Superresolution size determination in fluorescence microscopy: A comparison between spatially modulated illumination and confocal laser scanning microscopy

Abstract

Articles you may be interested inA sensitive and versatile laser scanning confocal optical microscope for single-molecule fluorescence at 77 K Rev. Sci. Instrum. 81, 113705 (2010); 10.1063/1.3499260 Diode-pumped solid state laser light sources for confocal laser scanning fluorescence microscopy Fluorescence spectroscopy and transmission electron microscopy of the same isolated semiconductor nanocrystals Appl.A low-temperature scanning confocal and near-field optical microscope Rev.Recently developed far field light optical methods are a powerful tool to analyze biological nanostructures and their dynamics, in particular including the interior of three-dimensionally conserved cells. In this article, the recently described method of spatially modulated illumination ͑SMI͒ microscopy has been further extended to the online determination of the extension of small, subwavelength sized, fluorescent objects ͑nanosizing͒. Using fluorescence excitation with 488 nm, the determination of fluorescent labeled object diameters down to 40 nm corresponding to about 1/12th of the wavelength used for one-photon excitation could be shown. The results of the SMI nanosizing procedure for a detailed, systematic variation of the object diameter are presented together with a fast algorithm for online size evaluation. In addition, we show a direct comparison of the diameter of ''colocalization volumes'' between SMI nanosizing and conventional confocal laser scanning microscopy.

A Fluorescence-Based Technique to Construct Size Distributions from Single-Object Measurements: Application to the Extrusion of Lipid Vesicles

“…This section will describe a fast and easy single-particle scheme to convert integrated intensity into physical dimensions of fluorescent objects, both above and below optical resolution. Intensity distributions have previously been used to reveal the size of such subresolution objects as colloidal beads (43,44) and even human genes (45), but these procedures have required specialized microscope setups. We present here a general method readily applicable to all objects with a defined geometry.…”

Lectures

“…This system has been applied to measure the size of nuclear macromolecular complexes 2 and of individual small gene regions. The potential of such ‘light nanoscopy’ approaches extends to the ‘in situ’ analysis of cellular protein–protein and protein–nucleic acid interactions, at a measurement accuracy about two orders of magnitude smaller than the observation volume of a confocal laser scanning microscope using a high numerical aperture objective lens 3 .…”

Single Molecule Localization Microscopy of Mammalian Cell Nuclei on the Nanoscale

“…To test such predictions, the spatial resolution of conventional light microscopy (limited to about 200 nm in the object plane and ca. 600 nm along the optical axis) is not sufficient; and combinations of fluorescence microscopy techniques may be employed ( Spöri et al, 2004 ; Rossberger et al, 2013 ). Electron and enhanced resolution light microscopy (for review see Huang et al, 2009 ; Cremer and Masters, 2013 ) have been applied to study the chromatin distribution in mammalian cell nuclei on the nanoscale ( Rouquette et al, 2010 ).…”