Journal
JOURNAL OF MICROSCOPY
Volume 231, Issue 1, Pages 156-167Publisher
WILEY
DOI: 10.1111/j.1365-2818.2008.02026.x
Keywords
absorption coefficient; cell thickness; cell volume; concentrated dyes; refractive index; surface metrology; surface topography
Categories
Ask authors/readers for more resources
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.
Authors
I am an author on this paper
Click your name to claim this paper and add it to your profile.
Reviews
Recommended
No Data Available