Second-harmonic imaging microscopy of living cells

Second harmonic generation (SHG) has been developed in our laboratories as a high-resolution nonlinear optical imaging microscopy for cellular membranes and intact tissues. SHG shares many of the advantageous features for microscopy of another more established nonlinear optical technique: two-photon excited fluorescence (TPEF). Both are capable of optical sectioning to produce three-dimensional images of thick specimens and both result in less photodamage to living tissue than confocal microscopy. SHG is complementary to TPEF in that it uses a different contrast mechanism and is most easily detected in the transmitted light optical path. It can be used to image membrane probes with high membrane specificity and displays extraordinary sensitivity in reporting membrane potential; it also has the ability to image highly ordered structural proteins without any exogenous labels.     

Second harmonic imaging of membrane potential of neurons with retinal

We present a method to optically measure and image the membrane potential of neurons, using the nonlinear optical phenomenon of second harmonic generation (SHG) with a photopigment retinal as the chromophore [second harmonic retinal imaging of membrane potential (SHRIMP)]. We show that all-trans retinal, when adsorbed to the plasma membrane of living cells, can report on the local electric field via its change in SHG. Using a scanning mode-locked Ti-sapphire laser, we collect simultaneous two-photon excited fluorescence (TPEF) and SHG images of retinal-stained kidney cells and cultured pyramidal neurons. Patch clamp experiments on neurons stained with retinal show an increase of 25% in SHG intensity per 100-mV depolarization. Our data are the first demonstration of optical measurements of membrane potential of mammalian neurons with SHG. SHRIMP could have wide applicability in neuroscience and, by modifying rhodopsin, could in principle be subject for developing genetically engineered voltage sensors.     

Imaging of cerebellar surface activation in vivo using voltage sensitive dyes

The understanding of the information processing performed by complex neuronal networks in the central nervous system will require techniques permitting the simultaneous monitoring of the electrical activity of neuronal ensembles. Voltage sensitive dyes offer the potential for non-invasive optical monitoring of the activity in large populations of neurons. In this report we describe the use of voltage sensitive dyes and image processing techniques to monitor in vivo the activation of parallel fibers and associated neuronal events produced by stimulation of the cerebellar cortex in the rat. Despite the temporal limitations of video processing a relatively brief set of neuronal events was successfully imaged. Using this methodology we demonstrate that the detected fluorescent light changes were highly correlated with the evoked extracellular field potentials. Graded surface stimulation produced graded spatial patterns consistent with known parallel fiber anatomy and physiology. The optical signals were dependent on the presence of the voltage sensitive dyes and were abolished by topical application of a local anesthetic agent. In essence, activation of a parallel fiber beam and associated activity were imaged at relatively high resolution.     

Imaging membrane potential in dendrites and axons of single neurons

This review focuses on the use of imaging techniques to record electrical signaling in the fine processes of neurons such as dendrites and axons. Voltage imaging began with the use and development of externally applied voltage-sensitive dyes. With the introduction of internally applied dyes and advances in detection technology, it is now possible to record supra-threshold action potential responses, as well as sub-threshold synaptic potentials, in fine neuronal processes including dendritic spines. The development of genetically coded sensors, as well as variants of laser scanning microscopy such as second harmonic generation, offers promise for further advances in this field. Through the use and further development of these methods, optical imaging of membrane potential will continue to be a valuable tool for investigators wishing to explore the electrical events underlying single neuronal computation.     

Mechanisms of membrane potential sensing with second-harmonic generation microscopy

We characterize the transmembrane voltage response of a novel second-harmonic generation (SHG) marker using a screening protocol with giant unilamellar vesicles. Two mechanisms are found to contribute to the voltage response: (1) an electro-optic-induced alteration of the molecular hyperpolarizability and (2) an electric-field-induced alteration of the degree of molecular alignment. We quantify the relative weights and of these contributions and provide an upper limit to their response time, which is found to be submillisecond. The identification of two voltage response mechanisms leads to new strategies for the molecular design of membrane potential markers.     

Second-harmonic generation sensitivity to transmembrane potential in normal and tumor cells

Second-harmonic generation (SHG) is emerging as a powerful tool for the optical measurement of transmembrane potential in live cells with high sensitivity and temporal resolution. Using a patch clamp, we characterize the sensitivity of the SHG signal to transmembrane potential for the RH 237 dye in various normal and tumor cell types. SHG sensitivity shows a significant dependence on the type of cell, ranging from 10 to 17% per 100 mV. Furthermore, in the samples studied, tumor cell lines display a higher sensitivity compared to normal cells. In particular, the SHG sensitivity increases in the cell line Balb/c3T3 by the transformation induced with SV40 infection of the cells. We also demonstrate that fluorescent labeling of the membrane with RH 237 at the concentration used for SHG measurements does not induce any measurable alteration in the electrophysiological properties of the cells investigated. Therefore, SHG is suitable for the investigation of outstanding questions in electrophysiology and neurobiology.     

Optical approaches to ontogeny of electrical activity and related functional organization during early heart development

Direct intracellular measurement of electrical events in the early embryonic heart is impossible because the cells are too small and frail to be impaled with microelectrodes; it is also not possible to apply conventional electrophysiological techniques to the early embryonic heart. For these reasons, complete understanding of the ontogeny of electrical activity and related physiological functions of the heart during early development has been hampered. Optical signals from voltage-sensitive dyes have provided a new powerful tool for monitoring changes in transmembrane voltage in a wide variety of living preparations. With this technique it is possible to make optical recordings from the cells that are inaccessible to microelectrodes. An additional advantage of the optical method for recording membrane potential activity is that electrical activity can be monitored simultaneously from many sites in a preparation. Thus, applying a multiple-site optical recording method with a 100- or 144-element photodiode array and voltage-sensitive dyes, we have been able to monitor, for the first time, spontaneous electrical activity in prefused cardiac primordia in the early chick embryos at the six- and the early seven-somite stages of development. We were able to determine that the time of initiation of the contraction is the middle period of the nine-somite stage. In the rat embryonic heart, the onset of spontaneous electrical activity and contraction occurs at the three-somite stage. In this review, a new view of the ontogenetic sequence of spontaneous electrical activity and related physiological functions such as ionic properties, pacemaker function, conduction, and characteristics of excitation-contraction coupling in the early embryonic heart are discussed.     

Optical mapping approaches to cardiac electrophysiological functions

Recently, optical methods for monitoring membrane potential with fast voltage-sensitive dyes have been introduced as a powerful tool for studying cardiac electrical functions. These methods offer two principal advantages over more conventional electrophysiological techniques. One is that optical recordings may be made from very small cells that are inaccessible to microelectrode impalement, and the other is that multiple sites/regions of a preparation can be monitored simultaneously to provide spatially resolved mapping of electrical activity. The former has made it possible to record spontaneous electrical activities in early embryonic precontractile hearts, and the latter has been applied for mapping of the propagation patterns of electrical activities in the cardiac tissue. In this article, optical studies of the electrophysiological function of the vertebrate heart are reviewed.     

G protein modulation of voltage-sensitive muscarinic receptor signalling in mouse pancreatic acinar cells

Submaximal stimulation of mouse pancreatic acinar cells by acetylcholine (ACh) generates periodic Ca2+ responses sensitive to the membrane potential. Monitoring the muscarinic Ca2+ responses using patch-clamp whole-cell current recordings, we examined the mechanism of guanine nucleotide-binding protein (G protein)-receptor interaction in terms of the membrane potential. The lowest ACh concentration able to elicit consistent repetitive spikes was 50 nM, in the presence of which hyperpolarization increased and depolarization decreased the spike frequency. The saturating concentration was 10 microM, this induced a sustained response insensitive to voltage. Internal guanosine 5'-tri- and diphosphates (GTP, GDP) depressed and potentiated the voltage sensitivity, respectively, but not for the response to a saturating ACh concentration (10 microM). Internal guanosine 5'-O-(3-thiotriphosphate) (GTPgammaS) abolished the voltage sensitivity. The results indicate that the ACh-induced Ca2+ response is sensitive to the membrane potential and that a close linkage exists between voltage sensitivity and the G protein association/dissociation cycle in the muscarinic receptor.     

Optically teasing apart neural swelling and depolarization

We measured birefringence, 90 degree scattered light, and voltage sensitive dye changes from lobster walking leg nerves. Systematic application of key chemical agents revealed separate cellular mechanisms underlying fast optical signals. Each agent exhibited mixed effects, some having a greater effect on cellular swelling and refractive index, and some altering membrane potential. Birefringence changes were tightly correlated with voltage sensitive dye signals and were perturbed by those agents that altered membrane potential. Signals from light scattered at 90 degrees corroborated the hypothesis that large angle scattering signals arise from changes in the interstitial spaces and were perturbed by those agents that altered cellular swelling and refractive index. We conclude that multiple cellular mechanisms can be exploited to measure rapid optical signals. Since birefringence produces much larger changes than scattering, the use of polarized light might lead to improvements in imaging neural activity with high temporal resolution, especially since birefringence changes corresponded closely to membrane potential.     

Quantitative second harmonic generation imaging and modeling of the optical clearing mechanism in striated muscle and tendon

We have investigated the mechanisms and capabilities of optical clearing in conjunction with second harmonic generation (SHG) imaging in tendon and striated muscle. Our approach combines three-dimensional (3-D) SHG imaging of the axial attenuation and directional response with Monte Carlo simulation (based on measured bulk optical properties) of the creation intensity and propagation through the tissues. Through these experiments and simulations, we show that reduction of the primary filter following glycerol treatment dominates the axial attenuation response in both muscle and tendon. However, these disparate tissue types are shown to clear through different mechanisms of the glycerol-tissue interaction. In the acellular tendon, glycerol application reduces scattering by both index matching as well as increasing the interfibril separation. This results in an overall enhancement of the 3-D SHG intensity, where good agreement is found between experiment and simulation. Through analysis of the axial response as a function of glycerol concentration in striated muscle, we conclude that the mechanism in this tissue arises from matching of the refractive index of the cytoplasm of the muscle cells with that of the surrounding higher-index collagenous perimysium. We further show that the proportional decrease in the scattering coefficient mu(s) with increasing glycerol fraction can be well-approximated by Mie theory.     

Second harmonic generation imaging microscopy studies of osteogenesis imperfecta

We have used quantitative second harmonic generation (SHG) imaging microscopy to investigate the collagen matrix organization in the oim mouse model for human osteogenesis imperfecta (OI). OI is a heritable disease in which the type I collagen fibrils are either abnormally organized or small, resulting in a clinical presentation of recurrent bone fractures and other pathologies related to collagen-comprised tissues. Exploiting the exquisite sensitivity of SHG to supramolecular assembly, we investigated whether this approach can be utilized to differentiate normal and oim tissues. By comparing SHG intensity, fibrillar morphology, polarization anisotropy, and signal directionality, we show that statistically different results are obtained for the wild type (WT) and disease states in bone, tendon, and skin. All these optical signatures are consistent with the collagen matrix in the oim tissues being more disordered, and these results are further consistent with the known weaker mechanical properties of the oim mouse. While the current work shows the ability of SHG to differentiate normal and diseased states in a mouse model, we suggest that our results provide a framework for using SHG as a clinical diagnostic tool for human OI. We further suggest that the SHG metrics described could be applied to other connective tissue disorders that are characterized by abnormal collagen assembly.     

Optical recording of fast neuronal membrane potential transients in acute mammalian brain slices by second-harmonic generation microscopy

Although nonlinear microscopy and fast (approximately 1 ms) membrane potential (Vm) recording have proven valuable for neuroscience applications, their potentially powerful combination has not yet been shown for studies of Vm activity deep in intact tissue. We show that laser illumination of neurons in acute rat brain slices intracellularly filled with FM4-64 dye generates an intense second-harmonic generation (SHG) signal from somatic and dendritic plasma membranes with high contrast >125 microm below the slice surface. The SHG signal provides a linear response to DeltaVm of approximately 7.5%/100 mV. By averaging repeated line scans (approximately 50), we show the ability to record action potentials (APs) optically with a signal-to-noise ratio (S/N) of approximately 7-8. We also show recording of fast Vm steps from the dendritic arbor at depths inaccessible with previous methods. The high membrane contrast and linear response of SHG to DeltaVm provides the advantage that signal changes are not degraded by background and can be directly quantified in terms of DeltaVm. Experimental comparison of SHG and two-photon fluorescence Vm recording with the best known probes for each showed that the SHG technique is superior for Vm recording in brain slice applications, with FM4-64 as the best tested SHG Vm probe.     

A review of NIR dyes in cancer targeting and imaging

The development of multifunctional agents for simultaneous tumor targeting and near infrared (NIR) fluorescence imaging is expected to have significant impact on future personalized oncology owing to the very low tissue autofluorescence and high tissue penetration depth in the NIR spectrum window. Cancer NIR molecular imaging relies greatly on the development of stable, highly specific and sensitive molecular probes. Organic dyes have shown promising clinical implications as non-targeting agents for optical imaging in which indocyanine green has long been implemented in clinical use. Recently, significant progress has been made on the development of unique NIR dyes with tumor targeting properties. Current ongoing design strategies have overcome some of the limitations of conventional NIR organic dyes, such as poor hydrophilicity and photostability, low quantum yield, insufficient stability in biological system, low detection sensitivity, etc. This potential is further realized with the use of these NIR dyes or NIR dye-encapsulated nanoparticles by conjugation with tumor specific ligands (such as small molecules, peptides, proteins and antibodies) for tumor targeted imaging. Very recently, natively multifunctional NIR dyes that can preferentially accumulate in tumor cells without the need of chemical conjugation to tumor targeting ligands have been developed and these dyes have shown unique optical and pharmaceutical properties for biomedical imaging with superior signal-to-background contrast index. The main focus of this article is to provide a concise overview of newly developed NIR dyes and their potential applications in cancer targeting and imaging. The development of future multifunctional agents by combining targeting, imaging and even therapeutic routes will also be discussed. We believe these newly developed multifunctional NIR dyes will broaden current concept of tumor targeted imaging and hold promise to make an important contribution to the diagnosis and therapeutics for the treatment of cancer.     

Fast optical recordings of membrane potential changes from dendrites of pyramidal neurons.

Understanding the biophysical properties of single neurons and how they process information is fundamental to understanding how the brain works. A technique that would allow recording of temporal and spatial dynamics of electrical activity in neuronal processes with adequate resolution would facilitate further research. Here, we report on the application of optical recording of membrane potential transients at many sites on neuronal processes of vertebrate neurons in brain slices using intracellular voltage-sensitive dyes. We obtained evidence that 1) loading the neurons with voltage-sensitive dye using patch electrodes is possible without contamination of the extracellular environment; 2) brain slices do not show any autofluorescence at the excitation/emission wavelengths used; 3) pharmacological effects of the dye were completely reversible; 4) the level of photodynamic damage already allows meaningful measurements and could be reduced further; 5) the sensitivity of the dye was comparable to that reported for invertebrate neurons; 6) the dye spread approximately 500 micron into distal processes within 2 h incubation period. This distance should increase with longer incubation; 7) the optically recorded action potential signals from basolateral dendrites (that are difficult or impossible to approach by patch electrodes) and apical dendrites show that both direct soma stimulation and synaptic stimulation triggered action potentials that originated near the soma. The spikes backpropagated into both basolateral dendrites and apical processes; the propagation was somewhat faster in the apical dendrites.     

A hybrid approach to measuring electrical activity in genetically specified neurons

The development of genetically encoded fluorescent voltage probes is essential to image electrical activity from neuronal populations. Previous green fluorescent protein (GFP)-based probes have had limited success in recording electrical activity of neurons because of their low sensitivity and poor temporal resolution. Here we describe a hybrid approach that combines a genetically encoded fluorescent probe (membrane-anchored enhanced GFP) with dipicrylamine, a synthetic voltage-sensing molecule that partitions into the plasma membrane. The movement of the synthetic voltage sensor is translated via fluorescence resonance energy transfer (FRET) into a large fluorescence signal (up to 34% change per 100 mV) with a fast response and recovery time (0.5 ms). Using this two-component approach, we were able to optically record action potentials from neuronal cell lines and trains of action potentials from primary cultured neurons. This hybrid approach may form the basis for a new generation of protein-based voltage probes.     

Optical studies of early developing cardiac and neural activities using voltage-sensitive dyes

Using optical methods for monitoring cellular electrical activity based on voltage-sensitive dyes, we have overcome several obstacles to the study of electrical function in the embryonic heart and central nervous system during early development. We have been able to monitor, for the first time, spontaneous electrical activity in the pre-fused cardiac primordia in early chick embryos at the 6- and early 7-somite stages of development and to follow the early development of electrical activity in the pre-contractile heart at the 7- to 9-somite stages. In addition, we have monitored neural responses in the early embryonic chick brain stem by optical means, and determined the spatial pattern of the response.     

Long-term optical recording of patterns of electrical activity in ensembles of cultured Aplysia neurons

1. Left upper quadrant (LUQ) cells isolated from the abdominal ganglion of Aplysia were maintained in culture to study how the cellular and synaptic properties of individual neurons contribute to the generation of patterns of electrical activity by neuronal ensembles. 2. Conventional microelectrodes were used to examine the spiking characteristics of individually cultured LUQ cells in vitro and to characterize their synaptic interactions. 3. In vitro, in contrast to in situ, LUQ neurons innervate other LUQ neurons. Intracellular recordings from pairs of LUQ cells showed that the prevalent type of postsynaptic potential was purely inhibitory. The other type of response was a dual-action postsynaptic potential, with inhibition followed by a delayed, slow excitation. 4. We established a set of criteria for the use of multiple-site optical recording techniques, in combination with impermeant probes of membrane potential, to observe the patterns of electrical activity generated by ensembles of co-cultured LUQ cells. 5. The spiking activity of individual cells within the neuronal ensembles was detected by means of the change in optical absorption of cells that were vitally stained with the dye RH155. The change in absorption was typically delta A congruent to 4 X 10(-4) per spike. We achieved a signal-to-noise (peak-to-peak) ratio of approximately 10 for a 50 X 50-microns photodetector field and an incident intensity of approximately 10 mW/cm2, close to the theoretical limit. 6. These conditions permitted, for the first time, continuous optical recording from cultured neurons for periods of up to 3 h with no discernible photodynamic damage or photobleaching. This long-term optical recording permitted examination of the different patterns of electrical activity generated by individual neuronal ensembles under several different experimental conditions. 7. An elaborate tracery of regenerated neurites present in these cultures resulted in individual photodetectors recording simultaneously the activity of multiple neurons. We reconstructed the temporal firing patterns for individual neurons within ensembles even with all the neurons active simultaneously and determined the functional connections in the ensemble by analyzing the temporal relationships between firing patterns of individual neurons. Excitatory as well as inhibitory functional interactions could be observed within the neuronal ensemble, the latter after the tonic activity of the neurons was increased by reducing the extracellular [Mg2+]. 8. Examination of the optical data from ensembles constructed from identified cells having characteristic physiological responses allowed us to address the question of how cellular and synaptic properties affect the patterns of electrical activity generated by neuronal ensembles.(ABSTRACT TRUNCATED AT 400 WORDS)     

Sensitivity of EEG and MEG measurements to tissue conductivity

Monitoring the electrical activity inside the human brain using electrical and magnetic field measurements requires a mathematical head model. Using this model the potential distribution in the head and magnetic fields outside the head are computed for a given source distribution. This is called the forward problem of the electro-magnetic source imaging. Accurate representation of the source distribution requires a realistic geometry and an accurate conductivity model. Deviation from the actual head is one of the reasons for the localization errors. In this study, the mathematical basis for the sensitivity of voltage and magnetic field measurements to perturbations from the actual conductivity model is investigated. Two mathematical expressions are derived relating the changes in the potentials and magnetic fields to conductivity perturbations. These equations show that measurements change due to secondary sources at the perturbation points. A finite element method (FEM) based formulation is developed for computing the sensitivity of measurements to tissue conductivities efficiently. The sensitivity matrices are calculated for both a concentric spheres model of the head and a realistic head model. The rows of the sensitivity matrix show that the sensitivity of a voltage measurement is greater to conductivity perturbations on the brain tissue in the vicinity of the dipole, the skull and the scalp beneath the electrodes. The sensitivity values for perturbations in the skull and brain conductivity are comparable and they are, in general, greater than the sensitivity for the scalp conductivity. The effects of the perturbations on the skull are more pronounced for shallow dipoles, whereas, for deep dipoles, the measurements are more sensitive to the conductivity of the brain tissue near the dipole. The magnetic measurements are found to be more sensitive to perturbations near the dipole location. The sensitivity to perturbations in the brain tissue is much greater when the primary source is tangential and it decreases as the dipole depth increases. The resultant linear system of equations can be used to update the initially assumed conductivity distribution for the head. They may be further exploited to image the conductivity distribution of the head from EEG and/or MEG measurements. This may be a fast and promising new imaging modality.