Dye distance mapping using waveguide evanescent field fluorescence microscopy and its application to cell biology.
Previous studies have measured the distance between cells and the substratum at sites of adhesion via the emission of a fluorescent dye and waveguide methods. Here, we demonstrate a novel approach to measure the position of fluorescent dyes above a waveguide surface in the 10-200 nm distance range throughout an entire area, yielding a 2D dye distance map or a 3D contour plot. The dye is located in a multilayered Langmuir Blodgett (LB) film or in the plasma membrane of a cell. Waveguide evanescent field fluorescence (WEFF) images obtained using two different waveguide modes are employed allowing, with a simple mathematical approach, the calculation of dye distance maps. Ultra-thin steps made using LB technology, adhesion distances and the bending of the plasma membrane between focal adhesions of osteoblastic cells are shown as examples. The errors are discussed. False color representation of a dye distance map with four osteoblasts. The inset represents an overexposed WEFF image of the same field of view.
Department of Physics and Astronomy, The University of Western Ontario (Western University), London, Ontario, N6A 3K7, Canada.|RheinMain University of Applied Sciences, Department of Engineering Sciences/Institute of Microtechnologies, Am Brückweg 26, 65428, Rüsselsheim, Germany.
Frederik
F
Fleissner
Department of Physics and Astronomy, The University of Western Ontario (Western University), London, Ontario, N6A 3K7, Canada.|RheinMain University of Applied Sciences, Department of Engineering Sciences/Institute of Microtechnologies, Am Brückweg 26, 65428, Rüsselsheim, Germany.
Michael
M
Morawitz
Department of Physiology and Pharmacology, Schulich School of Medicine & Dentistry, The University of Western Ontario (Western University), London, Ontario, N6A 5C1, Canada.
S Jeffrey
SJ
Dixon
RheinMain University of Applied Sciences, Department of Engineering Sciences/Institute of Microtechnologies, Am Brückweg 26, 65428, Rüsselsheim, Germany.
Uwe
U
Langbein
Department of Physics and Astronomy, The University of Western Ontario (Western University), London, Ontario, N6A 3K7, Canada. smittler@uwo.ca.
Silvia
S
Mittler
D016475:3T3 Cells; D000818:Animals; D001692:Biological Transport; D005456:Fluorescent Dyes / Q000378:metabolism*; D051379:Mice; D008856:Microscopy, Fluorescence / Q000379:methods*; D010006:Osteoblasts / Q000166:cytology / Q000378:metabolism
D005456:Fluorescent Dyes
LB films; Waveguide Evanescent Field Fluorescence (WEFF) Microscopy; cell-substrate contacts; dye location from waveguide surface; optical waveguide; osteoblasts