Abstract

We present a simple and cost-effective wide-field, depth-sectioning, fluorescence microscope utilizing a commercial multimedia projector to generate excitation patterns on the sample. Highly resolved optical sections of fluorescent pollen grains at 1.9μm axial resolution are constructed using the structured illumination technique. This requires grid excitation patterns to be scanned across the sample, which is straightforwardly implemented by creating slideshows of gratings at different phases, projecting them onto the sample, and synchronizing camera acquisition with slide transition. In addition to rapid dynamic pattern generation, the projector provides high illumination power and spectral excitation selectivity. We exploit these properties by imaging mouse neural cells in cultures multistained with Alexa 488 and Cy3. The spectral and structural neural information is effectively resolved in three dimensions. The flexibility and commercial availability of this light source is envisioned to open multidimensional imaging to a broader user base.

© 2007 Optical Society of America

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References

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    [CrossRef]
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    [CrossRef]
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    [CrossRef] [PubMed]
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    [CrossRef] [PubMed]
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    [CrossRef]
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2006 (3)

L. Krzewina and M. Kim, "Single exposure optical sectioning by color structured illumination microscopy," Opt. Lett. 31, 477-479 (2006).
[CrossRef] [PubMed]

M. Gustafsson, "Nonlinear structured-illumination microscopy: wide-field fluorescence imaging with theoretically unlimited resolution," Proc. Natl. Acad. Sci. U.S.A. 102, 13081-13086 (2006).
[CrossRef]

S. Monneret, M. Rauzi, and P. F. Lenne, "Highly flexible whole-field sectioning microscope with liquid-crystal light modulator," Pure Appl. Opt. 8, S461-S466 (2006).
[CrossRef]

2005 (1)

2004 (1)

L. H. Schaefer, D. Schuster, and J. Schaffer, "Structured illumination microscopy: artifact analysis and reduction utilizing a parameter optimization approach," J. Microsc. 216, 165-174 (2004).
[CrossRef] [PubMed]

2003 (1)

2002 (1)

S. E. D. Webb, Y. Gu, S. Lévêque-Fort, J. Siegel, M. J. Cole, K. Dowling, R. Jones, P. M. W. French, M. A. A. Neil, R. Juškaitis, L. O. D. Sucharov, T. Wilson, and M. J. Lever, "Wide-field time domain FLIM microscope with optical sectioning," Rev. Sci. Instrum. 73, 1898-1907 (2002).
[CrossRef]

2001 (1)

M. J. Cole, J. Siegel, S. E. D. Webb, R. Jones, K. Dowling, M. J. Dayel, D. Parsons-Karavassilis, P. M. W. French, M. J. Lever, L. O. D. Sucharov, M. A. A. Neil, R. Juskaitis, and T. Wilson, "Time-domain whole-field fluorescence lifetime imaging with optical sectioning," J. Microsc. 203, 246-257 (2001).
[CrossRef] [PubMed]

2000 (1)

J. T. Frohn, H. F. Knapp, and A. Stemmer, "True optical resolution beyond the Rayleigh limit achieved by standing wave illumination," Proc. Natl. Acad. Sci. U.S.A. 97, 7232-7236 (2000).
[CrossRef] [PubMed]

1999 (1)

R. Heintzmann and C. Cremer, "Lateral modulated excitation microscopy: improvement of resolution by using a diffraction grating," Proc. SPIE 3568, 185-196 (1999).
[CrossRef]

1997 (1)

Appl. Opt. (1)

J. Microsc. (2)

M. J. Cole, J. Siegel, S. E. D. Webb, R. Jones, K. Dowling, M. J. Dayel, D. Parsons-Karavassilis, P. M. W. French, M. J. Lever, L. O. D. Sucharov, M. A. A. Neil, R. Juskaitis, and T. Wilson, "Time-domain whole-field fluorescence lifetime imaging with optical sectioning," J. Microsc. 203, 246-257 (2001).
[CrossRef] [PubMed]

L. H. Schaefer, D. Schuster, and J. Schaffer, "Structured illumination microscopy: artifact analysis and reduction utilizing a parameter optimization approach," J. Microsc. 216, 165-174 (2004).
[CrossRef] [PubMed]

Opt. Lett. (3)

Proc. Natl. Acad. Sci. U.S.A. (2)

M. Gustafsson, "Nonlinear structured-illumination microscopy: wide-field fluorescence imaging with theoretically unlimited resolution," Proc. Natl. Acad. Sci. U.S.A. 102, 13081-13086 (2006).
[CrossRef]

J. T. Frohn, H. F. Knapp, and A. Stemmer, "True optical resolution beyond the Rayleigh limit achieved by standing wave illumination," Proc. Natl. Acad. Sci. U.S.A. 97, 7232-7236 (2000).
[CrossRef] [PubMed]

Proc. SPIE (1)

R. Heintzmann and C. Cremer, "Lateral modulated excitation microscopy: improvement of resolution by using a diffraction grating," Proc. SPIE 3568, 185-196 (1999).
[CrossRef]

Pure Appl. Opt. (1)

S. Monneret, M. Rauzi, and P. F. Lenne, "Highly flexible whole-field sectioning microscope with liquid-crystal light modulator," Pure Appl. Opt. 8, S461-S466 (2006).
[CrossRef]

Rev. Sci. Instrum. (1)

S. E. D. Webb, Y. Gu, S. Lévêque-Fort, J. Siegel, M. J. Cole, K. Dowling, R. Jones, P. M. W. French, M. A. A. Neil, R. Juškaitis, L. O. D. Sucharov, T. Wilson, and M. J. Lever, "Wide-field time domain FLIM microscope with optical sectioning," Rev. Sci. Instrum. 73, 1898-1907 (2002).
[CrossRef]

Other (4)

Texas Instruments, "DLP Technology," http://www.dlp.com/tech/what.aspx.

Answers.com, "DLP: information from answers.com," http://www.answers.com/topic/dlp.

Nikon MicroscopyU, "Confocal image gallery," http://www.microscopyu.com/galleries/confocal/pollen1.html.

Nikon MicroscopyU, "Confocal image gallery," http://www.microscopyu.com/galleries/confocal/pinegerminatedpollen.html.

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Figures (8)

Fig. 1
Fig. 1

(Color online) Optical setup. A sinusoidal grating pattern is projected by a multimedia projector onto a fluorescence microscopy to generate structured illumination. L1 and L2 are coupling lenses; Ex, DM, Em are the excitation, dichroic, and emission filters, respectively, and L3 is a tube lens.

Fig. 2
Fig. 2

(Color online) (a) Spectra of the output signal when pure red, green, blue, and white colors are displayed separately. (b) Light intensity of a single projected pixel taken at various camera exposure times showing increasing intensity stability for longer integration period t.

Fig. 3
Fig. 3

(Color online) (a) Binary grating patterns of 1-, 2-, 4-, 8-, and 16-projector-pixel strips projected onto the imaging planes of the microscope. (b) Cross section of the resulting image reflected off a mirror sample and taken with a CCD camera. The optical system truncates the high spatial frequencies, resulting to the sinusoidal edges.

Fig. 4
Fig. 4

(Color online) (a) Axial response of the optical. Experiment data (circles) are consistent with theory (solid) revealing a 1.9 and 8.0 μm resolution for system under NA = 1.3 and 0.55, respectively. (b) FWHM of the axial response decreases as a function of grid frequency resulting to maximum resolution at 474 lines / mm . The finite size of the projector pixel and optical magnification impose a practical limit of 125 lines / mm to the optical system.

Fig. 5
Fig. 5

(Color online) Images reconstructed using plane (a)–(c) and patterned illumination (e)–(g), taken at 6 μm axial intervals. Superior optical sectioning is achieved by grid illumination even more evident in the autofocus (maximum intensity projection) images (d), (h). The arrows highlight the ability of structured illumination to sharply delineate the spines and cleavages on the pollen grain, which are barely discernible in the wide-field (plane illumination) images.

Fig. 6
Fig. 6

Sectioned images of a pine pollen grain taken at 10 μm increments and the resulting three-dimensional reconstruction (rightmost column) for increasing number of image slices N s used in rendering.

Fig. 7
Fig. 7

(Color online) Optical sections of a pollen grain taken with two different filter sets. (a)–(c) taken with an FITC∕Alexa 488 filter [Ex: 450 490 nm , DM : > 505 nm , Em > 515 nm ], (e)–(g) Olympus U-MNG filter [Ex: 530 550 nm , DM : > 570 nm , Em : > 590 nm ]. The autofocus projections (d), (h) are assigned to the green and red channels of an RGB image, respectively, to reveal the three-dimensional dye distribution.

Fig. 8
Fig. 8

(Color online) Images of mice neural cell culture at different depths. The glial cells (red) are stained with Cy3 and observed using the U-MNG filter and while the neural cells (green) are stained with Alexa 488 and captured through an FITC∕Alexa 488 filter set. The colored image is reconstructed by assigning the Cy3 image to the red channel and the Alexa 488 to the green channel of the RGB image.

Equations (3)

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I sectioned = 3 2 [ ( I 1 I 2 ) 2 + ( I 1 I 3 ) 2 + ( I 2 I 3 ) 2 ] 1 / 2 ,
I wide field = 1 3 [ I 1 + I 2 + I 3 ] ,
I ( u ) | 2 J 1 ( 2 u γ ( 1 γ 2 ) ) ( 2 u γ ( 1 γ 2 ) ) | ,

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