Abstract

In optical microscopy the contrast of transparent objects achieved with conventional methods is often not satisfactory, for example for the automated recognition of cells. In this paper we present a nano-optical label-free approach for contrast enhancement based on photonic crystal slabs (PCS) as the specimen holder. Quasi-guided modes inside these structures cause an intrinsic color of the PCS, which strongly depends on the wavelength and the quality factor of the optical mode. Objects on the surface of the PCS experience a significant color and intensity contrast enhancement, as they change properties of the optical modes.

© 2012 OSA

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    [CrossRef] [PubMed]

2011 (2)

S. M. Shamah and B. T. Cunningham, “Label-free cell-based assays using photonic crystal optical biosensors,” Analyst (Lond.) 136(6), 1090–1102 (2011).
[CrossRef] [PubMed]

D. H. Rapoport, T. Becker, A. Madany Mamlouk, S. Schicktanz, and C. Kruse, “A novel validation algorithm allows for automated cell tracking and the extraction of biologically meaningful parameters,” PLoS ONE 6(11), e27315 (2011).
[CrossRef] [PubMed]

2010 (2)

2009 (1)

2008 (1)

2007 (1)

J. L. Guo, “Nanoimprint lithography: methods and material requirements,” J. Adv. Mater. 19(4), 495–513 (2007).
[CrossRef]

2005 (2)

B. P. Möhrle, K. Köhler, J. Jaehrling, R. Brock, and G. Gauglitz, “Label-free characterization of cell adhesion using reflectometric interference spectroscopy (RIfS),” Anal. Bioanal. Chem. 384(2), 407–413 (2005).
[CrossRef] [PubMed]

I. L. Medintz, H. T. Uyeda, E. R. Goldman, and H. Mattoussi, “Quantum dot bioconjugates for imaging, labelling and sensing,” Nat. Mater. 4(6), 435–446 (2005).
[CrossRef] [PubMed]

2004 (1)

B. T. Cunningham, P. Li, S. Schulz, B. Lin, C. Baird, J. Gerstenmaier, C. Genick, F. Wang, E. Fine, and L. Laing, “Label-free assays on the BIND system,” J. Biomol. Screen. 9(6), 481–490 (2004).
[CrossRef] [PubMed]

2003 (1)

D. J. Stephens and V. J. Allan, “Light microscopy techniques for live cell imaging,” Science 300(5616), 82–86 (2003).
[CrossRef] [PubMed]

2002 (1)

S. Fan and J. Joannopoulos, “Analysis of guided resonances in photonic crystal slabs,” Phys. Rev. B 65(23), 235112 (2002).
[CrossRef]

2001 (3)

A. S. Curtis, B. Casey, J. O. Gallagher, D. Pasqui, M. A. Wood, and C. D. Wilkinson, “Substratum nanotopography and the adhesion of biological cells. Are symmetry or regularity of nanotopography important?” Biophys. Chem. 94(3), 275–283 (2001).
[CrossRef] [PubMed]

B. Polisky, R. Jenison, S. Yang, and A. Haeberli, “Interference-based detection of nucleic acid targets on optically coated silicon,” Nat. Biotechnol. 19(1), 62–65 (2001).
[CrossRef] [PubMed]

R. Jenison, M. Rihanek, and B. Polisky, “Use of a thin film biosensor for rapid visual detection of PCR products in a multiplex format,” Biosens. Bioelectron. 16(9-12), 757–763 (2001).
[CrossRef] [PubMed]

1999 (1)

K. Giebel, C. Bechinger, S. Herminghaus, M. Riedel, P. Leiderer, U. Weiland, and M. Bastmeyer, “Imaging of cell/substrate contacts of living cells with surface plasmon resonance microscopy,” Biophys. J. 76(1), 509–516 (1999).
[CrossRef] [PubMed]

1988 (1)

B. Rothenhäusler and W. Knoll, “Surface-plasmon microscopy,” Nature 332(6165), 615–617 (1988).
[CrossRef]

1969 (1)

R. D. Allen, G. B. David, and G. Z. Nomarski, “The zeiss-Nomarski differential interference equipment for transmitted-light microscopy,” Z. Wiss. Mikrosk. 69(4), 193–221 (1969).
[PubMed]

1964 (1)

A. S. G. Curtis, “The mechanism of adhesion of cells to glass: a study by interference reflection microscopy,” J. Cell Biol. 20, 199–215 (1964).
[CrossRef] [PubMed]

1942 (2)

F. Zernike, “Phase contrast, a new method for the microscopic observation of transparent objects,” Physica 9(7), 686–698 (1942).
[CrossRef]

D. L. MacAdam, “Visual sensitivities to color differences in daylight,” J. Opt. Soc. Am. 32(5), 247–273 (1942).
[CrossRef]

1929 (1)

W. D. Wright, “A re-determination of the trichromatic coefficients of the spectral colours,” Trans. Opt. Soc. 30(4), 141–164 (1929).
[CrossRef]

Allan, V. J.

D. J. Stephens and V. J. Allan, “Light microscopy techniques for live cell imaging,” Science 300(5616), 82–86 (2003).
[CrossRef] [PubMed]

Allen, R. D.

R. D. Allen, G. B. David, and G. Z. Nomarski, “The zeiss-Nomarski differential interference equipment for transmitted-light microscopy,” Z. Wiss. Mikrosk. 69(4), 193–221 (1969).
[PubMed]

Baird, C.

B. T. Cunningham, P. Li, S. Schulz, B. Lin, C. Baird, J. Gerstenmaier, C. Genick, F. Wang, E. Fine, and L. Laing, “Label-free assays on the BIND system,” J. Biomol. Screen. 9(6), 481–490 (2004).
[CrossRef] [PubMed]

Bastmeyer, M.

K. Giebel, C. Bechinger, S. Herminghaus, M. Riedel, P. Leiderer, U. Weiland, and M. Bastmeyer, “Imaging of cell/substrate contacts of living cells with surface plasmon resonance microscopy,” Biophys. J. 76(1), 509–516 (1999).
[CrossRef] [PubMed]

Bechinger, C.

K. Giebel, C. Bechinger, S. Herminghaus, M. Riedel, P. Leiderer, U. Weiland, and M. Bastmeyer, “Imaging of cell/substrate contacts of living cells with surface plasmon resonance microscopy,” Biophys. J. 76(1), 509–516 (1999).
[CrossRef] [PubMed]

Becker, T.

D. H. Rapoport, T. Becker, A. Madany Mamlouk, S. Schicktanz, and C. Kruse, “A novel validation algorithm allows for automated cell tracking and the extraction of biologically meaningful parameters,” PLoS ONE 6(11), e27315 (2011).
[CrossRef] [PubMed]

Block, I. D.

Bog, U.

Brock, R.

B. P. Möhrle, K. Köhler, J. Jaehrling, R. Brock, and G. Gauglitz, “Label-free characterization of cell adhesion using reflectometric interference spectroscopy (RIfS),” Anal. Bioanal. Chem. 384(2), 407–413 (2005).
[CrossRef] [PubMed]

Casey, B.

A. S. Curtis, B. Casey, J. O. Gallagher, D. Pasqui, M. A. Wood, and C. D. Wilkinson, “Substratum nanotopography and the adhesion of biological cells. Are symmetry or regularity of nanotopography important?” Biophys. Chem. 94(3), 275–283 (2001).
[CrossRef] [PubMed]

Cunningham, B. T.

S. M. Shamah and B. T. Cunningham, “Label-free cell-based assays using photonic crystal optical biosensors,” Analyst (Lond.) 136(6), 1090–1102 (2011).
[CrossRef] [PubMed]

I. D. Block, P. C. Mathias, S. I. Jones, L. O. Vodkin, and B. T. Cunningham, “Optimizing the spatial resolution of photonic crystal label-free imaging,” Appl. Opt. 48(34), 6567–6574 (2009).
[CrossRef] [PubMed]

B. T. Cunningham, P. Li, S. Schulz, B. Lin, C. Baird, J. Gerstenmaier, C. Genick, F. Wang, E. Fine, and L. Laing, “Label-free assays on the BIND system,” J. Biomol. Screen. 9(6), 481–490 (2004).
[CrossRef] [PubMed]

Curtis, A. S.

A. S. Curtis, B. Casey, J. O. Gallagher, D. Pasqui, M. A. Wood, and C. D. Wilkinson, “Substratum nanotopography and the adhesion of biological cells. Are symmetry or regularity of nanotopography important?” Biophys. Chem. 94(3), 275–283 (2001).
[CrossRef] [PubMed]

Curtis, A. S. G.

A. S. G. Curtis, “The mechanism of adhesion of cells to glass: a study by interference reflection microscopy,” J. Cell Biol. 20, 199–215 (1964).
[CrossRef] [PubMed]

David, G. B.

R. D. Allen, G. B. David, and G. Z. Nomarski, “The zeiss-Nomarski differential interference equipment for transmitted-light microscopy,” Z. Wiss. Mikrosk. 69(4), 193–221 (1969).
[PubMed]

Fan, S.

S. Fan and J. Joannopoulos, “Analysis of guided resonances in photonic crystal slabs,” Phys. Rev. B 65(23), 235112 (2002).
[CrossRef]

Fine, E.

B. T. Cunningham, P. Li, S. Schulz, B. Lin, C. Baird, J. Gerstenmaier, C. Genick, F. Wang, E. Fine, and L. Laing, “Label-free assays on the BIND system,” J. Biomol. Screen. 9(6), 481–490 (2004).
[CrossRef] [PubMed]

Gallagher, J. O.

A. S. Curtis, B. Casey, J. O. Gallagher, D. Pasqui, M. A. Wood, and C. D. Wilkinson, “Substratum nanotopography and the adhesion of biological cells. Are symmetry or regularity of nanotopography important?” Biophys. Chem. 94(3), 275–283 (2001).
[CrossRef] [PubMed]

Gauglitz, G.

B. P. Möhrle, K. Köhler, J. Jaehrling, R. Brock, and G. Gauglitz, “Label-free characterization of cell adhesion using reflectometric interference spectroscopy (RIfS),” Anal. Bioanal. Chem. 384(2), 407–413 (2005).
[CrossRef] [PubMed]

Genick, C.

B. T. Cunningham, P. Li, S. Schulz, B. Lin, C. Baird, J. Gerstenmaier, C. Genick, F. Wang, E. Fine, and L. Laing, “Label-free assays on the BIND system,” J. Biomol. Screen. 9(6), 481–490 (2004).
[CrossRef] [PubMed]

Gerken, M.

Gerstenmaier, J.

B. T. Cunningham, P. Li, S. Schulz, B. Lin, C. Baird, J. Gerstenmaier, C. Genick, F. Wang, E. Fine, and L. Laing, “Label-free assays on the BIND system,” J. Biomol. Screen. 9(6), 481–490 (2004).
[CrossRef] [PubMed]

Geyer, U.

Giebel, K.

K. Giebel, C. Bechinger, S. Herminghaus, M. Riedel, P. Leiderer, U. Weiland, and M. Bastmeyer, “Imaging of cell/substrate contacts of living cells with surface plasmon resonance microscopy,” Biophys. J. 76(1), 509–516 (1999).
[CrossRef] [PubMed]

Goldman, E. R.

I. L. Medintz, H. T. Uyeda, E. R. Goldman, and H. Mattoussi, “Quantum dot bioconjugates for imaging, labelling and sensing,” Nat. Mater. 4(6), 435–446 (2005).
[CrossRef] [PubMed]

Gu, Z.

Y. Zhao, X. Zhao, and Z. Gu, “Photonic crystals in bioassays,” Adv. Funct. Mater. 20(18), 2970–2988 (2010).
[CrossRef]

Guo, J. L.

J. L. Guo, “Nanoimprint lithography: methods and material requirements,” J. Adv. Mater. 19(4), 495–513 (2007).
[CrossRef]

Haeberli, A.

B. Polisky, R. Jenison, S. Yang, and A. Haeberli, “Interference-based detection of nucleic acid targets on optically coated silicon,” Nat. Biotechnol. 19(1), 62–65 (2001).
[CrossRef] [PubMed]

Herminghaus, S.

K. Giebel, C. Bechinger, S. Herminghaus, M. Riedel, P. Leiderer, U. Weiland, and M. Bastmeyer, “Imaging of cell/substrate contacts of living cells with surface plasmon resonance microscopy,” Biophys. J. 76(1), 509–516 (1999).
[CrossRef] [PubMed]

Jaehrling, J.

B. P. Möhrle, K. Köhler, J. Jaehrling, R. Brock, and G. Gauglitz, “Label-free characterization of cell adhesion using reflectometric interference spectroscopy (RIfS),” Anal. Bioanal. Chem. 384(2), 407–413 (2005).
[CrossRef] [PubMed]

Jenison, R.

B. Polisky, R. Jenison, S. Yang, and A. Haeberli, “Interference-based detection of nucleic acid targets on optically coated silicon,” Nat. Biotechnol. 19(1), 62–65 (2001).
[CrossRef] [PubMed]

R. Jenison, M. Rihanek, and B. Polisky, “Use of a thin film biosensor for rapid visual detection of PCR products in a multiplex format,” Biosens. Bioelectron. 16(9-12), 757–763 (2001).
[CrossRef] [PubMed]

Joannopoulos, J.

S. Fan and J. Joannopoulos, “Analysis of guided resonances in photonic crystal slabs,” Phys. Rev. B 65(23), 235112 (2002).
[CrossRef]

Jones, S. I.

Kley, E.-B.

Knoll, W.

B. Rothenhäusler and W. Knoll, “Surface-plasmon microscopy,” Nature 332(6165), 615–617 (1988).
[CrossRef]

Köhler, K.

B. P. Möhrle, K. Köhler, J. Jaehrling, R. Brock, and G. Gauglitz, “Label-free characterization of cell adhesion using reflectometric interference spectroscopy (RIfS),” Anal. Bioanal. Chem. 384(2), 407–413 (2005).
[CrossRef] [PubMed]

Kruse, C.

D. H. Rapoport, T. Becker, A. Madany Mamlouk, S. Schicktanz, and C. Kruse, “A novel validation algorithm allows for automated cell tracking and the extraction of biologically meaningful parameters,” PLoS ONE 6(11), e27315 (2011).
[CrossRef] [PubMed]

Laing, L.

B. T. Cunningham, P. Li, S. Schulz, B. Lin, C. Baird, J. Gerstenmaier, C. Genick, F. Wang, E. Fine, and L. Laing, “Label-free assays on the BIND system,” J. Biomol. Screen. 9(6), 481–490 (2004).
[CrossRef] [PubMed]

Leiderer, P.

K. Giebel, C. Bechinger, S. Herminghaus, M. Riedel, P. Leiderer, U. Weiland, and M. Bastmeyer, “Imaging of cell/substrate contacts of living cells with surface plasmon resonance microscopy,” Biophys. J. 76(1), 509–516 (1999).
[CrossRef] [PubMed]

Lemmer, U.

Li, P.

B. T. Cunningham, P. Li, S. Schulz, B. Lin, C. Baird, J. Gerstenmaier, C. Genick, F. Wang, E. Fine, and L. Laing, “Label-free assays on the BIND system,” J. Biomol. Screen. 9(6), 481–490 (2004).
[CrossRef] [PubMed]

Lin, B.

B. T. Cunningham, P. Li, S. Schulz, B. Lin, C. Baird, J. Gerstenmaier, C. Genick, F. Wang, E. Fine, and L. Laing, “Label-free assays on the BIND system,” J. Biomol. Screen. 9(6), 481–490 (2004).
[CrossRef] [PubMed]

MacAdam, D. L.

Madany Mamlouk, A.

D. H. Rapoport, T. Becker, A. Madany Mamlouk, S. Schicktanz, and C. Kruse, “A novel validation algorithm allows for automated cell tracking and the extraction of biologically meaningful parameters,” PLoS ONE 6(11), e27315 (2011).
[CrossRef] [PubMed]

Mappes, T.

Mathias, P. C.

Mattoussi, H.

I. L. Medintz, H. T. Uyeda, E. R. Goldman, and H. Mattoussi, “Quantum dot bioconjugates for imaging, labelling and sensing,” Nat. Mater. 4(6), 435–446 (2005).
[CrossRef] [PubMed]

Medintz, I. L.

I. L. Medintz, H. T. Uyeda, E. R. Goldman, and H. Mattoussi, “Quantum dot bioconjugates for imaging, labelling and sensing,” Nat. Mater. 4(6), 435–446 (2005).
[CrossRef] [PubMed]

Möhrle, B. P.

B. P. Möhrle, K. Köhler, J. Jaehrling, R. Brock, and G. Gauglitz, “Label-free characterization of cell adhesion using reflectometric interference spectroscopy (RIfS),” Anal. Bioanal. Chem. 384(2), 407–413 (2005).
[CrossRef] [PubMed]

Müller, J.

Nazirizadeh, Y.

Nomarski, G. Z.

R. D. Allen, G. B. David, and G. Z. Nomarski, “The zeiss-Nomarski differential interference equipment for transmitted-light microscopy,” Z. Wiss. Mikrosk. 69(4), 193–221 (1969).
[PubMed]

Pasqui, D.

A. S. Curtis, B. Casey, J. O. Gallagher, D. Pasqui, M. A. Wood, and C. D. Wilkinson, “Substratum nanotopography and the adhesion of biological cells. Are symmetry or regularity of nanotopography important?” Biophys. Chem. 94(3), 275–283 (2001).
[CrossRef] [PubMed]

Polisky, B.

R. Jenison, M. Rihanek, and B. Polisky, “Use of a thin film biosensor for rapid visual detection of PCR products in a multiplex format,” Biosens. Bioelectron. 16(9-12), 757–763 (2001).
[CrossRef] [PubMed]

B. Polisky, R. Jenison, S. Yang, and A. Haeberli, “Interference-based detection of nucleic acid targets on optically coated silicon,” Nat. Biotechnol. 19(1), 62–65 (2001).
[CrossRef] [PubMed]

Rapoport, D. H.

D. H. Rapoport, T. Becker, A. Madany Mamlouk, S. Schicktanz, and C. Kruse, “A novel validation algorithm allows for automated cell tracking and the extraction of biologically meaningful parameters,” PLoS ONE 6(11), e27315 (2011).
[CrossRef] [PubMed]

Riedel, M.

K. Giebel, C. Bechinger, S. Herminghaus, M. Riedel, P. Leiderer, U. Weiland, and M. Bastmeyer, “Imaging of cell/substrate contacts of living cells with surface plasmon resonance microscopy,” Biophys. J. 76(1), 509–516 (1999).
[CrossRef] [PubMed]

Rihanek, M.

R. Jenison, M. Rihanek, and B. Polisky, “Use of a thin film biosensor for rapid visual detection of PCR products in a multiplex format,” Biosens. Bioelectron. 16(9-12), 757–763 (2001).
[CrossRef] [PubMed]

Rothenhäusler, B.

B. Rothenhäusler and W. Knoll, “Surface-plasmon microscopy,” Nature 332(6165), 615–617 (1988).
[CrossRef]

Schelle, D.

Schicktanz, S.

D. H. Rapoport, T. Becker, A. Madany Mamlouk, S. Schicktanz, and C. Kruse, “A novel validation algorithm allows for automated cell tracking and the extraction of biologically meaningful parameters,” PLoS ONE 6(11), e27315 (2011).
[CrossRef] [PubMed]

Schulz, S.

B. T. Cunningham, P. Li, S. Schulz, B. Lin, C. Baird, J. Gerstenmaier, C. Genick, F. Wang, E. Fine, and L. Laing, “Label-free assays on the BIND system,” J. Biomol. Screen. 9(6), 481–490 (2004).
[CrossRef] [PubMed]

Sekula, S.

Shamah, S. M.

S. M. Shamah and B. T. Cunningham, “Label-free cell-based assays using photonic crystal optical biosensors,” Analyst (Lond.) 136(6), 1090–1102 (2011).
[CrossRef] [PubMed]

Stephens, D. J.

D. J. Stephens and V. J. Allan, “Light microscopy techniques for live cell imaging,” Science 300(5616), 82–86 (2003).
[CrossRef] [PubMed]

Tünnermann, A.

Uyeda, H. T.

I. L. Medintz, H. T. Uyeda, E. R. Goldman, and H. Mattoussi, “Quantum dot bioconjugates for imaging, labelling and sensing,” Nat. Mater. 4(6), 435–446 (2005).
[CrossRef] [PubMed]

Vodkin, L. O.

Wang, F.

B. T. Cunningham, P. Li, S. Schulz, B. Lin, C. Baird, J. Gerstenmaier, C. Genick, F. Wang, E. Fine, and L. Laing, “Label-free assays on the BIND system,” J. Biomol. Screen. 9(6), 481–490 (2004).
[CrossRef] [PubMed]

Weiland, U.

K. Giebel, C. Bechinger, S. Herminghaus, M. Riedel, P. Leiderer, U. Weiland, and M. Bastmeyer, “Imaging of cell/substrate contacts of living cells with surface plasmon resonance microscopy,” Biophys. J. 76(1), 509–516 (1999).
[CrossRef] [PubMed]

Wilkinson, C. D.

A. S. Curtis, B. Casey, J. O. Gallagher, D. Pasqui, M. A. Wood, and C. D. Wilkinson, “Substratum nanotopography and the adhesion of biological cells. Are symmetry or regularity of nanotopography important?” Biophys. Chem. 94(3), 275–283 (2001).
[CrossRef] [PubMed]

Wood, M. A.

A. S. Curtis, B. Casey, J. O. Gallagher, D. Pasqui, M. A. Wood, and C. D. Wilkinson, “Substratum nanotopography and the adhesion of biological cells. Are symmetry or regularity of nanotopography important?” Biophys. Chem. 94(3), 275–283 (2001).
[CrossRef] [PubMed]

Wright, W. D.

W. D. Wright, “A re-determination of the trichromatic coefficients of the spectral colours,” Trans. Opt. Soc. 30(4), 141–164 (1929).
[CrossRef]

Yang, S.

B. Polisky, R. Jenison, S. Yang, and A. Haeberli, “Interference-based detection of nucleic acid targets on optically coated silicon,” Nat. Biotechnol. 19(1), 62–65 (2001).
[CrossRef] [PubMed]

Zernike, F.

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Figures (5)

Fig. 1
Fig. 1

(a) Schematic setup for surface contrast microscopy (SCM). Two polarization filters before and behind the photonic crystal slab (PCS) suppress background light and allow only guided-mode resonances (GMR), which have their origin in the quasi-guided modes, to pass. Any object on the surface influences the optical properties such as resonance position and quality factor of the GMR, which leads to a contrast of hue and intensity. (b) Transmission through a PCS with and without crossed polarization filters. The PCS is a 70-nm deep linear grating etched into a 300-nm Ta2O5 layer on a glass substrate and has a periodicity of 370 nm Without polarization filters the transmission spectrum is a superposition of the white background light and the GMRs. Here the GMRs do not induce any significant color or intensity changes. With polarization filters only the GMR can pass the second polarization filter and hence any change in optical properties of the GMR is visible in the transmission image.

Fig. 2
Fig. 2

Refractometric experiments with PCSs. (a) Transmission spectra with crossed polarization filters of the PCS with varying bulk refractive index (air and water-glycerol dilutions). A spectral position shift and a decrease in intensity are observed. (b) The color change caused by the spectral position shift is plotted in the CIE color space. A shift from yellow to red is observed. A 10x magnified MacAdam ellipse around the color point for air indicates the loci, which cannot be distinguished by the human eye.

Fig. 3
Fig. 3

Human cheek cell on PCS. (a) Bright field microscopy. (b) Phase contrast microscopy. (c) Surface contrast microscopy using a PCS with a periodicity of 370 nm and a 300 nm thick high index layer. (d) Surface contrast microscopy using a PCS with a periodicity of 350 nm and a 135 nm thick high index layer. The color of the PCS with and without object on its surface is a function of the PCS’s geometry. Whereas phase contrast microscopy gives overall optical information about the structure of the cell interior, surface contrast microscopy only images the parts of the cell that are close to the surface.

Fig. 4
Fig. 4

(a) Mold grown on the surface of the PCS with bright field microscopy and SCM. In area 1 only in the SCM image the transparent part of the mold is visible. In area 2 parts of the mold growing out of the plane exhibit lower contrast enhancement. (b) Lipid remains on the surface of the PCS. Phase contrast microscopy is limited to detecting the thicker parts, surface contrast microscopy also reveals details that are very thin and close to the surface.

Fig. 5
Fig. 5

Qualitative performance comparison of phase contrast microscopy and SCM using automated cell detection. (a) Typical phase contrast microscopy and SCM images with overlayed cell segmentation. (b) Detection error versus binning factor of images for both microscopy methods. Two segmentation methods (global threshold and active contours) were used. The overall error rate for SCM is up to 3.5 times lower using global threshold segmentation and up to 2 times using active contours segmentation compared to phase contrast microscopy. The binning of the image up to a factor of 32 delivers for the global threshold segmentation method error rates below 10%.

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