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Biomedical Optics Express

Biomedical Optics Express

  • Editor: Joseph A. Izatt
  • Vol. 4, Iss. 10 — Oct. 1, 2013
  • pp: 2087–2094
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Independent and simultaneous three-dimensional optical trapping and imaging

Maya Yevnin, Dror Kasimov, Yael Gluckman, Yuval Ebenstein, and Yael Roichman  »View Author Affiliations


Biomedical Optics Express, Vol. 4, Issue 10, pp. 2087-2094 (2013)
http://dx.doi.org/10.1364/BOE.4.002087


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Abstract

Combining imaging and control of multiple micron-scaled objects in three dimensions opens up new experimental possibilities such as the fabrication of colloidal-based photonic devices, as well as high-throughput studies of single cell dynamics. Here we utilize the dual-objectives approach to combine 3D holographic optical tweezers with a spinning-disk confocal microscope. Our setup is capable of trapping multiple different objects in three dimensions with lateral and axial accuracy of 8 nm and 20 nm, and precision of 20 nm and 200 nm respectively, while imaging them in four different fluorescence channels. We demonstrate fabrication of ordered two-component and three dimensional colloidal arrays, as well as trapping of yeast cell arrays. We study the kinetics of the division of yeast cells within optical traps, and find that the timescale for division is not affected by trapping.

© 2013 OSA

1. Introduction

A single laser beam brought to a tight focus by a high numerical aperture microscope objective lens constitutes an optical trap [9

9. A. Ashkin, J. M. Dziedzic, and T. Yamane, “Optical trapping and manipulation of single cells using infrared laser beams,” Nature 330(6150), 769–771 (1987). [CrossRef] [PubMed]

]. HOTs generalize this idea splitting a single laser beam, by means of a computer controlled diffractive optical element (DOE) [5

5. D. G. Grier, “A revolution in optical manipulation,” Nature 424(6950), 810–816 (2003). [CrossRef] [PubMed]

], to form multiple optical traps [5

5. D. G. Grier, “A revolution in optical manipulation,” Nature 424(6950), 810–816 (2003). [CrossRef] [PubMed]

,10

10. D. McGloin, G. C. Spalding, H. Melville, W. Sibbett, and K. Dholakia, “Three-dimensional arrays of optical bottle beams,” Opt. Commun. 225(4-6), 215–222 (2003). [CrossRef]

,11

11. N. B. Simpson, L. Allen, and M. J. Padgett, “Optical tweezers and optical spanners with Laguerre-Gaussian modes,” J. Mod. Opt. 43(12), 2485–2491 (1996). [CrossRef]

] in the sample plane. Using a spatial light modulator as the DOE to split the beam by a phase-only hologram is especially beneficial since it utilizes most of the laser power for trapping. In addition, it enables more sophisticated beam shaping and allows for real-time corrections of aberrations in the optical train [6

6. M. Polin, K. Ladavac, S. H. Lee, Y. Roichman, and D. G. Grier, “Optimized holographic optical traps,” Opt. Express 13(15), 5831–5845 (2005). [CrossRef] [PubMed]

,12

12. Y. Roichman, A. Waldron, E. Gardel, and D. G. Grier, “Optical traps with geometric aberrations,” Appl. Opt. 45(15), 3425–3429 (2006). [CrossRef] [PubMed]

,13

13. R. W. Bowman, A. J. Wright, and M. J. Padgett, “An SLM-based Shack–Hartmann wavefront sensor for aberration correction in optical tweezers,” J. Opt. 12(12), 124004 (2010). [CrossRef]

]. The position of the optical traps in a HOTs setup is determined by a computer controlled hologram which can encode positions of hundreds of traps in three dimensions, and change their location in a stepwise manner to manipulate trapped objects in three dimensions. When combined with a 3D imaging system, such as a confocal microscope the 3D positioning of objects can be characterized and corrected in real time. In the remainder of the article we will describe our combined HOTs-Confocal microscope setup, and characterize the accuracy and precision of our positioning both laterally and axially. In Sec. 4 we will give examples of experiments that are unique to our new setup: fabrication and 3D imaging of a 3D colloidal array, fabrication of a heterogeneous colloidal structure, and single cell division kinetics investigation of Saccharomyces Cerevisiae yeast cells.

2. Experimental

A schematic of our experimental setup is shown in Fig. 1
Fig. 1 A schematic diagram of the experimental setup. Lenses L1 and L2 expand the beam diameter to overfill the SLM active area; lenses L3 and L4 reduce it to overfill the objective’s back aperture while conserving power. The inverted objective lens focuses the beam to create the optical trap array. The upright objective is used for confocal imaging.
. We use an Olympus upright/inverted IX microscope to create a stable, well aligned two objectives setup. Through the bottom part of the microscope, which consists of a half of an inverted microscope we inset a HOTs setup driven by an Ytterbium doped fiber laser (Keopsys, KPS-KILAS-TRAPP-1083-20-PM-CO) with an emission wavelength of 1083nm and maximal power output of 20W. As is the common practice [6

6. M. Polin, K. Ladavac, S. H. Lee, Y. Roichman, and D. G. Grier, “Optimized holographic optical traps,” Opt. Express 13(15), 5831–5845 (2005). [CrossRef] [PubMed]

], the trapping laser beam is imprinted with a phase pattern generated by a liquid crystal spatial light modulator (SLM, Hamamatsu X10468-07). Optical traps are formed in the sample after being focused by a high numerical aperture objective lens (Olympus PlanApo, x60, NA = 1.42, oil immersion). Brightfield imaging is done by a CCD camera (Point Grey, Grasshopper) situated at the left port of the microscope, using transmitted illumination.

The top part of our setup consists of a half upright microscope through which confocal imaging is done (Andor, Revolution XD). The spinning disc confocal microscope includes a Yokogawa (CSU-X1) spinning disc and an Andor (iXon 897) EM-CCD camera, with 512 × 512 pixel resolution and single photon sensitivity. Fluorescence excitation is available at λ = 405 nm, 488 nm, 561 nm, 640 nm. To acquire three-dimensional images, the objective lens (Olympus UPlanL N, x60, NA = 1.25, oil immersion) is mounted on a piezoelectric scanner (Physik Instrumente, Pifoc P-721.LLQ) and consecutive slices of the sample are recorded with 100 nm axial resolution.

Preparation of yeast culture

Colonies of Saccharomyces Cerevisiae expressing fluorescent proteins (spc42-YFP/htb2-mCherry, kind gift from Dr. Iftach Nachman) were cultured in YPD agarose media (Formedium YPD broth) at 30̊ C for at least 24 h. In order to reduce the effects of cell to cell variation, we ensure that a clonal population of cells is generated by growing sparse colonies each originating from a single mother cell. One colony was removed to a liquid YPD media and cultured for 24 h in a 120-140 RPM shaker-incubator at 30̊ C. This process allows optimal growth of free yeast cells suspended in solution. About 1 ml was taken out of the culture, pelleted by centrifugation and frozen with 30% glycerol at −80̊ C for long term storage (fresh colonies were grown in a similar manner for each experiment from the frozen stock).

Sample preparation

Two types of colloidal silica beads, 1.5 µm in diameter with fluorescence excitation/emission of λ = 485/510 nm and λ = 569/585 nm (Kisker Biotech, PSI-G1.5 and PSI-R1.5) were suspended in an aqueous solution and sandwiched between two coverslips. Samples, 24 mm × 50 mm × 25 µm in size, were sealed and placed in a home built sample holder providing mechanical stability. Yeast cell samples were prepared similarly, using Bovine serum albumin coated coverslips to prevent cells from sticking to the glass.

Hydrogel preparation and device fabrication

In order to fabricate 3D colloidal arrays we prepare colloidal dispersions in an aqueous solution of 180: 12: 1 (wt/wt) acrylamide (99%, Sigma), N,N-methylenebisacrylamide (99%, Alfa Aesar), and diethoxyacetophenone (98%, Alfa Aesar). Once particles are trapped in location, ultraviolet illumination is used to polymerize this solution into a transparent polyacrylamide hydrogel. When the gelation process is terminated, the trapping laser is turned off and the colloidal structures remain stable [2

2. Y. Roichman and D. G. Grier, “Holographic assembly of quasicrystalline photonic heterostructures,” Opt. Express 13(14), 5434–5439 (2005). [CrossRef] [PubMed]

].

3. Characterization

Accurate positioning of particles in three dimensions requires a calibration between programmed position and resulting position. This calibration is usually done prior to trapping using adaptive optics by imaging the optical traps as they reflect from a mirror [17

17. Y. Roichman, I. Cholis, and D. G. Grier, “Volumetric imaging of holographic optical traps.,” Opt. Express 14(22), 10907–10912 (2006). [CrossRef] [PubMed]

]. This protocol works well for two dimensional trapping at a given axial displacement. However, the distance between traps scales with laser wavelength and with axial displacement. Moreover, spherical aberrations arising from light passing through media with different refractive indexes change the actual height of the optical traps. Theoretically [18

18. J. W. Goodman, Introduction to Fourier Optics, 2nd ed. (McGraw-Hill, 1996).

], the actual lateral distance between traps should change linearly when shifted axially in a linear manner, since the magnification of an image depends linearly on its distance from the focal plane. We correct for all these effects to achieve accurate 3D positioning using adaptive optics. In practice, this is done by imaging the distance between two trapped particles as a function of their axial position, thus forming a calibration curve translating actual distance to the distance programmed into the generating hologram. This calibration curve is then used to calculate holograms in which lateral distances do not depend on the trap’s axial position. In this manner we achieve sub pixel accuracies of fewer than 100 nm (Fig. 3(b)
Fig. 3 3D confocal images of trapped arrays. a) 5x3 array of Silica particles of two types: green fluorescing (in the center line), and red fluorescing (forming the outer rows). b) Volume rendering of a 2x2x2 array of trapped, green fluorescent, colloidal Silica particles, the particles are positioned within 100 nm of their desired position. c) S. Cerevisiae yeast cells trapped in a 3x3 array, the nuclei imaged in red and the cytoplasm in green. Each trapping site is comprised of two optical traps to ensure horizontal orientation of cells.
).

4. Applications

In the following section we will describe two unique applications for our combined HOTs-Confocal setup: simultaneous 3D imaging and trapping of various materials to produce free standing colloidal arrays, and fluorescence characterization of biological processes in localized cells.

Trapping of non-spherical objects, such as yeast cells requires at least two optical traps per lattice site to control the orientation of the trapped object. In Fig. 3(c) we show a 3x3 array of S. Cerevisiae yeast cells which were trapped using nine pairs of optical traps and imaged in two fluorescence channels to observe the nuclei (red) and the cytoplasm (green). It is interesting to note that mother and daughter cells remain attached long after division has occurred. This is seen in Fig. 3(c), were two cells become trapped in a single array location.

5. Discussion

In this article we presented a new optical trapping setup; a combination of holographic optical tweezers and a confocal microscope, allowing simultaneous 3D imaging and manipulation. This novel combination allows us to harness adaptive optics to optimize positioning accuracy in three dimensions. We presented two example applications which can benefit from these novel capacities, namely, micro-fabrication of all optical photonic devices, and parallel molecular kinetics measurements in live cells, on the single cell level.

The method presented here to fabricate colloidal particle arrays can be utilized in the future to produce all-optical photonic devices, if two additional requirements are met. First connections to light sources and detectors are required, and second propagating beams in the lateral direction need to be confined in the axial direction as well. Fiber connections to external light sources and detectors can be placed during sample cell assembly, the colloidal array can then be assembled around the input and output fibers to ensure optimal coupling to the colloidal device. Axial confinement can be achieved by using Bragg mirror coverslips designed to reflect the wavelength of light propagating in the device (e.g. 1.55 µm), and to transmit the wavelength of the trapping laser (e.g. 1.085 µm).

The four florescence channels present in our setup, and the high sensitivity of our camera can be used to study correlations in the position of up to four different biological molecules within a localized cell during processes such as drug intake and division. Performing such experiments in parallel, on an array of trapped cells, will enable assessment of population diversity.

Acknowledgments

Y.R would like to thank William Irvine for discussions originating this line of work, and Iftach Nachman for the fluorescently labeled Saccharomyces Cerevisiae cells. This work was supported in part by the James Franck German-Israeli Binational Program in Laser-Matter interactions, in part by the Marie Curie Reintegration Grant (PIRG04-GA-2008- 239378), and in part by the Israel Science Foundation (Grant Number 1271/08).

References and links

1.

K. Visscher and G. J. Brakenhoff, “Single beam optical trapping integrated in a confocal microscope for biological applications,” Cytometry 12(6), 486–491 (1991). [CrossRef] [PubMed]

2.

Y. Roichman and D. G. Grier, “Holographic assembly of quasicrystalline photonic heterostructures,” Opt. Express 13(14), 5434–5439 (2005). [CrossRef] [PubMed]

3.

K. Visscher, G. J. Brakenhoff, and J. J. Krol, “Micromanipulation by “multiple” optical traps created by a single fast scanning trap integrated with the bilateral confocal scanning laser microscope,” Cytometry 14(2), 105–114 (1993). [CrossRef] [PubMed]

4.

D. L. J. Vossen, A. van der Horst, M. Dogterom, and A. van Blaaderen, “Optical tweezers and confocal microscopy for simultaneous three-dimensional manipulation and imaging in concentrated colloidal dispersions,” Rev. Sci. Instrum. 75(9), 2960 (2004). [CrossRef]

5.

D. G. Grier, “A revolution in optical manipulation,” Nature 424(6950), 810–816 (2003). [CrossRef] [PubMed]

6.

M. Polin, K. Ladavac, S. H. Lee, Y. Roichman, and D. G. Grier, “Optimized holographic optical traps,” Opt. Express 13(15), 5831–5845 (2005). [CrossRef] [PubMed]

7.

F. Peng, B. Yao, S. Yan, W. Zhao, and M. Lei, “Trapping of low-refractive-index particles with azimuthally polarized beam,” J. Opt. Soc. Am. B 26(12), 2242 (2009). [CrossRef]

8.

M. P. MacDonald, L. Paterson, W. Sibbett, K. Dholakia, and P. E. Bryant, “Trapping and manipulation of low-index particles in a two-dimensional interferometric optical trap,” Opt. Lett. 26(12), 863–865 (2001). [CrossRef] [PubMed]

9.

A. Ashkin, J. M. Dziedzic, and T. Yamane, “Optical trapping and manipulation of single cells using infrared laser beams,” Nature 330(6150), 769–771 (1987). [CrossRef] [PubMed]

10.

D. McGloin, G. C. Spalding, H. Melville, W. Sibbett, and K. Dholakia, “Three-dimensional arrays of optical bottle beams,” Opt. Commun. 225(4-6), 215–222 (2003). [CrossRef]

11.

N. B. Simpson, L. Allen, and M. J. Padgett, “Optical tweezers and optical spanners with Laguerre-Gaussian modes,” J. Mod. Opt. 43(12), 2485–2491 (1996). [CrossRef]

12.

Y. Roichman, A. Waldron, E. Gardel, and D. G. Grier, “Optical traps with geometric aberrations,” Appl. Opt. 45(15), 3425–3429 (2006). [CrossRef] [PubMed]

13.

R. W. Bowman, A. J. Wright, and M. J. Padgett, “An SLM-based Shack–Hartmann wavefront sensor for aberration correction in optical tweezers,” J. Opt. 12(12), 124004 (2010). [CrossRef]

14.

J. C. Crocker and D. G. Grier, “Methods of digital video microscopy for colloidal studies,” J. Colloid Interface Sci. 179(1), 298–310 (1996). [CrossRef]

15.

Y. Gao and M. L. Kilfoil, “Accurate detection and complete tracking of large populations of features in three dimensions,” Opt. Express 17(6), 4685–4704 (2009). [CrossRef] [PubMed]

16.

G. M. Gibson, J. Leach, S. Keen, A. J. Wright, and M. J. Padgett, “Measuring the accuracy of particle position and force in optical tweezers using high-speed video microscopy,” Opt. Express 16(19), 14561–14570 (2008). [CrossRef] [PubMed]

17.

Y. Roichman, I. Cholis, and D. G. Grier, “Volumetric imaging of holographic optical traps.,” Opt. Express 14(22), 10907–10912 (2006). [CrossRef] [PubMed]

18.

J. W. Goodman, Introduction to Fourier Optics, 2nd ed. (McGraw-Hill, 1996).

19.

P. G. Lord and A. E. Wheals, “Rate of cell cycle initiation of yeast cells when cell size is not a rate-determining factor,” J. Cell Sci. 59, 183–201 (1983). [PubMed]

OCIS Codes
(090.1760) Holography : Computer holography
(120.4610) Instrumentation, measurement, and metrology : Optical fabrication
(140.7010) Lasers and laser optics : Laser trapping
(180.1790) Microscopy : Confocal microscopy

ToC Category:
Optical Traps, Manipulation, and Tracking

History
Original Manuscript: June 14, 2013
Revised Manuscript: July 30, 2013
Manuscript Accepted: July 30, 2013
Published: September 9, 2013

Virtual Issues
Optical Trapping and Applications (2013) Biomedical Optics Express

Citation
Maya Yevnin, Dror Kasimov, Yael Gluckman, Yuval Ebenstein, and Yael Roichman, "Independent and simultaneous three-dimensional optical trapping and imaging," Biomed. Opt. Express 4, 2087-2094 (2013)
http://www.opticsinfobase.org/boe/abstract.cfm?URI=boe-4-10-2087


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References

  1. K. Visscher and G. J. Brakenhoff, “Single beam optical trapping integrated in a confocal microscope for biological applications,” Cytometry12(6), 486–491 (1991). [CrossRef] [PubMed]
  2. Y. Roichman and D. G. Grier, “Holographic assembly of quasicrystalline photonic heterostructures,” Opt. Express13(14), 5434–5439 (2005). [CrossRef] [PubMed]
  3. K. Visscher, G. J. Brakenhoff, and J. J. Krol, “Micromanipulation by “multiple” optical traps created by a single fast scanning trap integrated with the bilateral confocal scanning laser microscope,” Cytometry14(2), 105–114 (1993). [CrossRef] [PubMed]
  4. D. L. J. Vossen, A. van der Horst, M. Dogterom, and A. van Blaaderen, “Optical tweezers and confocal microscopy for simultaneous three-dimensional manipulation and imaging in concentrated colloidal dispersions,” Rev. Sci. Instrum.75(9), 2960 (2004). [CrossRef]
  5. D. G. Grier, “A revolution in optical manipulation,” Nature424(6950), 810–816 (2003). [CrossRef] [PubMed]
  6. M. Polin, K. Ladavac, S. H. Lee, Y. Roichman, and D. G. Grier, “Optimized holographic optical traps,” Opt. Express13(15), 5831–5845 (2005). [CrossRef] [PubMed]
  7. F. Peng, B. Yao, S. Yan, W. Zhao, and M. Lei, “Trapping of low-refractive-index particles with azimuthally polarized beam,” J. Opt. Soc. Am. B26(12), 2242 (2009). [CrossRef]
  8. M. P. MacDonald, L. Paterson, W. Sibbett, K. Dholakia, and P. E. Bryant, “Trapping and manipulation of low-index particles in a two-dimensional interferometric optical trap,” Opt. Lett.26(12), 863–865 (2001). [CrossRef] [PubMed]
  9. A. Ashkin, J. M. Dziedzic, and T. Yamane, “Optical trapping and manipulation of single cells using infrared laser beams,” Nature330(6150), 769–771 (1987). [CrossRef] [PubMed]
  10. D. McGloin, G. C. Spalding, H. Melville, W. Sibbett, and K. Dholakia, “Three-dimensional arrays of optical bottle beams,” Opt. Commun.225(4-6), 215–222 (2003). [CrossRef]
  11. N. B. Simpson, L. Allen, and M. J. Padgett, “Optical tweezers and optical spanners with Laguerre-Gaussian modes,” J. Mod. Opt.43(12), 2485–2491 (1996). [CrossRef]
  12. Y. Roichman, A. Waldron, E. Gardel, and D. G. Grier, “Optical traps with geometric aberrations,” Appl. Opt.45(15), 3425–3429 (2006). [CrossRef] [PubMed]
  13. R. W. Bowman, A. J. Wright, and M. J. Padgett, “An SLM-based Shack–Hartmann wavefront sensor for aberration correction in optical tweezers,” J. Opt.12(12), 124004 (2010). [CrossRef]
  14. J. C. Crocker and D. G. Grier, “Methods of digital video microscopy for colloidal studies,” J. Colloid Interface Sci.179(1), 298–310 (1996). [CrossRef]
  15. Y. Gao and M. L. Kilfoil, “Accurate detection and complete tracking of large populations of features in three dimensions,” Opt. Express17(6), 4685–4704 (2009). [CrossRef] [PubMed]
  16. G. M. Gibson, J. Leach, S. Keen, A. J. Wright, and M. J. Padgett, “Measuring the accuracy of particle position and force in optical tweezers using high-speed video microscopy,” Opt. Express16(19), 14561–14570 (2008). [CrossRef] [PubMed]
  17. Y. Roichman, I. Cholis, and D. G. Grier, “Volumetric imaging of holographic optical traps.,” Opt. Express14(22), 10907–10912 (2006). [CrossRef] [PubMed]
  18. J. W. Goodman, Introduction to Fourier Optics, 2nd ed. (McGraw-Hill, 1996).
  19. P. G. Lord and A. E. Wheals, “Rate of cell cycle initiation of yeast cells when cell size is not a rate-determining factor,” J. Cell Sci.59, 183–201 (1983). [PubMed]

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