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

• Published in: Journal of microscopy (Oxford) 2008-07, Vol.231 (1), p.156-167
• Main Authors: MODEL, M.A, KHITRIN, A.K, BLANK, J.L
• Format: Article
• Language: English
• Subjects: Absorption; Absorption coefficient; Cell Line, Tumor; cell thickness; cell volume; Coloring Agents - metabolism; concentrated dyes; Fluorescent Dyes - metabolism; Humans; Microscopy - instrumentation; Microscopy, Confocal - instrumentation; Microscopy, Confocal - methods; Neurons - metabolism; Neurons - ultrastructure; refractive index; Refractometry; Saccharomyces cerevisiae - metabolism; Saccharomyces cerevisiae - ultrastructure; surface metrology; Surface Properties; surface topography
• ISSN: 0022-2720; 1365-2818
• DOI: 10.1111/j.1365-2818.2008.02026.x

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. Conversion of the image grey scale into the units of vertical distance requires knowledge of the absorption coefficient of the dye. The same method that is used for imaging can be adapted to measure the absorption coefficient of concentrated dyes. The solution to be analyzed is placed between a glass slide and a spherical lens of known radius. The absorption coefficient is determined from attenuation of transmitted intensity as a function of the distance to the centre. At the same time, the interference pattern in the reflected image allows measurement of the refractive index of the dye.