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Optics Express

Optics Express

  • Editor: Michael Duncan
  • Vol. 14, Iss. 16 — Aug. 7, 2006
  • pp: 7005–7013
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Living specimen tomography by digital holographic microscopy: morphometry of testate amoeba

Florian Charrière, Nicolas Pavillon, Tristan Colomb, Christian Depeursinge, Thierry J. Heger, Edward A.D. Mitchell, Pierre Marquet, and Benjamin Rappaz  »View Author Affiliations


Optics Express, Vol. 14, Issue 16, pp. 7005-7013 (2006)
http://dx.doi.org/10.1364/OE.14.007005


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Abstract

This paper presents an optical diffraction tomography technique based on digital holographic microscopy. Quantitative 2-dimensional phase images are acquired for regularly-spaced angular positions of the specimen covering a total angle of π, allowing to built 3-dimensional quantitative refractive index distributions by an inverse Radon transform. A 20× magnification allows a resolution better than 3 μm in all three dimensions, with accuracy better than 0.01 for the refractive index measurements. This technique is for the first time to our knowledge applied to living specimen (testate amoeba, Protista). Morphometric measurements are extracted from the tomographic reconstructions, showing that the commonly used method for testate amoeba biovolume evaluation leads to systematic under evaluations by about 50%.

© 2006 Optical Society of America

1. Introduction

Optical microscopy techniques nowadays offer non-contact, high-resolution and real-time cell imaging facilities. Though, the knowledge of the optical properties of the intracellular organelles is deemed to bring valuable information about cells morphology, cellular internal processes or organelles spatial distribution. A good review of the available techniques and refractive indices in the literature can be found in Ref. 1. Besides the techniques quoted in Ref. 1, different approaches developed to measure the refractive index (RI) of cells should be mentioned. Bereiter-Hahn et al. have studied the refractive index variation at the surface of mammalian cells in culture with quantitative reflection contrast microscopy.2

2. J. Bereiter-Hahn, Cecil H. Fox, and BO Thorell, “Quantitative reflection contrast microscopy of living cells,” J. Cell Biol. 82, 767–779 (1979). [CrossRef] [PubMed]

More recently, Curl et al. have compared height measurement achieved with a confocal microscope and optical path length (OPL) measurements with a phase-sensitive technique to deduce the integrated RI through a muscle cell.3

3. C. L. Curl, C. J. Bellair, T. Harris, B. E. Allman, P. J. Harris, A. G. Stewart, A. Roberts, K. A. Nugent, and L. M. D. Delbridge, “Refractive index measurement in viable cells using quantitative phase-amplitude microscopy and confocal microscopy,” Cyt. A 65, 88 (2005). [CrossRef]

Rappaz et al. have followed dynamically the integrated RI through neuronal cells during a hypotonic stress, comparing absolute phase measurements obtained with digital holographic microscopy for two different perfusion solutions.4

4. B. Rappaz, P. Marquet, E. Cuche, Y. Emery, C. Depeursinge, and P. Magistretti, “Measurement of the integral refractive index and dynamic cell morphometry of living cells with digital holographic microscopy,” Opt. Express 13, 9361–9373 (2005). [CrossRef] [PubMed]

However, these last two approaches allow to measure the 2D planar distribution of the RI integrated along the optical axis, making the visualization of individual intracellular organelles difficult.

A well-suited technique to address this particular problematic of measuring the 3-dimentionnal (3D) refractive index distribution of a cell, is the so-called optical diffraction tomography (ODT), which theoretical bases have been developed by Wolf5

5. E. Wolf, “Three-dimensional structure determination of semi-transparent object from holographic data,” Opt. Commun. 1, 153–156 (1969). [CrossRef]

and Dändliker6

6. R. Dändliker and K. Weiss, “Reconstruction of three-dimensional refractive index from scattered waves,“ Opt. Commun. 1, 323–328 (1970). [CrossRef]

in the early seventies. ODT allows, by recording the complex wavefront diffracted by a transparent or semi-transparent object under varying illumination angles, to reconstruct its 3D scattering potential. Lauer used for example phase-shifting interferometry (PSI) to record the Fourier spectrum of the diffracted wavefront, varying the illumination direction by changing the direction of the beam itself.7

7. V. Lauer, “New approach to optical diffraction tomography yielding a vector equation of diffraction tomography and a novel tomographic microscope,” J. Microsc. 205, 165–176 (2002). [CrossRef] [PubMed]

Vishnyakov and Levin combined PSI with a Linnik interferometer also allowing multiple angles illumination.89

8. G. N. Vishnyakov and G. G. Levin, “Optical microtomography of phase objects, ” Opt. Spectrosc. 85, 73–77 (1998).

Barty et al. developed a phase-retrieval algorithm, based on 3 intensity measurements performed on different focus planes, combined with a rotation of the specimen relatively to a fixed illumination beam.8

8. G. N. Vishnyakov and G. G. Levin, “Optical microtomography of phase objects, ” Opt. Spectrosc. 85, 73–77 (1998).

There are really few successful applications of ODT techniques to living specimen imaging, mainly due to the difficulty of measuring accurately the complex diffracted wavefront: Lauer used his tomographic microscope to observe bacteria and yeasts,7

7. V. Lauer, “New approach to optical diffraction tomography yielding a vector equation of diffraction tomography and a novel tomographic microscope,” J. Microsc. 205, 165–176 (2002). [CrossRef] [PubMed]

Noda et al. investigated green mold (Aspergillus oryzae),9

9. G. N. Vishnyakov, G. G. Levin, A. V. Likhachev, and V. V. Pikalov, “Phase Tomography of 3D Biological Microobjects: Numerical Simulation and Experimental Results,” Opt. Spectrosc. 87, 413–419 (1999).

and Vishnyakov and Levin observed human red blood cells and lymphocites.89

8. G. N. Vishnyakov and G. G. Levin, “Optical microtomography of phase objects, ” Opt. Spectrosc. 85, 73–77 (1998).

Note that quantitative RI data are found only in Refs. 8–9.

In a recent paper,12

12. F. Charrière, F. Montfort, J. Kühn, T. Colomb, A. Marian, E. Cuche, P. Marquet, and Ch. Depeursinge, “Cell refractive index tomography by digital holographic microscopy,” Opt. Lett. 31, 178–180 (2006). [CrossRef] [PubMed]

we have shown for the first time to our knowledge the quantitative 3D distribution of RI of a semi-transparent object, in our case a pollen grain, provided by backprojecting OPL values collected with digital holographic microscopy (DHM) on a series of projections of the preparation taken at various incidence angles. The accuracy of the RI determination was better than 0.01, and the 3D spatial resolution better than 1 μm in all 3D. Furthermore, DHM reconstructs the complex diffracted wavefront from a single hologram for each orientation of the specimen, while at least 3 images are required for PSI, reducing this way the acquisition time and the stability requirements for the system.

In the present paper, the system described in Ref. 12 has been further developed, to allow the first investigation of living biological specimens, thanks to a specifically designed observation chamber. The reconstructed 3D RI distributions, revealing intracellular structures, have permitted to accurately measure the volume of the specimen. The biological specimen observed, presented in Fig. 1, is a testate (or shelled) amoeba (“protozoa”, more specifically a protist belonging to the Amoebozoa), Hyalosphenia papilio, with a shell approximately 130 μm long, 70 μm wide and 35 μm deep.

Fig. 1. Images of the testate amoebae Hyalosphenia papilio: (a) bright-field microscope image illustrating the amoeba itself and its content, P pseudostome (opening through which the amoeba pseudopods emerge), AS algal symbionts, PV phagocytic vacuoles; (b) SEM image illustrating the shell.

2. Experimental setup

In the present work, the specimen is observed in a specifically designed chamber, principally composed by two microscope coverslips orthogonal to the optical axis. An aluminum frame maintains the two coverslips 4 mm distant, while fixations assure the watetightness of the chamber. One lateral side of the chamber is free to allow the introduction of a micropipette (MP) used to manipulate the specimen, or to change the perfusion medium during observation.4

4. B. Rappaz, P. Marquet, E. Cuche, Y. Emery, C. Depeursinge, and P. Magistretti, “Measurement of the integral refractive index and dynamic cell morphometry of living cells with digital holographic microscopy,” Opt. Express 13, 9361–9373 (2005). [CrossRef] [PubMed]

The manipulation method is similar to the so-called patch-clamp technique, in which thin MPs are used to maintain contact with a specific part of cells, for example the plasma membrane. These MPs are produced by the elongation of glass tubes during a controlled heating process. Their extremities, with a diameter around 1 micrometer, may then be polished to ensure a good contact with the cell membrane. Here, the specimen may be manipulated (translated or rotated) by one of these MPs, by using a little vacuum to maintain contact between the glass and the testate. To ensure good manipulation and rotation stability, the MP used during the final measurement had extremities about 10 μm, instead of the standard ones. The MP is fixed on a motorized rotating stage, and its position is adjusted with a micrometric xy-stage to place accurately its extremity on the rotation axis, minimizing therefore displacements of the specimen during rotation. A first micrometric xyz-stage allows moving the MP relatively to the chamber, to grab a specific specimen or to drag a selected specimen in a clean area of the preparation for observation, while a second xyz-stage allows moving simultaneously the MP and the chamber for a proper positioning in the field of view. Note that the chamber can be inclined relatively to the optical axis to allow picking up the desired specimen, in case it is laying on the bottom of the chamber due to gravity.

Fig. 2. Holographic microscope for transmission imaging: NF neutral density filter; PBS polarizing beam splitter; BE beam expander with spatial filter; λ/2 half-wave plate; MO microscope objective; FL field lens; M mirror; BS beam splitter; O object wave; R reference wave; MP micropipette; CS coverslip; S specimen; IL immersion liquid. Inset: a detail showing the off-axis geometry at the incidence on the CCD.

Glycerol (n= 1.473) is used here as immersion liquid, to minimize the number of the so-called 2π-jumps in the phase signal by minimizing the refractive index difference between the specimen and its surrounding medium, suppressing ambiguity during the unwrapping procedure involved in the reconstruction.12

12. F. Charrière, F. Montfort, J. Kühn, T. Colomb, A. Marian, E. Cuche, P. Marquet, and Ch. Depeursinge, “Cell refractive index tomography by digital holographic microscopy,” Opt. Lett. 31, 178–180 (2006). [CrossRef] [PubMed]

Note that the chamber is waterproof enough to also work with less viscous immersion media such as water or physiological solution.

3. Holograms processing and tomographic reconstruction

Digital holographic microscopy (DHM) provides quantitative measurement of the OPL distribution that enable to describe semi-transparent samples, such as living cells with a diffraction-limited transverse resolution and a sub-wavelength axial accuracy.22

22. P. Marquet, B. Rappaz, P. J. Magistretti, E. Cuche, Y. Emery, T. Colomb, and C. Depeursinge, “Digital holographic microscopy: a noninvasive contrast imaging technique allowing quantitative visualization of living cells with subwavelength axial accuracy,” Opt. Lett. 30, 468–470 (2005). [CrossRef] [PubMed]

The hologram processing, to reconstruct the complex diffracted wavefront, is described in details in Refs. 19 and 20. Briefly, it consists in multiplying the hologram by a digital reference wave simulating an illumination wave, then a propagation calculation within the Fresnel approximation allows to reconstruct a focused image of the specimen in a plane of coordinates, where a digital phase correction is applied to compensate for the wave front curvature induced by the objective lens and other aberrations of the optical imaging system.

In DHM, the reconstructed phase distribution depends on both specimen thickness and specimen RI distribution.22

22. P. Marquet, B. Rappaz, P. J. Magistretti, E. Cuche, Y. Emery, T. Colomb, and C. Depeursinge, “Digital holographic microscopy: a noninvasive contrast imaging technique allowing quantitative visualization of living cells with subwavelength axial accuracy,” Opt. Lett. 30, 468–470 (2005). [CrossRef] [PubMed]

If in first approximation the case of a weekly diffracting object is assumed, the optical path length of the collimated illuminating photons across the specimen is parallel to the optical axis. This assumption was found to be in very good agreement with the DHM data in the case of cellular imaging.23

23. P. Marquet, “Développement d’une nouvelle technique de microscopie optique tridimensionnelle, la microscopie holographique digitale. Perspective pour l’étude le la plasticité neuronale,” MD-PhD Thesis Dissertation (Chapt. 5), UNI-Lausanne, 2003.

The reconstructed phase distribution is therefore directly proportional to this optical path length:

φxy=2πλΔnxyzdz,
(1)

where z shows the optical axis direction, λ is the wavelength of the light source and Δn(x, y, z) is the difference between the 3D specimen RI spatial distribution and the RI of the surrounding medium. Consequently, φ(x,y) is only proportional to the projection of Δn(x, y, z) along the z-axis.

4. Results and discussion

Tomographic reconstructions were performed on 5 different Hyalosphenia papilio during this study. Central cuts in the reconstructed 3D refractive index volumes for 4 different amoebae are presented on Fig. 3 and Fig. 4. On these images, the cellular body of the amoebae themselves insides their testate can be easily discriminated thanks to the large difference (>0.2) between the cell RI and the RI inside the testate.

One can note on these images that the refractive index inside the testate is lower compared to the surrounding medium. This is due to the fact that glycerol (n=1.473) was used as immersion liquid: the mix of some remaining water inside the testate with the surrounding glycerol explains this decrease of the refractive index value. Note that if the measurement was performed immediately after the immersion of the amoeba in the glycerol, the refractive index inside the test corresponded directly to water (n=1.33), while in the presented measurements, higher values are obtained, indicating clearly the presence of glycerol inside the test. The presence of glycerol around the cellular body may slightly change its volume, but no dramatic changes were noticed during cells preparation and observation.

Fig. 3. Cuts in the tomographic reconstructions of 2 different Hyalosphenia papilio. Discrete values of the measured refractive index n are coded in false colors, the color-coding scales being displayed on the right part of each corresponding cut.

Morphometry measurements can be achieved on the tomographic reconstruction, to determine for example the biovolume of the amoebae. These volumes were evaluated by considering only the voxels with a refractive index value above an appropriate threshold. Note that the reconstructed volumes were first convoluted with a gaussian filter to minimize errors due to the reconstruction noise, and that the detected volumes distinct from the main cellular body were removed from counting.

The obtained results are presented in Table 1; the error ranges correspond to the optical resolution of the system. These volume measurements are compared in Table 1 with the estimation methods based on size measurements performed with light microscopy,17–18

17. D. Gilbert, C. Amblard, G. Bourdier, and A.-J. Francez, “The microbial loop at the surface of a peatland: Structure, function, and impact of nutrient input,” Microbial Ecol. 35, 83–93 (1998). [CrossRef]

where such biovolumes estimations are calculated from the observed individuals using the formula for an ellipsoid:

V=43πwld18,
(2)

where w, l and d are respectively the width, the length and the depth of the amoeba, and 1/8 is a form factor.

The estimated errors on the volume estimation with Eq. (2) displayed in Table 1 correspond to an accuracy of 2 μm in the size measurements. DHM tomography clearly delivers the most precise estimations of the biovolumes, i.e. with the lowest standard error. This shows the advantage of taking into account the actual shape of the cellular body instead not an ellipsoid approximation, in which slightly errors in the dimension measurement lead to large variation in the volume estimation.

Table 1. Estimated biovolumes for Hyalosphenia papilio.

table-icon
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The tomography also reveals that those volumes are under-estimated by nearly 50% with the actual standard method. This comparison illustrates the potential of DHM tomography for the precise and accurate estimation of biovolumes, which allows to determining biomasses. More generally, our results also show that in addition to the well-recognized problem of estimating the number of microorganisms in the environment,27

27. E. A. Paul and F. E. Clark, Soil microbiology and biochemistry, (Second Edition, Academic Press, San Diego, CA. 1996).

more attention should be devoted to the estimation of the biovolume and biomass of individual species. A reliable estimate of biomass is crucial for understanding of the role of different microorganisms in the microbial food webs,17

17. D. Gilbert, C. Amblard, G. Bourdier, and A.-J. Francez, “The microbial loop at the surface of a peatland: Structure, function, and impact of nutrient input,” Microbial Ecol. 35, 83–93 (1998). [CrossRef]

,18

18. E. A. D. Mitchell, D. Gilbert, A. Buttler, P. Grosvernier, C. Amblard, and J.-M. Gobat, “Structure of microbial communities in Sphagnum peatlands and effect of atmospheric carbon dioxide enrichment,” Microbial Ecol. 16, 187–199 (2003).

,28

28. M. Bölter, J. Bloem, K. Meiners, and R. Möller, “Enumeration and biovolume determination of microbial cells - a methodological review and recommendations for applications in ecological research,“ Biol. Fert. Soils 36, 249–259 (2002). [CrossRef]

or the effect of environmental stress like organic pollutants or heavy metals on selected organisms in ecotoxicology.

At present, the whole reconstruction process involved in DHM tomography, including specimen manipulation, holograms acquisition/processing and tomographic reconstruction still requires too many operator interventions to calculate systematically the biovolumes of large amount of amoeba. However, as suggested above, this technique could help to derive allometric relationships that would greatly increase the precision of biovolumes and biomass estimates.

Fig. 4. Animations [1.3MB (a), 1.7MB (b)] through the tomographic reconstructions of two selected Hyalosphenia papilio presenting some clearly visible inner structures tentatively identified as algal symbionts (AS) and phagocytic vacuoles (PV). The gray-level scales of the measured refractive index n are displayed on the right part of each corresponding cut.

The actual resolution in the tomographic reconstruction is mainly limited because of mechanical instabilities. Theoretically, the optical resolution in all three dimensions for the tomographic reconstructions is about 1.5 μm, but an inaccurate centering of the MP on its rotation axis leads to a precession movement of the specimen during rotation that tends to derogate from the final tomography resolution. Therefore, during the reconstruction, a re-centering numerical procedure based on the MP edges detection in the phase images has been applied. Even if it is not currently possible to identify accurately all the symbiotic algae, with diameter around 3 μm,29

29. Dr. Enrique Lara, Swiss Federal Research Institute WSL and Laboratory of Ecological Systems, Ecole Polytechnique Fédérale de Lausanne (EPFL), CH-1015 Lausanne, Switzerland. (personnal communication, 2006).

and other organelles present in the amoeba cellular body, some of these organelles (principally the phagocytic vacuoles, but also some algal symbionts) are visible in the tomographic reconstructed volumes. Due to discrete color scale, it may be difficult to notice them in Fig. 3, but the continuous color scale make their spherical shape clearly visible, as shown in Fig. 4, especially in the linked movies presenting animations through the reconstructed volumes, in which they appear like spheres in the cellular body. This shows that potentially, the tomographic reconstructions could also be used to solve the problem of symbiotic algae counting and volume estimate inside the amoeba cellular body.

5. Conclusion

Acknowledgements

This work has been supported by the Swiss National Science Foundation (grant n° 205320-103885/1) and by EU project RECIPE (Reconciling Commercial Exploitation of Peat With Biodiversity in Peatlands Ecosystems). RECIPE is partly supported by the European Commission (n° EVK2-2002-00269) and partly, for the Swiss partners EPFL and WSL-AR, by the Swiss Federal Office for Education and Science (SER n° 01.0438-1).

The SEM image of Fig. 1(b) was kindly provided by Dr. Jerry Kudenov and Keiko Kishaba, University of Alaska, Anchorage.

The authors also would like to thank the people at Lyncée Tec SA (www.lynceetec.com), PSE-A, CH-1015 Lausanne, for their enthusiasm and their constructive comments during the paper preparation.

References and links

1.

A. Dunn, Light scattering properties of cells, PhD Diss., Univ. of Texas, Austin, 1997.

2.

J. Bereiter-Hahn, Cecil H. Fox, and BO Thorell, “Quantitative reflection contrast microscopy of living cells,” J. Cell Biol. 82, 767–779 (1979). [CrossRef] [PubMed]

3.

C. L. Curl, C. J. Bellair, T. Harris, B. E. Allman, P. J. Harris, A. G. Stewart, A. Roberts, K. A. Nugent, and L. M. D. Delbridge, “Refractive index measurement in viable cells using quantitative phase-amplitude microscopy and confocal microscopy,” Cyt. A 65, 88 (2005). [CrossRef]

4.

B. Rappaz, P. Marquet, E. Cuche, Y. Emery, C. Depeursinge, and P. Magistretti, “Measurement of the integral refractive index and dynamic cell morphometry of living cells with digital holographic microscopy,” Opt. Express 13, 9361–9373 (2005). [CrossRef] [PubMed]

5.

E. Wolf, “Three-dimensional structure determination of semi-transparent object from holographic data,” Opt. Commun. 1, 153–156 (1969). [CrossRef]

6.

R. Dändliker and K. Weiss, “Reconstruction of three-dimensional refractive index from scattered waves,“ Opt. Commun. 1, 323–328 (1970). [CrossRef]

7.

V. Lauer, “New approach to optical diffraction tomography yielding a vector equation of diffraction tomography and a novel tomographic microscope,” J. Microsc. 205, 165–176 (2002). [CrossRef] [PubMed]

8.

G. N. Vishnyakov and G. G. Levin, “Optical microtomography of phase objects, ” Opt. Spectrosc. 85, 73–77 (1998).

9.

G. N. Vishnyakov, G. G. Levin, A. V. Likhachev, and V. V. Pikalov, “Phase Tomography of 3D Biological Microobjects: Numerical Simulation and Experimental Results,” Opt. Spectrosc. 87, 413–419 (1999).

10.

A. Barty, K.A. Nugent, A. Roberts, and D. Paganin, “Quantitative phase tomography,” Opt. Commun. 175, 329–336 (2000). [CrossRef]

11.

T. Noda, S. Kawata, and S. Minami, “Three-dimensional phase-contrast imaging by a computed-tomography microscope”, Appl. Opt. 31, 670–674 (1992). [CrossRef] [PubMed]

12.

F. Charrière, F. Montfort, J. Kühn, T. Colomb, A. Marian, E. Cuche, P. Marquet, and Ch. Depeursinge, “Cell refractive index tomography by digital holographic microscopy,” Opt. Lett. 31, 178–180 (2006). [CrossRef] [PubMed]

13.

E. A. D. Mitchell, A. Buttler, B. Warner, and J.-M. Gobat, “Ecology of testate amoebae (Protozoa: Rhizopoda) in Sphagnum-dominated peatlands in the Jura Mountains, Switzerland and France,” Ecoscience 6, 565–576 (1999).

14.

E. A. D. Mitchell, D. J. Charman, and B. G. Warner, “Testate amoebae analysis in ecological and paleoecological studies of wetlands: past, present and future,” Biodivers. Conserv. (to be published).

15.

H. Nguyen-Viet, N. Bernard, E. A. D. Mitchell, J. Cortet, P. M. Badot, and D. Gilbert, “Relationship between testate amoebae and atmospheric heavy metals (Pb, Cd, Zn, Ni, Cu, Mn and Fe) accumulated in the moss Barbula indica Hanoi, Vietnam,” Microbial Ecol. (to be published).

16.

R. E. Madrid and C. J. Felice, “Microbial biomass estimation,“ Crit. Rev. Biotechnol. , 25, 97–112 (2005). [CrossRef] [PubMed]

17.

D. Gilbert, C. Amblard, G. Bourdier, and A.-J. Francez, “The microbial loop at the surface of a peatland: Structure, function, and impact of nutrient input,” Microbial Ecol. 35, 83–93 (1998). [CrossRef]

18.

E. A. D. Mitchell, D. Gilbert, A. Buttler, P. Grosvernier, C. Amblard, and J.-M. Gobat, “Structure of microbial communities in Sphagnum peatlands and effect of atmospheric carbon dioxide enrichment,” Microbial Ecol. 16, 187–199 (2003).

19.

E. Cuche, P. Marquet, and C. Depeursinge, “Simultaneous amplitude-contrast and quantitative phase-contrast microscopy by numerical reconstruction of Fresnel off-axis holograms,” Appl. Opt. 38, 6994–7001 (1999). [CrossRef]

20.

T. Colomb, E. Cuche, F. Charrière, J. Kühn, N. Aspert, F. Montfort, P. Marquet, and Ch. Depeursinge, “Automatic procedure for aberration compensation in digital holographic microscopy and applications to specimen shape compensation,“ Appl. Opt. 45, 851–863 (2006). [CrossRef] [PubMed]

21.

F. Charrière, E. Cuche, P. Marquet, and C. Depeursinge, “Biological cell (pollen grain) refractive index tomography with digital holographic microscopy.“ in Three-Dimensional and Multidimensional Microscopy: Image Acquisition and Processing XIII, J.-A. Conchello, C.J. Cogswell, T. Wilson, eds., Proc. SPIE 6090, 22–29 (2006).

22.

P. Marquet, B. Rappaz, P. J. Magistretti, E. Cuche, Y. Emery, T. Colomb, and C. Depeursinge, “Digital holographic microscopy: a noninvasive contrast imaging technique allowing quantitative visualization of living cells with subwavelength axial accuracy,” Opt. Lett. 30, 468–470 (2005). [CrossRef] [PubMed]

23.

P. Marquet, “Développement d’une nouvelle technique de microscopie optique tridimensionnelle, la microscopie holographique digitale. Perspective pour l’étude le la plasticité neuronale,” MD-PhD Thesis Dissertation (Chapt. 5), UNI-Lausanne, 2003.

24.

A. C. Kak and M. Slaney. Principles of Computerized Tomographic Imaging. Soc. of Ind. and Appl. Math. SIAM , 2001.

25.

T. C. Wedberg, J. J. Stamnes, and W. Singer, “Experimental examination of the quantitative imaging properties of optical diffraction tomography,” J. Opt. Soc. Am. A 12, 493–500 (1995). [CrossRef]

26.

W. Singer, T. C. Wedberg, and J. J. Stamnes, “Comparison of the filtered backpropagation and the filtered backprojection algorithms for quantitative tomography,”Appl. Opt. 34, 6575–6581 (1995). [CrossRef] [PubMed]

27.

E. A. Paul and F. E. Clark, Soil microbiology and biochemistry, (Second Edition, Academic Press, San Diego, CA. 1996).

28.

M. Bölter, J. Bloem, K. Meiners, and R. Möller, “Enumeration and biovolume determination of microbial cells - a methodological review and recommendations for applications in ecological research,“ Biol. Fert. Soils 36, 249–259 (2002). [CrossRef]

29.

Dr. Enrique Lara, Swiss Federal Research Institute WSL and Laboratory of Ecological Systems, Ecole Polytechnique Fédérale de Lausanne (EPFL), CH-1015 Lausanne, Switzerland. (personnal communication, 2006).

OCIS Codes
(090.1760) Holography : Computer holography
(110.6880) Imaging systems : Three-dimensional image acquisition
(110.6960) Imaging systems : Tomography
(170.6900) Medical optics and biotechnology : Three-dimensional microscopy

ToC Category:
Holography

History
Original Manuscript: June 9, 2006
Revised Manuscript: July 20, 2006
Manuscript Accepted: July 24, 2006
Published: August 7, 2006

Virtual Issues
Vol. 1, Iss. 9 Virtual Journal for Biomedical Optics

Citation
Florian Charrière, Nicolas Pavillon, Tristan Colomb, Christian Depeursinge, Thierry J. Heger, Edward A. D. Mitchell, Pierre Marquet, and Benjamin Rappaz, "Living specimen tomography by digital holographic microscopy: morphometry of testate amoeba," Opt. Express 14, 7005-7013 (2006)
http://www.opticsinfobase.org/oe/abstract.cfm?URI=oe-14-16-7005


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References

  1. A. Dunn, Light scattering properties of cells, PhD Diss., Univ. of Texas, Austin, 1997.
  2. J. Bereiter-Hahn, CecilH. Fox and BO Thorell, "Quantitative reflection contrast microscopy of living cells," J. Cell Biol. 82, 767-779 (1979). [CrossRef] [PubMed]
  3. C. L. Curl, C. J. Bellair, T. Harris, B. E. Allman, P. J. Harris, A. G. Stewart, A. Roberts, K. A. Nugent and L. M. D. Delbridge, "Refractive index measurement in viable cells using quantitative phase-amplitude microscopy and confocal microscopy," Cyt. A 65, 88 (2005). [CrossRef]
  4. B. Rappaz, P. Marquet, E. Cuche, Y. Emery, C. Depeursinge, and P. Magistretti, "Measurement of the integral refractive index and dynamic cell morphometry of living cells with digital holographic microscopy," Opt. Express 13, 9361-9373 (2005). [CrossRef] [PubMed]
  5. E.  Wolf, "Three-dimensional structure determination of semi-transparent object from holographic data," Opt. Commun. 1, 153-156 (1969). [CrossRef]
  6. R.  Dändliker, K.  Weiss, "Reconstruction of three-dimensional refractive index from scattered waves," Opt. Commun. 1, 323-328 (1970). [CrossRef]
  7. V.  Lauer, "New approach to optical diffraction tomography yielding a vector equation of diffraction tomography and a novel tomographic microscope," J. Microsc. 205, 165-176 (2002). [CrossRef] [PubMed]
  8. G. N. Vishnyakov, G. G. Levin, "Optical microtomography of phase objects, " Opt. Spectrosc. 85, 73-77 (1998).
  9. G. N. Vishnyakov, G. G. Levin, A. V. Likhachev, V. V. Pikalov, "Phase Tomography of 3D Biological Microobjects: Numerical Simulation and Experimental Results," Opt. Spectrosc. 87, 413-419 (1999).
  10. A. Barty, K.A. Nugent, A. Roberts, D. Paganin, "Quantitative phase tomography," Opt. Commun. 175, 329-336 (2000). [CrossRef]
  11. T. Noda, S. Kawata and S. Minami, "Three-dimensional phase-contrast imaging by a computed-tomography microscope", Appl. Opt. 31, 670-674 (1992). [CrossRef] [PubMed]
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