Fluorescence interference contrast (FLIC) microscopy is a microscopic technique developed to achieve z-resolution on the nanometer scale.

FLIC occurs whenever fluorescent objects are in the vicinity of a reflecting surface (e.g. Si wafer). The resulting interference between the direct and the reflected light leads to a double sin2 modulation of the intensity, I, of a fluorescent object as a function of distance, h, above the reflecting surface. This allows for the nanometer height measurements.

FLIC microscope is well suited to measuring the topography of a membrane that contains fluorescent probes e.g. an artificial lipid bilayer, or a living cell membrane or the structure of fluorescently labeled proteins on a surface.

FLIC optical theory

General two layer system

The optical theory underlying FLIC was developed by Armin Lambacher and Peter Fromherz. They derived a relationship between the observed fluorescence intensity and the distance of the fluorophore from a reflective silicon surface.

, and the probability of measuring an emitted photon) a distance d gives the orientation of the transition dipole is proportional to the squared projection of the local electric field, which includes the effects of interference, on the direction of the transition dipole. given by , but the silicon surface does not perfectly reflect the incident light. Fresnel coefficients give the change in amplitude between an incident and reflected wave. The Fresnel coefficients, the indices of refraction of the two mediums and the polarization can be related by Snell's Law. The expressions for the reflection coefficients are: TE refers to the component of the electric field perpendicular to the plane of incidence and TM to the parallel component (The incident plane is defined by the plane normal and the propagation direction of the light). In cartesian coordinates, the local electric field is azimuthal to the plane of incidence. . Many of the parameters used above would vary in a normal experiment. The variation in the five following parameters should be included in this theoretical description.

• The coherence of the excitation light

• ) of excitation light

• ) of the excitation light

• ) of the fluorophore

• )

. Averaging over the first 4 parameters gives

is a distribution of the orientation angle of the fluorophore dipoles. The azimuthal. except that the parameter labels em are replaced with ex and in is replaced with out. The resulting fluorescence intensity measured is proportional to the product of the excitation probability and emission probability

and the distance of the fluorophore above the reflective surface. The fact that it is not an equality relation will have a significant effect on the experimental procedure.

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 10nm, oxide thickness around 50). . 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.

Analysis

The basic analysis involves fitting) to be a free parameter values into a histogram. The statistical error comes from two sources: the error in fitting of the optical theory to the data and the uncertainty in the thickness of the oxide layer. Systematic error.

References