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1 September 2007 Integral refractive index determination of living suspension cells by multifocus digital holographic phase contrast microscopy
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Abstract
A method for the determination of the integral refractive index of living cells in suspension by digital holographic microscopy is described. Digital holographic phase contrast images of spherical cells in suspension are recorded, and the radius as well as the integral refractive index are determined by fitting the relation between cell thickness and phase distribution to the measured phase data. The algorithm only requires information about the refractive index of the suspension medium and the image scale of the microscope system. The specific digital holographic microscopy advantage of subsequent focus correction allows a simultaneous investigation of cells in different focus planes. Results obtained from human pancreas and liver tumor cells show that the integral cellular refractive index decreases with increasing cell radius.

1.

Introduction

Digital holographic microscopy 1, 2, 3, 4 and interferometric phase contrast methods5, 6 enable a quantitative marker-free dynamic analysis of living cells. Knowledge of the integral cellular refractive index allows the determination of the cell thickness and for adherently grown cells, the cell shape from the obtained phase contrast images.3, 7 Previously published methods for the determination of the refractive index of single cells require a preparation of the sample,7 an exchange of the cell culture medium,8 or specific microfluidic equipment,9, 10 which may affect the sample, is time consuming, or often not applicable. Here, a method for the determination of the cellular refractive index by digital holographic microscopy is described that requires only cells in suspension and a calibrated image scale. Therefore, the method is easy to handle and opens up an effective way of single-cell integral refractive index determination. The combination with the specific digital holographic microscopy feature of subsequent numerical focus correction provides increased data acquisition by simultaneous recording of cells in different focus planes.

2.

Setup for Digital Holographic Microscopy

Figure 1 depicts the scheme of the applied digital holographic microscopy system. An inverse microscope arrangement enables the investigation of living cells in culture/buffer medium. Microscope lenses (Zeiss LD Achroplan 40×0.6Korr , Zeiss LD Neofluar 63×0.75Korr ) are applied to magnify the object wave. The reconstruction of the digitally captured off-axis holograms is performed by a nondiffractive reconstruction method.2, 11 Due to the applied algorithm, the reconstructed holographic images do not contain the disturbing terms “twin image” and “zero order.” Furthermore, a reconstruction of holograms with a sharply focused image of the sample is possible, which is of particular advantage for the alignment of the experimental setup.7

Fig. 1

Scheme for an inverse off-axis digital holographic microscopy setup with frequency-doubled Nd:YAG laser (λ=532nm) .

054009_1_025705jbo1.jpg

3.

Refractive Index Determination of Spherical Cells in Suspension

The relation between the measured phase distribution Δφcell that is affected by the cell thickness d(x,y) in comparison to the surrounding buffer medium is:

Eq. 1

Δφcell(x,y)=2πλ(ncellnmedium)d(x,y),
with the integral cellular refractive index ncell , the known homogenous refractive index of the buffer medium nmedium , and the wavelength λ of the applied laser light. For cells in suspension located at x=x0 , y=y0 with a spherical shape and radius R (see Fig. 2 ), the cell thickness d(x,y) is

Fig. 2

Principle for refractive index determination of spherical cells in suspension. R is the cell radius; ncell the integral cellular refractive index; and nmedium the refractive index of the buffer medium.

054009_1_025705jbo2.jpg

Eq. 2

d(x,y)={2[R2(xx0)2(yy0)2]12for(xx0)2+(yy0)2R20for(xx0)2+(yy0)2> R2.
Insertion of Eq. 1 into Eq. 2 yields:

Eq. 3

Δφcell(x,y)={4πλ[R2(xx0)2(yy0)2]12(ncellnmedium)for(xx0)2+(yy0)2R20for(xx0)2+(yy0)2> R2.
with the unknown parameters ncell , R , x0 , and y0 . For the described experiments, Eq. 3 is fitted line-wise in the x direction with the Gauß-Newton method12 to the measured phase data of spherical suspension cells by iterative calculation of ncell , Rx(y)=[R2(yy0)2]12 , and x0 . The image scale was calibrated by a transparent USAF 1951 resolution test chart. Figure 3 illustrates the evaluation process by a representative result that has been obtained from a trypsinized human pancreas tumor cell (PaTu 8988 T) with spherical shape. The refractive index of the cell culture medium Dulbecco’s modified eagle medium (DMEM) containing 5% Fetal calf serum (FCS) and 5% horse serum is determined to nmedium=1.337±0.001 with an Abbe refractometer. Figure 3a shows the phase contrast image of the cell, coded to 256 gray levels (8bit) . The data Δφcell(x,y) for the fitting process is selected by a threshold value that specifies the phase noise in the area around the cell. In Fig. 3b, the fit of Eq. 3 to the data along the cross section in the x direction that is marked by the dashed line in Fig. 3a is depicted. Figures 3c and 3d represent the pseudo 3-D plots of the phase distribution in Fig. 3a in comparison to the same data achieved by the line-wise fitted data from Eq. 3. The mean value of the cell refractive index ncell=1.372±0.002 is used for further analysis. The uncertainty for ncell is estimated by the standard deviation obtained from all line fits. The cell radius is obtained by determination of R=Rx(v=v0)Rfit,max from all fitted lines (for Fig. 3, Rfit,max=10.2±0.1μm ). The uncertainty for R is calculated by the standard deviation of ±5 neighboring lines to the central line at R=Rfit,max . Figure 3e shows the absolute values of the phase difference between the measured phase contrast data in Fig. 3c and the fitted data in Fig. 3d (mean value=0.3rad ), which indicates a homogeneous distribution of the fitting errors.

Fig. 3

Refractive index determination of suspension cells: (a) reconstructed quantitative digital holographic phase contrast image of a spherical trypsinized pancreas tumor cell located at (x0,y0) ; (b) phase data Δφ along the cross section marked by the dashed line in (a) and the fitted data corresponding to Eq. 3; (c) rendered pseudo 3-D plot of the phase distribution in (a) that is used for the determination of the integral refractive index; (d) rendered pseudo 3-D plot of data that is obtained by line-wise fitting of Eq. 3; (e) rendered pseudo 3-D plot of the phase difference data between (c) and (d).

054009_1_025705jbo3.jpg

4.

Results

First, investigations on simulated phase data of cells with a constant refractive index (ncell,sim=1.38) and different cell radii ( R=0.510μm , N=64 ) according to Eq. 3 were carried out to determine the resolution of the described fit-algorithm for refractive index measurement. For all simulations, a cell radius independent refractive index ncell,sim=1.38001±0.00001 was obtained. Further experiments on homogeneous beads ( Cytodex 1, GE Healthcare, Germany, N=27 ) in water (nwater=1.334±0.001) resulted in a radius independent refractive index ncell,beads=1.3377±0.0004 .

Three different human pancreas tumor cell lines13, 14 [PaTu 8988 T (N=28) , PaTu 8988 S (N=15) , and PaTu 8988 T pLXIN E-Cadherin (N=20) ] were investigated in comparison to a human liver tumor cell line15 (HepG2, N=55 ). The cells were trypsinized, and for each cell line, the parameters ncell and Rfit,max were determined with the described algorithm (nmedium=1.337±0.001) . For data evaluation, only cells with spherical shape were selected, which were observed in the main fraction of the recorded holographic phase contrast images for all investigated cell lines. Figures 4a and 4b depict the refractive index ncell in dependence of Rfit,max for pancreas and liver tumor cells. Within the range of uncertainty, no significant differences between the data of the three different types of pancreas tumor cells are observed [Fig. 4a]. Furthermore, Fig. 4a shows that the cellular refractive index of the cells decreases with increasing Rfit,max . The data obtained from the liver tumor cells [Fig. 4b] shows the same behavior. For the pancreas tumor cells, the mean refractive index determined to ncell¯=1.375±0.004 for a mean cell radius Rfit,max¯=9.1±1.1 , while for the liver tumor cells ncell¯=1.369±0.005 with Rfit,max¯=9.1±0.9 is obtained. To verify the fitting process, the cell radius Rfit,max is plotted versus the radius Rlat of the cells that is determined from the phase contrast images by the image scale. The results are shown in Figs. 4c and 4d for PaTu 8988 XX and HepG2 cells. A proportional relation is obtained that is in correspondence with the results of the fitting algorithm. The decrease of the refractive index with increasing cell radius, and thus the cell volume, may be explained by cellular water content.16 Figures 5a and 5b show the histogram plots of the refractive index for both cell lines. The maximum numbers of cells is located near the mean value of the refractive index.

Fig. 4

Refractive index determination of PaTu 8988 XX cells and HepG2 cells. (a) and (b) Integral cellular refractive index ncell of PaTu 8988 XX cells and HepG2 cells versus the cell radius Rfit,max obtained from the fit of Eq. 3. (c) and (d) Cell radius Rfit,max obtained from Eq. 3 versus the cell radius Rlat that is determined from the lateral image scale for both PaTu 8988 XX cells and HepG2 cells.

054009_1_025705jbo4.jpg

Fig. 5

Histogram of the refractive index data for (a) PaTu 8988 XX cells and (b) HepG2 cells.

054009_1_025705jbo5.jpg

5.

Conclusions

In summary, an algorithm for the marker-free retrieval of the integral refractive index of living cells from digital holographic phase contrast images is presented that can be applied to cell lines with a mainly spherical shape, like the investigated trypsinized PaTu 8988 XX and HepG2 tumor cells in suspension. The mean error for ncell is determined to 0.005. The algorithm allows a refractive index analysis of cells without additional sample preparation. Due to the multifocus ability of digital holographic microscopy, an increased data acquisition can be achieved by simultaneous recording of cells located in different focal planes of the suspension. Furthermore, it is demonstrated that the integral refractive index of cells depends on the cell size. This information is important, e.g., for the utilization of optical tweezers and related optical manipulation systems, where the individual cellular refractive index represents a parameter for calculation of the applied forces.17, 18, 19 In addition, the method opens up new perspectives for the quantitative monitoring of cell swelling processes.16 Here, e.g., water absorption/permeability effects a decrease of the cellular refractive index that leads to an underestimation of the cell thickness in digital holographic phase contrast microscopy due to Eq. 1. In this way, the precision of such measurements can be significantly improved. In conclusion, the proposed method allows the application of digital holographic microscopy in an enhanced variety of life cell analytical approaches.

Acknowledgments

The authors acknowledge financial support by the German Federal Ministry of Education and Research (BMBF) and Deutsche Forschungsgemeinschaft (DFG), and thank Andreas Bauwens, Institute of Medical Physics and Biophysics, University of Muenster, Germany, for the support during the experiments with the beads.

References

1. 

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). 0003-6935 Google Scholar

2. 

D. Carl, B. Kemper, G. Wernicke, and G. von Bally, “Parameter-optimized digital holographic microscope for high-resolution living cell analysis,” Appl. Opt., 43 6536 –6544 (2004). https://doi.org/10.1364/AO.43.006536 0003-6935 Google Scholar

3. 

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 sub wavelength accuracy,” Opt. Lett., 30 468 –470 (2005). https://doi.org/10.1364/OL.30.000468 0146-9592 Google Scholar

4. 

C. J. Mann, L. Yu, C. M. Lo, and M. K. Kim, “High-resolution quantitative phase-contrast microscopy by digital holography,” Opt. Express, 13 8693 –8698 (2005). https://doi.org/10.1364/OPEX.13.008693 1094-4087 Google Scholar

5. 

G. Popescu, L. Deflores, J. C. Vanghan, K. Badizadegen, H. Iwai, R. R. Dasari, and M. S. Feld, “Fourier phase microscopy for investigations of biological structures and dynamics,” Opt. Lett., 29 2503 –2505 (2004). https://doi.org/10.1364/OL.29.002503 0146-9592 Google Scholar

6. 

T. Ikeda, P. Popescu, P. Dasari, and M. S. Feld, “Hilbert phase microscopy for investigating fast dynamics in transparent systems,” Opt. Lett., 30 1165 –1167 (2005). https://doi.org/10.1364/OL.30.001165 0146-9592 Google Scholar

7. 

B. Kemper, D. Carl, J. Schnekenburger, I. Bredebusch, M. Schäfer, W. Domschke, and G. von Bally, “Investigations on living pancreas tumor cells by digital holographic microscopy,” J. Biomed. Opt., 11 034005 (2006). https://doi.org/10.1117/1.2204609 1083-3668 Google Scholar

8. 

B. Rappaz, P. Marquet, E. Cuche, Y. Emery, C. Depeursinge, and P. J. Magistretti, “Measurement of the integral refractive index and dynamic cell morpholometry of living cells with digital holographic microscopy,” Opt. Express, 13 9361 –9373 (2005). https://doi.org/10.1364/OPEX.13.009361 1094-4087 Google Scholar

9. 

X. J. Liang, A. Q. Liu, X. M. Zhang, P. H. Yap, T. C. Ayi, and H. S. Yoon, “Determination of refractive index for single living cell using integrated biochip,” 1712 –1715 (2005). Google Scholar

10. 

W. Z. Zhong, X. M. Zhang, A. Q. Liu, C. S. Lim, P. H. Yap, and H. M. M. Hosseini, “Refractive index measurement of single living cells using on-chip Fabry-Pérot cavity,” Appl. Phys. Lett., 89 203901 (2006). https://doi.org/10.1063/1.2387965 0003-6951 Google Scholar

11. 

M. Liebling, T. Blu, and M. Unser, “Complex-wave retrieval from a single off-axis hologram,” J. Opt. Soc. Am. A, 21 367 –377 (2004). https://doi.org/10.1364/JOSAA.21.000367 0740-3232 Google Scholar

12. 

Å. Björk, Numerical Methods for Least Squares Problems, Society of Industrial & Applied Mathematics, Philadelphia, PA (1996). Google Scholar

13. 

H. P. Elsässer, U. Lehr, B. Agricola, and H. F. Kern, “Establishment and characterization of two cell lines with different grade of differentiation derived from one primary human pancreatic adenocarcinoma,” Virchows Arch. B, 61 295 –306 (1992). 0340-6075 Google Scholar

14. 

J. Schnekenburger, J. Mayerle, B. Krüger, I. Buchwalow, F. U. Weiss, E. Albrecht, V. E. Samoilova, W. Domschke, and M. M. Lerch, “Protein tyrosine phosphatase k and SHP-1 are involved in the regulation of cell-cell contacts at adherens junctions in the exocrine pancreas,” Gut, 54 1445 –1455 (2005). 0017-5749 Google Scholar

15. 

D. P. Aden, A. Fogel, S. Plotkin, I. Damjanov, and B. B. Knowles, “Controlled synthesis of HBsAg in a different human liver carcinoma-derived cell line,” Nature (London), 282 615 –616 (1979). https://doi.org/10.1038/282615a0 0028-0836 Google Scholar

16. 

J. Farinas and A. S. Verkman, “Cell volume and plasma membrane osmotic permeability in epiphelial cell layers measured by interferometry,” Biophys. J., 71 3511 –3522 (1996). 0006-3495 Google Scholar

17. 

A. Ashkin, “Optical trapping and manipulation of neutral particles using lasers,” Proc. Natl. Acad. Sci. U.S.A., 94 4853 –4860 (1997). https://doi.org/10.1073/pnas.94.10.4853 0027-8424 Google Scholar

18. 

J. Guck, R. Ananthakrishnan, T. J. Moon, C. C. Cunningham, and J. Käs, “The optical strecher—a novel, noninvasive tool to manipulate biological materials,” Biophys. J., 81 767 –784 (2001). 0006-3495 Google Scholar

19. 

Z. Hu, J. Wang, and J. Liang, “Manipulation and arrangement of biological and dielectric particles by a lensend fiber probe,” Opt. Express, 12 4123 –4128 (2004). https://doi.org/10.1364/OPEX.12.004123 1094-4087 Google Scholar
©(2007) Society of Photo-Optical Instrumentation Engineers (SPIE)
Björn Kemper, Sebastian Kosmeier, Patrik Langehanenberg, Gert von Bally, Ilona Bredebusch, Wolfram Domschke, and Jürgen Schnekenburger "Integral refractive index determination of living suspension cells by multifocus digital holographic phase contrast microscopy," Journal of Biomedical Optics 12(5), 054009 (1 September 2007). https://doi.org/10.1117/1.2798639
Published: 1 September 2007
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