Abstract

In this work, we present a method providing real-time, low cost, three-dimensional imaging in a three-dimensional optical micromanipulation system. The three-dimensional imaging system is based on a small form factor LED based projector. The projector is used to dynamically shape the rear illumination light in a counter-propagating beam-trapping setup. This allows us to produce stereoscopic images, from which the human brain can construct a three-dimensional image, or alternatively image analysis can be applied by a computer, thereby obtaining true three-dimensional coordinates in real-time for the trapped objects.

© 2008 Optical Society of America

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References

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  1. D. G. Grier, "A revolution in optical manipulation," Nature 424, 810-816 (2003).
    [CrossRef] [PubMed]
  2. A. Ashkin, "Acceleration and trapping of particles by radiation pressure," Phys. Rev. Lett. 24, 156 (1970).
    [CrossRef]
  3. P. J. Rodrigo, V. R. Daria, and J. Glückstad, "Real-time three-dimensional optical micromanipulation of multiple particles and living cells," Opt. Lett. 29, 2270-2272 (2004).
    [CrossRef] [PubMed]
  4. A. Isomura, N. Magome, M. I. Kohira, and K. Yoshikawa, "Toward the stable optical trapping of a droplet with counter laser beams under microgravity," Chem. Phys. Lett. 429, 321-325 (2006).
    [CrossRef]
  5. S. -H. Lee and D. G. Grier, "Holographic microscopy of holographically trapped three-dimensional structures," Opt. Express 15, 1505-1512 (2007).
    [CrossRef] [PubMed]
  6. I. Perch-Nielsen, P. Rodrigo, and J. Glückstad, "Real-time interactive 3D manipulation of particles viewed in two orthogonal observation planes," Opt. Express 13, 2852-2857 (2005).
    [CrossRef] [PubMed]
  7. P. J. Rodrigo, I. R. Perch-Nielsen, C. A. Alonzo, and J. Glückstad, "GPC-based optical micromanipulation in 3D real-time using a single spatial light modulator," Opt. Express 14, 13107-13112 (2006).
    [CrossRef] [PubMed]
  8. J. S. Dam, P. J. Rodrigo, I. R. Perch-Nielsen, and J. Glückstad, "Fully automated beam-alignment and single stroke guided manual alignment of counter-propagating multi-beam based optical micromanipulation systems," Opt. Express 15, 7968-7973 (2007).
    [CrossRef] [PubMed]
  9. S. Delica, and C. M. Blanca, "Wide-field depth-sectioning fluorescence microscopy using projector-generated patterned illumination," Appl. Opt. 46, 7237-7243 (2007).
    [CrossRef] [PubMed]
  10. E. C. Samson and C. M. Blanca, "Dynamic contrast enhancement in widefield microscopy using projector-generated illumination patterns," New J. Phys. 9363 (2007).
    [CrossRef]
  11. P. J. Rodrigo, I. R. Perch-Nielsen, and J. Glückstad, "Three-dimensional forces in GPC-based counterpropagating-beam traps," Opt. Express 14, 5812-5822 (2006).
    [CrossRef] [PubMed]
  12. J. S. Dam, P. J. Rodrigo, I. R. Perch-Nielsen, C. A. Alonzo, and J. Glückstad, "Computerized "drag-and-drop" alignment of GPC-based optical micromanipulation system," Opt. Express 15, 1923-1931 (2007).
    [CrossRef] [PubMed]
  13. K. Burton, "An aperture-shifting light-microscopic method for rapidly quantifying positions of cells in 3D matrices," Cytometry Part A 54, 125-131 (2003).
    [CrossRef]

2007

2006

2005

2004

2003

D. G. Grier, "A revolution in optical manipulation," Nature 424, 810-816 (2003).
[CrossRef] [PubMed]

K. Burton, "An aperture-shifting light-microscopic method for rapidly quantifying positions of cells in 3D matrices," Cytometry Part A 54, 125-131 (2003).
[CrossRef]

1970

A. Ashkin, "Acceleration and trapping of particles by radiation pressure," Phys. Rev. Lett. 24, 156 (1970).
[CrossRef]

Alonzo, C. A.

Ashkin, A.

A. Ashkin, "Acceleration and trapping of particles by radiation pressure," Phys. Rev. Lett. 24, 156 (1970).
[CrossRef]

Blanca, C. M.

E. C. Samson and C. M. Blanca, "Dynamic contrast enhancement in widefield microscopy using projector-generated illumination patterns," New J. Phys. 9363 (2007).
[CrossRef]

S. Delica, and C. M. Blanca, "Wide-field depth-sectioning fluorescence microscopy using projector-generated patterned illumination," Appl. Opt. 46, 7237-7243 (2007).
[CrossRef] [PubMed]

Burton, K.

K. Burton, "An aperture-shifting light-microscopic method for rapidly quantifying positions of cells in 3D matrices," Cytometry Part A 54, 125-131 (2003).
[CrossRef]

Dam, J. S.

Daria, V. R.

Delica, S.

Glückstad, J.

Grier, D. G.

Isomura, A.

A. Isomura, N. Magome, M. I. Kohira, and K. Yoshikawa, "Toward the stable optical trapping of a droplet with counter laser beams under microgravity," Chem. Phys. Lett. 429, 321-325 (2006).
[CrossRef]

Kohira, M. I.

A. Isomura, N. Magome, M. I. Kohira, and K. Yoshikawa, "Toward the stable optical trapping of a droplet with counter laser beams under microgravity," Chem. Phys. Lett. 429, 321-325 (2006).
[CrossRef]

Lee, S. -H.

Magome, N.

A. Isomura, N. Magome, M. I. Kohira, and K. Yoshikawa, "Toward the stable optical trapping of a droplet with counter laser beams under microgravity," Chem. Phys. Lett. 429, 321-325 (2006).
[CrossRef]

Perch-Nielsen, I.

Perch-Nielsen, I. R.

Rodrigo, P.

Rodrigo, P. J.

Samson, E. C.

E. C. Samson and C. M. Blanca, "Dynamic contrast enhancement in widefield microscopy using projector-generated illumination patterns," New J. Phys. 9363 (2007).
[CrossRef]

Yoshikawa, K.

A. Isomura, N. Magome, M. I. Kohira, and K. Yoshikawa, "Toward the stable optical trapping of a droplet with counter laser beams under microgravity," Chem. Phys. Lett. 429, 321-325 (2006).
[CrossRef]

Appl. Opt.

Chem. Phys. Lett.

A. Isomura, N. Magome, M. I. Kohira, and K. Yoshikawa, "Toward the stable optical trapping of a droplet with counter laser beams under microgravity," Chem. Phys. Lett. 429, 321-325 (2006).
[CrossRef]

Cytometry Part A

K. Burton, "An aperture-shifting light-microscopic method for rapidly quantifying positions of cells in 3D matrices," Cytometry Part A 54, 125-131 (2003).
[CrossRef]

Nature

D. G. Grier, "A revolution in optical manipulation," Nature 424, 810-816 (2003).
[CrossRef] [PubMed]

New J. Phys.

E. C. Samson and C. M. Blanca, "Dynamic contrast enhancement in widefield microscopy using projector-generated illumination patterns," New J. Phys. 9363 (2007).
[CrossRef]

Opt. Express

Opt. Lett.

Phys. Rev. Lett.

A. Ashkin, "Acceleration and trapping of particles by radiation pressure," Phys. Rev. Lett. 24, 156 (1970).
[CrossRef]

Supplementary Material (2)

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» Media 2: GIF (109 KB)     

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Figures (6)

Fig. 1.
Fig. 1.

The output from the projector is collected by a 25 mm focal length lens (L1), which forms a focused image on the diffuser. This image (displayed by the projector) is relayed by a 4-f system to the back focal plane of the lower objective. L2 has a focal length of 150 mm and L3 125 mm. An aperture is placed approximately 2-f from the diffuser. The objectives are both long working distance 50× Olympus PlanIR objectives with a numerical aperture of 0.55. L4 is an Olympus microscope lens with a focal length of 180 mm. The green line symbolizes the counter-propagating trapping laser beam set being reflected in dichroic mirrors (dashed lines). The lower image to the left shows how an empty scene looks without the diffusive filter in place. Notice the unevenness of the illumination. After insertion of a diffusive filter, the homogeneity problem is completely solved as shown in the right image. Not only does it make for nicer images, it also simplifies any subsequent image analysis greatly.

Fig. 2.
Fig. 2.

The sketch on the left shows the principle of how corrective lenses can be used to displace the observation plane of the used microscope objectives. CCD indicates a color camera; FW1 and FW2 are filter wheels with a selection of corrective lenses to insert in the optical path. DM are dichroic mirrors which reflect the laser light while allowing visible light to pass. FOC indicates the focal plane of the upper objective. The red line shows the uncorrected image path, and the black line shows how the corrective lenses displace the observed plane. The green lines are the trapping laser beam sets. The table on the right shows focus displacement and relative magnification changes as a function of the lenses inserted in the optical path.

Fig. 3.
Fig. 3.

The projector is displaying a red and a blue circle (A) at the back focal plane of the lower microscope objective. The dashed circle shows the back aperture of the objective, and the blue and red dots are the image displayed there. This custom shaped illumination makes the particle (which in this example is placed before the observation plane) give two displaced shadows (C) in the observed plane.

Fig. 4.
Fig. 4.

The left image shows particles of varying sizes (diameters 2 µm, 4.5 µm and 10 µm) being trapped at different z-locations. The dashed inserts show the illumination pattern displayed in the back focal plane. If a set of red/blue 3D goggles is worn, the image can be interpreted by the brain as a 3D structure. The right image is the same image after image analysis (thresholding) has been applied. The analyzed image has negative colors, i.e. a blue dot where the blue light is missing (where the left image shows red).

Fig. 5.
Fig. 5.

(AVI: 1.0 MB) Four frames from movie showing how a rotating illumination can give a 3D impression of the scene. The effect is similar to watching a 3D object with one eye closed while moving ones head in circles to gain 3D information. Note that nothing in the scene is moving, only the illumination pattern changes. Frames with different illumination patterns can be combined to produce color coded stereoscopic images. The red/blue image is a combination of the left illuminated frame (colored red) and right illuminated frame (colored blue). [GIF: contrast enhanced version] [Media 1][Media 2]

Fig. 6.
Fig. 6.

The images show the effect of an aperture (placed in the white light module as shown in Fig. 1) imaged out of focus under different illuminations. The left image shows how the imaged aperture appears sharp under low NA illumination, and shift by the angle of illumination. The use of such an aperture allow us to do time-multiplexing of different illumination patterns while using subsequent image analysis to determine how each video frame was illuminated.

Equations (2)

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[ 1 f 1 0 1 ] · [ 1 0 1 f 1 1 ] · [ 1 f 1 + f 2 d 1 d 2 0 1 ] · [ 1 0 1 f cor 2 1 ] · [ 1 d 2 0 1 ] · [ 1 0 1 f cor 1 ] · [ 1 d 1 0 1 ] · [ 1 f 2 + z 0 1 ] · ( 0 θ ) = ( x y )
z = x blue x red 2 tan θ

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