Laboratory For Fluorescence Dynamics Research Articles

A laser-scanning confocal microscopy study of carrageenan in red algae from seaweed farms near the Caribbean entrance of the Panama Canal

Kappaphycus alvarezii (Doty) Doty ex P.C. Silva, a red macroalga, is a commercial source of carrageenan, a widely used polysaccharide compound important in the food and pharmaceutical industries, in nanotechnology, and in pharmacological applications. Carrageenan is found mainly in the cell wall and in the intercellular matrix. This is the first study to propose the characterization of carrageenans in vitro, using the auto-fluorescence properties of the alga treated with different polyamines: putrescine, spermidine, and spermine. This study suggests a four-phase cultivation sequence for seaweed farmers to enhance and assess the potential carrageenan yield of their crops. In phase 1, seedlings were treated with each of the polyamines. Explants were subsequently transferred through two additional culture phases before being planted on the sea farms in phase 4 and then harvested after 60 days for analysis. Images from transverse sections of 11 representative cultured K. alvarezii samples were obtained at 561 nm excitation wavelength for both the cell center and the cell wall of each sample. Spectral data were also analyzed using the spectral phasor algorithm of SimFCS developed at the Laboratory for Fluorescence Dynamics ( www.lfd.uci.edu ). We report on the identification of several spectral fluorescence emission fingerprints from different auto-fluorescence compounds spatially mapped using this technique. These fingerprints have the potential to improve strain selection of explants for enhanced carrageenan yield in seaweed farming operations as well as to enable wholesale pricing to correspond with crop quality.

Lactose Glicolipids and their Capabilities to form Giant Vesicles and Tubules

Vesicles shape transformations have attracted a attention of researchers due to the possible application the different shapes may have as template systems or vehicles for drug delivery. The common spherical shape of closed bilayer, namely vesicle or liposome, can change into less common structures like starfish, pears, stomatocytes, discocytes or tubules, depending on the bilayer composition or on several parameters like temperature, pH, osmotic pressure, etc. At specific compositions, Giant Unilamellar Vesicles (GUVs) can coexist with nanotubules. There are previous reports of these abilities (vesicle formation and nanotubule transformation) for mixtures of anionic double chain glycophospholipids (incuding galactose, mannose and glucose) with either two hydrocarbon or two perfluorocarbon chains. A ternary system (mixture of sphingomyelin, phosphatidylcholine and cholesterol) for generating emerging long tubular nanostructures has been reported, demonstrating that composition is different in the tubes and in the vesicles and tube split-off depends on phase separation. These results indicate that lipid organization is dependent critically on both membrane curvature and phase separation. We have synthesized a series of glycolipids derived from lactose linked through a glycerol moiety to two alkyl chains of different length (8, 12 and 16 methylene units). Using these glycolipids in combination with POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine), we studied its vesicle-forming capabilities (with and without cholesterol). Using the electroformation method we prepared GUVs and characterized them by two-photon microscopy. Our results showed that all derivatives in combination with cholesterol and POC have the capability to form GUVs and those derivatives with longer alkyl chains besides also form tubular structures coexisting with the giant liposomes when mixed with cholesterol.The authors would like to thank to Fondecyt 1140454 (S.S.) and 1120196 (G.G.) and Beca de postgrado Conicyt 21120554 (C.S.) and Laboratory for Fluorescence Dynamics.

Background-Free Super-Resolution Microscopy of Subcellular Structures by Lifetime Tuning and Photons Separation

Background-Free Super-Resolution Microscopy of Subcellular Structures by Lifetime Tuning and Photons Separation

Fluorescence Fluctuation Microscopy Techniques to Study mRNA Synthesis and Dynamics

1627-Pos Board B578 Probing Short-Range Protein Brownian Motion in the Cytoplasm of Living Cells Carmine Di Rienzo 1,2 , Enrico Gratton 3 , Fabio Beltram 1,2 , Francesco Cardarelli 2 . NEST, Scuola Normale Superiore, Pisa, Italy, 2 Center for Nanotechnology Innovation @NEST, Istituto Italiano di Tecnologia, Pisa, Italy, 3 Laboratory for Fluorescence Dynamics, Department of Biomedical Engineering., University of California, Irvine, CA, USA. The translational motion of small molecules in cells appears to be suppressed compared to what is observed in dilute solutions. Although, the rotation of small proteins is almost unhindered, pointing out a local aqueous environment. Different theoretical models provide explanations for this apparent discrepancy but with predictions that drastically depend on the nanoscale organization assumed for macromolecular crowding agents. A conclusive experimental test of the nature of the translational motion in cells is still missing owing to the lack of techniques capable of probing protein motion with the required temporal and spatial resolution. We show that fluorescence-fluctuation analysis of raster scans at variable time scales can provide this information. By using GFP, we measure protein translational motion at the unprecedented time-scale of 1 micro- second, unveiling unobstructed Brownian motion from 25 to 100 nanometers, and partially-suppressed diffusion above 100 nm. Experiments on in vitro model systems attribute this effect to the presence of relatively immobile structures rather than to diffusing crowding agents. In this regard, internal membranes (e.g. the ER sheets, vesicles, Golgi apparatus, etc.) appear to be the more likely candidates as selective disruption of the microtubules network by treatment with Nocodazole did not significantly alter GFP behavior in the cytoplasm. Also, the same measurement in a structurally-different (e.g devoid of membranes) intra- cellular environment, such as the nucleoplasm, yields a different behavior, in which GFP motion is never coincident with that in a dilute solution. Finally, we believe the present findings coupled with use of genetically-encoded fluores- cent markers pave the way to novel studies of biomolecular processes in live cells at the physiologically-relevant spatio-temporal scale. Supported by grants NIH P41-GM103540 and NIH P50-GM076516 (grants to EG), MIUR under FIRB- RBAP11X42L and Fondazione Monte dei Paschi di Siena (grants to FB).

Diffusion Mapping in Living Cells using Camera-Based Correlation Spectroscopy and Phasor Analysis

Diffusion Mapping in Living Cells using Camera-Based Correlation Spectroscopy and Phasor Analysis

Spectroscopic Properties of Intrinsic Proteins in Collagen Samples by using Gold-Nanoparticles and Two-Photon Excited Fluorescence Microscopy

2011-Pos Board B741 Spectroscopic Properties of Intrinsic Proteins in Collagen Samples by using Gold-Nanoparticles and Two-Photon Excited Fluorescence Microscopy Manuela Gabriel 1 , Enrico Gratton 2 , Laura Cecilia Estrada 1,3 . Quantum Electronics Lab, Physics Department, University of Buenos Aires, Buenos Aires, Argentina, 2 Laboratory for Fluorescence Dynamics, Department of Biomedical Engineering, University of California, Irvine, CA, USA, 3 National Council of Scientific and Technical Research (CONICET), Buenos Aires, Argentina. Multiphoton microscopy provides optical sectioning for high-resolution imag- ing. In biological systems, most multiphoton microscopy studies have relied on two-photon excited fluorescence (TPEF) to produce images. In particular, TPEF from structural proteins has emerged as an invaluable tool for 3D imag- ing. However, depending on the fluorophores involved, the TPEF could be extremely weak, limiting TPEF use for thick-tissue applications. The physics and optical properties of noble-metal nanoparticles (NPs) is currently attracting much attention. The signal enhancement of molecules near a metal surface arises from the interaction with surface plasmon (SP) modes. These interactions also result in the shortening of the excited-state life- time improving the molecules’ photo-stability. The optical properties of the environment near a metallic NP are affected by the near-field electromag- netic-field. In the case of fluorescent molecules located at very short distances from a metallic surface, non-radiative energy transfer to SPs in the metal takes place; however, emission enhancement due to SP coupling still occurs at optimal distances from the NP. In this study, we use a broad range of excitation wavelengths (~800-900 nm) to demonstrate that absorption enhancement can also be achieved in unstained samples by using a combination of gold-NPs and a TPEF. The TPEF images of gold-NPs/collagen samples were used to determine the collagen absorption spectra and confirm enhancement factors >100, which provide promising new applications in fields such as tissue imaging, and fluorescence-engineering based on surface-enhancement effects. Our results suggest that even weakly intrinsic signals from structural proteins can be extremely-enhanced due to SPs coupling. The use of metallic NPs and TPEF in combination provides complementary in- formation that allows noninvasive, spatially localized, in-vivo characterization in unstained samples and tissues. Work supported by NIH grants P50GM076516 and P41GM103540, PICT0457- 2010 and UbaCyT20020100100719.

Determining the Quantitative Dynamics of Nucleic Acids in Live Cells through Rics and iMSD Approaches

361-Pos Board B116 Determining the Quantitative Dynamics of Nucleic Acids in Live Cells through Rics and iMSD Approaches Stephen P. Mieruszynski 1 , Michelle A. Digman 2 , Enrico Gratton 2 , Mark Jones 1 . Health and Science, University of Western Sydney, Richmond, Australia, Biomedical Engineering Department, Laboratory for Fluorescence Dynamics, University of California, Irvine, CA, USA. Addressing the mobility and dynamics of DNA within live cells requires the characterization of individual particles in real time. Two such techniques enabling the quantification of the dynamics and means of motion of fluorescently labeled particles include the Raster Image Correlation Spectroscopy (RICS) and the image-based Mean Square Displacement (iMSD) approaches. In this study, the RICS and iMSD approaches were applied in order to elucidate the mobility and means of motion of DNA lipoplexes formed from varying sized DNA (20bp- 5.5kbp). The RICS approach demonstrated that two species of mobility were pre- sent within the cytoplasm. Across the DNA fragment sizes, the slower species was not size dependent and were statistically the same. Whereas, faster moving particles which demonstrated a size dependent mobility. Nuclear localized DNA demonstrated to be significantly dynamic compared to other previously pub- lished work, ranging from 1.22 mm2/s (5.5kbp) to 3.67 mm2/s (21bp), consistent to nuclear proteins and RNA. The iMSD approach applied enabled the discrim- ination between different means of motion including random diffusion (RD), active transport (AT), confined diffusion (CD), anomalous subdiffusion (AS) and transient confinement (TC). Motion through RD and TC had demonstrated a size dependency across the DNA sizes assessed. Whereas, motion through AT, CD and AS did not demonstrate size dependency additionally these other means of motion had a higher mobility rate than that of RD.

Fast Spatiotemporal Correlation Spectroscopy to Determine Protein Lateral Diffusion Laws in Live Cell Membranes

Fast Spatiotemporal Correlation Spectroscopy to Determine Protein Lateral Diffusion Laws in Live Cell Membranes

Phasor Plots and Spectral Phasor Analysis of Laurdan and Prodan for Membrane Heterogeneity Studies: New Frontiers in Membrane Biophysics

449-Pos Board B204 Phasor Plots and Spectral Phasor Analysis of Laurdan and Prodan for Membrane Heterogeneity Studies: New Frontiers in Membrane Biophysics Leonel S. Malacrida 1 , Arturo Briva 1 , Carrisa M. Vetromile 2 , Enrico Gratton 3 , Ana Denicola 4 , David M. Jameson 2 . Departamento de Fisiopatologi´a, Hospital de Cli´nicas, Universidad de la Republica, Montevideo, Uruguay, 2 Department of Cell and Molecular Biology, John A. Burns School of Medicine, University of Hawaii, Honolulu, HI, USA, 3 Laboratory for Fluorescence Dynamics, University of California, Irvine, CA, USA, 4 Laboratorio de Fisicoqui´mica Biolo´gica, Facultad de Ciencias, Universidad de la Republica, Montevideo, Uruguay. Since its introduction by Weber [1], fluorophores in the PRODAN series have contributed to our understanding of hydration and packing in biological mem- branes. Here we apply methods based on lifetime determinations and phasor plots as well as steady-state measurements using the spectral phasor approach, for analysis of the behavior of LAURDAN and PRODAN in vesicles. The life- time Phasor approach (Jameson et al., [2]) uses a plot of M.sin(F) versus M.cos(F), where M is the modulation ratio and F is the phase angle taken from frequency domain fluorometry. With Spectral phasors, introduced by Fer- eidouni et al [3], the steady-state fluorescence spectrum is Fourier transformed, resulting in two coordinates in x and y used for a scatter plot (Spectral phasor). The temporal Phasor approach shows significant improvement compared with older methods as regards discrimination of the effects of temperature, choles- terol content and drug addition, in our membrane model systems. This approach is very convenient for characterization of complex systems wherein lifetime heterogeneity and relaxation processes are present. The Spectral phasor approach is a very useful method for characterization of subtle changes in membrane hydration and packing. The major advantage of both methods is that they provide a model-less approach, which is relevant to complex studies on native systems, where endogenous fluorescence can introduce undesired mistakes. Examples of the application of both methods to membrane systems will be given. [1] Weber et al, Biochemistry, 1979. [2] Jameson et al, Appl. Spectosc. Rev., 1991. [3] Fereidouni et al, Opt. Express, 2012.

Tracking the Mechanical Dynamics of Stem Cell Chromatin

1030-Plat Tracking the Mechanical Dynamics of Stem Cell Chromatin Elizabeth Hinde 1 , Francesco Cardarelli 2 , Aaron Chen 3 , Michelle Khine 3 , Enrico Gratton 1 . Laboratory for Fluorescence Dynamics, University fo California, Irvine, CA, USA, 2 Center for Nanotechnology Innovation @NEST, Istituto Italiano di Tecnologia, Pisa, Italy, 3 University fo California, Irvine, CA, USA. A plastic chromatin structure has emerged as fundamental to the self-renewal and pluripotent capacity of embryonic stem (ES) cells. Direct measurement of chromatin dynamics in vivo is however challenging as high spatiotemporal resolution is required. Here we present a new tracking based method which can detect chromatin fiber movement and quantify the mechanical dynamics of chromatin in live cells. We use this method to study how the mechanical properties of chromatin movement in hESCs are modulated spatiotemporally during differentiation into cardiomyocytes (CM). Notably, we find that plurip- otency is associated with a highly discrete, energy-dependent frequency of chromatin movement, that we refer to as a ‘‘breathing’’ state. We find that this ‘‘breathing’’ state is strictly dependent on the metabolic state of the cell and is progressively silenced during differentiation, thus representing a hall- mark of pluripotency maintenance. This is a result that could not have been observed without the nanometer resolution provided by this novel tracking method.

Cerebral Oxygenation During Anesthesia

Cerebral Oxygenation During Anesthesia: Correlation With Blood Pressure and Cardiac OutputWilliam W. Mantulin∗ and Lingzhong Meng∗∗University of California IrvineBeckman Laser Institute,∗ Laboratory for Fluorescence Dynamics∗ and Department of Anesthesiology∗∗Irvine, CADuring surgery, the anesthesiologist is tasked with monitoring, manipulating and maintaining the patient's key physiological parameters, especially of critical organs such as the brain. Generally, cerebral tissue oxygenation is not explicitly tracked, but a measure of Mean Arterial Pressure (MAP) is considered an adequate surrogate marker for sufficient cardiac output to satisfy cerebral oxygen demand. Avoidance of cerebral ischemia is an important consideration. Consequently, we have used a tissue oximeter (Oxipex TS; ISS, Inc.) to non-invasively and quantitatively monitor cerebral oxygenation (bilateral; frontal lobes) during surgery in more than two dozen patients (under IRB approved protocols). If blood pressure declines after inducing anesthesia, then the anesthesiologist pharmacologically intervenes with a pressor such as Ephedrine or Phenylephrine. Phenylephrine acts as a vasoconstrictor, whereas ephedrine stimulates cardiac output. We were intrigued by our preliminary observations that cerebral oxygenation (and oxygenated Hemoglobin concentrations) declined with administration of Phenylephrine, but remained unchanged with Ephedrine. Both pressors produced the anticipated increase in blood pressure. We sought a correlation of this observation through a direct monitor of cardiac output with a Doppler Esophageal device (Cardio Q, Deltex Inc.). The data clearly showed that administration of Phenylephrine induced a decrease in cardiac output and a correlation with (a decreasing) cerebral oxygenation. The cerebral blood volume and tissue oxygen saturation tracked with the blood pressure. Our observations are now engendering a reexamination amongst clinicians as to how best to understand the interplay between cardiac output, mean arterial pressure and the use of increased blood pressure as a means to better perfuse and oxygenate critical organs such as the brain.

Abstract P5-01-12: Specific Tumor Component Spectrum as a Predictor of Response to Neoadjuvant Chemotherapy in Breast Cancer

Abstract BACKGROUND: Monitoring individual tumor response to neoadjuvant chemotherapy could provide surrogate markers for oncologists to adjust therapeutics in order to minimize collateral damage from ineffective therapies. Diffuse optical spectroscopy imaging (DOSI), by increasing the wavelength range and resolution, has been employed to characterize malignant tumor and its response to neoadjuvant chemotherapy since it measures the abundance in the four major chromophores (lipid, oxy-hemoglobin, deoxy-hemoglobin and water). Through the application of a spectral analysis method, Self-Referencing Differential Spectroscopy (SRDS) analysis, which accounts for only intersubject variability, a novel optical intrinsic biomarker which is unique to cancer was revealed. By fitting the difference spectrum between tumor and normal tissues to the four component basis spectra, the residuals disclose a spectral fingerprint called specific tumor component (STC) spectrum which contains specific absorption bands which separate normal from abnormal tissue and benign from malignant tumors. PATIENTS AND METHODS: 14 patients were enrolled in this study and all the subjects provided informed written consent according to an institution-approved protocol. Measurements were taken pre-, post-neoajuvant chemotherapy and the midpoint in the treatment. The chemotherapy regimen is 2-4 cycles of doxorubicin-cyclophosphamide (A/C), followed by 2-4 cycles of carboplatin-taxol/abraxane-avastin (nabTC). Tumor classification was confirmed by pathology reports and 8 out of 14 patients were classified as complete responders (CR); the rest as noneresponders (NR). Pre and post-treatment STC spectra were compared in different pathological responding categories. Data were analyzed by using custom software designed for Matlab. The concentrations of NIR absorbers will be calculated from a spectral model of tissue absorption by using the basis spectra. For the SRDS method, the absorption spectra will be further analyzed by using custom software (Elantest; Laboratory for Fluorescence Dynamics, Irvine, Calif, www.lfd.edu). Details of SRDS method has been described previously. RESULTS: The shape of STC spectra for non-responders was well preserved since insusceptible tumor structure will remain basically unaffected during the chemotherapy. Whereas in the completed responders, the therapy-induced changes in tumor microvasculature, edema, and necrosis lead to a significant dissimilarity in STC spectra. CONCLUSION: STC spectrum, as an endogenous in vivo biomarker, can predict pathological response in breast tumors treated with neoadjuvant chemotherapy. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P5-01-12.

Receptor-Ligand Interactions in the Plasma Membrane of Live Cells Resolved in Space and Time by N&B Analysis

3898-Pos Receptor-Ligand Interactions in the Plasma Membrane of Live Cells Resolved in Space and Time by N&B Analysis Christian Hellriegel 1 , Valeria R. Caiolfa 2,1 , Nicolai Sidenius 3 , Enrico Gratton 4 , Moreno Zamai 2,1 . CNIC, Madrid, Spain, 2 San Raffaele Scientific Institute, Milano, Italy, IFOM- Italian Cancer Research Foundation FIRC, Milano, Italy, Laboratory for Fluorescence Dynamics, University of California Irvine, Irvine, CA, USA. In this presentation we show how we push the Number and Brightness analysis (N&B) to the limits of applicability. We demonstrate that by N&B we can observe how a GFP labeled membrane receptor (namely uPAR) dimerizes upon ligand binding in live cells. We show how we obtain real time, spatially and temporally resolved images of the molecular reorganization of uPAR in the cell membrane. These results are backed by extensive simulations, and by well-defined live cell calibration experiments (using monomeric and dimeric GFP-uPAR constructs). N&B quantifies the amplitudes of fluorescence intensity fluctuations as individ- ual fluorescent species diffuse in and out of a pixel in a series of images. The basic idea is that the amplitude fluctuations of a diffusing molecule labeled with two dyes (e.g. a dimer, or a bound ligand-receptor pair) will be twice as large as the amplitudes of a molecule with only one dye (i.e. a monomer, or the unbound ligands and receptors), simply because the doubly labeled object is twice as bright as the individual one. N&B is related to fluctuation spectroscopy such as fluorescence correlation spectroscopy FCS and photon counting histogram, PCH. These methods can re- solve molecule-molecule interactions, but are usually restricted to the acquisi- tion at one specific pixel. N&B was described recently for 2-photon scanning microscopy. There, N&B was typically used to distinguish between mobile molecules and large aggregates in cells, using time-sequences of about 50- 100 frames (typically 512x512 pixels at 4s/frame). However, when attempting to distinguish between monomers and dimers, as the smallest possible increment of molecule-molecule interactions, the experi- menter is confronted with low signal-to-noise ratios and long-term perturba- tions (cell movement, vesicle trafficking). In this work we describe how we have resolved these issues.

Visualization and analysis of apolipoprotein A-I interaction with binary phospholipid bilayers

Apolipoprotein A-I (apoA-I) interaction with specific cell lipid domains was suggested to trigger cholesterol and phospholipid efflux. We analyzed here apoA-I interaction with dimyristoylphosphatidylcholine/distearoylphosphatidylcholine (DMPC/DSPC) bilayers at a temperature showing phase coexistence. Solid and liquid-crystalline domains were visualized by two-photon fluorescence microscopy on giant unilamellar vesicles (GUVs) labeled with 6-dodecanoyl-2-dimethyl-amino-naphthalene (Laurdan). A decrease of vesicle size was detected as long as they were incubated with lipid-free apoA-I, together with a shape deformation and a relative enrichment in DSPC. Selective lipid removal mediated by apoA-I from different domains was followed in real time by changes in the Laurdan generalized polarization. The data show a selective interaction of apoA-I with liquid-crystalline domains, from which it removes lipids, at a molar ratio similar to the domain compositions. Next, apoA-I was incubated with DMPC/DSPC small unilamellar vesicles, and products were isolated and quantified. Protein solubilized both lipids but formed complexes relatively enriched in the liquid component. We also show changes in the GUV morphology when cooling down. Our results suggest that the most efficient reaction between apoA-I and DMPC/DSPC occurs in particular bilayer conditions, probably when small fluid domains are nucleated within a continuous gel phase and interfacial packing defects are maximal.

Direct Evidence That Neural Cell Adhesion Molecule (NCAM) Polysialylation Increases Intermembrane Repulsion and Abrogates Adhesion

Molecular force measurements quantified the impact of polysialylation on the adhesive properties both of membrane-bound neural cell adhesion molecule (NCAM) and of other proteins on the same membrane. These results show quantitatively that NCAM polysialylation increases the range and magnitude of intermembrane repulsion. The repulsion is sufficient to overwhelm both homophilic NCAM and cadherin attraction at physiological ionic strength, and it abrogates the protein-mediated intermembrane adhesion. The steric repulsion is ionic strength dependent and decreases substantially at high monovalent salt concentrations with a concomitant increase in the intermembrane attraction. The magnitude of the repulsion also depends on the amount of polysialic acid (PSA) on the membranes, and the PSA-dependent attenuation of cadherin adhesion increases with increasing PSA-NCAM:cadherin ratios. These findings agree qualitatively with independent reports based on cell adhesion studies and reveal the likely molecular mechanism by which NCAM polysialylation regulates cell adhesion and intermembrane space.

A Key Point Mutation (V156E) Affects the Structure and Functions of Human Apolipoprotein A-I

A Key Point Mutation (V156E) Affects the Structure and Functions of Human Apolipoprotein A-I