Mapping of real-time morphological changes in the neuronal cytoskeleton with label-free wide-field second-harmonic imaging: a case study of nocodazole.

We demonstrate the use of wide-field high-throughput second-harmonic (SH) microscopy for investigating cytoskeletal morphological changes on the single-cell level. The method allows for real-time, in vitro, label-free measurements of cytoskeletal changes that can, under certain conditions, be quantified in terms of orientational distribution or in terms of changes in the number of microtubules. As SH generation is intrinsically sensitive to noncentrosymmetrically structured microtubules, but not to isotropic or centrosymmetric materials, we use it to probe the microtubule structure in the cytoskeleton when it undergoes dynamic changes induced by the application of nocodazole, a well-known microtubule-destabilizing drug that reversibly depolymerizes microtubules. In addition, the orientational directionality of microtubules in neurites and cell bodies is determined label-free using SH polarimetry measurements. Finally, we use spatiotemporal SH imaging to show label-free, real-time nocodazole-induced morphological changes in neurons of different age and in a single axon.

Endogenous SHG and 2PEF coherence imaging of substructures in neurons in 3D.

Neuronal morphology, long-distance transport and signalling critically depend on the organization of microtubules in the cytoskeleton. Second harmonic generation (SHG) imaging has been recognized as a potentially powerful tool for in situ label-free neuroimaging with specific sensitivity to microtubules. We study here the structural organization of microtubules in living neurons using a wide-field multiphoton microscope that performs 3D imaging using a structured illumination. This microscope allows label-free high throughput imaging of living mammalian neurons. We show that we can image structural correlations by taking advantage of the structured illumination and the coherence of the emitted light. The result allows us to study the microtubule organization throughout the development of the neuron and to differentiate between the regions of the cytoskeleton in the matured neuron.

Optical imaging of surface chemistry and dynamics in confinement.

We imaged the interfacial structure and dynamics of water in a microscopically confined geometry, in three dimensions and on millisecond time scales, with a structurally illuminated wide-field second harmonic microscope. The second harmonic images reported on the orientational order of interfacial water, induced by charge-dipole interactions between water molecules and surface charges. The images were converted into surface potential maps. Spatially resolved surface acid dissociation constant (pKa,s) values were determined for the silica deprotonation reaction by following pH-induced chemical changes on the curved and confined surfaces of a glass microcapillary immersed in aqueous solutions. These values ranged from 2.3 to 10.7 along the wall of a single capillary because of surface heterogeneities. Water molecules that rotate along an oscillating external electric field were also imaged.

Characterization of the interface of binary mixed DOPC:DOPS liposomes in water: The impact of charge condensation.

Solutions of liposomes composed of binary mixtures of anionic dioleoylphosphatidylserine (DOPS) and zwitterionic dioleoylphosphatidylcholine (DOPC) are investigated with label-free angle-resolved (AR) second harmonic scattering (SHS) and electrophoretic mobility measurements. The membrane surface potential is extracted from the AR-SHS response. The surface potential changes from -10 to -145 mV with varying DOPS content ( from 0% to 100%) and levels off already at ∼ 10 % DOPS content. The ζ-potential shows the same trend but with a drastically lower saturation value (-44 mV). This difference is explained by the formation of a condensed layer of Na+ counterions around the outer leaflet of the liposome as predicted by charge condensation theories for polyelectrolyte systems.

Electrolytes induce long-range orientational order and free energy changes in the H-bond network of bulk water.

Electrolytes interact with water in many ways: changing dipole orientation, inducing charge transfer, and distorting the hydrogen-bond network in the bulk and at interfaces. Numerous experiments and computations have detected short-range perturbations that extend up to three hydration shells around individual ions. We report a multiscale investigation of the bulk and surface of aqueous electrolyte solutions that extends from the atomic scale (using atomistic modeling) to nanoscopic length scales (using bulk and interfacial femtosecond second harmonic measurements) to the macroscopic scale (using surface tension experiments). Electrolytes induce orientational order at concentrations starting at 10 µM that causes nonspecific changes in the surface tension of dilute electrolyte solutions. Aside from ion-dipole interactions, collective hydrogen-bond interactions are crucial and explain the observed difference of a factor of 6 between light water and heavy water.

Probing rotational and translational diffusion of nanodoublers in living cells on microsecond time scales.

Nonlinear microscopes have seen an increase in popularity in the life sciences due to their molecular and structural specificity, high resolution, large penetration depth, and volumetric imaging capability. Nonetheless, the inherently weak optical signals demand long exposure times for live cell imaging. Here, by modifying the optical layout and illumination parameters, we can follow the rotation and translation of noncentrosymetric crystalline particles, or nanodoublers, with 50 µs acquisition times in living cells. The rotational diffusion can be derived from variations in the second harmonic intensity that originates from the rotation of the nanodoubler crystal axis. We envisage that by capitalizing on the biocompatibility, functionalizability, stability, and nondestructive optical response of the nanodoublers, novel insights on cellular dynamics are within reach.

High throughput second harmonic imaging for label-free biological applications.

Second harmonic generation (SHG) is inherently sensitive to the absence of spatial centrosymmetry, which can render it intrinsically sensitive to interfacial processes, chemical changes and electrochemical responses. Here, we seek to improve the imaging throughput of SHG microscopy by using a wide-field imaging scheme in combination with a medium-range repetition rate amplified near infrared femtosecond laser source and gated detection. The imaging throughput of this configuration is tested by measuring the optical image contrast for different image acquisition times of BaTiO3 nanoparticles in two different wide-field setups and one commercial point-scanning configuration. We find that the second harmonic imaging throughput is improved by 2-3 orders of magnitude compared to point-scan imaging. Capitalizing on this result, we perform low fluence imaging of (parts of) living mammalian neurons in culture.

Label-free second harmonic and hyper Rayleigh scattering with high efficiency.

We present a method to perform hyper Rayleigh scattering from aqueous solutions and second harmonic scattering measurements from unlabeled interfaces of liposomes and nanoparticles in dilute solutions. The water and interfacial response can be measured on a millisecond timescale, thus opening up the possibility to measure label-free time dependent transport processes in biological (membrane) systems.

A priori information and optimisation in polarimetry.

Polarimetric measurements are designed to obtain information pertaining to the system under study, however noise in the system limits the precision and hence information obtainable. Exploitation of a priori knowledge of the system allows for an improvement in the precision of experimental data. In this vein we present a framework for system design and optimisation based upon the Fisher information matrix, which allows easy incorporation of such a priori information. As such the proposed figure of merit is more complete than the commonly used condition number. Conditions of equivalence are considered, however a number of examples highlight the failings of the condition number under more general scenarios. Bounds on the achievable informational gains via multiple polarimeter arms are also given. Finally we present analytic results concerning error distribution in a Mueller matrix polar decomposition, allowing for a more accurate noise analysis in polarimetric experiments.