Abstract

The confocal detection principle is extended to a highly parallel optical system that continuously analyzes thousands of concurrent sample locations. This is achieved through the use of a holographic laser illumination multiplexer combined with a confocal pinhole array before a prism dispersive element used to provide spectroscopic information from each confocal volume. The system is demonstrated to detect and identify single fluorescent molecules from each of several thousand independent confocal volumes in real time.

© 2008 Optical Society of America

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  1. J. B. Pawley, Handbook of Biological Confocal Microscopy, 3rd ed. (Springer, 2006).
    [CrossRef]
  2. S. Weiss, Science 283, 1676 (1999).
    [CrossRef] [PubMed]
  3. W. E. Moerner and L. Kandor, Anal. Chem. 61, A1217 (1989).
    [CrossRef]
  4. G. Q. Xiao, T. R. Corle, and G. S. Kino, Appl. Phys. Lett. 53, 716 (1988).
    [CrossRef]
  5. T. Wilson, J. N. Gannaway, and P. Johnson, J. Microsc. 118, 390 (1980).
    [CrossRef]
  6. Q. S. Handley and T. J. Jovin, Appl. Spectrosc. 55, 1115 (2001).
    [CrossRef]
  7. J. Jung and A. Van Orden, J. Am. Chem. Soc. 128, 1240 (2006).
    [CrossRef] [PubMed]
  8. H.-S. Chon, G. Park, S.-B. Lee, S. Yoon, J. Kim, J.-H. Lee, and K. An, J. Opt. Soc. Am. A 24, 60 (2007).
    [CrossRef]
  9. B. Kress, Digital Diffractive Optics: an Introduction to Planar Diffractive Optics and Related Technology, (Wiley, 2000).
  10. M. Eisner, N. Lindlein, and J. Schwider, Opt. Lett. 23, 748 (1998).
    [CrossRef]
  11. C. J. R. Sheppard and C. J. Cogswell, Scanning 13, 240 (1991).
    [CrossRef]
  12. M. Foquet, J. Korlach, W. Zipfel, W. W. Webb, and H. G. Craighead, Anal. Chem. 74, 1415 (2002).
    [CrossRef] [PubMed]
  13. T. D. Corrigan, S. Guo, R. J. Phaneuf, and H. Szmacinski, J. Fluoresc. 15, 777 (2005).
    [CrossRef] [PubMed]
  14. M. Levene, J. Korlach, S. W. Turner, M. Foquet, H. G. Craighead, and W. W. Webb, Science 299, 682 (2003).
    [CrossRef] [PubMed]

2007 (1)

2006 (1)

J. Jung and A. Van Orden, J. Am. Chem. Soc. 128, 1240 (2006).
[CrossRef] [PubMed]

2005 (1)

T. D. Corrigan, S. Guo, R. J. Phaneuf, and H. Szmacinski, J. Fluoresc. 15, 777 (2005).
[CrossRef] [PubMed]

2003 (1)

M. Levene, J. Korlach, S. W. Turner, M. Foquet, H. G. Craighead, and W. W. Webb, Science 299, 682 (2003).
[CrossRef] [PubMed]

2002 (1)

M. Foquet, J. Korlach, W. Zipfel, W. W. Webb, and H. G. Craighead, Anal. Chem. 74, 1415 (2002).
[CrossRef] [PubMed]

2001 (1)

1999 (1)

S. Weiss, Science 283, 1676 (1999).
[CrossRef] [PubMed]

1998 (1)

1991 (1)

C. J. R. Sheppard and C. J. Cogswell, Scanning 13, 240 (1991).
[CrossRef]

1989 (1)

W. E. Moerner and L. Kandor, Anal. Chem. 61, A1217 (1989).
[CrossRef]

1988 (1)

G. Q. Xiao, T. R. Corle, and G. S. Kino, Appl. Phys. Lett. 53, 716 (1988).
[CrossRef]

1980 (1)

T. Wilson, J. N. Gannaway, and P. Johnson, J. Microsc. 118, 390 (1980).
[CrossRef]

An, K.

Chon, H.-S.

Cogswell, C. J.

C. J. R. Sheppard and C. J. Cogswell, Scanning 13, 240 (1991).
[CrossRef]

Corle, T. R.

G. Q. Xiao, T. R. Corle, and G. S. Kino, Appl. Phys. Lett. 53, 716 (1988).
[CrossRef]

Corrigan, T. D.

T. D. Corrigan, S. Guo, R. J. Phaneuf, and H. Szmacinski, J. Fluoresc. 15, 777 (2005).
[CrossRef] [PubMed]

Craighead, H. G.

M. Levene, J. Korlach, S. W. Turner, M. Foquet, H. G. Craighead, and W. W. Webb, Science 299, 682 (2003).
[CrossRef] [PubMed]

M. Foquet, J. Korlach, W. Zipfel, W. W. Webb, and H. G. Craighead, Anal. Chem. 74, 1415 (2002).
[CrossRef] [PubMed]

Eisner, M.

Foquet, M.

M. Levene, J. Korlach, S. W. Turner, M. Foquet, H. G. Craighead, and W. W. Webb, Science 299, 682 (2003).
[CrossRef] [PubMed]

M. Foquet, J. Korlach, W. Zipfel, W. W. Webb, and H. G. Craighead, Anal. Chem. 74, 1415 (2002).
[CrossRef] [PubMed]

Gannaway, J. N.

T. Wilson, J. N. Gannaway, and P. Johnson, J. Microsc. 118, 390 (1980).
[CrossRef]

Guo, S.

T. D. Corrigan, S. Guo, R. J. Phaneuf, and H. Szmacinski, J. Fluoresc. 15, 777 (2005).
[CrossRef] [PubMed]

Handley, Q. S.

Johnson, P.

T. Wilson, J. N. Gannaway, and P. Johnson, J. Microsc. 118, 390 (1980).
[CrossRef]

Jovin, T. J.

Jung, J.

J. Jung and A. Van Orden, J. Am. Chem. Soc. 128, 1240 (2006).
[CrossRef] [PubMed]

Kandor, L.

W. E. Moerner and L. Kandor, Anal. Chem. 61, A1217 (1989).
[CrossRef]

Kim, J.

Kino, G. S.

G. Q. Xiao, T. R. Corle, and G. S. Kino, Appl. Phys. Lett. 53, 716 (1988).
[CrossRef]

Korlach, J.

M. Levene, J. Korlach, S. W. Turner, M. Foquet, H. G. Craighead, and W. W. Webb, Science 299, 682 (2003).
[CrossRef] [PubMed]

M. Foquet, J. Korlach, W. Zipfel, W. W. Webb, and H. G. Craighead, Anal. Chem. 74, 1415 (2002).
[CrossRef] [PubMed]

Kress, B.

B. Kress, Digital Diffractive Optics: an Introduction to Planar Diffractive Optics and Related Technology, (Wiley, 2000).

Lee, J.-H.

Lee, S.-B.

Levene, M.

M. Levene, J. Korlach, S. W. Turner, M. Foquet, H. G. Craighead, and W. W. Webb, Science 299, 682 (2003).
[CrossRef] [PubMed]

Lindlein, N.

Moerner, W. E.

W. E. Moerner and L. Kandor, Anal. Chem. 61, A1217 (1989).
[CrossRef]

Park, G.

Pawley, J. B.

J. B. Pawley, Handbook of Biological Confocal Microscopy, 3rd ed. (Springer, 2006).
[CrossRef]

Phaneuf, R. J.

T. D. Corrigan, S. Guo, R. J. Phaneuf, and H. Szmacinski, J. Fluoresc. 15, 777 (2005).
[CrossRef] [PubMed]

Schwider, J.

Sheppard, C. J. R.

C. J. R. Sheppard and C. J. Cogswell, Scanning 13, 240 (1991).
[CrossRef]

Szmacinski, H.

T. D. Corrigan, S. Guo, R. J. Phaneuf, and H. Szmacinski, J. Fluoresc. 15, 777 (2005).
[CrossRef] [PubMed]

Turner, S. W.

M. Levene, J. Korlach, S. W. Turner, M. Foquet, H. G. Craighead, and W. W. Webb, Science 299, 682 (2003).
[CrossRef] [PubMed]

Van Orden, A.

J. Jung and A. Van Orden, J. Am. Chem. Soc. 128, 1240 (2006).
[CrossRef] [PubMed]

Webb, W. W.

M. Levene, J. Korlach, S. W. Turner, M. Foquet, H. G. Craighead, and W. W. Webb, Science 299, 682 (2003).
[CrossRef] [PubMed]

M. Foquet, J. Korlach, W. Zipfel, W. W. Webb, and H. G. Craighead, Anal. Chem. 74, 1415 (2002).
[CrossRef] [PubMed]

Weiss, S.

S. Weiss, Science 283, 1676 (1999).
[CrossRef] [PubMed]

Wilson, T.

T. Wilson, J. N. Gannaway, and P. Johnson, J. Microsc. 118, 390 (1980).
[CrossRef]

Xiao, G. Q.

G. Q. Xiao, T. R. Corle, and G. S. Kino, Appl. Phys. Lett. 53, 716 (1988).
[CrossRef]

Yoon, S.

Zipfel, W.

M. Foquet, J. Korlach, W. Zipfel, W. W. Webb, and H. G. Craighead, Anal. Chem. 74, 1415 (2002).
[CrossRef] [PubMed]

Anal. Chem. (2)

W. E. Moerner and L. Kandor, Anal. Chem. 61, A1217 (1989).
[CrossRef]

M. Foquet, J. Korlach, W. Zipfel, W. W. Webb, and H. G. Craighead, Anal. Chem. 74, 1415 (2002).
[CrossRef] [PubMed]

Appl. Phys. Lett. (1)

G. Q. Xiao, T. R. Corle, and G. S. Kino, Appl. Phys. Lett. 53, 716 (1988).
[CrossRef]

Appl. Spectrosc. (1)

J. Am. Chem. Soc. (1)

J. Jung and A. Van Orden, J. Am. Chem. Soc. 128, 1240 (2006).
[CrossRef] [PubMed]

J. Fluoresc. (1)

T. D. Corrigan, S. Guo, R. J. Phaneuf, and H. Szmacinski, J. Fluoresc. 15, 777 (2005).
[CrossRef] [PubMed]

J. Microsc. (1)

T. Wilson, J. N. Gannaway, and P. Johnson, J. Microsc. 118, 390 (1980).
[CrossRef]

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

Opt. Lett. (1)

Scanning (1)

C. J. R. Sheppard and C. J. Cogswell, Scanning 13, 240 (1991).
[CrossRef]

Science (2)

S. Weiss, Science 283, 1676 (1999).
[CrossRef] [PubMed]

M. Levene, J. Korlach, S. W. Turner, M. Foquet, H. G. Craighead, and W. W. Webb, Science 299, 682 (2003).
[CrossRef] [PubMed]

Other (2)

B. Kress, Digital Diffractive Optics: an Introduction to Planar Diffractive Optics and Related Technology, (Wiley, 2000).

J. B. Pawley, Handbook of Biological Confocal Microscopy, 3rd ed. (Springer, 2006).
[CrossRef]

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Figures (4)

Fig. 1
Fig. 1

Schematic diagram of reversed confocal illumination and spectroscopy scheme. The images from multiple HPMs are superimposed at the illumination confocal plane. To exploit optimum dichroic filter design (narrow excitation bandwidths and high fluorescence throughput), the usual positions of the illumination and collection paths have been reversed. A pinhole array is placed at the confocal plane of the collection path to reject out-of-focus light.

Fig. 2
Fig. 2

Multiple-spot illumination pattern at the front focal plane of the microscope objective lens. This pattern was generated using 488 nm laser light divided into 5114 beams by a computer-generated HPM. The image was acquired using a thin film of fluorescent polymer spin coated on a glass substrate placed in the sample plane of the system with the prism removed. The inset is a magnified section from the corner of the optical field. Undiffracted laser light (0.2% of the total) appears near the center of the pattern. The overall variation in spot intensity is within ± 15 % .

Fig. 3
Fig. 3

Point-spread function of Alexa-488 fluorescence light collected at the image plane for optimal focus as well as ± 1 and 2 μ m defocus. The 16 μ m camera pixel size corresponds to 0.266 μ m in the sample plane.

Fig. 4
Fig. 4

Example time traces (left) and spectra (right) collected from one observation volume at different times. Each of the four colors represents one spectral channel from a multicomponent analysis of the dispersed light collected from the observation volume. In the spectral plots, the solid colored curves represent reference spectra collected from each of the four fluoropohores in a calibration process. In each plot the black curve with error bars represents the photon flux integrated over the duration of the burst as a function of relative spectral position. The shown fluorescence bursts represent integrated burst SNR ratios between 20 and 35.

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