Spatial Cumulant Analysis to Study D2-Like Dopamine Receptor Dynamics on Plasma Membrane

Abstract

information encoded in the nanosecond fluorescence lifetime [2] or variations of the depletion power [3]. We show here that in the case of SIM it is possible to use SPLIT to analyze the information encoded into the images acquired at different illumination patterns. In particular an important aspect of SPLIT is that the Phasor Plot provides a visual, intuitive and direct evaluation of the acquired data [3].We demonstrate that knowledge of the illumination pattern can be used to perform SPLIT analysis and to reconstruct a super-resolved image bypassing the traditional Fourier reconstruction.As an application, we use the SPLIT-SIM approach to perform super-resolution imaging of chromatin-related structures.

Proposal for simultaneous analysis of fluorescence intensity fluctuations and resonance energy transfer (IFRET) measurements

“…Extensions to both Number and Brightness as well as Photon Counting Histogram techniques, termed ccN&B [63] and dual color PCH [64], respectively, measure the cross correlation of fluorescence amplitude fluctuations from two different colors, obtained from repeated measurements over time at a fixed position in a sample. More recently, Faust et al have extended molecular brightness based methods which measure fluctuations in the spatial domain with multi-color analysis in a technique called Two-Color Spatial Cumulant Analysis (2c-SpCA) [65]. Unfortunately, to date none of these multi-color approaches have incorporated a measurement of RET efficiency into their analysis procedure, and are therefore constrained to either resolving mixtures of non-interacting species to qualitatively probe the presence of heteromeric interactions in the presence of RET, or quantifying the hetero-oligomerization of protein complexes when no RET is occurring between the fluorescent probes.…”