The 3D structure and the local environment of stimuli-responsive microgels were investigated with the superresolution fluorescence microscopy method PAINT using Nile Red as solvatochromic dye.
We describe a new concept of multimodal super-resolution imaging which combines the cumulant analysis from Super-resolution Optical Fluctuation Imaging (SOFI) with the imprinting of three-dimensional, spectral or other information into peculiar Point-Spread Function (PSF) patterns. This concept allows for encoding multidimensional or multimodal information into a single image plane and to extract this information by an appropriate spatio-temporal correlation analysis of emitter fluctuations. Here, we develop the general theory of this concept, and present proof-of-principle experiments of three-dimensional super-resolution imaging.
Stimuli-sensitive poly(N-isopropylacrylamide) (PNIPAM) microgels have various prospective practical applications and uses in fundamental research. In this work, we use single particle tracking of fluorescently labeled PNIPAM microgels as a showcase for tuning microgel size by a rapid non-stirred precipitation polymerization procedure. This approach is well suited for prototyping new reaction compositions and conditions or for applications that do not require large amounts of product. Microgel synthesis, particle size and structure determination by dynamic and static light scattering are detailed in the protocol. It is shown that the addition of functional comonomers can have a large influence on the particle nucleation and structure. Single particle tracking by wide-field fluorescence microscopy allows for an investigation of the diffusion of labeled tracer microgels in a concentrated matrix of non-labeled microgels, a system not easily investigated by other methods such as dynamic light scattering.
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