Abstract

Fluorescence Lifetime Imaging Microscopy (FLIM) is a powerful technique that is increasingly being used in the life sciences during the past decades. However, a broader application of FLIM requires more cost-effective and user-friendly solutions. We demonstrate the use of a simple CCD/CMOS lock-in imager for fluorescence lifetime detection. The SwissRanger SR-2 time-of-flight detector, originally developed for 3D vision, embeds all the functionalities required for FLIM in a compact system. The further development of this technology and its combination with light-emitting- and laser diodes could drive a wider spreading of the use of FLIM including high-throughput applications.

© 2005 Optical Society of America

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References

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  1. J. R. Lakowicz, Principles of Fluorescence Spectroscopy (Kluwer Academic/Plenum Publishers, New York 1999).
  2. E. A. Jares-Erijman and T. M. Jovin, "FRET imaging," Nat. Biotechnol. 21, 1387-1395 (2003).
    [CrossRef] [PubMed]
  3. F. S. Wouters, P. J. Verveer, and P. I. Bastiaens, "Imaging biochemistry inside cells," Trends Cell Biol. 11, 203-211 (2001).
    [CrossRef] [PubMed]
  4. A. Esposito and F. S. Wouters, "Fluorescence Lifetime Imaging Microscopy" in Current Protocols in Cell Biology, Juan S. Bonifacino, Mary Dasso, Joe B. Harford, Jennifer Lippincott-Schwartz, and Kenneth M. Yamada, eds. 2004).
    [CrossRef]
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    [CrossRef] [PubMed]
  6. A. C. Mitchell, J. E. Wall, J. G. Murray, and C. G. Morgan, "Direct modulation of the effective sensitivity of a CCD detector: a new approach to time-resolved fluorescence imaging," J. Microsc. 206, 225-232 (2002).
    [CrossRef] [PubMed]
  7. A. C. Mitchell, J. E. Wall, J. G. Murray, and C. G. Morgan, "Measurement of nanosecond time-resolved fluorescence with a directly gated interline CCD camera," J. Microsc. 206, (2002).
    [CrossRef] [PubMed]
  8. K. Nishikata, Y. Kimura, Y. Takai, T. Ikuta, and R. Shimizu, "Real-time lock-in imaging by a newly developed high-speed image-processing charged coupled device video camera," Rev. Sci. Instrum. 74, 1393-1396 (2003).
    [CrossRef]
  9. T. Oggier, M. Lehmann, R. Kaufmann, M. Schweizer, M. Richter, P. Metzler, G. Lang, F. Lustenberger, and N. Blanc, "An all-solid-state optical range camera for 3D real-time imaging with sub-centimeter depth resolution (SwissRanger)," in Optical Design and Engineering, L. Mazuray, P.J. Rogers and R. Wartmann, eds., Proc. SPIE 5249, 534-545 (2004).
    [CrossRef]
  10. R. Lange, P. Seitz, A. Biber, and R. Schwarte, "Time-of-flight range imaging with a custom solid state image sensor," in Laser Metrology and Inspection, H.J. Tiziani and P.K. Rastogi, eds., Proc. SPIE 3823, 180-191 (1999).
    [CrossRef]
  11. O. Zapata-Hommer and O. Griesbeck, "Efficiently folding and circularly permuted variants of the Sapphire mutant of GFP," BMC Biotechnol. 3, (2003).
    [CrossRef] [PubMed]
  12. T. W. Gadella, Jr., T. M. Jovin, and R. M. Clegg, "Fluorescence Lifetime Imaging Microscopy (FLIM) -Spatial-Resolution of Microstructures on the Nanosecond Time-Scale," Biophys. Chem. 48, 221-239 (1993).
    [CrossRef]
  13. E. B. van Munster and T. W. Gadella, Jr., "Suppression of photobleaching-induced artifacts in frequencydomain FLIM by permutation of the recording order," Cytometry 58A, (2004).
  14. R. M. Clegg and P. C. Schneider, "Fluorescence lifetime-resolved imaging microscopy: a general description of lifetime-resolved imaging measurements" in Fluorescence Microscopy and Fluorescent Probes, J. Slavik, ed. (Plenum Press, New York 1996).
  15. J. Philip and K. Carlsson, "Theoretical investigation of the signal-to-noise ratio in fluorescence lifetime imaging," J. Opt. Soc. Am. A Opt. Image Sci. Vis. 20, (2003).
    [CrossRef] [PubMed]
  16. H. C. Gerritsen, M. A. Asselbergs, A. V. Agronskaia, and W. G. Van Sark, "Fluorescence lifetime imaging in scanning microscopes: acquisition speed, photon economy and lifetime resolution," J. Microsc. 206, (2002).
    [CrossRef] [PubMed]

Biophys. Chem. (1)

T. W. Gadella, Jr., T. M. Jovin, and R. M. Clegg, "Fluorescence Lifetime Imaging Microscopy (FLIM) -Spatial-Resolution of Microstructures on the Nanosecond Time-Scale," Biophys. Chem. 48, 221-239 (1993).
[CrossRef]

BMC Biotechnol. (1)

O. Zapata-Hommer and O. Griesbeck, "Efficiently folding and circularly permuted variants of the Sapphire mutant of GFP," BMC Biotechnol. 3, (2003).
[CrossRef] [PubMed]

Current Protocols in Cell Biology (1)

A. Esposito and F. S. Wouters, "Fluorescence Lifetime Imaging Microscopy" in Current Protocols in Cell Biology, Juan S. Bonifacino, Mary Dasso, Joe B. Harford, Jennifer Lippincott-Schwartz, and Kenneth M. Yamada, eds. 2004).
[CrossRef]

Cytometry (1)

E. B. van Munster and T. W. Gadella, Jr., "Suppression of photobleaching-induced artifacts in frequencydomain FLIM by permutation of the recording order," Cytometry 58A, (2004).

Fluorescence Microscopy and Fluorescent (1)

R. M. Clegg and P. C. Schneider, "Fluorescence lifetime-resolved imaging microscopy: a general description of lifetime-resolved imaging measurements" in Fluorescence Microscopy and Fluorescent Probes, J. Slavik, ed. (Plenum Press, New York 1996).

J. Microsc. (3)

H. C. Gerritsen, M. A. Asselbergs, A. V. Agronskaia, and W. G. Van Sark, "Fluorescence lifetime imaging in scanning microscopes: acquisition speed, photon economy and lifetime resolution," J. Microsc. 206, (2002).
[CrossRef] [PubMed]

A. C. Mitchell, J. E. Wall, J. G. Murray, and C. G. Morgan, "Direct modulation of the effective sensitivity of a CCD detector: a new approach to time-resolved fluorescence imaging," J. Microsc. 206, 225-232 (2002).
[CrossRef] [PubMed]

A. C. Mitchell, J. E. Wall, J. G. Murray, and C. G. Morgan, "Measurement of nanosecond time-resolved fluorescence with a directly gated interline CCD camera," J. Microsc. 206, (2002).
[CrossRef] [PubMed]

J. Opt. Soc. Am. A Opt. Image Sci. Vis. (1)

J. Philip and K. Carlsson, "Theoretical investigation of the signal-to-noise ratio in fluorescence lifetime imaging," J. Opt. Soc. Am. A Opt. Image Sci. Vis. 20, (2003).
[CrossRef] [PubMed]

Nat. Biotechnol. (1)

E. A. Jares-Erijman and T. M. Jovin, "FRET imaging," Nat. Biotechnol. 21, 1387-1395 (2003).
[CrossRef] [PubMed]

Proc. SPIE (2)

T. Oggier, M. Lehmann, R. Kaufmann, M. Schweizer, M. Richter, P. Metzler, G. Lang, F. Lustenberger, and N. Blanc, "An all-solid-state optical range camera for 3D real-time imaging with sub-centimeter depth resolution (SwissRanger)," in Optical Design and Engineering, L. Mazuray, P.J. Rogers and R. Wartmann, eds., Proc. SPIE 5249, 534-545 (2004).
[CrossRef]

R. Lange, P. Seitz, A. Biber, and R. Schwarte, "Time-of-flight range imaging with a custom solid state image sensor," in Laser Metrology and Inspection, H.J. Tiziani and P.K. Rastogi, eds., Proc. SPIE 3823, 180-191 (1999).
[CrossRef]

Rev. Sci. Instrum. (1)

K. Nishikata, Y. Kimura, Y. Takai, T. Ikuta, and R. Shimizu, "Real-time lock-in imaging by a newly developed high-speed image-processing charged coupled device video camera," Rev. Sci. Instrum. 74, 1393-1396 (2003).
[CrossRef]

Spectrochim. Acta A Mol. Biomol. Spectro (1)

S. Landgraf, "Application of semiconductor light sources for investigations of photochemical reactions," Spectrochim. Acta A Mol. Biomol. Spectrosc. 57, 2029-2048 (2001).
[CrossRef] [PubMed]

Trends Cell Biol. (1)

F. S. Wouters, P. J. Verveer, and P. I. Bastiaens, "Imaging biochemistry inside cells," Trends Cell Biol. 11, 203-211 (2001).
[CrossRef] [PubMed]

Other (1)

J. R. Lakowicz, Principles of Fluorescence Spectroscopy (Kluwer Academic/Plenum Publishers, New York 1999).

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Figures (3)

Fig. 1.
Fig. 1.

The lock-in imager sensor. Panel A shows microphotography of the sensor. Each single pixel, arranged in a 124×160 array has a dimension of about 40μm × 55μm. Each pixel has two gates that are controlled with voltages in opposite phase (Panel B). Thus, the photoelectrons generated in the photosensitive area, will accumulate in the two storage areas according to the relative phase of the photon flux and the gate potentials.

Fig. 2.
Fig. 2.

Response of the lock-in imager. Panel A and B show the average intensity at each detected phase. The grey curve represents the average intensity (I0) and the circles are the experimental points connected by a spline curve (dashed). Gray circles correspond to images acquired by the injection of the additional delay by the external delay unit. The left side panels (A, C and E) represent measurements of a reflective foil, while B, D and F refer to a fluorescent slide acquisition. C and D depict the demodulation of the signal measured over the entire illuminated field of view; E and F show the correspondent phases. The latter are inhomogeneous over the field of view (arrows). Considering the lifetime of the samples, i.e. 0 ns and 4.8 ns for the reflective foil and fluorescent slide, respectively, the initial phase of the detection is shown to be constant, while the demodulations suffer from a color-effect of the lock-in imager.

Fig. 3.
Fig. 3.

Fluorescence lifetime sensing. The lock-in imager distinguishes compounds with different fluorescent lifetimes. Panel A shows the phase-lifetime maps and distributions of: EGFP in solution (gray line), a fluorescent slide (dashed curve) and DNA-bound GelStar (black solid line) in solution. The lifetimes were 2.6±0.4 ns, 4.8±0.4 ns and 6.6±0.7 ns, respectively. Both the phase- (panel B, gray curve) and demodulation- (black line) lifetimes can be measured at a modulation frequency of 20MHz. A Turbo-Sapphire GFP bead showed values of 2.67±0.09 ns and 3.7±0.2 ns, respectively. Panel B inset (R6G) shows the phase (4.3±0.2) and modulation (4.3±0.4) lifetime of the mono-exponential decaying fluorophore standard Rhodamine 6G.

Equations (4)

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{ ϕ = arctan ( S 3 π 2 S π 2 S 0 S π ) ϕ m = ( S 3 π 2 S π 2 ) 2 + ( S 0 S π ) 2 m ( S 0 + S π 2 + S π + S 3 π 2 )
{ τ ϕ = ω 1 tan ϕ τ m = ω 1 1 m 2 1
{ F DC = k = 0 7 S 4 F SIN = k = 0 7 S 4 sin ( 4 ) F COS = k = 0 7 S 4 cos ( 4 )
{ ϕ = arctan ( F SIN F COS ) ϕ m = F SIN 2 + F COS 2 m F DC

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