Abstract

We demonstrate Diffraction Phase Cytometry (DPC) as a single shot, full-field, high throughput quantitative phase imaging modality, dedicated to analyzing whole blood smears. Utilizing a commercial CD as a sample substrate, along with dynamic spatial filtering via a liquid crystal spatial light modulator, we have developed a compact instrument capable of making quantitative, physiologically relevant measurements. To illustrate the ability of the system to function as a highly sensitive cytometer we imaged a large number (N=1,537) of live human erythrocytes in whole blood without preparation. We retrieved a comprehensive set of geometrical parameters including cell volume and surface area, which are not directly available using existing cytometers. Furthermore, we retrieved the minimum cylindrical diameter, through which red blood cells can pass, and deliver oxygen. These initial results prove the concept for an inexpensive lab-on-a-chip blood screening device.

© 2009 Optical Society of America

Full Article  |  PDF Article

References

  • View by:
  • |
  • |
  • |

  1. F. Brochard and J. F. Lennon, "Frequency spectrum of the flicker phenomenon in erythrocytes," J. Physique 36, 1035-1047 (1975).
    [CrossRef]
  2. N. Gov, A. G. Zilman and S. Safran, "Cytoskeleton confinement and tension of red blood cell membranes," Phys. Rev. Lett. 90, 228101 (2003).
    [CrossRef] [PubMed]
  3. P. B. Canham and A. C. Burton, "Distribution of Size and Shape in Populations of Normal Human Red Cells," Circ. Res. 22, 405-422 (1968).
    [PubMed]
  4. K. G. E. a H. J. Meiselman, "Effects of Pressure on Red Blood Cell Geometry during Micropipette Aspiration," Cytometry 23, 22-27 (1996).
    [CrossRef]
  5. W. Groner, N. Mohandas, and M. Bessis, "New Optical Technique for Measuring Erythrocyte Deformability with Ektacytometer," Clin. Chem. 26, (1980).
    [PubMed]
  6. D. J. Stephens and V. J. Allan, "Light Microscopy Techniques for Live Cell Imaging," Science 300, 82-86 (2003).
    [CrossRef] [PubMed]
  7. G. Popescu, Methods in Nano Cell Biology, 90, 87, P. J. Bhanu, ed., (Elsevier, 2008).
  8. G. Popescu, T. Ikeda, R. R. Dasari, and M. S. Feld, "Diffraction phase microscopy for quantifying cell structure and dynamics," Opt. Lett. 31, 775-777 (2006).
    [CrossRef] [PubMed]
  9. Z. Wang, L. J. Millet, M. U. Gillette, and G. Popescu, "Jones phase microscopy of transparent and anisotropic samples," Opt. Lett. 33, 1270-1272 (2008).
    [CrossRef] [PubMed]
  10. F. Charriere et al., "Amplitude point-spread function measurement of high-NA microscope objectives by digital holographic microscopy," Opt. Lett. 32, 2456-2458 (2007).
    [CrossRef] [PubMed]
  11. J. B. Bain, Blood Cells, A Practical Guide (Blackwell Science, 2002).
  12. Y. L. Jin, J. Y. Chen, L. Xu, and P. N. Wang, "Refractive index measurement for biomaterial samples by total internal reflection," Phys. Med. Biol. 51, 371-379 (2006).
    [CrossRef]
  13. M. Hammer, D. Schweitzer, B. Michel, E. Thamm, and A. Kolb, "Single scattering by red blood cells," Appl. Opt. 37, 7410-7418 (1998).
    [CrossRef]
  14. R. C. Gonzalez, R. E. Woods, and S. L. Eddins, Digital Image Processing Using MATLAB (Pearson Prentice Hall, NJ, 2004).
  15. B. Rappaz et al., "Comparative Study of Human Ertythrocytes by Digital Holographic Microscopy," Confocal Microscopy, and Impedance Volume Analyzer, Cyto. A. 73A, 895-903 (2008).
  16. R. P. Rand and A. C. Burton, "Area and volume changes in hemolysis of single erythrocytes," J. Cell. Comp. Physiol. 61, 245-253 (1963).
    [CrossRef] [PubMed]

2008 (2)

B. Rappaz et al., "Comparative Study of Human Ertythrocytes by Digital Holographic Microscopy," Confocal Microscopy, and Impedance Volume Analyzer, Cyto. A. 73A, 895-903 (2008).

Z. Wang, L. J. Millet, M. U. Gillette, and G. Popescu, "Jones phase microscopy of transparent and anisotropic samples," Opt. Lett. 33, 1270-1272 (2008).
[CrossRef] [PubMed]

2007 (1)

2006 (2)

G. Popescu, T. Ikeda, R. R. Dasari, and M. S. Feld, "Diffraction phase microscopy for quantifying cell structure and dynamics," Opt. Lett. 31, 775-777 (2006).
[CrossRef] [PubMed]

Y. L. Jin, J. Y. Chen, L. Xu, and P. N. Wang, "Refractive index measurement for biomaterial samples by total internal reflection," Phys. Med. Biol. 51, 371-379 (2006).
[CrossRef]

2003 (2)

D. J. Stephens and V. J. Allan, "Light Microscopy Techniques for Live Cell Imaging," Science 300, 82-86 (2003).
[CrossRef] [PubMed]

N. Gov, A. G. Zilman and S. Safran, "Cytoskeleton confinement and tension of red blood cell membranes," Phys. Rev. Lett. 90, 228101 (2003).
[CrossRef] [PubMed]

1998 (1)

1996 (1)

K. G. E. a H. J. Meiselman, "Effects of Pressure on Red Blood Cell Geometry during Micropipette Aspiration," Cytometry 23, 22-27 (1996).
[CrossRef]

1980 (1)

W. Groner, N. Mohandas, and M. Bessis, "New Optical Technique for Measuring Erythrocyte Deformability with Ektacytometer," Clin. Chem. 26, (1980).
[PubMed]

1975 (1)

F. Brochard and J. F. Lennon, "Frequency spectrum of the flicker phenomenon in erythrocytes," J. Physique 36, 1035-1047 (1975).
[CrossRef]

1968 (1)

P. B. Canham and A. C. Burton, "Distribution of Size and Shape in Populations of Normal Human Red Cells," Circ. Res. 22, 405-422 (1968).
[PubMed]

1963 (1)

R. P. Rand and A. C. Burton, "Area and volume changes in hemolysis of single erythrocytes," J. Cell. Comp. Physiol. 61, 245-253 (1963).
[CrossRef] [PubMed]

Allan, V. J.

D. J. Stephens and V. J. Allan, "Light Microscopy Techniques for Live Cell Imaging," Science 300, 82-86 (2003).
[CrossRef] [PubMed]

Bessis, M.

W. Groner, N. Mohandas, and M. Bessis, "New Optical Technique for Measuring Erythrocyte Deformability with Ektacytometer," Clin. Chem. 26, (1980).
[PubMed]

Brochard, F.

F. Brochard and J. F. Lennon, "Frequency spectrum of the flicker phenomenon in erythrocytes," J. Physique 36, 1035-1047 (1975).
[CrossRef]

Burton, A. C.

P. B. Canham and A. C. Burton, "Distribution of Size and Shape in Populations of Normal Human Red Cells," Circ. Res. 22, 405-422 (1968).
[PubMed]

R. P. Rand and A. C. Burton, "Area and volume changes in hemolysis of single erythrocytes," J. Cell. Comp. Physiol. 61, 245-253 (1963).
[CrossRef] [PubMed]

Canham, P. B.

P. B. Canham and A. C. Burton, "Distribution of Size and Shape in Populations of Normal Human Red Cells," Circ. Res. 22, 405-422 (1968).
[PubMed]

Charriere, F.

Chen, J. Y.

Y. L. Jin, J. Y. Chen, L. Xu, and P. N. Wang, "Refractive index measurement for biomaterial samples by total internal reflection," Phys. Med. Biol. 51, 371-379 (2006).
[CrossRef]

Dasari, R. R.

Feld, M. S.

Gillette, M. U.

Gov, N.

N. Gov, A. G. Zilman and S. Safran, "Cytoskeleton confinement and tension of red blood cell membranes," Phys. Rev. Lett. 90, 228101 (2003).
[CrossRef] [PubMed]

Groner, W.

W. Groner, N. Mohandas, and M. Bessis, "New Optical Technique for Measuring Erythrocyte Deformability with Ektacytometer," Clin. Chem. 26, (1980).
[PubMed]

Hammer, M.

Ikeda, T.

Jin, Y. L.

Y. L. Jin, J. Y. Chen, L. Xu, and P. N. Wang, "Refractive index measurement for biomaterial samples by total internal reflection," Phys. Med. Biol. 51, 371-379 (2006).
[CrossRef]

Kolb, A.

Lennon, J. F.

F. Brochard and J. F. Lennon, "Frequency spectrum of the flicker phenomenon in erythrocytes," J. Physique 36, 1035-1047 (1975).
[CrossRef]

Michel, B.

Millet, L. J.

Mohandas, N.

W. Groner, N. Mohandas, and M. Bessis, "New Optical Technique for Measuring Erythrocyte Deformability with Ektacytometer," Clin. Chem. 26, (1980).
[PubMed]

Popescu, G.

Rand, R. P.

R. P. Rand and A. C. Burton, "Area and volume changes in hemolysis of single erythrocytes," J. Cell. Comp. Physiol. 61, 245-253 (1963).
[CrossRef] [PubMed]

Rappaz, B.

B. Rappaz et al., "Comparative Study of Human Ertythrocytes by Digital Holographic Microscopy," Confocal Microscopy, and Impedance Volume Analyzer, Cyto. A. 73A, 895-903 (2008).

Safran, S.

N. Gov, A. G. Zilman and S. Safran, "Cytoskeleton confinement and tension of red blood cell membranes," Phys. Rev. Lett. 90, 228101 (2003).
[CrossRef] [PubMed]

Schweitzer, D.

Stephens, D. J.

D. J. Stephens and V. J. Allan, "Light Microscopy Techniques for Live Cell Imaging," Science 300, 82-86 (2003).
[CrossRef] [PubMed]

Thamm, E.

Wang, P. N.

Y. L. Jin, J. Y. Chen, L. Xu, and P. N. Wang, "Refractive index measurement for biomaterial samples by total internal reflection," Phys. Med. Biol. 51, 371-379 (2006).
[CrossRef]

Wang, Z.

Xu, L.

Y. L. Jin, J. Y. Chen, L. Xu, and P. N. Wang, "Refractive index measurement for biomaterial samples by total internal reflection," Phys. Med. Biol. 51, 371-379 (2006).
[CrossRef]

Zilman, A. G.

N. Gov, A. G. Zilman and S. Safran, "Cytoskeleton confinement and tension of red blood cell membranes," Phys. Rev. Lett. 90, 228101 (2003).
[CrossRef] [PubMed]

Appl. Opt. (1)

Circ. Res. (1)

P. B. Canham and A. C. Burton, "Distribution of Size and Shape in Populations of Normal Human Red Cells," Circ. Res. 22, 405-422 (1968).
[PubMed]

Clin. Chem. (1)

W. Groner, N. Mohandas, and M. Bessis, "New Optical Technique for Measuring Erythrocyte Deformability with Ektacytometer," Clin. Chem. 26, (1980).
[PubMed]

Cyto. A. (1)

B. Rappaz et al., "Comparative Study of Human Ertythrocytes by Digital Holographic Microscopy," Confocal Microscopy, and Impedance Volume Analyzer, Cyto. A. 73A, 895-903 (2008).

Cytometry (1)

K. G. E. a H. J. Meiselman, "Effects of Pressure on Red Blood Cell Geometry during Micropipette Aspiration," Cytometry 23, 22-27 (1996).
[CrossRef]

J. Cell. Comp. Physiol. (1)

R. P. Rand and A. C. Burton, "Area and volume changes in hemolysis of single erythrocytes," J. Cell. Comp. Physiol. 61, 245-253 (1963).
[CrossRef] [PubMed]

J. Physique (1)

F. Brochard and J. F. Lennon, "Frequency spectrum of the flicker phenomenon in erythrocytes," J. Physique 36, 1035-1047 (1975).
[CrossRef]

Opt. Lett. (3)

Phys. Med. Biol. (1)

Y. L. Jin, J. Y. Chen, L. Xu, and P. N. Wang, "Refractive index measurement for biomaterial samples by total internal reflection," Phys. Med. Biol. 51, 371-379 (2006).
[CrossRef]

Phys. Rev. Lett. (1)

N. Gov, A. G. Zilman and S. Safran, "Cytoskeleton confinement and tension of red blood cell membranes," Phys. Rev. Lett. 90, 228101 (2003).
[CrossRef] [PubMed]

Science (1)

D. J. Stephens and V. J. Allan, "Light Microscopy Techniques for Live Cell Imaging," Science 300, 82-86 (2003).
[CrossRef] [PubMed]

Other (3)

G. Popescu, Methods in Nano Cell Biology, 90, 87, P. J. Bhanu, ed., (Elsevier, 2008).

J. B. Bain, Blood Cells, A Practical Guide (Blackwell Science, 2002).

R. C. Gonzalez, R. E. Woods, and S. L. Eddins, Digital Image Processing Using MATLAB (Pearson Prentice Hall, NJ, 2004).

Cited By

OSA participates in CrossRef's Cited-By Linking service. Citing articles from OSA journals and other participating publishers are listed here.

Alert me when this article is cited.


Figures (4)

Fig. 1.
Fig. 1.

DPC setup. SLM, Spatial Light Modulator; L1, lens; focal length, f1=300 mm. GV, Grey Value of pixels; Ø, Diameter of pinholes.

Fig. 2.
Fig. 2.

Unbiased height frequency distribution for RBCs and a control sample of 4.5 μm polystyrene microspheres.

Fig. 3.
Fig. 3.

Frequency distributions a) Volume distribution, the dashed line shows volume data from Rappaz et al. [15] , b) Surface Area; inset shows the calculated surface mesh for a slightly deformed cell, c) Sphericity, d) Minimum Cylindrical Diameter; dashed lines show data from Canham and Burton [3].

Fig. 4.
Fig. 4.

Red lines are linear fits with R2 values shown in the legend a) Volume vs. Diameter, b) Surface Area vs. Diameter, c) Surface Area vs. Volume with constant MCD lines shown in black, d) Sphericity Index vs. Volume, quartered to show that cells with larger volume are thinner.

Tables (1)

Tables Icon

Table 1: Comparison of DPC data with subject R.S. from Canham and Burton [3].

Equations (3)

Equations on this page are rendered with MathJax. Learn more.

h x y = 2 πλ Δ ϕ ( x , y ) / Δn ,
Sphericity = 4.84 V 2 / 3 SA
V = SA * MCD π MCD 3 12

Metrics