Near-Field Fluorescence Microscopy: An Optical Nanotool to Study Protein Organization at the Cell Membrane

Abstract

The ability to study the structure and function of cell membranes and membrane components is fundamental to understanding cellular processes. This requires the use of methods capable of resolving structures with nanometer-scale resolution in intact or living cells. Although fluorescence microscopy has proven to be an extremely versatile tool in cell biology, its diffraction-limited resolution prevents the investigation of membrane compartmentalization at the nanometer scale. Near-field scanning optical microscopy (NSOM) is a relatively unexplored technique that combines both enhanced spatial resolution of probing microscopes and simultaneous measurement of topographic and optical signals. Because of the very small nearfield excitation volume, background fluorescence from the cytoplasm is effectively reduced, enabling the visualization of nano-scale domains on the cell membrane with single molecule detection sensitivity at physiologically relevant packing densities. In this article we discuss technological aspects concerning the implementation of NSOM for cell membrane studies and illustrate its unique advantages in terms of spatial resolution, background suppression, sensitivity, and surface specificity for the study of protein clustering at the cell membrane. Furthermore, we demonstrate reliable operation under physiological conditions, without compromising resolution or sensitivity, opening the road toward truly live cell imaging with unprecedented detail and accuracy.

10000 times volume reduction for fluorescence correlation spectroscopy using nano-antennas

“…They use optical phenomena such as stimulated emission depletion [3,4], and interference fringes in the excitation beam [5]. Other approaches rely on the use of near-field phenomena like total internal reflection [6], near-field fluorescence microscopy [7,8], and surface plasmon coupled emission [9,10], or mechanically confined compartments such as sub-microfluidic channels [11] or nanoholes [12][13][14][15].…”

Live endothelial cells imaged by Scanning Near‐field Optical Microscopy (SNOM): capabilities and challenges

“…Recently, SNOM has been applied in material science and for biological samples measurement, where both the transmission and reflection SNOM configuration can be used . This method seems to be a very efficient for nanometric exploration of tissue sections, chromosomes, erythrocytes, biological membranes as well as cells . Of particular importance in life sciences is the possibility to conduct the SNOM investigations in liquid environments, that gives the opportunity to observe biological samples under physiological conditions.…”

Implementation of NSOM to Biological Samples

“…In such a case, the NSOM microscope can be a very useful tool due to its high-resolution especially when combined with uorescent techniques. The latter one in conjunction with labeling methods could bring information about distribution of molecules of interest like, for example, surface receptors present in a cellular membrane [2,11]. Moreover, the measurements of optical properties in liquid environments will give the opportunity to observe changes of living cells in their natural environment [12].…”