Abstract

Capturing and quantifying dynamic changes in three-dimensional cellular geometries on fast time scales is a challenge because of mechanical limitations of imaging systems as well as of the inherent tradeoffs between temporal resolution and image quality. We have combined a custom high-speed two-photon microscopy approach with a novel image segmentation method, the weighted directional adaptive-threshold (WDAT), to quantify the dimensions of intercellular spaces of cells under compressive stress on timescales previously inaccessible. The adaptation of a high-speed two-photon microscope addressed the need to capture events occurring on short timescales, while the WDAT method was developed to address artifacts of standard intensity-based analysis methods when applied to this system. Our novel approach is demonstrated by the enhanced temporal analysis of the three-dimensional cellular and extracellular deformations that accompany compressive loading of airway epithelial cells.

© 2008 Optical Society of America

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  1. D. J. Tschumperlin, G. Dai, I. V. Maly, T. Kikuchi, L. H. Laiho, A. K. McVittie, K. J. Haley, C. M. Lilly, P. T. So, D. A. Lauffenburger, R. D. Kamm, and J. M. Drazen, "Mechanotransduction through growth-factor shedding into the extracellular space," Nature 429, 83-86 (2004).
    [CrossRef] [PubMed]
  2. D. J. Tschumperlin, J. D. Shively, M. A. Swartz, E. S. Silverman, K. J. Haley, G. Raab, and J. M. Drazen, "Bronchial epithelial compression regulates MAP kinase signaling and HB-EGF-like growth factor expression," Am. J. Physiol. Lung Cell Mol. Physiol. 282, L904-911 (2002).
  3. K. H. Kim, C. Buehler, and P. T. C. So, "High-speed, two-photon scanning microscope," Appl. Opt. 38, 6004-6009 (1999).
    [CrossRef]
  4. T. P. Padera, B. R. Stoll, P. T. C. So, and R. K. Jain, "Conventional and high-speed intravital multiphoton laser scanning microscopy of microvasculature, lymphatics, and leukocyte-endothelial interactions," Mol. Imaging 1, 9-15 (2002).
    [CrossRef]
  5. T. Kikuchi, J. D. Shively, J. S. Foley, J. M. Drazen, and T. D. J., "Differentiation-dependent responsiveness of bronchial epithelial cells to IL-4/13 stimulation," Am. J. Physiol. Lung Cell Mol. Physiol. 287, L119-126 (2004).
    [CrossRef]
  6. B. Horn, Robot vision, MIT Press ed., MIT electrical engineering and computer science series (MIT Press; McGraw-Hill, Cambridge, Mass., 1986), pp. x, 509 p.
  7. B. Jähne, Digital Image Processing: Concepts, Algorithms, and Scientific Applications, 6th Ed. (Springer-Verlag Telos, New York, 1997).
  8. R. C. Gonzalez and R. E. Woods, Digital Image Processing (Prentice Hall, Upper Saddle River, NJ, 2002).
  9. D. A. Forsyth and J. Ponce, Computer Vision: A modern approach (Prentice Hall, Upper Saddle River, NJ, 2003).
  10. C. K. Chow and T. Kaneko, "Automatic boundary detection of the left ventricle from cineangiograms," Comput. Biomed. Res. 5, 388-410 (1972).
    [CrossRef] [PubMed]
  11. N. Kojic, M. Kojic, and D. J. Tschumperlin, "Computational modeling of extracellular mechanotransduction," Biophys. J. 90, 4261-4270 (2006).
    [CrossRef] [PubMed]

2006

N. Kojic, M. Kojic, and D. J. Tschumperlin, "Computational modeling of extracellular mechanotransduction," Biophys. J. 90, 4261-4270 (2006).
[CrossRef] [PubMed]

2004

D. J. Tschumperlin, G. Dai, I. V. Maly, T. Kikuchi, L. H. Laiho, A. K. McVittie, K. J. Haley, C. M. Lilly, P. T. So, D. A. Lauffenburger, R. D. Kamm, and J. M. Drazen, "Mechanotransduction through growth-factor shedding into the extracellular space," Nature 429, 83-86 (2004).
[CrossRef] [PubMed]

T. Kikuchi, J. D. Shively, J. S. Foley, J. M. Drazen, and T. D. J., "Differentiation-dependent responsiveness of bronchial epithelial cells to IL-4/13 stimulation," Am. J. Physiol. Lung Cell Mol. Physiol. 287, L119-126 (2004).
[CrossRef]

2002

D. J. Tschumperlin, J. D. Shively, M. A. Swartz, E. S. Silverman, K. J. Haley, G. Raab, and J. M. Drazen, "Bronchial epithelial compression regulates MAP kinase signaling and HB-EGF-like growth factor expression," Am. J. Physiol. Lung Cell Mol. Physiol. 282, L904-911 (2002).

T. P. Padera, B. R. Stoll, P. T. C. So, and R. K. Jain, "Conventional and high-speed intravital multiphoton laser scanning microscopy of microvasculature, lymphatics, and leukocyte-endothelial interactions," Mol. Imaging 1, 9-15 (2002).
[CrossRef]

1999

1972

C. K. Chow and T. Kaneko, "Automatic boundary detection of the left ventricle from cineangiograms," Comput. Biomed. Res. 5, 388-410 (1972).
[CrossRef] [PubMed]

Buehler, C.

Chow, C. K.

C. K. Chow and T. Kaneko, "Automatic boundary detection of the left ventricle from cineangiograms," Comput. Biomed. Res. 5, 388-410 (1972).
[CrossRef] [PubMed]

Dai, G.

D. J. Tschumperlin, G. Dai, I. V. Maly, T. Kikuchi, L. H. Laiho, A. K. McVittie, K. J. Haley, C. M. Lilly, P. T. So, D. A. Lauffenburger, R. D. Kamm, and J. M. Drazen, "Mechanotransduction through growth-factor shedding into the extracellular space," Nature 429, 83-86 (2004).
[CrossRef] [PubMed]

Drazen, J. M.

D. J. Tschumperlin, G. Dai, I. V. Maly, T. Kikuchi, L. H. Laiho, A. K. McVittie, K. J. Haley, C. M. Lilly, P. T. So, D. A. Lauffenburger, R. D. Kamm, and J. M. Drazen, "Mechanotransduction through growth-factor shedding into the extracellular space," Nature 429, 83-86 (2004).
[CrossRef] [PubMed]

T. Kikuchi, J. D. Shively, J. S. Foley, J. M. Drazen, and T. D. J., "Differentiation-dependent responsiveness of bronchial epithelial cells to IL-4/13 stimulation," Am. J. Physiol. Lung Cell Mol. Physiol. 287, L119-126 (2004).
[CrossRef]

D. J. Tschumperlin, J. D. Shively, M. A. Swartz, E. S. Silverman, K. J. Haley, G. Raab, and J. M. Drazen, "Bronchial epithelial compression regulates MAP kinase signaling and HB-EGF-like growth factor expression," Am. J. Physiol. Lung Cell Mol. Physiol. 282, L904-911 (2002).

Foley, J. S.

T. Kikuchi, J. D. Shively, J. S. Foley, J. M. Drazen, and T. D. J., "Differentiation-dependent responsiveness of bronchial epithelial cells to IL-4/13 stimulation," Am. J. Physiol. Lung Cell Mol. Physiol. 287, L119-126 (2004).
[CrossRef]

Haley, K. J.

D. J. Tschumperlin, G. Dai, I. V. Maly, T. Kikuchi, L. H. Laiho, A. K. McVittie, K. J. Haley, C. M. Lilly, P. T. So, D. A. Lauffenburger, R. D. Kamm, and J. M. Drazen, "Mechanotransduction through growth-factor shedding into the extracellular space," Nature 429, 83-86 (2004).
[CrossRef] [PubMed]

D. J. Tschumperlin, J. D. Shively, M. A. Swartz, E. S. Silverman, K. J. Haley, G. Raab, and J. M. Drazen, "Bronchial epithelial compression regulates MAP kinase signaling and HB-EGF-like growth factor expression," Am. J. Physiol. Lung Cell Mol. Physiol. 282, L904-911 (2002).

Jain, R. K.

T. P. Padera, B. R. Stoll, P. T. C. So, and R. K. Jain, "Conventional and high-speed intravital multiphoton laser scanning microscopy of microvasculature, lymphatics, and leukocyte-endothelial interactions," Mol. Imaging 1, 9-15 (2002).
[CrossRef]

Kamm, R. D.

D. J. Tschumperlin, G. Dai, I. V. Maly, T. Kikuchi, L. H. Laiho, A. K. McVittie, K. J. Haley, C. M. Lilly, P. T. So, D. A. Lauffenburger, R. D. Kamm, and J. M. Drazen, "Mechanotransduction through growth-factor shedding into the extracellular space," Nature 429, 83-86 (2004).
[CrossRef] [PubMed]

Kaneko, T.

C. K. Chow and T. Kaneko, "Automatic boundary detection of the left ventricle from cineangiograms," Comput. Biomed. Res. 5, 388-410 (1972).
[CrossRef] [PubMed]

Kikuchi, T.

D. J. Tschumperlin, G. Dai, I. V. Maly, T. Kikuchi, L. H. Laiho, A. K. McVittie, K. J. Haley, C. M. Lilly, P. T. So, D. A. Lauffenburger, R. D. Kamm, and J. M. Drazen, "Mechanotransduction through growth-factor shedding into the extracellular space," Nature 429, 83-86 (2004).
[CrossRef] [PubMed]

T. Kikuchi, J. D. Shively, J. S. Foley, J. M. Drazen, and T. D. J., "Differentiation-dependent responsiveness of bronchial epithelial cells to IL-4/13 stimulation," Am. J. Physiol. Lung Cell Mol. Physiol. 287, L119-126 (2004).
[CrossRef]

Kim, K. H.

Kojic, M.

N. Kojic, M. Kojic, and D. J. Tschumperlin, "Computational modeling of extracellular mechanotransduction," Biophys. J. 90, 4261-4270 (2006).
[CrossRef] [PubMed]

Kojic, N.

N. Kojic, M. Kojic, and D. J. Tschumperlin, "Computational modeling of extracellular mechanotransduction," Biophys. J. 90, 4261-4270 (2006).
[CrossRef] [PubMed]

Laiho, L. H.

D. J. Tschumperlin, G. Dai, I. V. Maly, T. Kikuchi, L. H. Laiho, A. K. McVittie, K. J. Haley, C. M. Lilly, P. T. So, D. A. Lauffenburger, R. D. Kamm, and J. M. Drazen, "Mechanotransduction through growth-factor shedding into the extracellular space," Nature 429, 83-86 (2004).
[CrossRef] [PubMed]

Lauffenburger, D. A.

D. J. Tschumperlin, G. Dai, I. V. Maly, T. Kikuchi, L. H. Laiho, A. K. McVittie, K. J. Haley, C. M. Lilly, P. T. So, D. A. Lauffenburger, R. D. Kamm, and J. M. Drazen, "Mechanotransduction through growth-factor shedding into the extracellular space," Nature 429, 83-86 (2004).
[CrossRef] [PubMed]

Lilly, C. M.

D. J. Tschumperlin, G. Dai, I. V. Maly, T. Kikuchi, L. H. Laiho, A. K. McVittie, K. J. Haley, C. M. Lilly, P. T. So, D. A. Lauffenburger, R. D. Kamm, and J. M. Drazen, "Mechanotransduction through growth-factor shedding into the extracellular space," Nature 429, 83-86 (2004).
[CrossRef] [PubMed]

Maly, I. V.

D. J. Tschumperlin, G. Dai, I. V. Maly, T. Kikuchi, L. H. Laiho, A. K. McVittie, K. J. Haley, C. M. Lilly, P. T. So, D. A. Lauffenburger, R. D. Kamm, and J. M. Drazen, "Mechanotransduction through growth-factor shedding into the extracellular space," Nature 429, 83-86 (2004).
[CrossRef] [PubMed]

McVittie, A. K.

D. J. Tschumperlin, G. Dai, I. V. Maly, T. Kikuchi, L. H. Laiho, A. K. McVittie, K. J. Haley, C. M. Lilly, P. T. So, D. A. Lauffenburger, R. D. Kamm, and J. M. Drazen, "Mechanotransduction through growth-factor shedding into the extracellular space," Nature 429, 83-86 (2004).
[CrossRef] [PubMed]

Padera, T. P.

T. P. Padera, B. R. Stoll, P. T. C. So, and R. K. Jain, "Conventional and high-speed intravital multiphoton laser scanning microscopy of microvasculature, lymphatics, and leukocyte-endothelial interactions," Mol. Imaging 1, 9-15 (2002).
[CrossRef]

Raab, G.

D. J. Tschumperlin, J. D. Shively, M. A. Swartz, E. S. Silverman, K. J. Haley, G. Raab, and J. M. Drazen, "Bronchial epithelial compression regulates MAP kinase signaling and HB-EGF-like growth factor expression," Am. J. Physiol. Lung Cell Mol. Physiol. 282, L904-911 (2002).

Shively, J. D.

T. Kikuchi, J. D. Shively, J. S. Foley, J. M. Drazen, and T. D. J., "Differentiation-dependent responsiveness of bronchial epithelial cells to IL-4/13 stimulation," Am. J. Physiol. Lung Cell Mol. Physiol. 287, L119-126 (2004).
[CrossRef]

D. J. Tschumperlin, J. D. Shively, M. A. Swartz, E. S. Silverman, K. J. Haley, G. Raab, and J. M. Drazen, "Bronchial epithelial compression regulates MAP kinase signaling and HB-EGF-like growth factor expression," Am. J. Physiol. Lung Cell Mol. Physiol. 282, L904-911 (2002).

Silverman, E. S.

D. J. Tschumperlin, J. D. Shively, M. A. Swartz, E. S. Silverman, K. J. Haley, G. Raab, and J. M. Drazen, "Bronchial epithelial compression regulates MAP kinase signaling and HB-EGF-like growth factor expression," Am. J. Physiol. Lung Cell Mol. Physiol. 282, L904-911 (2002).

So, P. T.

D. J. Tschumperlin, G. Dai, I. V. Maly, T. Kikuchi, L. H. Laiho, A. K. McVittie, K. J. Haley, C. M. Lilly, P. T. So, D. A. Lauffenburger, R. D. Kamm, and J. M. Drazen, "Mechanotransduction through growth-factor shedding into the extracellular space," Nature 429, 83-86 (2004).
[CrossRef] [PubMed]

So, P. T. C.

T. P. Padera, B. R. Stoll, P. T. C. So, and R. K. Jain, "Conventional and high-speed intravital multiphoton laser scanning microscopy of microvasculature, lymphatics, and leukocyte-endothelial interactions," Mol. Imaging 1, 9-15 (2002).
[CrossRef]

K. H. Kim, C. Buehler, and P. T. C. So, "High-speed, two-photon scanning microscope," Appl. Opt. 38, 6004-6009 (1999).
[CrossRef]

Stoll, B. R.

T. P. Padera, B. R. Stoll, P. T. C. So, and R. K. Jain, "Conventional and high-speed intravital multiphoton laser scanning microscopy of microvasculature, lymphatics, and leukocyte-endothelial interactions," Mol. Imaging 1, 9-15 (2002).
[CrossRef]

Swartz, M. A.

D. J. Tschumperlin, J. D. Shively, M. A. Swartz, E. S. Silverman, K. J. Haley, G. Raab, and J. M. Drazen, "Bronchial epithelial compression regulates MAP kinase signaling and HB-EGF-like growth factor expression," Am. J. Physiol. Lung Cell Mol. Physiol. 282, L904-911 (2002).

Tschumperlin, D. J.

N. Kojic, M. Kojic, and D. J. Tschumperlin, "Computational modeling of extracellular mechanotransduction," Biophys. J. 90, 4261-4270 (2006).
[CrossRef] [PubMed]

D. J. Tschumperlin, G. Dai, I. V. Maly, T. Kikuchi, L. H. Laiho, A. K. McVittie, K. J. Haley, C. M. Lilly, P. T. So, D. A. Lauffenburger, R. D. Kamm, and J. M. Drazen, "Mechanotransduction through growth-factor shedding into the extracellular space," Nature 429, 83-86 (2004).
[CrossRef] [PubMed]

D. J. Tschumperlin, J. D. Shively, M. A. Swartz, E. S. Silverman, K. J. Haley, G. Raab, and J. M. Drazen, "Bronchial epithelial compression regulates MAP kinase signaling and HB-EGF-like growth factor expression," Am. J. Physiol. Lung Cell Mol. Physiol. 282, L904-911 (2002).

Am. J. Physiol. Lung Cell Mol. Physiol.

T. Kikuchi, J. D. Shively, J. S. Foley, J. M. Drazen, and T. D. J., "Differentiation-dependent responsiveness of bronchial epithelial cells to IL-4/13 stimulation," Am. J. Physiol. Lung Cell Mol. Physiol. 287, L119-126 (2004).
[CrossRef]

D. J. Tschumperlin, J. D. Shively, M. A. Swartz, E. S. Silverman, K. J. Haley, G. Raab, and J. M. Drazen, "Bronchial epithelial compression regulates MAP kinase signaling and HB-EGF-like growth factor expression," Am. J. Physiol. Lung Cell Mol. Physiol. 282, L904-911 (2002).

Appl. Opt.

Biophys. J.

N. Kojic, M. Kojic, and D. J. Tschumperlin, "Computational modeling of extracellular mechanotransduction," Biophys. J. 90, 4261-4270 (2006).
[CrossRef] [PubMed]

Comput. Biomed. Res.

C. K. Chow and T. Kaneko, "Automatic boundary detection of the left ventricle from cineangiograms," Comput. Biomed. Res. 5, 388-410 (1972).
[CrossRef] [PubMed]

Mol. Imaging

T. P. Padera, B. R. Stoll, P. T. C. So, and R. K. Jain, "Conventional and high-speed intravital multiphoton laser scanning microscopy of microvasculature, lymphatics, and leukocyte-endothelial interactions," Mol. Imaging 1, 9-15 (2002).
[CrossRef]

Nature

D. J. Tschumperlin, G. Dai, I. V. Maly, T. Kikuchi, L. H. Laiho, A. K. McVittie, K. J. Haley, C. M. Lilly, P. T. So, D. A. Lauffenburger, R. D. Kamm, and J. M. Drazen, "Mechanotransduction through growth-factor shedding into the extracellular space," Nature 429, 83-86 (2004).
[CrossRef] [PubMed]

Other

B. Horn, Robot vision, MIT Press ed., MIT electrical engineering and computer science series (MIT Press; McGraw-Hill, Cambridge, Mass., 1986), pp. x, 509 p.

B. Jähne, Digital Image Processing: Concepts, Algorithms, and Scientific Applications, 6th Ed. (Springer-Verlag Telos, New York, 1997).

R. C. Gonzalez and R. E. Woods, Digital Image Processing (Prentice Hall, Upper Saddle River, NJ, 2002).

D. A. Forsyth and J. Ponce, Computer Vision: A modern approach (Prentice Hall, Upper Saddle River, NJ, 2003).

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Figures (10)

Fig. 1.
Fig. 1.

Schematic of the high-speed two-photon microscope imaging system: Infrared excitation pulsed beam (optical path depicted in red) is scanned through a polygonal mirror (fast, horizontal) and a galvanometric mirror (slow, vertical) to generate raster scanning on a sample. The emitted fluorescence (optical path depicted in green) is collected by the objective lens and detected by PMT after spectral filtration by a dichroic mirror and a barrier filter.

Fig. 2.
Fig. 2.

Schematic showing the interface of the two-photon imaging system with transwell cell culture insert. The HSTPM in interfaced to an upright microscope, hence the transwell is inverted on a custom stage. A controlled pressure source provides pneumatic pressure via a tube going through the plug on the apical side of the cells. The bottom of the transwell (area ~0.33cm2) is covered with 100μL of media containing fluorescent FITC-dextran, which diffuses into the LIS of the cells (bounded by tight junctions apically). For convenience, we define the z-axis as the baso-apical depth axis, and the x and y axes as the corresponding planar axes.

Fig. 3.
Fig. 3.

Cultured normal human bronchial epithelial cells, with FITC-dextran (white) in the extracellular space. (a) Stack of 2-D images obtained by the high-speed two-photon microscope, scale bar is 10 microns. (b) Schematic of two neighboring cells, dashed line indicates an imaging z-plane. (c) Reconstructed image in the z-x plane, bar is 5 microns.

Fig. 4.
Fig. 4.

Comparison of image segmentation algorithms. White regions correspond to the LIS, dark regions correspond to intracellular space. (a) Original, raw image. (b) Image analyzed using flat thresholding. (c) Image analyzed using standard adaptive thresholding. (d) Image analyzed using the WDAT algorithm.

Fig. 5.
Fig. 5.

Comparison of flat threshold settings. Analysis of the raw image in Fig. 4(a) (here in lower right corner) for a range of flat thresholds chosen based on the mean (average) intensity value of the raw image and the corresponding standard deviation of the mean. Upper left corner: threshold=mean intensity value-0.5*(standard deviation of the mean). Upper right corner: threshold=mean intensity value of the raw image. Black and white images: white is LIS and black is intracellular space.

Fig. 6.
Fig. 6.

Detailed comparison of adaptive thresholding and WDAT. The raw intensity image of a ~30×20μm region is shown on the left of each segmentation image for comparison. (a) Adaptive thresholding with a high relative threshold parameter results in patchy, overly-sparse LIS structures. (b) Adaptive thresholding with a moderate relative threshold is superior to standard thresholding, but the artificially high local average intensities at LIS intersections results in misclassified pixels at these regions. (c) Adaptive thresholding with a low threshold results in small patches intracellular regions being incorrectly classified as LIS space (d) The WDAT method addresses both of these LIS segmentation issues.

Fig. 7.
Fig. 7.

Outline of the WDAT image analysis procedure. Images are preprocessed with a median filter to remove shot noise (step 1), local averages are calculated using weighted directional averaging filters (step 2), a threshold is applied relative to each local average (step 3), segmentation results are combined (step 4), and removal of regions uncharacteristic of LIS regions (small, disconnected regions) is performed on the combined segmentation (step 5).

Fig. 8.
Fig. 8.

Comparison of cell and extracellular fluorescent labeling. Left: combined pseudocolor image where the inside of the cell was labeled with CellTracker Green (green) and the extracellular space with a TexasRed dextran (red). Middle top: grayscale TexasRed image. Middle bottom: grayscale CellTracker Green image. Right top: analyzed TexasRed image (white is LIS). Right bottom: analyzed, inverted CellTracker image (white is LIS). The pixel agreement in the segmentation between the analyzed Celltracker and TexasRed images was 95%.

Fig. 9.
Fig. 9.

Timecourse of changes in LIS dimensions. Analyzed and raw images for 3 consecutive 1micron sections for t=0 (pre-collapse) and 20, 600 seconds after application of a constant 30cmH2O transcellular pressure gradient; the white region is LIS, determined using WDAT algorithm. The corresponding LIS volume change plot is shown, normalized to the initial LIS volume, obtained by summing all of the white pixels (LIS) for a stack at a given time point and normalizing to the t=0 value.

Fig. 10.
Fig. 10.

Full LIS image stacks at 0 and 600 seconds. Top row: WDAT analyzed images for consecutive 1micron sections (white is LIS). Middle row: original raw images. Bottom row: combined image (original and inverted analyzed image, black is LIS) to demonstrate the effectiveness of WDAT algorithm in identifying the LIS.

Equations (5)

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

I h x y ¯ = Δ = N N 1 Q e Δ 2 2 σ 2 I ( x + Δ , y )
I v x y ¯ = Δ = N N 1 Q e Δ 2 2 σ 2 I x y + Δ
I d 2 x y ¯ = Δ = N N 1 Q e Δ 2 2 σ 2 I x Δ y + Δ
I d 2 x y ¯ = Δ = N N 1 Q e Δ 2 2 σ 2 I x Δ y + Δ
LIS x y = { 1 if I x y > T + I h x y ¯ 1 if I x y > T + I v x y ¯ 1 if I x y > T + I d 1 x y ¯ 1 if I x y > T + I d 2 x y ¯ 0 otherwise

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