Cell biology beyond the diffraction limit: near-field scanning optical microscopy

Abstract

Throughout the years, fluorescence microscopy has proven to be an extremely versatile tool for cell biologists to study live cells. Its high sensitivity and non-invasiveness, together with the ever-growing spectrum of sophisticated fluorescent indicators, ensure that it will continue to have a prominent role in the future. A drawback of light microscopy is the fundamental limit of the attainable spatial resolution – ∼250 nm – dictated by the laws of diffraction. The challenge to break this diffraction limit has led to the development of several novel imaging techniques. One of them, near-field scanning optical microscopy (NSOM), allows fluorescence imaging at a resolution of only a few tens of nanometers and, because of the extremely small near-field excitation volume, reduces background fluorescence from the cytoplasm to the extent that single-molecule detection sensitivity becomes within reach. NSOM allows detection of individual fluorescent proteins as part of multimolecular complexes on the surface of fixed cells, and similar results should be achievable under physiological conditions in the near future.

Optical Antenna-Based Fluorescence Correlation Spectroscopy to Probe the Nanoscale Dynamics of Biological Membranes

“…Reducing this gap between optical resolution and the size of lipid rafts to gain better insights on membrane organization at the nanoscale is currently a field of active research. With the advent of super-resolution optical microscopy approaches such as single molecule localization methods [53][54][55] , stimulated emission depletion (STED) microscopy [56][57][58] and nearfield scanning optical microscopy (NSOM) 8,[59][60][61][62] , it is now becoming clearer that lipids and proteins can indeed organize in nanometric compartments on the cell membrane, albeit a 6 consensus in terms of their sizes and dynamics has not yet been reached. In terms of dynamic measurements at the nanoscale, NSOM has been combined with FCS to show anomalous diffusion of ganglioside GM1 on living cell membranes at sizes smaller than 120nm.…”

Super-resolution measurements related to uncertainty relations in optical and biological fluorescence systems

“…There are different optical [8][9][10][11][12][13][14][15] and biological [2][3][4][5][6][7][16][17][18][19][20][21][22][23][24][25][26][27][28] systems in which uncertainty relations can be used to obtain super-resolution measurements. In the NSOM method super-resolution measurements are made on evanescent waves in which the wavevector in the propagation direction becomes imaginary increasing the perpendicular wavevectors components.…”

“…I describe, shortly, in the next Section the optical super-resolution methods known as Near Field Scanning Optical Microscopes ( NSOM) [8][9][10][11][12][13], hyperlens system [14,15] , and structured illumination microscopy (SIM) [2,4]. I show that the common feature of these methods is their relation to the above uncertainty relation.…”

Preparation and characteristic analysis of nanofacula array

“…NSOM breaks through the optical diffraction limit and achieves unprecedented resolution 3 . Also, it retains the unusual characteristics of optical microscope, such as non-invasion, stability, low cost, as well as the optical contrast mechanism similar to traditional optics, which gives NSOM considerable versatility 4 , 5 . However, the low time resolution is the key factor that limit the development of NSOM in the biologic research, because of that long scanning time is required for large area and high resolution imaging, which cannot meet the study of single molecule localization and dynamic process in cells 6 .…”