Refractive Index of Cells Measured through a Microscope with Variable-Angle TIR

Abstract

The refractive index in the interior of single cells is interesting because it both indirectly senses the concentration of solutes in the cytoplasm, and also directly affects the evanescent field depth in quantitative studies using total internal reflection (TIR) fluorescence. However, only a few studies have directly measured the intracellular refractive index, notably that of Bereiter-Hahn et al (1979) using quantitative interference reflection contrast (IRC) microscopy. IRC microscopy produces sharp images but does contain some inherent (but partly surmountable) ambiguities, particularly in distinguishing refractive index effects from distance -to-substrate effects. We present an alternative: direct microscopic imaging of a collimated light beam reflected from the substrate/buffer/cell interference at variable angles of incidence. Above the TIR critical angle (which is a strong function of refractive index), the reflection is 100%, but in the immediate sub-critical angle zone, the reflection intensity is a very strong ascending function of incidence angle. By analyzing the angular position of that edge at each location in the field of view, the local refractive index can be estimated. In addition, by analyzing the steepness of the edge, the distance -to-substrate can be separately determined. We show how this non-fluorescence technique can be implemented in a modified objective-based TIR setup and we present results on both calibration samples and on chromaffin cells in primary culture. The optical technique suffers from rather poor lateral resolution, so subcellular organelles cannot be seen easily. However, the technique as combined with appropriate theory and calibration provides reasonable results for both refractive index (∼1.375) and for distance-to-substrate (∼100 nm) for the cells. Supported by NIH RO1-GM110289 to RWH and DA.