Correlative Microscopy of Living Cells between Fluorescence and Quantitative Phase Imaging with a High Resolution Wavefront Sensor

Abstract

Phase visualization of cells is used for decades, with setups such as Zernike phase contrast or Nomarski-DIC equipments. Those techniques use the fact that light passing through a sample accumulates phase shift. Nevertheless, they are quite impossible to give information on the quantitative phase shift of each pixel of the image and are commonly used only to increase contrast. We describe here the use of quadri-wave lateral shearing interferometry [1] for wavefront sensing, in order to measure quantitatively the local phase shift within a sample, with a high sensitivity.We use a high resolution wavefront sensor and we get a 300x400 sampling points on the sample, with both phase and intensity information. The method is simple and can be implemented on a conventional microscope: its native bright-field illumination system is used as the light source, and the wavefront sensor has only to be mounted on a video port, with the possibility to combine phase and fluorescence imaging.Correlative microscopy between wide-field fluorescence and optical phase on COS-7 living cells is considered, either on regular substrate or fibronectin-coated micro-patterns. The phase visualization of intracellular organelles is clearly possible with a high contrast, and targeted fluorescence allows a precise and simultaneous identification of each component. A multidimensional image is then obtained by combining those two images. Many organelles, such as mitochondria and endocytosis vesicles, can be specifically determined by their relative phase shift and shape, with a high degree of confidence. Refractive index anisotropy is also used to visualize phase shift of anisotropic components, such as cytoskeleton, with different polarizations of transmitted light.[1] Bon, Maucort, Wattellier and Monneret, “Quadriwave lateral shearing interferometry for quantitative phase microscopy of living cells,” Opt. Express 17, 13080-13094 (2009).