Measurement of the absorption of concentrated dyes and their use for quantitative imaging of surface topography

We propose a method to image the surface topography of transparent objects. The space between the object and the opposite closely positioned surface (such as a cover glass or a slide) is filled with a strongly absorbing dye. The contrast is generated by recording a transmission image at a wavelength where the dye absorbs. Since the transmitted intensity depends on the depth of the dye layer, it carries information about the relief of the tested surface. With sufficiently concentrated dyes, nanometre unevenness of a surface can be detected. By using less‐concentrated solutions, it is possible to image and measure larger objects, such as biological cells. At the present stage, biological applications of the method are only semi‐quantitative, but the method still provides detailed information about cell shapes that is not readily obtainable with other imaging techniques.

[1]  B. Wiesner,et al.  Cell volume kinetics of adherent epithelial cells measured by laser scanning reflection microscopy: determination of water permeability changes of renal principal cells. , 2001, Biophysical journal.

[2]  G. Kroesen,et al.  Measurement of the complex refractive index of liquids in the infrared using spectroscopic attenuated total reflection ellipsometry: correction for depolarization by scattering. , 1995, Applied Optics.

[3]  M. Model,et al.  Concentrated dyes as a source of two‐dimensional fluorescent field for characterization of a confocal microscope , 2008, Journal of microscopy.

[4]  Zhihong Liu,et al.  Fluorescein–formaldehyde oligomeric pH indicator: Facile synthesis, characterization, and potential application , 2005 .

[5]  L. Michaelis.  The Reversible Polymerization and Molecular Aggregation. , 1950 .

[6]  A. Penzkofer,et al.  Optical constants measurements of strongly absorbing media. , 1986, Applied optics.

[7]  S. Timoshenko,et al.  THEORY OF PLATES AND SHELLS , 1959 .

[8]  M J Lab,et al.  Cell volume measurement using scanning ion conductance microscopy. , 2000, Biophysical journal.

[9]  W. Reichert,et al.  Integrated optical attenuated total reflection spectrometry of aqueous superstrates using prism-coupled polymer waveguides. , 1990, Analytical chemistry.

[10]  A S Verkman,et al.  Cell volume and plasma membrane osmotic water permeability in epithelial cell layers measured by interferometry. , 1996, Biophysical journal.

[11]  T. Litman,et al.  Measurement of Cell Volume Changes by Fluorescence Self-Quenching , 2002, Journal of Fluorescence.

[12]  Kyriacos A Athanasiou,et al.  Development and validation of vertical scanning interferometry as a novel method for acquiring chondrocyte geometry. , 2005, Journal of biomedical materials research. Part A.

[13]  K. Nugent,et al.  Quantitative phase microscopy: A new tool for investigating the structure and function of unstained live cells , 2004, Clinical and experimental pharmacology & physiology.

[14]  Chau-Hwang Lee,et al.  Noninterferometric differential confocal microscopy with 2-nm depth resolution , 1997 .

[15]  M. Kubista,et al.  Absorption and fluorescence properties of fluorescein , 1995 .

[16]  K. Healy,et al.  Quantification of the surface density of a fluorescent label with the optical microscope. , 2000, Journal of biomedical materials research.

[17]  R. Wepf,et al.  Noninvasive measurement of cell volume changes by negative staining. , 2005, Journal of biomedical optics.

[18]  S. Doglia,et al.  Technical report: Cell thickness measurements by confocal fluorescence microscopy on C3H10T1/2 and V79 cells. , 1998, International journal of radiation biology.

[19]  M. Model,et al.  A standard for calibration and shading correction of a fluorescence microscope. , 2001, Cytometry.

[20]  Takeo Kanade,et al.  Reconstructing specimens using DIC microscope images , 2003, IEEE Trans. Syst. Man Cybern. Part B.

[21]  David J. Whitehouse,et al.  Surfaces and their Measurement , 2002 .

[22]  M. Ashfold.  Absorption and Fluorescence , 1995 .

[23]  A. Penzkofer,et al.  Refractive-index measurement of absorbing condensed media. , 1984, Applied optics.

[24]  Use of a wedge cuvette in thin layer photometry and its application to oximetry , 2004, Pflügers Archiv.

[25]  M. Friebel,et al.  Determination of the complex refractive index of highly concentrated hemoglobin solutions using transmittance and reflectance measurements. , 2005, Journal of biomedical optics.

[26]  D. Axelrod.  Cell-substrate contacts illuminated by total internal reflection fluorescence , 1981, The Journal of cell biology.

[27]  R. Grygorczyk,et al.  Evaluation of rapid volume changes of substrate‐adherent cells by conventional microscopy 3D imaging , 2004, Journal of microscopy.

[28]  P. Forsgren,et al.  Confocal microscopy: important considerations for accurate imaging. , 2002, Methods in Cell Biology.

[29]  P. Bongrand,et al.  A novel role for E- and P-selectins: shape control of endothelial cell monolayers. , 1994, Journal of cell science.

[30]  Gerald T. Cooney,et al.  Fluid refractive index measurements using rough surface and prism minimum deviation techniques , 2004 .