No abstract

Amphiphilic donor-acceptor meso-ethynyl porphyrins with polar pyridinium electron-acceptor head groups and hydrophobic dialkyl-aniline electron donors have high molecular hyperpolarizabilities (as measured by hyper-Rayleigh scattering) and high affinities for biological membranes. When bound to water droplets in dodecane, or to the plasma membranes of living cells, they can be used for second harmonic generation (SHG) microscopy; an incident light of wavelength 840 nm generates a strong frequency-doubled signal at 420 nm. Copper(II) and nickel(II) porphyrin complexes give similar SHG signals to those of the free-base porphyrins, while exhibiting no detectable two-photon excited fluorescence.

Nonlinear optical imaging has revolutionized microscopy for the life sciences. Second harmonic generation (SHG), the younger sibling of two-photon excited fluorescence (2PF), is a technique that can produce high resolution images from deep inside biological tissues. Second harmonic light is generated by the coherent scattering of an ensemble of aligned chromophores in a focused, pulsed laser beam. SHG is only generated at the focal spot, reducing the background signal, and requires ordered chromophores, so is highly structure-specific. In contrast to two-photon fluorescence, the physical process that creates the signal does not require the formation of excited states, allowing elimination of harmful photochemistry. While the SHG of native proteins and biopolymers is well known, the use of exogenous dyes can provide SHG contrast from areas without a sufficiently high intrinsic quadratic hyperpolarizability, β. Dyes for SHG primarily target lipid bilayers; a trait that, combined with sensitivity to transmembrane potential, allows monitoring of action potentials in a variety of excitable cells, most importantly mammalian neurons. This article summarizes the principles of SHG imaging and explores approaches for maximizing the SHG signal from a biological specimen. We survey methods of optimizing the optical set-up, enhancing the β of the dye and achieving biological compatibility. In conclusion, we examine novel applications of SHG imaging and highlight promising directions for the development of the field.

NMDA receptors are important for synaptic plasticity, including long-term potentiation (LTP) and long-term depression (LTD).To help investigate the precise location of the NMDA receptors that are required for different types of synaptic plasticity, we synthesized a caged form of the use-dependent NMDA receptor antagonist MK801, which we loaded into individual neurons in vitro, followed by compartment-specific uncaging. We used this method to investigate timing-dependent plasticity at layer 4-layer 2/3 synapses of mouse barrel cortex. Somatodendritic photorelease of MK801 in the postsynaptic neuron produced a use-dependent block of synaptic NMDA receptor-mediated currents and prevented the induction of LTP. Compartment-specific photorelease of MK801 in the presynaptic neuron showed that axonal, but not somatodendritic, presynaptic NMDA receptors are required for induction of LTD. The rate of use-dependent block of postsynaptic NMDA receptor current was slower following induction of LTD, consistent with a presynaptic locus of expression. Thus, this new caged compound has demonstrated the axonal location of NMDA receptors required for induction and the presynaptic locus of expression of LTD at layer 4-layer 2/3 synapses.

Numerous dyes are available or under development for probing the structural and functional properties of biological membranes. Exogenous chromophores adopt a range of orientations when bound to membranes, which have a drastic effect on their biophysical behavior. Here, we present a method that employs optical anisotropy data from three polarization-imaging techniques to establish the distribution of orientations adopted by molecules in monolayers and bilayers. The resulting probability density functions, which contain the preferred molecular tilt μ and distribution breadth γ, are more informative than an average tilt angle [φ]. We describe a methodology for the extraction of anisotropy data through an image-processing technology that decreases the error in polarization measurements by about a factor of four. We use this technique to compare di-4-ANEPPS and di-8-ANEPPS, both dipolar dyes, using data from polarized 1-photon, 2-photon fluorescence and second-harmonic generation imaging. We find that di-8-ANEPPS has a lower tilt but the same distributional width. We find the distribution of tilts taken by di-4-ANEPPS in two phospholipid membrane models: giant unilamellar vesicles and water-in-oil droplet monolayers. Both models result in similar distribution functions with average tilts of 52° and 47°, respectively.

The established approach to design a molecule with strong second-order nonlinear optical (NLO) activity is to connect an electron-donor to an electron-acceptor via a p-conjugated bridge, to generate push-pull system. Surprisingly, we have found that dyes with large first hyperpolarizabilities, and which exhibit strong second harmonic generation (SHG), can be created just by attaching an electron-donor to a porphyrin. The free-base porphyrin core is sufficiently electron-deficient that the hyperpolarizability does not increase on addition of a pyridinium electron-acceptor.

We report the synthesis of four new cationic dipolar push-pull dyes, together with an evaluation of their photophysical and photobiological characteristics pertinent for imaging membranes by fluorescence and second harmonic generation. All four dyes consist of an N,N-diethylaniline electron-donor conjugated to a pyridinium electron-acceptor via a thiophene bridge, with either vinylene (-CH=CH-) or ethynylene 10 (-CC-) linking groups, and with either singly-charged or doubly-charged pyridinium terminals. The absorption and fluorescence behavior of these dyes was compared to a commercially available fluorescent membrane stain, the styryl dye FM4-64. The hyperpolarizabilities of all dyes were compared using hyper-Rayleigh scattering at 800 nm. Cellular uptake, localization, toxicity and phototoxicity were evaluated using tissue cell cultures (HeLa, SK-OV-3 and MDA-231). Replacing the central alkene bridge 15 of FM4-64 with a thiophene does not substantially change the absorption, fluorescence or hyperpolarizability, whereas changing the vinylene-links to ethynylenes shifts the absorption and fluorescence to shorter wavelengths, and reduces the hyperpolarizability by about a factor of two. SHG and fluorescence imaging experiments in live cells showed that the doubly-charged thiophene dyes localize in plasma membranes, and exhibit lower internalization rates compared to FM4-64, resulting in 20 less signal from the cell cytosol. At a typical imaging concentration of 1 µM, the doubly-charged dyes showed no significant light or dark toxicity, whereas the singly-charged dyes are phototoxic even at 0.5 µM, and toxic in the dark at concentrations above 5 µM. The doubly-charged dyes showed phototoxicity at concentrations greater than 10 µM, although they do not generate singlet oxygen, indicating that the phototoxicity is type I rather than type II. Our data demonstrate that the doubly-charged thiophene dyes 25 are more effective than FM4-64 as nonlinear optical imaging agents for live cells.

Neurons communicate by using electrical signals, mediated by transient changes in the voltage across the plasma membrane. Optical techniques for visualizing these transmembrane potentials could revolutionize the field of neurobiology by allowing the spatial profile of electrical activity to be imaged in real time with high resolution, along individual neurons or groups of neurons within their native networks.1, 2 Second harmonic generation (SHG) is one of the most promising methods for imaging membrane potential, although so far this technique has only been demonstrated with a narrow range of dyes.3 Here we show that SHG from a porphyrin-based membrane probe gives a fast electro-optic response to an electric field which is about 5–10 times greater than that of conventional styryl dyes. Our results indicate that porphyrin dyes are promising probes for imaging membrane potential.