Image enhancement of the in vivo leukocyte-endothelium contact zone using optical sectioning microscopy

Zhe Shen, Herbert Herling Lipowsky

Research output: Contribution to journalArticle

8 Citations (Scopus)

Abstract

A major determinant of the strength of leukocyte [white blood cell (WBC)] to endothelium [endothelial cell (EC)] adhesion is the contact area formed between the two cells, which is often obscured by out-of-focus information inherent to intravital microscopy. To improve visualization of the WBC-EC contact zone, techniques of optical sectioning microscopy were developed to enhance brightfield images of WBC-EC adhesion in postcapillary venules of the mesentery of the rat. A 50x/1.0 NA objective was held in a piezoelectric mount that was computer-driven, and video images were obtained by digitizing images from a CCD camera while focusing through the vertical direction in 1 μm steps over a depth of 16 μm. Using measurements of the microscope's optical transfer function, deconvolution of the central image was performed in the Fourier domain using the technique of singular value decomposition with Tikhonov-Miller regulation to remove out-of-focus information. Measurement of the length of the WBC-EC contact zone (Lc) in the original images yielded values on the order of 4.32 ± 1.08 μm (mean ± SD). The enhanced images showed a significantly 35% smaller Lc equal to 2.78 μm 0.70 μm. Topical application of the chemoattractant f-met-leu-phe resulted in a 26% increase in Lc to 3.49 ± 0.72 μm, thus suggesting that upregulation of adhesion molecules on the WBC membrane results in the recruitment of additional membrane area from surface ruffles into the zone of adhesion. Other advantages of the deconvolution were to visualize structural characteristics of the microvascular wall and parenchymal tissue in greater detail. Thus, brightfield optical sectioning microscopy may provide a valuable tool for in vivo studies of the microvasculature, and serves as a useful alternative to fluorescence microscopy without the undesirable effects of exogenous fluorophores and exposure to ultraviolet radiation.

Original languageEnglish (US)
Pages (from-to)521-535
Number of pages15
JournalAnnals of Biomedical Engineering
Volume25
Issue number3
DOIs
StatePublished - Jan 1 1997

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Image enhancement
Optical microscopy
Endothelial cells
Blood
Cell adhesion
Deconvolution
Adhesion
Fluorophores
Fluorescence microscopy
Optical transfer function
Singular value decomposition
Cell membranes
CCD cameras
Ultraviolet radiation
Rats
Microscopes
Visualization
Cells
Tissue
Membranes

All Science Journal Classification (ASJC) codes

  • Biomedical Engineering

Cite this

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title = "Image enhancement of the in vivo leukocyte-endothelium contact zone using optical sectioning microscopy",
abstract = "A major determinant of the strength of leukocyte [white blood cell (WBC)] to endothelium [endothelial cell (EC)] adhesion is the contact area formed between the two cells, which is often obscured by out-of-focus information inherent to intravital microscopy. To improve visualization of the WBC-EC contact zone, techniques of optical sectioning microscopy were developed to enhance brightfield images of WBC-EC adhesion in postcapillary venules of the mesentery of the rat. A 50x/1.0 NA objective was held in a piezoelectric mount that was computer-driven, and video images were obtained by digitizing images from a CCD camera while focusing through the vertical direction in 1 μm steps over a depth of 16 μm. Using measurements of the microscope's optical transfer function, deconvolution of the central image was performed in the Fourier domain using the technique of singular value decomposition with Tikhonov-Miller regulation to remove out-of-focus information. Measurement of the length of the WBC-EC contact zone (Lc) in the original images yielded values on the order of 4.32 ± 1.08 μm (mean ± SD). The enhanced images showed a significantly 35{\%} smaller Lc equal to 2.78 μm 0.70 μm. Topical application of the chemoattractant f-met-leu-phe resulted in a 26{\%} increase in Lc to 3.49 ± 0.72 μm, thus suggesting that upregulation of adhesion molecules on the WBC membrane results in the recruitment of additional membrane area from surface ruffles into the zone of adhesion. Other advantages of the deconvolution were to visualize structural characteristics of the microvascular wall and parenchymal tissue in greater detail. Thus, brightfield optical sectioning microscopy may provide a valuable tool for in vivo studies of the microvasculature, and serves as a useful alternative to fluorescence microscopy without the undesirable effects of exogenous fluorophores and exposure to ultraviolet radiation.",
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Image enhancement of the in vivo leukocyte-endothelium contact zone using optical sectioning microscopy. / Shen, Zhe; Lipowsky, Herbert Herling.

In: Annals of Biomedical Engineering, Vol. 25, No. 3, 01.01.1997, p. 521-535.

Research output: Contribution to journalArticle

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