,” Proc. SPIE 5289B, 322-329 (2004).
[CrossRef]

S. Pelet, M. J. R. Previte, L. H. Laiho, and P. T. C. So, “A fast global algorithm for fluorescence lifetime imaging microscopy based on image segmentation,” Biophys. J. 87, 2807-2817(2004).
[CrossRef] [PubMed]

2003 (3)

P. R. Barber, B. Vojnovic, G. Atkin, F. M. Daley, S. A. Everett, G. D. Wilson, and J. D. Gilbey, “Applications of cost-effective spectral imaging microscopy in cancer research,” J. Phys. D 36, 1729-1738 (2003).
[CrossRef]

M. Kress, T. Meier, R. Steiner, F. Dolp, R. Erdmann, U. Ortmann, and A. Rück, “Time-resolved microspectrofluorometry and fluorescence lifetime imaging of photosensitizers using picosecond pulsed diode lasers in laser scanning microscopes,” J Biomed. Opt. 8, 26-32 (2003).
[CrossRef] [PubMed]

D. Dudley, W. M. Duncan, and J. Slaughter, “Emerging digital micromirror device (DMD) applications,” Proc. SPIE 4985, 14-25 (2003).
[CrossRef]

2002 (1)

2001 (2)

N. Ramanujam, J. X. Chen, K. Gossage, R. Richards-Kortum, and B. Chance, “Fast and noninvasive fluorescence imaging of biological tissues in vivo using a flying spot scanner,” IEEE Trans. Biomed. Eng. 48, 1034-1041 (2001).
[CrossRef] [PubMed]

S. P. Chan, Z. J. Fuller, J. N. Demas, and B. A. De Graff, “Optimized gating scheme for rapid lifetime determinations of single-exponential luminescence lifetimes,” Anal. Chem. 73, 4486-4490 (2001).
[CrossRef] [PubMed]

2000 (1)

N. Gat, “Imaging spectroscopy using tunable filters: a review,” Proc. SPIE 4056, 50-64 (2000).
[CrossRef]

1999 (2)

1998 (1)

Adv. Biochem. Eng./Biotechnol. (1)

T. Zimmermann, “Spectral imaging and linear unmixing in light microscopy,” Adv. Biochem. Eng./Biotechnol. 95, 245-265 (2005).

Anal. Chem. (2)

K. K. Sharman and A. Periasamy, “Error analysis of the rapid lifetime determination method for double-exponential decays and new windowing schemes,” Anal. Chem. 71, 947-952(1999).
[CrossRef] [PubMed]

S. P. Chan, Z. J. Fuller, J. N. Demas, and B. A. De Graff, “Optimized gating scheme for rapid lifetime determinations of single-exponential luminescence lifetimes,” Anal. Chem. 73, 4486-4490 (2001).
[CrossRef] [PubMed]

Appl. Spectrosc. (2)

Biophys. J. (1)

S. Pelet, M. J. R. Previte, L. H. Laiho, and P. T. C. So, “A fast global algorithm for fluorescence lifetime imaging microscopy based on image segmentation,” Biophys. J. 87, 2807-2817(2004).
[CrossRef] [PubMed]

Cytometry (1)

Q. S. Hanley, K. A. Lidke, R. Heintzmann, D. J. Arngt-Jovin, and T. M. Jovin, “Fluorescence lifetime imaging in an optically sectioning programmable array microscope (PAM),” Cytometry 67A, 112-118 (2005).
[CrossRef]

IEEE Trans. Biomed. Eng. (1)

N. Ramanujam, J. X. Chen, K. Gossage, R. Richards-Kortum, and B. Chance, “Fast and noninvasive fluorescence imaging of biological tissues in vivo using a flying spot scanner,” IEEE Trans. Biomed. Eng. 48, 1034-1041 (2001).
[CrossRef] [PubMed]

J Biomed. Opt. (1)

M. Kress, T. Meier, R. Steiner, F. Dolp, R. Erdmann, U. Ortmann, and A. Rück, “Time-resolved microspectrofluorometry and fluorescence lifetime imaging of photosensitizers using picosecond pulsed diode lasers in laser scanning microscopes,” J Biomed. Opt. 8, 26-32 (2003).
[CrossRef] [PubMed]

J. Phys. D (1)

P. R. Barber, B. Vojnovic, G. Atkin, F. M. Daley, S. A. Everett, G. D. Wilson, and J. D. Gilbey, “Applications of cost-effective spectral imaging microscopy in cancer research,” J. Phys. D 36, 1729-1738 (2003).
[CrossRef]

Nat. Biotechnol. (1)

R. A. Hoebe, C. H. Van Oven, T. W. J. Gadella, P. B. Dhonukshe, C. J. F. Van Noorden, and E. M. M. Manders, “Controlled light-exposure microscopy reduces photobleaching and phototoxicity in fluorescence live-cell imaging,” Nat. Biotechnol. 25, 249-253 (2007).
[CrossRef] [PubMed]

Opt. Lett. (2)

Photochem. Photobiol. Sci. (1)

D. Elson, J. Requejo-Isidro, I. Munro, F. Reavell, J. Siegel, K. Suhling, P. Tadrous, R. Benninger, P. Lanigan, J. McGinty, C. Talbot, B. Treanor, S. Webb, A. Sandison, A. Wallace, D. Davis, J. Lever, M. Neil, D. Phillips, G. Stamp, and P. French, “Time domain fluorescence lifetime imaging applied to biological tissue,” Photochem. Photobiol. Sci. 3, 795-801 (2004).
[CrossRef] [PubMed]

Phys. Med. Biol. (1)

G. Nishimura and M. Tamura, “Artefacts in the analysis of temporal response functions measured by photon counting,” Phys. Med. Biol. 50, 1327-1342 (2005).
[CrossRef] [PubMed]

Proc. SPIE (4)

R. Höfling and E. Ahl, “ALP: universal DMD controller for metrology and testing,” Proc. SPIE 5289B, 322-329 (2004).
[CrossRef]

D. Dudley, W. M. Duncan, and J. Slaughter, “Emerging digital micromirror device (DMD) applications,” Proc. SPIE 4985, 14-25 (2003).
[CrossRef]

N. Gat, “Imaging spectroscopy using tunable filters: a review,” Proc. SPIE 4056, 50-64 (2000).
[CrossRef]

M. Bouhifd, M. P. Whelan, and M. Aprahamian, “Fluorescence imaging spectroscopy utilising acousto-optic tunable filters,” Proc. SPIE 5826, 185-193 (2005).
[CrossRef]

Technol. Cancer Res. Treat. (1)

B. W. Pogue, S. L. Gibbs, B. Chen, and M. Savellano, “Fluorescence imaging in vivo: raster scanned point source imaging provides more accurate quantification than broad beam geometries,” Technol. Cancer Res. Treat. 3, 15-21 (2004).
[PubMed]

Other (2)

J. R. Lakowicz, Principles of Fluorescence Spectroscopy (Kluwer Academic, 1999).

A. Bednarkiewicz and M. P. Whelan, “Global analysis of microscopic fluorescence lifetime images using spectral segmentation and a digital micromirror spatial illuminator,” J. Biomed. Opt. (to be published).

Cited By

OSA participates in CrossRef's Cited-By Linking service. Citing articles from OSA journals and other participating publishers are listed here.

Alert me when this article is cited.


Figures (6)

Fig. 1
Fig. 1

Experimental setup. Light from the laser diode (LD) was expanded to illuminate the DMD through the variable gray filter (VGF). The pattern generated by the DMD was projected onto the sample, and whole-field fluorescence was collected by either a spectrophotometer (HSI experiments) or a photomultiplier PMT (for further TCSPC photon counting dedicated for FLIM experiments).

Fig. 2
Fig. 2

(a) Image of the HSI phantom consisting of spots of green (G) and orange (O) dyes on a white-paper background (P). (b) Fluorescence spectra of the dyes and paper used for the phantom and the RGB spectral bands used for spectral separation and visualization.

Fig. 3
Fig. 3

On-line processed HSI image of the phantom (a) and the image divided into three components according to indications given in Fig. 2b. Images (b), (d), and (c) show R, G, and B planes corresponding to orange dye, green dye, and paper fluorescence, respectively.

Fig. 4
Fig. 4

(a) White-light image of a phantom ( 14 mm × 12 mm in size) for FLIM measurements and (b) fluorescence decay curves acquired a priori for orange ( 1.303 ns ) and green ( 0.812 ns ) fluorescence dyes as well as for the paper ( 0.503 ns ).

Fig. 5
Fig. 5

(a) False colored FLIM image constructed using the RLA method and RGB encoding; (b) histogram of associated fluorescence decay times.

Fig. 6
Fig. 6

(a) False colored FLIM image constructed using the LMA method and RGB encoding; (b) histogram of associated fluorescence decay times.

Equations (1)

Equations on this page are rendered with MathJax. Learn more.

t = Δ t / ln ( D 1 / D 0 ) ,

Metrics