OSA's Digital Library

Virtual Journal for Biomedical Optics

Virtual Journal for Biomedical Optics

| EXPLORING THE INTERFACE OF LIGHT AND BIOMEDICINE

  • Editors: Andrew Dunn and Anthony Durkin
  • Vol. 8, Iss. 4 — May. 22, 2013
« Show journal navigation

Simultaneous angular multiplexing optical projection tomography at shifted focal planes

Lingling Chen, Natalie Andrews, Sunil Kumar, Paul Frankel, James McGinty, and Paul M. W. French  »View Author Affiliations


Optics Letters, Vol. 38, Issue 6, pp. 851-853 (2013)
http://dx.doi.org/10.1364/OL.38.000851


View Full Text Article

Acrobat PDF (650 KB)





Browse Journals / Lookup Meetings

Browse by Journal and Year


   


Lookup Conference Papers

Close Browse Journals / Lookup Meetings

Article Tools

Share
Citations

Abstract

We describe an angular multiplexing technique for optical projection tomography that improves resolution, signal-to-noise ratio, and imaging speed by ameliorating the trade-off between spatial resolution and depth of field and improving the light collection efficiency. Here we demonstrate that imaging at two orthogonal angular projections simultaneously, focused on shifted planes in the sample, improves the average spatial resolution by 20% and the light collection efficiency by a factor of 4, thereby enabling increased acquisition speed and reduced light dose.

© 2013 Optical Society of America

There is an increasing trend in biomedical research toward in situ studies, both ex vivo and in vivo, employing three-dimensional (3D) structural and functional imaging. For samples such as small animals, embryos, and engineered tissue in the “mesoscopic” (110mm) regime, a variety of imaging techniques have been developed, including optical projection tomography (OPT) [1

1. J. Sharpe, U. Ahlgren, P. Perry, B. Hill, A. Ross, J. Hecksher-Sorensen, R. Baldock, and D. Davidson, Science 296, 541 (2002). [CrossRef]

], scanning laser optical tomography (SLOT) [2

2. R. Lorbeer, M. Heidrich, C. Lorbeer, D. F. R. Ojeda, G. Bicker, H. Meyer, and A. Heisterkamp, Opt. Express 19, 5419 (2011). [CrossRef]

], selective plane illumination microscopy [3

3. J. Huisken, J. Swoger, F. Del Bene, J. Wittbrodt, and E. H. K. Stelzer, Science 305, 1007 (2004). [CrossRef]

], and ultramicroscopy [4

4. H. U. Dodt, U. Leischner, A. Schierloh, N. Jahrling, C. P. Mauch, K. Deininger, J. M. Deussing, M. Eder, W. Zieglgansberger, and K. Becker, Nat. Methods 4, 331 (2007). [CrossRef]

].

OPT, also known as optical computed tomography (CT) [5

5. N. Krstajic and S. J. Doran, Phys. Med. Biol. 51, 2055 (2006). [CrossRef]

], is the optical equivalent of x-ray CT, in which the 3D structure of a rotating sample is reconstructed from a series of wide-field 2D projections acquired at different angles. Typically a filtered backprojection (FBP) algorithm is used for image reconstruction [6

6. A. C. Kak and M. Slaney, Principles of Computerized Tomographic Imaging (SIAM, 1988).

], although iterative techniques have also been developed [7

7. M. A. Haidekker, Comput. Methods Programs Biomed. 80, 225 (2005). [CrossRef]

]. A key advantage of OPT compared to x-ray CT for imaging biological tissue is the use of optical radiation with its rich spectroscopic contrast, including absorption, fluorescence intensity, and fluorescence lifetime [8

8. J. McGinty, K. B. Tahir, R. Laine, C. B. Talbot, C. Dunsby, M. A. A. Neil, L. Quintana, J. Swoger, J. Sharpe, and P. M. W. French, J. Biophoton. 1, 390 (2008). [CrossRef]

]. Unfortunately, compared to x rays, the longer wavelength of optical radiation also leads to strong optical scattering and diffraction, which compromises the assumption of straight-ray projection in the FBP image reconstruction. To address the issue of scattering, it is necessary to apply OPT to samples that have been rendered transparent by chemical clearing [1

1. J. Sharpe, U. Ahlgren, P. Perry, B. Hill, A. Ross, J. Hecksher-Sorensen, R. Baldock, and D. Davidson, Science 296, 541 (2002). [CrossRef]

,9

9. T. Alanentalo, A. Asayesh, H. Morrison, C. E. Lorén, D. Holmberg, J. Sharpe, and U. Ahlgren, Nat. Methods 4, 31 (2007). [CrossRef]

] or that are inherently transparent [10

10. U. J. Birk, M. Rieckher, N. Konstantinides, A. Darrell, A. Sarasa-Renedo, H. Meyer, N. Tavernarakis, and J. Ripoll, Biomed. Opt. Express 1, 87 (2010). [CrossRef]

,11

11. A. Bassi, L. Fieramonti, C. D’Andrea, M. Mione, and G. Valentini, J. Biomed. Opt. 16, 100502 (2011). [CrossRef]

]. To accommodate the constraints imposed by diffraction-limited imaging, OPT is typically configured such that the volume to be imaged is confined within the depth of field (DOF) of the imaging system, thereby approximating parallel ray projection. This constraint results in a trade-off between sample size and achievable resolution as the DOF scales inversely with the square of the numerical aperture (NA) of the imaging lens while the optical resolution improves proportionally with the NA. The potential application of OPT to intact mesoscopic samples for biomedical research, especially for in vivo studies, has stimulated interest in optimizing the image quality, resolution, and acquisition time of OPT [12

12. J. R. Walls, J. G. Sled, J. Sharpe, and R. M. Henkelman, Phys. Med. Biol. 50, 4645 (2005). [CrossRef]

14

14. L. Chen, J. McGinty, H. B. Taylor, L. Bugeon, J. R. Lamb, M. J. Dallman, and P. M. W. French, Opt. Express 20, 7323 (2012). [CrossRef]

].

In many “standard” OPT systems this trade-off is mitigated by arranging for the DOF to extend through half of the sample by locating the focal plane (FP) a quarter of the way into the sample, as depicted in Fig. 1(a), rather than at the axis of rotation. Thus the front half of the sample is imaged under the parallel ray approximation, which interrogates the entire sample during the course of a full rotation. To realize higher-resolution imaging over larger volumes, the DOF for a given NA can be effectively extended by axially scanning the FP through the sample [15

15. Q. Miao, J. Hayenga, M. G. Meyer, T. Neumann, A. C. Nelson, and E. J. Seibel, Opt. Lett. 35, 3363 (2010). [CrossRef]

]. While this can provide a significant resolution improvement for OPT of smaller samples, it is less attractive in the mesoscopic regime due to the larger focal scanning (millimeter) distances required and the concomitant issues of scanning speed and stability.

Fig. 1. Schematic of (a) “standard” and (b) dual-axis OPT system setup (inset shows optimal DOFs and the FPs of two imaging systems). DOF, depth of field; EF, emission filter; AP, aperture; L1, 25 mm focal length lens; L2, 50 mm focal length lens; FP, focal plane; ϕ, sample diameter.

Here we demonstrate angularly multiplexed OPT using the experimental configuration depicted in Fig. 1(b). In brief, the sample was mounted under a rotation stage (T-NM17A200, Zaber Technologies, Inc.) and suspended in a refractive-index matched environment. A commercial 473 nm laser (Cobolt Blues™) was used to provide wide-field excitation. The sample was imaged onto two CCD cameras (Clara, Andor Technology plc, 1040×1392, 6.45 μm pixel size, cooled to 20°C) using two orthogonal imaging systems (L1, 25 mm achromatic doublet lens; L2, 50 mm achromatic doublet lens and an adjustable aperture) via appropriate EFs. The effective NA was adjusted using the apertures (AP) in the back FPs of lenses L1. The maximum field of view (FOV) of the system was 3.35mm×4.49mm.

Figures 2 and 3 show data from a cylindrical phantom of 3 mm diameter comprising a low concentration suspension of fluorescent beads in 2% agarose, with an average bead diameter of 4.2 μm and excitation/emission maxima at 505 and 515 nm, respectively (F8859, Life Technologies Ltd.). This was suspended in a water-filled cuvette and imaged through a 520±35nm EF. Image data were acquired with an NA of 0.024 for the “standard” OPT system with a DOF of 1.4 mm and with an NA of 0.033 for each arm of the dual-axis system, which gave a DOF of 0.78 mm. Images were acquired by the CCDs at 0.9° angular intervals over a full rotation of the sample with 0.5 s integration time for both systems and reconstructed using an FBP algorithm. Figure 2 shows the resulting images of a bead located 0.43 mm from the rotation axis. For the “standard” single-axis OPT system, the FP was located 0.69 mm from the axis of rotation, while for the dual-axis OPT system the FPs were located at 0.37 and 1.05 mm, respectively, from the rotation axis. Since the beads are smaller than the optical resolution, these reconstructed images indicate the resolution of the OPT systems. The resolution improvement of the dual-axis OPT system is confirmed by the normalized line sections through the bead center shown in Fig. 3, with accompanying Gaussian fits providing full width half-maxima (FWHM) of 13.4 and 10.8 μm for standard and dual-axis OPT systems, respectively. Figures 2 and 3 also highlight the significant improvement in signal-to-background ratio that results from both the 3.8× increase in light collection efficiency and the improved resolution.

Fig. 2. Reconstructed XY and XZ image slices of a bead acquired with standard OPT (a), (b) and dual-axis OPT (c), (d).
Fig. 3. Line plots and Gaussian fits through the Y axis of the reconstructed bead in Fig. 2 for the dual-axis (D, red line) and standard (S, black lines) OPT systems with the latter showing relative (solid) and normalized (dashed) line intensity data.

To further analyze the resolution improvement, a number of beads at different distances from the axis of rotation (0.17, 0.34, 0.43, 0.65, 0.98, and 1.39 mm) were analyzed and the measured average FWHM of these beads in the standard and the dual-axis OPT systems were 13.8±0.8μm and 11.1±1.2μm, respectively, showing a 20% improvement. The theoretical resolution (FWHM of Airy pattern) at best focus (i.e., limited by the NA of the objective lenses) was 11.1 and 8.1 μm for NAs of 0.024 and 0.033, respectively. The observed values are larger than the theoretical calculations because these OPT systems operate just within the sampling limit of the PSF and the reconstruction process involves interpolation. Nevertheless, we have demonstrated a significant improvement in image resolution and signal-to-background ratio that can be further improved using imaging detectors with more resolution elements and appropriate magnification.

To further illustrate the impact of the dual-axis OPTsystem, we performed imaging on the tail section of a fluorescent Casper:Fli-EGFP transgenic zebrafish imaged at 54 days postfertilization (dpf). Images were acquired at 200 angular projections with a CCD integration time of 0.1 s for both systems to maintain the total acquisition time at 2.5min (made up of 20 s of photon detection and 131 s of stage movement, CCD setup, and readout). Figures 4(a) and 4(b) show autoscaled maximum intensity projections (MIPs) of the reconstruction from the dual-axis and the standard OPT systems, respectively, while Fig. 4(c) shows the same MIP for the standard OPT reconstruction displayed on the same intensity scale as Fig. 4(a). We note that, unlike zebrafish embryos, mature fish exhibit significant optical scattering. Nevertheless, we can obtain images near the surface of the fish that illustrate significant improvement in the signal level and contrast for the dual-axis system, as confirmed by the line sections shown in Fig. 4(d). We note this improved imaging efficiency could be used to reduce the acquisition time of the dual-axis OPT system to achieve the signal-to-noise ratio of the standard approach, but with a proportional reduction in illumination dose.

Fig. 4. Reconstructed (maximum intensity projection) images of the tail of a 54 dpf zebrafish acquired with (a) dual-axis and (b), (c) standard OPT plotted for comparison with normalized intensity (a), (b) and absolute intensity (a), (c) scales. (d) Intensity line profiles from (a), (b) as indicated, where the standard OPT intensity data have been multiplied by 2 for clarity.

In conclusion, we have shown how angular multiplexing can improve the image resolution and collection efficiency of OPT by demonstrating a dual-axis OPT system acquiring image data at orthogonal angular projections that permits imaging with increased NA for a given sample. This approach can be used to image absorption as well as fluorescence and can be extended to more than two imaging axes, thereby further reducing image acquisition time and the corresponding light dose while improving spatial resolution and enhancing the ability to image larger samples. It could also be adapted for SLOT to improve the spatial resolution. We note that the orthogonal imaging systems could be configured to collect light from the same sample volume to simply increase the imaging speed, e.g., for intravital imaging.

The authors gratefully acknowledge funding from the UK Engineering and Physical Sciences Research Council (EPSRC). Lingling Chen acknowledges a Lee Family Scholarship, and Natalie Andrews acknowledges a studentship supported by EPSRC and AstraZeneca.

References

1.

J. Sharpe, U. Ahlgren, P. Perry, B. Hill, A. Ross, J. Hecksher-Sorensen, R. Baldock, and D. Davidson, Science 296, 541 (2002). [CrossRef]

2.

R. Lorbeer, M. Heidrich, C. Lorbeer, D. F. R. Ojeda, G. Bicker, H. Meyer, and A. Heisterkamp, Opt. Express 19, 5419 (2011). [CrossRef]

3.

J. Huisken, J. Swoger, F. Del Bene, J. Wittbrodt, and E. H. K. Stelzer, Science 305, 1007 (2004). [CrossRef]

4.

H. U. Dodt, U. Leischner, A. Schierloh, N. Jahrling, C. P. Mauch, K. Deininger, J. M. Deussing, M. Eder, W. Zieglgansberger, and K. Becker, Nat. Methods 4, 331 (2007). [CrossRef]

5.

N. Krstajic and S. J. Doran, Phys. Med. Biol. 51, 2055 (2006). [CrossRef]

6.

A. C. Kak and M. Slaney, Principles of Computerized Tomographic Imaging (SIAM, 1988).

7.

M. A. Haidekker, Comput. Methods Programs Biomed. 80, 225 (2005). [CrossRef]

8.

J. McGinty, K. B. Tahir, R. Laine, C. B. Talbot, C. Dunsby, M. A. A. Neil, L. Quintana, J. Swoger, J. Sharpe, and P. M. W. French, J. Biophoton. 1, 390 (2008). [CrossRef]

9.

T. Alanentalo, A. Asayesh, H. Morrison, C. E. Lorén, D. Holmberg, J. Sharpe, and U. Ahlgren, Nat. Methods 4, 31 (2007). [CrossRef]

10.

U. J. Birk, M. Rieckher, N. Konstantinides, A. Darrell, A. Sarasa-Renedo, H. Meyer, N. Tavernarakis, and J. Ripoll, Biomed. Opt. Express 1, 87 (2010). [CrossRef]

11.

A. Bassi, L. Fieramonti, C. D’Andrea, M. Mione, and G. Valentini, J. Biomed. Opt. 16, 100502 (2011). [CrossRef]

12.

J. R. Walls, J. G. Sled, J. Sharpe, and R. M. Henkelman, Phys. Med. Biol. 50, 4645 (2005). [CrossRef]

13.

J. R. Walls, J. G. Sled, J. Sharpe, and R. M. Henkelman, Phys. Med. Biol. 52, 2775 (2007). [CrossRef]

14.

L. Chen, J. McGinty, H. B. Taylor, L. Bugeon, J. R. Lamb, M. J. Dallman, and P. M. W. French, Opt. Express 20, 7323 (2012). [CrossRef]

15.

Q. Miao, J. Hayenga, M. G. Meyer, T. Neumann, A. C. Nelson, and E. J. Seibel, Opt. Lett. 35, 3363 (2010). [CrossRef]

OCIS Codes
(170.6900) Medical optics and biotechnology : Three-dimensional microscopy
(170.6960) Medical optics and biotechnology : Tomography

ToC Category:
Medical Optics and Biotechnology

History
Original Manuscript: January 2, 2013
Manuscript Accepted: January 29, 2013
Published: March 11, 2013

Virtual Issues
Vol. 8, Iss. 4 Virtual Journal for Biomedical Optics

Citation
Lingling Chen, Natalie Andrews, Sunil Kumar, Paul Frankel, James McGinty, and Paul M. W. French, "Simultaneous angular multiplexing optical projection tomography at shifted focal planes," Opt. Lett. 38, 851-853 (2013)
http://www.opticsinfobase.org/vjbo/abstract.cfm?URI=ol-38-6-851


Sort:  Author  |  Year  |  Journal  |  Reset  

References

  1. J. Sharpe, U. Ahlgren, P. Perry, B. Hill, A. Ross, J. Hecksher-Sorensen, R. Baldock, and D. Davidson, Science 296, 541 (2002). [CrossRef]
  2. R. Lorbeer, M. Heidrich, C. Lorbeer, D. F. R. Ojeda, G. Bicker, H. Meyer, and A. Heisterkamp, Opt. Express 19, 5419 (2011). [CrossRef]
  3. J. Huisken, J. Swoger, F. Del Bene, J. Wittbrodt, and E. H. K. Stelzer, Science 305, 1007 (2004). [CrossRef]
  4. H. U. Dodt, U. Leischner, A. Schierloh, N. Jahrling, C. P. Mauch, K. Deininger, J. M. Deussing, M. Eder, W. Zieglgansberger, and K. Becker, Nat. Methods 4, 331 (2007). [CrossRef]
  5. N. Krstajic and S. J. Doran, Phys. Med. Biol. 51, 2055 (2006). [CrossRef]
  6. A. C. Kak and M. Slaney, Principles of Computerized Tomographic Imaging (SIAM, 1988).
  7. M. A. Haidekker, Comput. Methods Programs Biomed. 80, 225 (2005). [CrossRef]
  8. J. McGinty, K. B. Tahir, R. Laine, C. B. Talbot, C. Dunsby, M. A. A. Neil, L. Quintana, J. Swoger, J. Sharpe, and P. M. W. French, J. Biophoton. 1, 390 (2008). [CrossRef]
  9. T. Alanentalo, A. Asayesh, H. Morrison, C. E. Lorén, D. Holmberg, J. Sharpe, and U. Ahlgren, Nat. Methods 4, 31 (2007). [CrossRef]
  10. U. J. Birk, M. Rieckher, N. Konstantinides, A. Darrell, A. Sarasa-Renedo, H. Meyer, N. Tavernarakis, and J. Ripoll, Biomed. Opt. Express 1, 87 (2010). [CrossRef]
  11. A. Bassi, L. Fieramonti, C. D’Andrea, M. Mione, and G. Valentini, J. Biomed. Opt. 16, 100502 (2011). [CrossRef]
  12. J. R. Walls, J. G. Sled, J. Sharpe, and R. M. Henkelman, Phys. Med. Biol. 50, 4645 (2005). [CrossRef]
  13. J. R. Walls, J. G. Sled, J. Sharpe, and R. M. Henkelman, Phys. Med. Biol. 52, 2775 (2007). [CrossRef]
  14. L. Chen, J. McGinty, H. B. Taylor, L. Bugeon, J. R. Lamb, M. J. Dallman, and P. M. W. French, Opt. Express 20, 7323 (2012). [CrossRef]
  15. Q. Miao, J. Hayenga, M. G. Meyer, T. Neumann, A. C. Nelson, and E. J. Seibel, Opt. Lett. 35, 3363 (2010). [CrossRef]

Cited By

Alert me when this paper is cited

OSA is able to provide readers links to articles that cite this paper by participating in CrossRef's Cited-By Linking service. CrossRef includes content from more than 3000 publishers and societies. In addition to listing OSA journal articles that cite this paper, citing articles from other participating publishers will also be listed.

Figures

Fig. 1. Fig. 2. Fig. 3.
 
Fig. 4.
 

« Previous Article  |  Next Article »

OSA is a member of CrossRef.

CrossCheck Deposited