This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. A C C E P T E D M A N U S C R I P T ACCEPTED MANUSCRIPT High-Precision FLIM-FRET in fixed and living cells reveals heterogeneity in a simple CFP-YFP fusion proteinMichael Millington, [a, b] G. Joan Grindlay, [c] Kirsten Altenbach, [a, d] Robert K. Neely, [a, b] Walter Kolch, [ c, e] Mojca Bencina , [ f ] Nick D. Read, [a, d] Anita C. Jones, [a, b] David T. F.Dryden, [a, b]

We describe the calibration of a technique allowing quantitative measurements of industrial coatings with thicknesses as small as 10-20 nm. Wax films doped with fluorescent rhodamine dye have been deposited by an electrospray method onto an optically flat surface of aluminium-coated glass. The films were of 220-450 nm peak thickness, which we measured with an optical profilometer using laser triangulation. Possession of a set of films allowing an absolute calibration of the fluorescence intensity versus thickness to be achieved for application in trials of the fluorescence method for measuring coating thicknesses of 10-20 nm under industrial process conditions.

Hot stamping foils are used in the printing mdustiy to achieve metallic effects on packaging, pseudo-holographic images for security applications, and other products. The performance of the foil in the stamping process is in part determined by the thickness of the release coating on the carrier foil. This coating is too thin (approximately lOnm) for successful application of conventional measurement methods. We describe a fluorescence-based optical technique to measure the thickness of this release coating on-line. A fluorescent rhodaniine dye added to the release coating allows excitation and detection in the visible part of the spectrum. Multimode optical fibre is used for excitation beam delivery and signal collection from a probe head situated on the coating machine. We outhne the system calibration and show some representative results from industrial trials.

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