Long-term measurement of spontaneous membrane fluctuations over a wide dynamic range in the living cell by low-coherent quantitative phase microscopy

Abstract

Surface topography and its dynamic fluctuations in live cultured cells were obtained by low-coherent quantitative phase microscopy (LC-QPM), using a reflection-type interference microscope employing the digital holographic technique with a low-coherent light source. Owing to the low coherency of the light-source, only the light reflected at a specific sectioning height of the sample generates interference fringes on the CCD camera. Because the digital holographic technique enables us to quantitatively measure the intensity and phase of the optical field, a nanometer-scale surface profile of a living cell can be obtained by capturing the light reflected by the cell membrane. The lateral and the vertical spatial resolution was 0.56 microns and 0.93 microns, respectively, and the mechanical stability of the phase measurement was better than 2 nanometers. The measurements were made at fast (21 frames/sec) and slow (2 frames/sec, time-lapse) frame rates and the slow measurements were performed over a period of 10 minutes. The temporal fluctuations of the cell membrane were analyzed by the mean-square-displacement (MSD) as a function of the time-difference τ. By merging the fast and slow data, the MSDs from τ = 50 msec to τ = 300 sec were obtained and wide-dynamic-range measurements of the MSDs from 2 nm 2 to over 100000 nm 2 were demonstrated. The results show significant differences among different cell types under various conditions.