Quantifying F-actin patches in single melanoma cells using total-internal reflection fluorescence microscopy.

Total-internal reflection fluorescence (TIRF) microscope is a unique technique for selective excitation of only those fluorophore molecules in a cellular environment, which are located at the sub-diffraction axial distance of a cell's contact-area. Despite this prominent feature of the TIRF microscope, making quantitative use of this technique has been a challenge, since the excitation intensity strongly depends on the axial position of a fluorophore molecule. Here, we present an easy-implemented data analysis method to quantitatively characterize the fluorescent signal, without considering the intensity-value. We use F-actin patches in single-melanoma cells as an example and define two quantities of elongation and surface density for F-actin patches at the contact-area of a melanoma cell. The elongation parameter can evaluate the dispersion of F-actin patches at the contact-area of a cell and is useful to classify the attaching, spreading, and expanding stages of a cell. Following that, we present the profile of the surface density of F-actin patches as a quantity to probe the spatio-temporal distribution of the F-actin patches at the contact-area of a cell. The data analysis methods that are proposed here will also be applicable in the image analysis of the other advanced optical microscopic methods.

Tuning fluorophore excitation in a total-internal-reflection-fluorescence microscopy.

In a total-internal-reflection-fluorescence-microscopy method, there is anisotropy in the polarized evanescent wave. Since the evanescent wave is used as an excitation field, the mentioned anisotropy is a disadvantage in using the total-internal-reflection-fluorescence-microscopy technique. Therefore, by theoretical and analytical approaches, and based on the Fresnel coefficients, the effect of three dielectrics media on the anisotropy of the evanescent wave is investigated. Following that, a proper combination of the cover glass, oil immersion, and prism for both living and non-living samples is suggested that not only enhances the intensity of the evanescent wave, but also and importantly, decreases the essential anisotropy of the evanescent wave.