GFP is a selective non-linear optical sensor of electrophysiological processes in Caenorhabditis elegans

Electrophysiology of the nematode Caenorhabditis elegans has the potential to bridge the wealth of information on the molecular biology and anatomy of this organism with the responses of selected cells and cellular neural networks associated with a behavioral response. In this paper we report that the nonlinear optical phenomenon of second harmonic generation (SHG) can be detected using green fluorescent protein (GFP) chimeras expressed in selected cells of living animals. Alterations in the SHG signal as a result of receptor ligand interactions and mechanical stimulation of the mechanosensory cells indicate that this signal is very sensitive to membrane potential. The results suggest that this approach to membrane potential measurements in C. elegans and in other biological systems could effectively couple data on selective locations within specific cells with functional responses that are associated with behavioral and sensory processes.     

Nonlinear optical properties of potential sensitive styryl dyes.

The nonlinear optical properties of dyes that alter their optical characteristics rapidly with membrane potential are described. The second harmonic signals from these dyes characterized in this paper are among the largest that have been detected to date. Structural conclusions are drawn from the second harmonic signals generated by the Langmuir Blodgett monolayers used in these measurements. Our results indicate that with appropriate instrumentation second harmonic signals could readily be detected from living cells stained with these dyes.     

High-resolution nonlinear optical imaging of live cells by second harmonic generation

By adapting a laser scanning microscope with a titanium sapphire femtosecond pulsed laser and transmission optics, we are able to produce live cell images based on the nonlinear optical phenomenon of second harmonic generation (SHG). Second harmonic imaging (SHIM) is an ideal method for probing membranes of living cells because it offers the high resolution of nonlinear optical microscopy with the potential for near-total avoidance of photobleaching and phototoxicity. The technique has been implemented on three cell lines labeled with membrane-staining dyes that have large nonlinear optical coefficients. The images can be obtained within physiologically relevant time scales. Both achiral and chiral dyes were used to compare image formation for the case of single- and double-leaflet staining, and it was found that chirality plays a significant role in the mechanism of contrast generation. It is also shown that SHIM is highly sensitive to membrane potential, with a depolarization of 25 mV resulting in an approximately twofold loss of signal intensity.     

Optical microscopy in photosynthesis

Emerging as well as the most frequently used optical microscopy techniques are reviewed and image contrast generation methods in a microscope are presented, focusing on the nonlinear contrasts such as harmonic generation and multiphoton excitation fluorescence. Nonlinear microscopy presents numerous advantages over linear microscopy techniques including improved deep tissue imaging, optical sectioning, and imaging of live unstained samples. Nonetheless, with the exception of multiphoton excitation fluorescence, nonlinear microscopy is in its infancy, lacking protocols, users and applications; hence, this review focuses on the potential of nonlinear microscopy for studying photosynthetic organisms. Examples of nonlinear microscopic imaging are presented including isolated light-harvesting antenna complexes from higher plants, starch granules, chloroplasts, unicellular alga Chlamydomonas reinhardtii, and cyanobacteria Leptolyngbya sp. and Anabaena sp. While focusing on nonlinear microscopy techniques, second and third harmonic generation and multiphoton excitation fluorescence microscopy, other emerging nonlinear imaging modalities are described and several linear optical microscopy techniques are reviewed in order to clearly describe their capabilities and to highlight the advantages of nonlinear microscopy.     

Electro-Optical Imaging Microscopy of Dye-Doped Artificial Lipidic Membranes

Artificial lipidic bilayers are widely used as a model for the lipid matrix in biological cell membranes. We use the Pockels electro-optical effect to investigate the properties of an artificial lipidic membrane doped with nonlinear molecules in the outer layer. We report here what is believed to be the first electro-optical Pockels signal and image from such a membrane. The electro-optical dephasing distribution within the membrane is imaged and the signal is shown to be linear as a function of the applied voltage. A theoretical analysis taking into account the statistical orientation distribution of the inserted dye molecules allows us to estimate the doped membrane nonlinearity. Ongoing extensions of this work to living cell membranes are discussed.     

Proposed test for detection of nonlinear responses in biological preparations exposed to RF energy

Demodulation of amplitude modulated radio frequency (RF) energy has been proposed as a mechanism for the biological responses to these fields. The experiment proposed here tests whether the electric and magnetic structures of biological cells exhibit the nonlinear responses necessary for demodulation. A high Q cavity and very low noise amplification can be used to detect ultraweak nonlinear responses that appear as a second harmonic of a RF field incident on the sample. Nonlinear fields scattered from metabolically active biological cells grown in monolayer or suspended in medium can be distinguished from nonlinearities of the apparatus. Estimates for the theoretical signal sensitivity and analysis of system noise indicate the possibility of detecting a microwave signal at 1.8 GHz (2nd harmonic of 900 MHz) as weak as one microwave photon per cell per second. The practical limit, set by degradation of the cavity Q, is extremely low compared to the much brighter thermal background, which has its peak in the infrared at a wavelength of about 17 microm and radiates 10(10) infrared photons per second per cell in the narrow frequency band within 0.5% of the peak. The system can be calibrated by introduction of known quantities of nonlinear material, e.g., a Schottky diode. For an input power of 160 microW at 900 MHz incident on such biological material, the apparatus is estimated to produce a robust output signal of 0.10 mV at 1.8 GHz if detected with a spectrum analyzer and a 30-dB gain low noise amplifier. The experimental threshold for detection of nonlinear interaction phenomena is 10(10) below the signal produced by a Schottky diode, giving an unprecedented sensitivity to the measurement of nonlinear energy conversion processes in living tissue.     

Sensitivity of second harmonic generation from styryl dyes to transmembrane potential

In this article we present results from the simultaneous nonlinear (second harmonic generation and two-photon excitation fluorescence) imaging and voltage clamping of living cells. Specifically, we determine the sensitivity to transmembrane potential of second harmonic generation by ANEP-chromophore styryl dyes as a function of excitation wavelength and dye structure. We have measured second harmonic sensitivities of up to 43% per 100 mV, more than a factor of four better than the nominal voltage sensitivity of the dyes under "one-photon" fluorescence. We find a dependence of voltage sensitivity on excitation wavelength that is consistent with a two-photon resonance, and there is a significant dependence of voltage sensitivity on the structure of the nonchromophore portion of the dyes.     

Characteristics of the generation of transmembrane current higher harmonics in lipid bilayers in glycerol and 1,2-propane diol solutions

It is shown that structural rearrangements of bilayer lipid membrane induced by glycerol and 1,2-propane diol result in noncompensated second harmonic of transmembrane current having quadratic dependence on input voltage and a weak dependence on frequency. The third harmonic has cubic dependence on input voltage and decreases with an increase in frequency. The mechanism is discussed according to which generation of non-compensated second harmonic is due to the formation of the intermediate lipid phase with viscoelastic properties differing from the initial bilayer in the process of structural rearrangements. The presence of this phase conditions the possibility of appearance of noncompensated second harmonic.     

Harmonic Nanoparticles for Regenerative Research

In this visualized experiment, protocol details are provided for in vitro labeling of human embryonic stem cells (hESC) with second harmonic generation nanoparticles (HNPs). The latter are a new family of probes recently introduced for labeling biological samples for multi-photon imaging. HNPs are capable of doubling the frequency of excitation light by the nonlinear optical process of second harmonic generation with no restriction on the excitation wavelength.Multi-photon based methodologies for hESC differentiation into cardiac clusters (maintained as long term air-liquid cultures) are presented in detail. In particular, evidence on how to maximize the intense second harmonic (SH) emission of isolated HNPs during 3D monitoring of beating cardiac tissue in 3D is shown. The analysis of the resulting images to retrieve 3D displacement patterns is also detailed.     

SHG nanoprobes: advancing harmonic imaging in biology

Second harmonic generating (SHG) nanoprobes have recently emerged as versatile and durable labels suitable for in vivo imaging, circumventing many of the inherent drawbacks encountered with classical fluorescent probes. Since their nanocrystalline structure lacks a central point of symmetry, they are capable of generating second harmonic signal under intense illumination - converting two photons into one photon of half the incident wavelength - and can be detected by conventional two-photon microscopy. Because the optical signal of SHG nanoprobes is based on scattering, rather than absorption as in the case of fluorescent probes, they neither bleach nor blink, and the signal does not saturate with increasing illumination intensity. When SHG nanoprobes are used to image live tissue, the SHG signal can be detected with little background signal, and they are physiologically inert, showing excellent long-term photostability. Because of their photophysical properties, SHG nanoprobes provide unique advantages for molecular imaging of living cells and tissues with unmatched sensitivity and temporal resolution.     

Second harmonic generation of glucose oxidase at the air/water interface

We present a study of the adsorption of the glucose oxidase enzyme (GOx) at the air/water interface, using the nonlinear optical technique of surface second harmonic generation (SSHG). Resonant SSHG experiments were achieved by probing the pi-pi* transition of the flavin adenine dinucleotide (FAD) chromophores embedded in the GOx protein. Because of the subsequent resonance enhancement of the signal, the second harmonic (SH) wave arising from the GOx entities adsorbed at the interface was detectable for protein bulk aqueous concentrations as low as 70 nM. The protein adsorption was followed, and, at high GOx coverage, a change in the orientation of the FAD chromophore was observed, indicating either a rearrangement or a reorientation of the protein at the interface. Inasmuch as GOx is negatively charged at the biological pH of 7, its interactions with charged surfactants were also investigated. As expected, spreading positively charged surfactants onto a partial protein monolayer was found to increase the GOx surface concentration, whereas in the case of negatively charged surfactants, the GOx surface concentration decreased until the SH signal went back to the pure buffer solution response level. With the increasing GOx surface concentration, the rearrangement or reorientation of the protein was also observed.     

Nonlinear broadband doubling of the extraordinary wave frequency in inhomogeneous magnetoactive plasma

The nonlinear resonance doubling of radio wave frequencies in inhomogeneous plasma is studied as applied to the ionosphere under the conditions of the phase synchronism between an extraordinary pump wave and its second harmonic. The synchronism is not related to plasma resonances, but is determined by the magnetic field and plasma electron density in the transparency region. The generation efficiency of the second harmonic of a transversely propagating wave is calculated for a wide frequency band lying higher than the lower hybrid resonance frequency. It is shown that this effect is physically analogous to the generation of the second harmonic of laser radiation in a nonlinear crystal. The generation efficiency of the second harmonic is determined for inhomogeneous ionospheric plasma in which the synchronism condition is satisfied in a limited frequency range. It is shown that this effect can be used for remote nonlinear diagnostics of the upper ionospheric plasma, in which the characteristic size of the synchronism region can reach several kilometers. It is proposed to use a combination of satellite and ground-based ion probes in experiments on transionospheric probing. Even if the frequency of the wave emitted from the satellite is lower than the critical frequency in the ionosphere, the frequency of its second harmonic can exceed the critical frequency, so that it can be recorded by a ground-based ion probe or a specially designed receiver. The reflected second-harmonic signal can also be detected at the satellite by using a broadband radio-frequency spectrometer.     

Theoretical foundations of detection of terahertz radiation in laser-plasma interactions

A theory is developed enabling one to calculate the temporal profile and spectrum of a terahertz wave packet from the energy of the second harmonic of optical radiation generated during the nonlinear interaction between terahertz and circularly polarized laser pulses in the skin layer of an overdense plasma. It is shown that the spectral and temporal characteristics of the envelope of the second harmonic of optical radiation coincide with those of the terahertz pulse only at small durations of the detecting laser radiation. For long laser pulses, the temporal profile and spectrum of the second harmonic are mainly determined by the characteristics of optical radiation at the carrier frequency.     

Detecting Subtle Plasma Membrane Perturbation in Living Cells Using Second Harmonic Generation Imaging

The requirement of center asymmetry for the creation of second harmonic generation (SHG) signals makes it an attractive technique for visualizing changes in interfacial layers such as the plasma membrane of biological cells. In this article, we explore the use of lipophilic SHG probes to detect minute perturbations in the plasma membrane. Three candidate probes, Di-4-ANEPPDHQ (Di-4), FM4-64, and all-trans-retinol, were evaluated for SHG effectiveness in Jurkat cells. Di-4 proved superior with both strong SHG signal and limited bleaching artifacts. To test whether rapid changes in membrane symmetry could be detected using SHG, we exposed cells to nanosecond-pulsed electric fields, which are believed to cause formation of nanopores in the plasma membrane. Upon nanosecond-pulsed electric fields exposure, we observed an instantaneous drop of ∼50% in SHG signal from the anodic pole of the cell. When compared to the simultaneously acquired fluorescence signals, it appears that the signal change was not due to the probe diffusing out of the membrane or changes in membrane potential or fluidity. We hypothesize that this loss in SHG signal is due to disruption in the interfacial nature of the membrane. The results show that SHG imaging has great potential as a tool for measuring rapid and subtle plasma membrane disturbance in living cells.     

Quantitative characterization of aqueous solutions probed by the third-harmonic generation microscopy

Third-harmonic microscopy is one of the emerging techniques for noninvasive microscopic imaging of biological structures. We use a novel technique for nonlinear optical material characterization and study the effect of different environment and the structural sensitivity of the third harmonic. In particular, a transformation of collagen in solution is observed for the first time using third-harmonic generation. We also study the ultimate limits of the third harmonic to detect micro- and nanoscopic features inside living cells and find that structures as small as 50 nm can be detected using the current level of technology.     

Harmonics of outer hair cell motility.

The voltage-dependent mechanical activity of outer hair cells (OHC) from the organ of Corti is considered responsible for the peripheral auditory system's enhanced ability to detect and analyze sound. Nonlinear processes within the inner ear are presumed to be characteristic of this enhancement process. Harmonic distortion in the OHC mechanical response was analyzed under whole-cell voltage clamp. It is shown that the OHC produces DC, fundamental and second harmonic length changes in response to sinusoidal transmembrane voltage stimulation. Mechanical second harmonic distortion decreases with frequency, whereas the predicted transmembrane second harmonic voltage increases with frequency. Furthermore, the phase of the second harmonic distortion does not correspond to the phase of the predicted transmembrane voltage. In contradistinction, it has been previously shown (Santos-Sacchi, J. 1992. Neuroscience. 12:1906-1916) that fundamental voltage and evoked mechanical responses share magnitude and phase characteristics. OHC length changes are modeled as resulting from voltage-dependent cell surface area changes. The model suggests that the observed harmonic responses in the mechanical response are consistent with the nonlinearity of the voltage-to-length change (V-delta L) function. While these conclusions hold for the data obtained with the present voltage clamp protocol and help to understand the mechanism of OHC motility, modeling the electromechanical system of the OHC in the in vivo state indicates that the mechanical nonlinearity of the OHC contributes minimally to mechanical distortion. That is, in vivo, at moderate sound pressure levels and below, the dominant factor which contributes to nonlinearities of the OHC mechanical response resides within the nonlinear, voltage-generating, stereociliar transduction process.     

Multimodal nonlinear optical imaging of collagen arrays

We report multimodal nonlinear optical imaging of fascia, a rich collagen type I sheath around internal organs and muscle. We show that second harmonic generation (SHG), third harmonic generation (THG) and Coherent anti-Stokes Raman scattering (CARS) microscopy techniques provide complementary information about the sub-micron architecture of collagen arrays. Forward direction SHG microscopy reveals the fibrillar arrangement of collagen type I structures as the main matrix component of fascia. SHG images detected in the backward direction as well as images of forward direction CARS microscopy show that the longitudinal collagen fiber bundles are further arranged in sheet-like bands. Forward-THG microscopy reveals the optically homogeneous content of the collagen sheet on a spatial scale of the optical wavelength. This is supported by the fact that the third harmonic signal is observed only at the boundaries between the sheets as well as by the CARS data obtained in both directions. The observations made with THG and CARS microscopy are explained using atomic force microscopy images.     

Characterization and application of a new optical probe for membrane lipid domains

In this article, we characterize the fluorescence of an environmentally sensitive probe for lipid membranes, di-4-ANEPPDHQ. In large unilamellar lipid vesicles (LUVs), its emission spectrum shifts up to 30 nm to the blue with increasing cholesterol concentration. Independently, it displays a comparable blue shift in liquid-ordered relative to liquid-disordered phases. The cumulative effect is a 60-nm difference in emission spectra for cholesterol containing LUVs in the liquid-ordered state versus cholesterol-free LUVs in the liquid-disordered phase. Given these optical properties, we use di-4-ANEPPDHQ to image the phase separation in giant unilamellar vesicles with both linear and nonlinear optical microscopy. The dye shows green and red fluorescence in liquid-ordered and -disordered domains, respectively. We propose that this reflects the relative rigidity of the molecular packing around the dye molecules in the two phases. We also observe a sevenfold stronger second harmonic generation signal in the liquid-disordered domains, consistent with a higher concentration of the dye resulting from preferential partitioning into the disordered phase. The efficacy of the dye for reporting lipid domains in cell membranes is demonstrated in polarized migrating neutrophils.     

Optical signals from surface and T system membranes in skeletal muscle fibers. Experiments with the potentiometric dye NK2367

Absorbance signals were recorded from cut single skeletal muscle fibers stained with the nonpenetrating potentiometric dye NK2367 and mounted in a three-vaseline-gap voltage clamp. The characteristics of the optical signals recorded under current and voltage-clamp conditions were studied at various wavelengths between 500 and 800 nm using unpolarized light. Our results indicate that the absorbance signals recorded with this dye reflect potential changes across both the surface and T system membranes and that the relative contribution of each of these membrane compartments to the total optical change is strongly wavelength dependent. A peak intensity change was detected at 720 nm for the surface membrane signal and at 670 nm for the T system. Evidence for this wavelength-dependent separation derives from an analysis of the kinetics and voltage dependence of the optical signals at different wavelengths, and results obtained in detubulated fibers. The 670-nm optical signal was used to demonstrate the lack of potential control in the T system by the voltage clamp and the effect of a tetrodotoxin (TTX)-sensitive sodium conductance on tubular depolarization.