Abstract

We introduce the use of a birefringent crystal with lensless digital holography to create an on-chip differential interference contrast (DIC) microscope. Using an incoherent source with a large aperture, in-line holograms of micro-objects are created, which interact with a uniaxial crystal and an absorbing polarizer, encoding differential interference contrast information of the objects on the chip. Despite the fact that a unit fringe magnification and an incoherent source with a large aperture have been used, holographic digital processing of such holograms rapidly recovers the differential phase contrast image of the specimen over a large field-of-view of ~24 mm2.

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2008 (1)

X. Cui, M. Lew, and C. Yang, “Quantitative differential interference contrast microscopy based on structured-aperture interference,” Appl. Phys. Lett. 93(9), 091113 (2008).
[CrossRef]

2007 (2)

2006 (5)

2005 (1)

2004 (1)

2002 (2)

G. Pedrini and H. Tiziani, “Short-coherence digital microscopy by use of a lensless holographic imaging system,” Appl. Opt. 41(22), 4489–4496 (2002).
[CrossRef] [PubMed]

E. D. Barone-Nugent, A. Barty, and K. A. Nugent, “Quantitative phase-amplitude microscopy I: optical microscopy,” J. Microsc. 206(3), 194–203 (2002).
[CrossRef] [PubMed]

2001 (1)

W. Xu, M. H. Jericho, I. A. Meinertzhagen, and H. J. Kreuzer, “Digital in-line holography for biological applications,” Proc. Natl. Acad. Sci. U.S.A. 98(20), 11301–11305 (2001).
[CrossRef] [PubMed]

2000 (1)

K. Buse and M. Luennemann, “3D imaging: wave front sensing utilizing a birefringent crystal,” Phys. Rev. Lett. 85(16), 3385–3387 (2000).
[CrossRef] [PubMed]

1992 (1)

1985 (1)

1978 (1)

1967 (1)

1955 (1)

G. Nomarski, “Differential microinterferometer with polarized light,” J. Phys. Radium 16, 9s–13s (1955).

1942 (1)

F. Zernike, “Phase-contrast, a new method for microscopic observation of transparent objects. Part II,” Physica 9(10), 974–986 (1942).
[CrossRef]

Alferi, D.

Avendaño-Alejo, M.

Badizadegan, K.

Barone-Nugent, E. D.

E. D. Barone-Nugent, A. Barty, and K. A. Nugent, “Quantitative phase-amplitude microscopy I: optical microscopy,” J. Microsc. 206(3), 194–203 (2002).
[CrossRef] [PubMed]

Barty, A.

E. D. Barone-Nugent, A. Barty, and K. A. Nugent, “Quantitative phase-amplitude microscopy I: optical microscopy,” J. Microsc. 206(3), 194–203 (2002).
[CrossRef] [PubMed]

Bernet, S.

Boyer, K.

Buse, K.

K. Buse and M. Luennemann, “3D imaging: wave front sensing utilizing a birefringent crystal,” Phys. Rev. Lett. 85(16), 3385–3387 (2000).
[CrossRef] [PubMed]

Choi, W.

Cui, X.

X. Cui, M. Lew, and C. Yang, “Quantitative differential interference contrast microscopy based on structured-aperture interference,” Appl. Phys. Lett. 93(9), 091113 (2008).
[CrossRef]

Cullen, D.

Dasari, R. R.

De Nicola, S.

De Petrocellis, L.

Feld, M. S.

Ferraro, P.

Fienup, J. R.

Finizio, A.

Fürhapter, S.

Garcia-Sucerquia, J.

Haddad, W.

Ikeda, T.

Jericho, M. H.

J. Garcia-Sucerquia, W. Xu, M. H. Jericho, and H. J. Kreuzer, “Immersion digital in-line holographic microscopy,” Opt. Lett. 31(9), 1211–1213 (2006).
[CrossRef] [PubMed]

W. Xu, M. H. Jericho, I. A. Meinertzhagen, and H. J. Kreuzer, “Digital in-line holography for biological applications,” Proc. Natl. Acad. Sci. U.S.A. 98(20), 11301–11305 (2001).
[CrossRef] [PubMed]

Jesacher, A.

Kim, M.

Kreuzer, H. J.

J. Garcia-Sucerquia, W. Xu, M. H. Jericho, and H. J. Kreuzer, “Immersion digital in-line holographic microscopy,” Opt. Lett. 31(9), 1211–1213 (2006).
[CrossRef] [PubMed]

W. Xu, M. H. Jericho, I. A. Meinertzhagen, and H. J. Kreuzer, “Digital in-line holography for biological applications,” Proc. Natl. Acad. Sci. U.S.A. 98(20), 11301–11305 (2001).
[CrossRef] [PubMed]

Lew, M.

X. Cui, M. Lew, and C. Yang, “Quantitative differential interference contrast microscopy based on structured-aperture interference,” Appl. Phys. Lett. 93(9), 091113 (2008).
[CrossRef]

Lo, C. M.

Longworth, J.

Lue, N.

Luennemann, M.

K. Buse and M. Luennemann, “3D imaging: wave front sensing utilizing a birefringent crystal,” Phys. Rev. Lett. 85(16), 3385–3387 (2000).
[CrossRef] [PubMed]

Mann, C.

Maurer, C.

McPherson, A.

Meinertzhagen, I. A.

W. Xu, M. H. Jericho, I. A. Meinertzhagen, and H. J. Kreuzer, “Digital in-line holography for biological applications,” Proc. Natl. Acad. Sci. U.S.A. 98(20), 11301–11305 (2001).
[CrossRef] [PubMed]

Nomarski, G.

G. Nomarski, “Differential microinterferometer with polarized light,” J. Phys. Radium 16, 9s–13s (1955).

Nugent, K. A.

E. D. Barone-Nugent, A. Barty, and K. A. Nugent, “Quantitative phase-amplitude microscopy I: optical microscopy,” J. Microsc. 206(3), 194–203 (2002).
[CrossRef] [PubMed]

Park, Y. K.

Pedrini, G.

Piano, E.

Pierattini, G.

Pontiggia, C.

Popescu, G.

Psaltis, D.

Repetto, L.

Rhodes, C.

Ritsch-Marte, M.

Rosete-Aguilar, M.

Sheridan, J. T.

Sherman, G. C.

Sirat, G.

Situ, G.

Solem, H.

Tiziani, H.

Xu, W.

J. Garcia-Sucerquia, W. Xu, M. H. Jericho, and H. J. Kreuzer, “Immersion digital in-line holographic microscopy,” Opt. Lett. 31(9), 1211–1213 (2006).
[CrossRef] [PubMed]

W. Xu, M. H. Jericho, I. A. Meinertzhagen, and H. J. Kreuzer, “Digital in-line holography for biological applications,” Proc. Natl. Acad. Sci. U.S.A. 98(20), 11301–11305 (2001).
[CrossRef] [PubMed]

Yang, C.

X. Cui, M. Lew, and C. Yang, “Quantitative differential interference contrast microscopy based on structured-aperture interference,” Appl. Phys. Lett. 93(9), 091113 (2008).
[CrossRef]

Yu, L.

Zernike, F.

F. Zernike, “Phase-contrast, a new method for microscopic observation of transparent objects. Part II,” Physica 9(10), 974–986 (1942).
[CrossRef]

Appl. Opt. (3)

Appl. Phys. Lett. (1)

X. Cui, M. Lew, and C. Yang, “Quantitative differential interference contrast microscopy based on structured-aperture interference,” Appl. Phys. Lett. 93(9), 091113 (2008).
[CrossRef]

J. Microsc. (1)

E. D. Barone-Nugent, A. Barty, and K. A. Nugent, “Quantitative phase-amplitude microscopy I: optical microscopy,” J. Microsc. 206(3), 194–203 (2002).
[CrossRef] [PubMed]

J. Opt. Soc. Am. (1)

J. Opt. Soc. Am. A (1)

J. Phys. Radium (1)

G. Nomarski, “Differential microinterferometer with polarized light,” J. Phys. Radium 16, 9s–13s (1955).

Opt. Express (3)

Opt. Lett. (6)

Phys. Rev. Lett. (1)

K. Buse and M. Luennemann, “3D imaging: wave front sensing utilizing a birefringent crystal,” Phys. Rev. Lett. 85(16), 3385–3387 (2000).
[CrossRef] [PubMed]

Physica (1)

F. Zernike, “Phase-contrast, a new method for microscopic observation of transparent objects. Part II,” Physica 9(10), 974–986 (1942).
[CrossRef]

Proc. Natl. Acad. Sci. U.S.A. (1)

W. Xu, M. H. Jericho, I. A. Meinertzhagen, and H. J. Kreuzer, “Digital in-line holography for biological applications,” Proc. Natl. Acad. Sci. U.S.A. 98(20), 11301–11305 (2001).
[CrossRef] [PubMed]

Other (2)

M. Pluta, Specialized Methods, Vol. 2 of Advanced light microscopy (Elsevier, New York, 1989), Chap. 7.

G. Popescu, “Quantitative phase imaging of nanoscale cell structure and dynamics,” Methods in Cell Biology, Edited by B. Jena (Elsevier, 2008)

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Figures (7)

Fig. 1
Fig. 1

DIC microscopy by lensless holographic imaging: (a) Holographic on-chip DIC microscopy setup using incoherent illumination with a large aperture (diameter D ~50-100 µm) and a uniaxial birefringent crystal together with z1 >> z2 (typically z1~5-10 cm and z2~1mm); (b) Differential phase interference due to double refraction phenomenon by a thin birefringent crystal plate (e.g., thickness ~0.18 mm). Angular sensitivity of the shear distance (δ) and the DC background intensity IDC are illustrated in (c) and (d), respectively. Note that the orientation of the first polarizer is adjusted to control the differential phase contrast while the second polarizer (i.e., the analyzer) is fixed at −45° with respect to the crystal orientation. Cross-polarizer and parallel polarizer configurations can be made equivalent to each other in terms of achieving optimum DIC performance, depending on the phase bias term to be even or odd multiples of π, respectively (refer to Section 3).

Fig. 5
Fig. 5

Supplementary. Reconstruction results of several well-defined micro-objects are illustrated to better quantify the resolution of the presented lensfree incoherent holographic imaging platform. The cross-sectional profiles along the dashed lines are shown on the images. For test feature 1, the FWHM values of the linear gap between the squares are 1.43μm and 0.6μm for the amplitude reconstruction and 40X objective-lens (NA = 0.6) microscope images, respectively. The same gap cannot be resolved by the 10X objective-lens (NA = 0.2). This indicates that the ~0.6 μm wide gap has been imaged with a spatial resolution of <1.5 μm using our incoherent lensfree holography approach. For test feature 2, the FWHM is measured on the cross-section across the letter “L”, and the values for amplitude reconstruction and 40X microscope images are 2.6 μm and 2.0 μm, respectively. The FWHM of the spacing between squares in test feature 3 is 1.80 μm and 1.95μm for amplitude reconstruction and 40X images, respectively. For all test features, we utilized z1 = ~3 cm, z2 = ~0.6 mm, F = ~1, D = 50 µm and spatially incoherent source at λ0=470 nm with a FWHM spectral bandwidth of ~5nm. Exposure time in these experiments was ~25 ms. Scale bars are 5 µm for the reconstructed images as well as their microscope comparisons, and scale bars are 50 μm for the raw holograms on the left column.

Fig. 7
Fig. 7

Supplementary. Amplitude and phase reconstruction results of the test features at 470 nm, 550 nm and 630 nm central wavelengths are illustrated. For this experiment, F ~1 and a 50 μm aperture at z1 = ~3cm is utilized. Source bandwidth is kept constant at ~5 nm. Scale bars, 5 μm. Notice that in the recovered phase images, the gap between the squares is imaged to be larger than the recovered amplitude images. The reason for this discrepancy is the fact that for the etched objects on glass, the edges do not obey a phase object criterion as they significantly scatter light, which cast a strong signature in the reconstructed amplitude images, yielding a better estimation of the gap between the square features in the amplitude domain rather than the phase.

Fig. 2
Fig. 2

Reconstructed lensless DIC images of micro-objects. Top Row: 5 & 10 μm sized melamine (n = 1.68) beads in a medium (n = 1.524, Norland Optical Adhesive 65). The sample was illuminated at 550 nm (~18 nm FWHM bandwidth). A 50 μm aperture (at z1 = 10 cm) and 0.18 mm-thick quartz plate (δ ~1 μm) were used. Bottom Row: White blood cells in a blood smear sample are imaged. The sample was illuminated at 670 nm (~18 nm FWHM bandwidth) through a 50 μm aperture (z1 = 10 cm) and 0.3 mm-thick quartz plate (δ ~2 μm) was used for the DIC image. The shear directions are indicated in the figures with dashed lines. Conventional bright-field microscope images of the same FOV are presented for comparison. The scale bars are 20 μm.

Fig. 3
Fig. 3

Reconstruction results of lensless DIC microscopy for 5 and 10 µm particles over a wide FOV of ~24 mm2 are demonstrated. The main figure shows the raw holograms of the micro-beads and the sub-figures (a1-a6) show the reconstructed DIC amplitude images at different areas of the FOV. The sample was illuminated at 550 nm (~18 nm FWHM bandwidth) with a 50 µm aperture (z1 = 10 cm). A 0.18mm-thick quartz crystal plate and two absorbing polarizer films were used. The scale bars for the subfigures are 20 μm.

Fig. 4
Fig. 4

Caenorhabditis elegans imaging: (a) Microscope image (with a 10X objective lens – NA ~0.25); (b) Reconstructed lensfree DIC image; (c) Reconstructed regular lensfree holographic image; (d-f) same as (a-c), except for another C. elegans sample. Imaging conditions are the same as in Fig. 2. The scale bars are 50 μm.

Fig. 6
Fig. 6

Supplementary. Amplitude reconstruction images are shown for the same test features as in Suppl. Figure 5 using the same incoherent source wavelength (470 nm), pinhole size (50 μm), F ~1, and pinhole-to-cell distance (z1 = ~3cm), with 5 nm and 20 nm source bandwidths. Integration times for the detection are less than 30ms. Scale bars, 5 μm.

Equations (1)

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| ψ ( x , y , z 2 ) | = 1 2 | ψ ( x , y , z 2 ) | × | Δ φ |

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