Optical Sectioning for Reflection Interference Microscopy: Quantitative Imaging at Soft Interfaces

Reflection interference contrast microscopy (RICM, also known as interference reflection microscopy) and related techniques have become of wide interest to the biophysical, soft matter, and biochemistry communities owing to their exquisite sensitivity for characterizing thin films or individual nanoscopic objects adsorbed onto surfaces, or for monitoring cell–substrate interactions. Over the recent years, striking progress has been made to improve the sensitivity and the quantitative analysis of RICM. Its use in more complex environments, with spurious reflections stemming from a variety of structures in the sample, remains however challenging. In this paper, we demonstrate two optical sectioning methods that effectively reduce such background and can be readily implemented in a conventional RICM setup: line confocal detection and structured illumination microscopy. We characterize experimentally the benefits to image quality and demonstrate the use of the methods for quantitative imaging of complex biological and biomimetic samples: cellular membranes, thin organic films, biofunctional surfaces. We then discuss the benefits of each method and provide guidelines to arbitrate between sectioning and signal-to-noise ratio. Finally, we provide a detailed description of our experimental setup and a home-written image acquisition and processing software that should allow the interested reader to duplicate such a setup on a home-built or commercial microscope.