Confocal Fluorescence Anisotropy and FRAP Imaging of α-Synuclein Amyloid Aggregates in Living Cells

We assessed the intracellular association states of the Parkinson's disease related protein α-synuclein (AS) in living cells by transfection with a functional recombinant mutant protein (AS-C4) bearing a tetracysteine tag binding the fluorogenic biarsenical ligands FlAsH and ReAsH, The aggregation states of AS-C4 were assessed by in situ microscopy of molecular translational mobility with FRAP (fluorescence recovery after photobleaching) and of local molecular density with confocal fluorescence anisotropy (CFA). FRAP recovery was quantitative and rapid in regions of free protein, whereas AS in larger aggregates was>80% immobile. A small 16% recovery characterized by an apparent diffusion constant of 0.03–0.04 µm2/s was attributed to the dynamics of smaller, associated forms of AS-C4 and the exchange of mobile species with the larger immobile aggregates. By CFA, the larger aggregates exhibited high brightness and very low anisotropy, consistent with homoFRET between closely packed AS, for which a Förster distance (Ro) of 5.3 nm was calculated. Other bright regions had high anisotropy values, close to that of monomeric AS, and indicative of membrane-associated protein with both low mobility and low degree of association. The anisotropy-fluorescence intensity correlations also revealed regions of free protein or of small aggregates, undetectable by conventional fluorescence imaging alone. The combined strategy (FRAP+CFA) provides a highly sensitive means for elucidating both the dynamics and structural features of protein aggregates and other intracellular complexes in living cells, and can be extended to other amyloid systems and to drug screening protocols

Effects of incomplete decay in fluorescence lifetime estimation

Fluorescence lifetime imaging has emerged as an important microscopy technique, where high repetition rate lasers are the primary light sources. As fluorescence lifetime becomes comparable to intervals between consecutive excitation pulses, incomplete fluorescence decay from previous pulses can superimpose onto the subsequent decay measurements. Using a mathematical model, the incomplete decay effect has been shown to lead to overestimation of the amplitude average lifetime except in mono-exponential decays. An inverse model is then developed to correct the error from this effect and the theoretical simulations are tested by experimental results

Quantum Dots as Templates for Self-Assembly of Photoswitchable Polymers: Small, Dual-Color Nanoparticles Capable of Facile Photomodulation

A photomodulatable amphiphilic polymer has been synthesized with a backbone of poly[isobutylene-alt-maleic anhydride] and pendant dodecyl alkyl chains, Lucifer Yellow (LY) fluorescent probes, and diheteroarylethenes photochromic (PC) groups. The latter serve as reversible UV-activated FRET acceptors for the LY donors. We characterized the spectral and switching properties of the polymer in an organic solvent (CHCl₃). In an aqueous medium the polymer forms polymersomes, constituting fluorescence probes ∼75 nm in diameter. Self-assembly of the polymer on the surface of a quantum dot (QD) serving as a template creates a dual-color photoswitchable nanoparticle (psNP) with improved properties due to the increase in polymer density and efficiency of PC photoconversion. The psNP exhibits a second QD red emission band that functions as an internal standard requiring only a single excitation wavelength, and is much reduced in size (<20 nm diameter) compared to the polymersomes. The QD template also greatly increases the depth of modulation by photochromic FRET of the LY emission monitored by both steady-state and time-resolved (lifetime) fluorescence (from 20%→70%, and from 12%→55%, respectively).Facultad de Ciencias ExactasInstituto de Investigaciones Fisicoquímicas Teóricas y Aplicada

Confocal and multiphoton imaging of intracellular Ca²⁺

This chapter compares the imaging capabilities of a range of systems including multiphoton microscopy in regard to measurements of intracellular Ca&lt;sup&gt;2+&lt;/sup&gt; within living cells. In particular, the excitation spectra of popular fluorescent Ca&lt;sup&gt;2+&lt;/sup&gt; indicators are shown during 1P and 2P excitation. The strengths and limitations of the current indicators are discussed along with error analysis which highlights the value of matching the Ca&lt;sup&gt;2+&lt;/sup&gt; affinity of the dye to a particular aspect of Ca&lt;sup&gt;2+&lt;/sup&gt; signaling. Finally, the combined emission spectra of Ca&lt;sup&gt;2+&lt;/sup&gt; and voltage sensitive dyes are compared to allow the choice of the optimum combination to allow simultaneous intracellular Ca&lt;sup&gt;2+&lt;/sup&gt; and membrane voltage measurement