Fluorescence Interference Contrast Microscopy - Experimental Setup

Experimental Setup

A silicon wafer is typically used as the reflective surface in a FLIC experiment. An oxide layer is then thermally grown on top of the silicon wafer to act as a spacer. On top of the oxide is placed the fluorescently labeled specimen, such as a lipid membrane, a cell or membrane bound proteins. With the sample system built, all that is needed is an epifluorescence microscope and a CCD camera to make quantitative intensity measurements.

The silicon dioxide thickness is very important in making accurate FLIC measurements. As mentioned before, the theoretical model describes the relative fluorescence intensity measured versus the fluorophore height. The fluorophore position cannot be simply read off of a single measured FLIC curve. The basic procedure is to manufacture the oxide layer with at least two known thicknesses (the layer can be made with photolithographic techniques and the thickness measured by ellipsometry). The thicknesses used depends on the sample being measured. For a sample with fluorophore height in the range of 10 nm, oxide thickness around 50 nm would be best because the FLIC intensity curve is steepest here and would produce the greatest contrast between fluorophore heights. Oxide thickness above a few hundred nanometers could be problematic because the curve begins to get smeared out by polychromatic light and a range of incident angles. A ratio of measured fluorescence intensities at different oxide thicknesses is compared to the predicted ratio to calculate the fluorophore height above the oxide .

The above equation can then be solved numerically to find . Imperfections of the experiment, such as imperfect reflection, nonnormal incidence of light and polychromatic light tend to smear out the sharp fluorescence curves. The spread in incidence angle can be controlled by the numerical aperture (N.A.). However, depending on the numerical aperture used, the experiment will yield good lateral resolution (x-y) or good vertical resolution (z), but not both. A high N.A. (~1.0) gives good lateral resolution which is best if the goal is to determine long range topography. Low N.A. (~0.001), on the other hand, provides accurate z-height measurement to determine the height of a fluorescently labeled molecule in a system.