Search

Duplication ofPMP22causes Charcot-Marie-Tooth disease type 1A (CMT1A) and is known to disrupt the lipid metabolism in myelinating Schwann cells by unknown mechanisms. By using two CMT1A mouse models overexpressing humanPMP22, we discovered thatPMP22dose-dependently downregulates genes that are involved in lipid and cholesterol metabolism. Lipidomic analysis on CMT1A mouse sciatic nerves confirmed lipid metabolic abnormalities primarily associated with cholesterol and sphingolipids. We observed similar lipidomic profiles and downregulation of genes associated with lipid metabolism in human CMT1A patient induced pluripotent stem cell-derived Schwann cell precursors (iPSC-SCPs). We confirmed these findings by demonstrating altered lipid raft dynamics and plasma membrane fluidity in CMT1A iPSC-SCPs. Additionally, we identified impaired cholesterol incorporation in the plasma membrane due to altered lipid storage homeostasis in CMT1A iPSC-SCPs, which could be modulated by changing the lipid composition of the cell culture medium. These findings suggest that PMP22 plays a role in regulating the lipid composition of the plasma membrane and lipid storage homeostasis. Targeting lipid metabolism may hold promise as a potential treatment for CMT1A patients.Graphical abstractHighlightsPMP22copy number causes a dose-dependent suppression of cholesterol and lipid biosynthesis in peripheral nerves of CMT1A miceLipid composition is altered in the sciatic nerves of CMT1A mice and in the membranes of patient derived iPSC-SCPs, with a significant reduction in sphingolipidsCMT1A iPSC-SCPs show decreased plasma membrane lipids required for regulating lipid raft dynamics, membrane fluidity, and membrane orderLipid storage misregulation is key in the pathogenesis of CMT1A

Distinct developmental pathologies, including autism spectrum disorder and schizophrenia, exhibit impaired reward-motivated behavior. Key to proper reward-motivated behavior is dopamine-mediated modulation of striatal activity. The glycine alpha 2 receptor (GlyRα2) is the single functionally expressed glycine receptor in adult striatum, and is therefore ideally positioned to modulate striatal behavior and cellular activity. Here, we report excessive appetitive conditioning in male GlyRα2 knockout mice. We next show that depletion of GlyRα2 enhances dopamine-induced increases in the activity of putative dopamine D1-expressing striatal projection neurons, while not affecting dopamine neuron activity. Moreover, we found that excessive locomotor responses to amphetamine in GlyRα2 KO mice correlate with immediate early gene c-fos expression in the dorsal striatum. 3-D modeling revealed an increase in the number of activated cell ensembles in the striatum in response to D-amphetamine in GlyRα2 KO mice. Taken together, we show that depletion of GlyRα2 impairs reward-motivated behavior and altered striatal signal integration. This sheds important light onto the cellular mechanisms that underlie reward function, and pave the way towards novel therapeutics for the treatment of e.g. schizophrenia and addiction.Significance statementThe glycine receptor alpha 2 has long been studied for its role in development, with expression assumed to decline throughout adulthood in favor of the glycine receptor alpha 1 and 3. Yet, we showed that in the dorsal striatum, the glycine alpha 2 receptor is the only functionally expressed glycine receptor at adult age (Molchanova et al., 2017).In the present work, we show for the first time that the glycine alpha 2 receptor crucially affects striatal cell activity, which lies at the basis of reward-motivated behaviors, and which is impaired in many psychiatric pathologies. Indeed, a link between the mutations in the glycine alpha 2 receptor and autism as well as schizophrenia has been described, but a functional role for the glycine alpha 2 receptor in adult brain structures that are involved in psychiatric pathologies, was never shown before.

The absence of photobleaching, blinking, and saturation combined with a high contrast provides unique advantages of higher-harmonic generating nanoparticles over fluorescent probes, allowing for prolonged correlation spectroscopy studies. We apply the coherent intensity fluctuation model to study the mobility of second harmonic generating nanoparticles. A concise protocol is presented for quantifying the diffusion coefficient from a single spectroscopy measurement without the need for separate point-spread-function calibrations. The technique's applicability is illustrated on nominally 56 nm LiNbO3 nanoparticles. We perform label-free raster image correlation spectroscopy imaging in aqueous suspension and spatiotemporal image correlation spectroscopy in A549 human lung carcinoma cells. In good agreement with the expected theoretical result, the measured diffusion coefficient in water at room temperature is (7.5 +/- 0.3) mu m(2)/s. The diffusion coefficient in the cells is more than 10(3) times lower and heterogeneous, with an average of (3.7 +/- 1.5) X 10(-3) mu m(2)/s. This research was supported by the Research Foundation Flanders (Fonds Wetenschappelijk Onderzoek, project G092915FWO) and by the Interuniversity Attraction Poles Program (IAP FS2 P7/05, Functional Supramolecular Systems) initiated by the Belgian Science Policy Office. The authors are grateful to Prof. Dr. J. D'Haen for the SEM image, to Dr. N. Smisdom for fruitful discussions, and to Dr. R. Paesen and Dr. N. Smisdom for the design of the polarization unit. Research in P.P.'s laboratory was supported by the Office of Science, Office of Basic Energy Sciences, of the U.S. Department of Energy under Contract No. DE-AC02-05CH11231.

The lipid organization of microbubbles is important in many applications. By monitoring the photoselection and emission spectrum of the fluorescent probe Laurdan in perfluorobutane gas-filled DPPC microbubbles with a two-photon laser scanning microscope, we observed a transition to a more rigid lipid organization in 30 minutes to several hours. The authors thank IOF funding of Hasselt University and the support from Interuniversity Attraction Poles Program (IAP FS2 P7/05, Functional Supramolecular Systems) initiated by the (Belgian Science Policy Office). The authors thank the province of Limburg for the support through IMPTECHNO. A. E. acknowledges support by European Commission funding under Horizon 2020 programme, FET-OPEN project AMPHORA, grant agreement 766456. S. S. is an SB PhD fellow at the Research Foundation Flanders (FWO). The authors are grateful to Dr R. Paesen for the development of the polarization control unit in the confocal laser scanning microscope. The authors thank H. Penxten for the technical assistance with the fluorescence spectroscopy measurements.

We present a general analytical model for the intensity fluctuation autocorrelation function for second and third harmonic generating point scatterers. Expressions are derived for a stationary laser beam and for scanning beam configurations for specific correlation methodologies. We discuss free translational diffusion in both three and two dimensions. At low particle concentrations, the expressions for fluorescence are retrieved, while at high particle concentrations a rescaling of the function parameters is required for a stationary illumination beam, provided that the phase shift per unit length of the beam equals zero.

The adverse health effects of particulate matter exposure are a generally accepted concern. Dramatic statistical figures suggest that fine dust is a main environmental risk in Europe and can be held accountable for hundreds of thousands of deaths per year [1]. Locating and tracking these nanometer sized particles, however, is not straight forward: In epidemiological and toxicology research only measurements based on labels [2] such as radionuclide markers have been applied. In this paper we present a direct, label-free optical contrast mechanism to detect carbon nanoparticles immersed in aqueous environments [3]. The virtue of this technique is its ability to perform in body fluids such as urine but also in cells and tissues. The mechanism is based on white light (WL) generation upon illumination with femtosecond pulsed near-infrared and is therefore non-incandescence related. We demonstrate the technique in various biological settings with dry and suspended carbon black particles (CB), a widely used model compound for soot [4]. Our approach allows for the unequivocal localization of CB alongside of common fluorophores and markers and can be performed on multiphoton laser-scanning microscopy platforms, a system commonly available in research laboratories. [1] European Environment Agency (2015). Press release. [2] Kong et al. Int. J. Mol. Sci. 2013, 14, (11), 22529-22543 [3] Bove and Steuwe et al. Nano letters, 2016, (16) , pages 3173-3178 [4] Arnal et al. Combust. Sci. Technol. 2012, 184, (7-8), 1191-1206.

Carbonaceous particle exposure and air pollution in general lead to a multitude of adverse human health effects and pose multiple challenges in terms of exposure, risk and safety assessment. Highly desirable for fast screening are label-free approaches for detecting these particle types in biological or medical context. We report a powerful approach for detecting carbonaceous particles using photothermal pump-probe microscopy, which directly probes their strong light absorption. The principle and reliability of this approach is demonstrated by examining 4 different carbon black (CB) species modeling soot with diameters ranging from 13 to 500 nm. Our results show that the proposed approach is applicable to a large number of CB types as well as black carbon. As the particles show a strong absorption over a wide spectral range as compared to other absorbing species, we can image CB particles almost background free. Our pump-probe approach allows label-free optical detection and unambiguous localization of CB particles in (bio)fluids and 3D cellular environments. In combination with fluorescence microscopy, this method allows for simultaneous colocalization of CB with different cellular components using fluorophores as shown here for human lung fibroblasts. We further demonstrate the versatility of pump-probe detection in a flow cell. Fonds Wetenschappelijk Onderzoek, Grant/Award numbers: 11ZB115N, 12R6315N, G082317; Onderzoeksraad, KU Leuven, Grant/Award number: C14/15/053, OT/12/059; H2020 European Research Council, Grant/Award number: ERC-2012-StG 310898

Although adverse health effects of carbon black (CB) exposure are generally accepted, a direct, label-free approach for detecting CB particles in fluids and at the cellular level is still lacking. Here, we report nonincandescence related white-light (WL) generation by dry and suspended carbon black particles under illumination with femtosecond (fs) pulsed near-infrared light as a powerful tool for the detection of these carbonaceous materials. This observation is done for four different CB species with diameters ranging from 13 to 500 nm, suggesting this WL emission under fs near-infrared illumination is a general property of CB particles. As the emitted radiation spreads over the whole visible spectrum, detection is straightforward and flexible. The unique property of the described WL emission allows optical detection and unequivocal localization of CB particles in fluids and in cellular environments while simultaneously colocalizing different cellular components using various specific fluorophores as shown here using human lung fibroblasts. The experiments are performed on a typical multiphoton laser-scanning microscopy platform, widely available in research laboratories. This research was supported by the Interuniversity Attraction Poles Program (P7/05) initiated by the Belgian Science Policy Office. H.B. acknowledges funding from Research Foundation Flanders (Fonds Wetenschappelijk Onderzoek, FWO) for a doctoral Fellowship: 11ZB115N. C.S. is supported by a postdoctoral FWO fellowship: 12R6315N. The authors also thank FWO for the research Grant G082113. M.R. acknowledges financial support from the KU Leuven Research Fund (C14/15/053, OT/12/059). M.A. thanks the Province of Limburg (Belgium) for the financial support within the tUL IMPULS FASE II program, allowing for the upgrading of the laser source used in this work. W. Baekeland is acknowledged for his support with the recording of the single photon extinction and emission spectra. H.B. gratefully acknowledges the assistance of Mrs. P. Bex and Mr. J. Janssen.

Statins have attracted interest as a treatment option for multiple sclerosis (MS) because of their pleiotropic antiinflammatory and immunomodulatory effects. However, contradictory results have been described when they are applied to oligodendrocytes (OLGs), the cell type predominantly affected in MS. In this study we focus on the in vitro effect of statins on process outgrowth in OLN-93 cells, a well-characterized OLG-derived cell line, and primary cultures of neonatal rat OLGs. Application of the lipophilic simvastatin, as low as 0.1-1 μM, disturbs process formation of both cell types, leading to less ramified cells. We show that both protein isoprenylation and cholesterol synthesis are required for the normal differentiation of OLGs. It is further demonstrated that the expression of 2',3'-cyclic-nucleotide-3' phosphodiesterase (CNP) and tubulin is lowered, concomitant with a reduction of membrane-bound CNP as well as tubulin. Therefore, we propose that lack of isoprenylation of CNP could help to explain the altered morphological and biochemical differentiation state of treated OLGs. Moreover, expression of specific myelin markers, such as myelin basic protein, myelin-associated glycoprotein, and myelin oligodendrocyte glycoprotein, was compromised after treatment. We conclude that simvastatin treatment has detrimental effects on OLG process outgrowth, the prior step in (re)myelination, thereby mortgaging long-term healing of MS lesions.

The authors thank Mrs. R. Beenaerts, Mr. J. Janssen, Mr. P. Pirotte, Mr. J. Soogen, Mr. R. van Werde for expert laboratory assistance, Prof. Dr. I. Lambrichts and Mr.M. Jans for logistics and fixation procedures, Mr. K. Vanstreels and Mr. B. Ruttens for assistance with the scanning electron microscope, Prof. Dr. R. Valcke for the use of his spectrophotometers, Mr. J. Duchateau, Mr. F. Horemans, Mrs. V. Vrindts for cleaning the diamond substrates, Ir. L. Naelaerts (KHLIM) for lending stereomicroscopes. S. W. is a post-doctoral research fellow of the Research Foundation – Flanders (FWO-Vlaanderen). This work was funded by the Research Council of the UHasselt, tUL, the K.U. Leuven (GOA/2006/02) and by a PhD grant of the Institute for the Promotion of Innovation through Science and Technology in Flanders (IWT-Vlaanderen). Support by IAP P6/27 Functional Supramolecular Systems (BELSPO) and by the FWOonderzoeksgemeenschap “Scanning andWide Field Microscopy of (Bio)-organic Systems” is gratefully acknowledged.

In the framework of developing a fast and label-free immunosensor for C-reactive protein (CRP) detection, H-terminated nanocrystalline diamond (NCD) was functionalised with anti-CRP antibodies that were physically adsorbed to the surface. Impedance spectroscopy was used to electronically detect real-time CRP recognition. Different impedance behaviours were observed after CRP addition as compared to after FITC-labelled ssDNA addition at low (100 Hz) as well as at high frequencies (1 MHz). Physical interpretations of the observed impedance changes were obtained by fitting the data to an equivalent electrical circuit. Concentrations of 1 μM CRP were recognised with a reaction time of 30 minutes. (© 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim)

This article reviews the Current state-of-the art of diamond-based DNA sensors. Some general concepts involved in biosensors are introduced and applied to DNA sensors. The properties of chemically vapor deposited (CVD) diamond relevant for this application are summed up, with special attention for the stability and bio-compatibility of the material. Several routes to functionalize the diamond Surface are considered. The physical properties of the obtained DNA layers are discussed in terms of surface density and molecular conformation. Possible read-out strategies are evaluated, including optical and electronic sensing. With diamond-based DNA sensors, real-time and label-free sensing is achieved.

Z -scan fluorescence correlation spectroscopy (FCS) uses multiple focus planes to determine diffusion coefficients in supported phospholipid bilayers (SPBs) without the need for extrinsic calibration. The approach can be used to discriminate diffusion modes. In this work, Z -scan FCS was applied to study the diffusion of the fluorescent lipid analogue DiI–C 18 (5) in the top membrane of giant unilamellar vesicles (GUVs) in the electroformation chamber. In contrast to SPBs, the intrinsic estimate of the radial waist might differ from the one obtained by extrinsic calibration. We describe a pragmatic approach to verify the validity of the Z -scan approach under these conditions.

The covalent attachment method for DNA on nanocrystalline diamond (NCD), involving the introduction of COOH functionalities on the surface by photoattachment of 10-undecenoic acid (10-UDA), followed by the 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC)-mediated coupling to NH 2-labeled ssDNA, is evaluated in terms of stability, density, and functionality of the resulting biological interface. This is of crucial importance in DNA biosensor development. The covalent nature of DNA attachment will infer the necessary stability and favorable orientation to the ssDNA probe molecules. Using confocal fluorescence microscopy, the influence of buffer type for the removal of excess 10-UDA and ssDNA, the probe ssDNA length, the probe ssDNA concentration, and the presence of the COOH-linker on the density and functionality of the ssDNA probe layer were investigated. It was determined that the most homogeneously dense and functional DNA layer was obtained when 300 pmol of short ssDNA was applied to COOH-modified NCD samples, while H-terminated NCD was resistant for DNA attachment. Exploiting this surface functionality dependence of the DNA attachment efficiency, a shadow mask was applied during the photochemical introduction of the COOH-functionalities, leaving certain regions on the NCD H-terminated. The subsequent DNA attachment resulted in a fluorescence pattern corresponding to the negative of the shadow mask. Finally, NCD surfaces covered with mixtures of the 10-UDA linker molecule and a similar molecule lacking the COOH functionality, functioning as a lateral spacer, were examined for their suitability in preventing nonspecific adsorption to the surface and in decreasing steric hindrance. However, purely COOH-modified NCD samples, patterned with H-terminated regions and treated with a controlled amount of probe DNA, proved the most efficient in fulfilling these tasks.

Mimicking the selectivity and sensitivity of biological systems for sensor devices is of increasing interest in biomedical, environmental and chemical analysis. Synthetic materials with imprinted nanocavities, acting as highly selective artificial receptors, are a tailor-made solution in obtaining such a sensor. Incorporation of such molecularly imprinted polymers (MIPs) in a platform suitable for electrochemical measurements, can offer high sensitivity together with device miniaturization and an electronic read-out. As a proof of principle, a MIP-based sensor for l -nicotine has been developed. To this end, the molecular structure of l -nicotine was imprinted in a polymer matrix of polymethacrylic acid (PMAA). Subsequently, microparticles of the imprinted polymer were immobilized on thin films of the conjugated polymer OC1C10-PPV. These films were incorporated in an impedimetric sensing device. Using electrochemical impedance spectroscopy, the real part of the impedance was monitored for various concentrations. This setup allows for the detection of l -nicotine from 1 to 10 nM and is insensitive for the resembling molecule l -cotinine.

The SAC8.5, a low-cost Peltier-cooled black and white 8-bit CCD camera for astronomy, was evaluated for its use in imaging microscopy. Two camera-microscope configurations were used: an epifluorescence microscope (Nikon Eclipse TE2000-U) and a bottom port laser scanning confocal microscope system (Zeiss LSCM 510 META). Main advantages of the CCD camera over the currently used photomultiplier detection in the scanning setup are fast image capturing, stable background, an improved signal-to-noise ratio and good linearity. Based on DAPI-labelled Chinese Hamster Ovarian cells, the signal-to-noise ratio was estimated to be 4 times higher with respect to the currently used confocal photomultiplier detector. A linear relationship between the fluorescence signal and the FITC-inulin concentrations ranging from 0.05 to 1.8 mg mL(-1) could be established. With the SAC8.5 CCD camera and using DAPI, calcein-AM and propidium iodide we could also distinguish between viable, apoptotic and necrotic cells: exposure to CdCl(2) caused necrosis in A6 cells. Additional examples include the observation of wire-like mitochondrial networks in Mito Tracker Green-loaded Madin-Darby canine kidney cells. Furthermore, it is straightforward to interface the SAC8.5 with automated shutters to prevent rapid fluorophore photobleaching via easy to use astrovideo software. In this study, we demonstrate that the SAC8.5 black and white CCD camera is an easy-to-implement and cost-conscious addition to quantitative fluorescence microfluorimetry on living tissues and is suitable for teaching laboratories.

We have studied by kinetic Chl-fluorescence imaging (Chl-FI) Nicotiana benthamiana plants infected with the Italian strain of the pepper mild mottle tobamovirus (PMMoV-I). We have mapped leaf photosynthesis at different points of the fluorescence induction curve as well as at different post-infection times. Images of different fluorescence parameters were obtained to investigate which one could discriminate control from infected leaves in the absence of symptoms. The non-photochemical quenching (NPQ) of excess energy in photosystem II (PSII) seems to be the most adequate chlorophyll fluorescence parameter to assess the effect of tobamoviral infection on the chloroplast. Non-symptomatic mature leaves from inoculated plants displayed a very characteristic time-varying NPQ pattern. In addition, a correlation between NPQ amplification and virus localization by tissue-print was found, suggesting that an increase in the local NPQ values is associated with the areas invaded by the pathogen. Changes in chloroplast ultrastructure in non-symptomatic leaf areas showing different NPQ levels were also investigated. A gradient of ultrastructural modifications was observed among the different areas.

The plasma membrane of eukaryotic cells exhibits lateral inhomogeneities, mainly containing cholesterol and sphingomyelin, which provide liquid-ordered microdomains (lipid "rafts") that segregate membrane components. Rafts are thought to modulate the biological functions of molecules that become associated with them, and as such, they appear to be involved in a variety of processes, including signal transduction, membrane sorting, cell adhesion and pathogen entry. Although still a matter of ongoing debate, evidence in favor of the presence of these microdomains is gradually accumulating but a consensus on issues like their size, lifetime, composition, and biological significance has yet to be reached. Here, we provide an overview of the evidence supporting the presence of rafts in oligodendrocytes, the myelin-producing cells of the central nervous system, and discuss their functional significance. the myelin membrane differs fundamentally from the plasma membrane, both in lipid and protein composition. Moreover, since myelin membranes are unusually enriched in glycosphingolipids, questions concerning the biogenesis and functional relevance of microdomains thus appear of special interest in oligodendrocytes. The current picture of rafts in oligodendrocytes is mainly based on detergent methods. The robustness of such data is discussed and alternative methods that may provide complementary data are indicated. (c) 2006 Wiley-Liss, Inc.

Time-resolved fluorescence spectroscopy and microscopy in both time and frequency domains provide very useful and accurate information on dynamic processes. Good quality data are essential in obtaining reliable parameter estimates. Distortions of the fluorescence response due to artifacts may have disastrous consequences. We provide here a concise overview of potential difficulties encountered under daily laboratory circumstances in the use of time- and frequency-domain equipment as well as practical remedies against common error conditions, elucidated with several graphs to aid the researcher in visual inspection and quality-control of collected data. A range of artifacts due to sample preparation or to optical and electronic pitfalls are discussed, as are remedies against them. Also recommended data analysis strategies are described.

Classification of harvested apples when predicting their storage potential is an important task. This paper describes how chlorophyll a fluorescence images taken in blue light through a red filter, can be used to classify apples. In such an image, fluorescence appears as a relatively homogenous area broken by a number of small nonfluorescing spots, corresponding to normal corky tissue patches, lenticells, and to damaged areas that lower the quality of the apple. The damaged regions appear more longish, curved or boat-shaped compared to the roundish, regular lenticells. We propose an apple classification method that employs a hierarchy of two neural networks. The first network classifies each spot according to geometrical criteria and the second network uses this information together with global attributes to classify the apple. The system reached 95% accuracy using a test material classified by an expert for "bad" and "good" apples.

20 pags, 2 tabs, After recalling the basic relations relevant to both steady-state and time-resolved fluorescence polarization, it is shown how the values of steady-state polarized intensities recorded experimentally usually need to be corrected for systematic effects and errors, caused by instrumentation and sample properties. A list of selected reference values of steady-state fluorescence anisotropy and polarization is given. Attention is also paid to analysis of time-resolved fluorescence anisotropy data obtained by pulse fluorometry or phase and modulation fluorometry techniques. Recommendations for checking the accuracy of measure ments are provided together with a list of selected time-resolved fluorescence anisotropy data as reported in the literature. © 2013 IUPAC., Support by the Spanish Ministry of Science and Innovation 2010CTQ16457 (AUA) and the IAP P6/27 (BELSPO) Functional Supramolecular Systems (MvdV) is thankfully acknowledged.

By combining near-infrared intensity-modulated illumination with frequency domain detection methods, we have obtained the first detailed optical images of thick tissues (1–3 cm). The raw data of these images are characterized by millimetre resolution and high contrast. The method employs a pulsed laser coupled to a cross-correlation frequency domain detection scheme. The near-infrared imaging method is non-invasive and inexpensive, uses non-ionizing radiation and is potentially fast enough for real-time imaging in the seconds time range. In the frequency domain, diffusional wave optics provides the framework for a description of the light propagation in tissues. Our approach results in a theoretically and experimentally simple way to exploit the advantages of time resolution in optical imaging of inhomogeneous highly scattering materials. At any given modulation frequency, the light wavefront advances at constant velocity in a spherical wave. Objects with scattering or absorption coefficients that are different from the surrounding medium cause a deformation of the propagating wavefront that can be accurately measured using frequency domain detection methods. The visualization (on a computer screen) of these wavefront deformations provides a projection of the interior's image.

Statins have attracted interest as a treatment option for multiple sclerosis (MS) because of their pleiotropic antiinflammatory and immunomodulatory effects. However, contradictory results have been described when they are applied to oligodendrocytes (OLGs), the cell type predominantly affected in MS. In this study we focus on the in vitro effect of statins on process outgrowth in OLN-93 cells, a well-characterized OLG-derived cell line, and primary cultures of neonatal rat OLGs. Application of the lipophilic simvastatin, as low as 0.1-1 μM, disturbs process formation of both cell types, leading to less ramified cells. We show that both protein isoprenylation and cholesterol synthesis are required for the normal differentiation of OLGs. It is further demonstrated that the expression of 2',3'-cyclic-nucleotide-3' phosphodiesterase (CNP) and tubulin is lowered, concomitant with a reduction of membrane-bound CNP as well as tubulin. Therefore, we propose that lack of isoprenylation of CNP could help to explain the altered morphological and biochemical differentiation state of treated OLGs. Moreover, expression of specific myelin markers, such as myelin basic protein, myelin-associated glycoprotein, and myelin oligodendrocyte glycoprotein, was compromised after treatment. We conclude that simvastatin treatment has detrimental effects on OLG process outgrowth, the prior step in (re)myelination, thereby mortgaging long-term healing of MS lesions.

Detailed practical information is provided with emphasis on mapping cytosolic and mitochondrial pH, mitochondrial Na(+), and briefly also aspects related to mitochondrial Ca(2+) measurements in living cells, as grown on (un)coated glass coverslips. This chapter lists (laser scanning confocal) microscope instrumentation and setup requirements for proper imaging conditions, cell holders, and an easy-to-use incubator stage. For the daily routine of preparing buffer and calibration solutions, extensive annotated protocols are provided. In addition, detailed measurement and image analysis protocols are given to routinely obtain optimum results with confidence, while avoiding a number of typical pitfalls.

Detailed practical information is provided with emphasis on mapping cytosolic and mitochondrial pH, mitochondrial Na(+), and briefly also aspects related to mitochondrial Ca(2+) measurements in living cells, as grown on (un)coated glass coverslips. This chapter lists (laser scanning confocal) microscope instrumentation and setup requirements for proper imaging conditions, cell holders, and an easy-to-use incubator stage. For the daily routine of preparing buffer and calibration solutions, extensive annotated protocols are provided. In addition, detailed measurement and image analysis protocols are given to routinely obtain optimum results with confidence, while avoiding a number of typical pitfalls.

Label-free detection of DNA molecules on chemically vapor-deposited diamond surfaces is achieved with spectroscopic ellipsometry in the infrared and vacuum ultraviolet range. This nondestructive method has the potential to yield information on the average orientation of single as well as double-stranded DNA molecules, without restricting the strand length to the persistence length. The orientational analysis based on electronic excitations in combination with information from layer thicknesses provides a deeper understanding of biological layers on diamond. The pi-pi* transition dipole moments, corresponding to a transition at 4.74 eV, originate from the individual bases. They are in a plane perpendicular to the DNA backbone with an associated n-pi* transition at 4.47 eV. For 8-36 bases of single- and double-stranded DNA covalently attached to ultra-nanocrystalline diamond, the ratio between in- and out-of-plane components in the best fit simulations to the ellipsometric spectra yields an average tilt angle of the DNA backbone with respect to the surface plane ranging from 45 degrees to 52 degrees . We comment on the physical meaning of the calculated tilt angles. Additional information is gathered from atomic force microscopy, fluorescence imaging, and wetting experiments. The results reported here are of value in understanding and optimizing the performance of the electronic readout of a diamond-based label-free DNA hybridization sensor.

Many membrane proteins and lipids are partially confined in substructures ranging from tens of nanometers to micrometers in size. Evidence for heterogeneities in the membrane of oligodendrocytes, i.e. the myelin-producing cells of the central nervous system, is almost exclusively based on detergent methods. However, as application of detergents can alter the membrane phase behaviour, it is important to investigate membrane heterogeneities in living cells. Here, we report on the first investigations of the diffusion behavior of the myelin-specific protein MOG (myelin oligodendrocyte glycoprotein) in OLN-93 as studied by the recently developed RICS (raster-scanning image correlation spectroscopy) technique. We implemented RICS on a standard confocal laser-scanning microscope with one-photon excitation and analog detection. Measurements on FITC-dextran were used to evaluate the performance of the system and the data analysis procedure.

Most challenging in the development of DNA sensors is the ability to distinguish between fully complementary target ssDNA (single-strand DNA) and 1-mismatch ssDNA. To deal with this problem, we performed impedance spectroscopy on DNA-functionalized nanocrystalline diamond (NCD) layers during hybridization and denaturation. In both reactions, a difference in behavior was observed for 1-mismatch target DNA and complementary target DNA in real-time. During real-time hybridization, a decrease of the impedance was observed at lower frequencies when the complementary target DNA was added, while the addition of 1-mismatch target ssDNA caused no significant change. Fitting these results to an electrical circuit demonstrates that this is correlated with a decrease of the depletion zone in the space charge region of the diamond. During real-time denaturation, differentiation between 1-mismatch and complementary target DNA was possible at higher frequencies. Denaturation of complementary DNA showed the longest exponential decay time of the impedance, while the decay time during 1-mismatch denaturation was the shortest. The real-time hybridization and denaturation experiments were carried out on different NCD samples in various buffer solutions at temperatures between 20 and 80 degrees C. It was revealed that the best results were obtained using a Microhyb hybridization buffer at 80 degrees C and 10x PCR buffer at 30 degrees C for hybridization and 0.1 M NaOH at temperatures above 40 degrees C for denaturation. We demonstrate that the combination of real-time hybridization spectra and real-time denaturation spectra yield important information on the type of target. This approach may allow a reliable identification of the mismatch sequence, which is the most biologically relevant.

A series of fluorophores with single-exponential fluorescence decays in liquid solution at 20 °C were measured independently by nine laboratories using single-photon timing and multi-frequency phase and modulation fluorometry instruments with lasers as excitation source. The dyes that can serve as fluorescence lifetime standards for time-domain and frequency-domain measurements are all commercially available, are photostable under the conditions of the measurements, and are soluble in solvents of spectroscopic quality (methanol, cyclohexane, and water). These lifetime standards are anthracene, 9-cyanoanthracene, 9, 10-diphenylanthracene, N-methylcarbazole, coumarin 153, erythrosin B, N-acetyl-L-tryptophanamide (NATA), 1, 4-bis(5-phenyloxazol-2-yl)benzene (POPOP), 2, 5-diphenyloxazole (PPO), rhodamine B, rubrene, N-(3-sulfopropyl)acridinium (SPA), and 1, 4-diphenylbenzene (p-terphenyl). At 20 °C the fluorescence lifetimes vary from 89 ps to 31.2 ns, depending on fluorescent dye and solvent, which is a useful range for modern pico- and nanosecond time-domain or mega- to gigahertz frequency-domain instrumentation. The decay times are independent of the excitation and emission wavelengths. Dependent on the structure of the dye and the solvent, the excitation wavelengths used range from 284 nm to 575 nm, the emission from 330 nm to 630 nm. These lifetime standards may be used to either calibrate or test the resolution of time- and frequency-domain instrumentation or as reference compounds to eliminate the color effect in photomultiplier tubes. Statistical analyses by means of two-sample charts indicate that there is no laboratory bias in the lifetime determinations. Moreover, statistical tests show that there is an excellent correlation between the lifetimes estimated by the time-domain and frequency-domain fluorometries. Comprehensive tables compiling the results for twenty {; ; ; ; fluorescence lifetime standard/solvent}; ; ; ; combinations are given.

The plasma membrane of various mammalian cell types is heterogeneous in structure and may contain microdomains, which can impose constraints on the lateral diffusion of its constituents. Fluorescence correlation spectroscopy (FCS) can be used to investigate the dynamic properties of the plasma membrane of living cells. Very recently, Wawrezinieck et al. (Wawrezinieck, L., H. Rigneault, D. Marguet, and P. F. Lenne. 2005. Biophys. J. 89:4029-4042) described a method to probe the nature of the lateral microheterogeneities of the membrane by varying the beam size in the FCS instrument. The dependence of the width of the autocorrelation function at half-maximum, i.e., the diffusion time, on the transverse area of the confocal volume gives information on the nature of the imposed confinement. We describe an alternative approach that yields essentially the same information, and can readily be applied on commercial FCS instruments by measuring the diffusion time and the particle number at various relative positions of the cell membrane with respect to the waist of the laser beam, i.e., by performing a Z-scan.

Laser assisted confocal microscopy has made a lot of progress over the past few years. Laser systems have become more modular and compact. There is an ever-increasing number of available laser excitation lines as well as an improvement in user friendliness and ease of use. At the same time, expansion of Web resources has provided easy access to a wealth of information. Our goal is both to aid the experienced and novice microscopist in quickly locating and sorting through the relevant laser information and to provide a means of avoiding common problems and pitfalls in the use of laser excitation in the various fluorescence techniques such as fluorescence correlation spectroscopy (FCS), fluorescence lifetime imaging microscopy (FLIM), fluorescence loss in photobleaching (FLIP), fluorescence recovery after photobleaching (FRAP), optical coherence tomography (OCT), second harmonic generation (SHG), single molecule detection (SMD), and single particle tracking (SPT). In this chapter we describe the characteristic properties of a number of lasers commonly used in fluorescence microscopy.

A model for analyzing Forster resonance energy transfer (FRET) data in relation to the cell plasma membrane raft hypothesis is developed to take into account: (a) the distribution of FRET donors and acceptors at the surface of probing antibody fragments specific for a putative raft component; (b) partitioning of the raft component between raft and non-raft areas of the membrane; and (c) the dependence of the raft partition on the expression level of the considered component. Analysis of relevant FRET data in the literature according to this model provides support for the raft hypothesis.

Chemical vapour deposited (CVD) diamond is a very promising material for biosensor fabrication owing both to its chemical inertness and the ability to make it electrical semiconducting that allows for connection with integrated circuits. For biosensor construction, a biochemical method to immobilize nucleic acids to a diamond surface has been developed. Nanocrystalline diamond is grown using microwave plasma-enhanced chemical vapour deposition (MPECVD). After hydrogenation of the surface, 10-undecenoic acid, an ω-unsaturated fatty acid, is tethered by 254 nm photochemical attachment. This is followed by 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide (EDC)-mediated attachment of amino (NH 2 )-modified dsDNA. The functionality of the covalently bound dsDNA molecules is confirmed by fluorescence measurements, PCR and gel electrophoresis during 35 denaturation and rehybridisation steps. The linking method after the fatty acid attachment can easily be applied to other biomolecules like antibodies and enzymes.

Il semble de mieux en mieux etabli que la membrane plasmique de nombreux types cellulaires de mammiferes est heterogene et composee de microdomaines lipidiques (radeaux lipidiques ou lipid rafts). Dans ce travail, nous nous sommes interesses a l'organisation membranaire des oligodendrocytes, les cellules myelinisantes du systeme nerveux central. Nous avons utilise la spectroscopie a correlation de fluorescence (fluorescence correlation spectroscopy, FCS) pour mesurer la diffusion laterale d'un lipide et d'une proteine dans la lignee cellulaire oligodendrogliale OLN-93. Le lipide etait la sphingomyeline marquee de facon fluorescente (Bodipy FL-C5 SM). La proteine etait la glycoproteine de la myeline oligodendrocytaire (MOG). Pour mesurer la diffusion laterale de la MOG, les cellules OLN-93 ont ete transfectees a l'aide d'une fusion vecteur MOG-EGFP (enhanced green fluorescent protein). Les mesures ont ete realisees a temperature ambiante. Les donnees de FCS ont ete analysees pour etudier la diffusion en deux dimensions (2D) selon trois modeles independants, incluant chacun une fraction de triplet : (a) 2D a un composant (2D1Cα), (b) 2D a diffusion anormale et (c) 2D a deux composants (2D2C). Nos resultats preliminaires suggerent que, dans le cas de la sphingomyeline, les meilleures tendances sont obtenues avec le modele 2D2C. Dans le cas de la MOG-EGFP, les modeles 2D2C et 2D1Cα donnent des previsions de qualites similaires. Les estimations parametriques obtenues avec le modele 2D1Cα ont cependant une deviation standard plus faible. Le parametre d'anormalite pour la MOG-EGFP est de 0,59 ′ 0,01.

In the work reported here, we investigated the interaction between the semiconducting polymer MDMO-PPV and antibodies against the fluorescent dyes fluorescein isothiocyanate (FITC) and Cy5. The antibodies are adsorbed physically onto thin polymer films on gold electrodes, as seen in AFM images of these films. By tuning the antibody concentration, the contact angle of distilled water with the film can be made to vary between 95 degrees and 50 degrees, showing that different surface densities of antibody can be obtained. That these biosensor films specifically bind their antigenic fluorescent molecules from PBS buffer solution is demonstrated by confocal fluorescence microscopy. Specific antigen-antibody recognition is demonstrated by lack of cross-sensitivity between the two antibodies and their antigens. In a biosensor prototype based on differential impedance spectroscopy, these polymer films show a clear response to 1 ppb antigen solution, with a time constant of 2-3 min.

In ischemic or hypoxic tissues, elevated Ca2+levels have emerged as one of the main damaging agents among other Ca2+-independent mechanisms of cellular injury. Because mitochondria, besides the endoplasmic reticulum, play a key role in the maintainance of cellular Ca2+homeostasis, alterations in the mitochondrial Ca2+content ([Ca2+]m) were monitored in addition to changes in cytosolic Ca2+concentration ([Ca2+]i) during metabolic inhibition (MI) in renal epithelial Madin-Darby canine kidney (MDCK) cells. [Ca2+]iand [Ca2+]mwere monitored via, respectively, fura 2 and rhod 2 measurements. MI induced an increase in [Ca2+]ireaching 631 ± 78 nM in ∼20 min, followed by a decrease to 118 ± 9 nM in the next ∼25 min. A pronounced drop in cellular ATP levels and a rapid increase in intracellular Na+concentrations in the first 20 min of MI excluded Ca2+efflux in the second phase via plasma membrane ATPases or Na+/Ca2+exchangers (NCE). Mitochondrial rhod 2 intensities increased to 434 ± 46% of the control value during MI, indicating that mitochondria sequester Ca2+during MI. The mitochondrial potential (ΔΨm) was lost in 20 min of MI, excluding mitochondrial Ca2+uptake via the ΔΨm-dependent mitochondrial Ca2+uniporter after 20 min of MI. Under Na+-free conditions, or when CGP-37157, a specific inhibitor of the mitochondrial NCE, was used, no drop in [Ca2+]iwas seen during MI, whereas the MI-induced increase in mitochondrial rhod 2 fluorescence was strongly reduced. To our knowledge, this study is the first to report that in metabolically inhibited renal epithelial cells mitochondria take up Ca2+via the NCE acting in the reverse mode.

Soluble copolymers of camptothecin (CPT), based on poly[N-(2-hydroxypropyl) methacrylamide] (pHPMA), were obtained by conjugation through the degradable spacers -Gly-Phe-Leu-Gly- or -Gly-6-aminohexanoyl-Gly-. We investigated to what extent passive accumulation and retention of hydroxypropyl methacrylamide copolymer of CPT (pHPMA-CPT) in tumors and modulation of the drug release influence efficacy. Release of CPT in vivo was detected by time-resolved phase-shift fluorescence imaging on tumor specimens, based on the evidence that free and bound drug had different fluorescence lifetimes in solution. HT-29 murine specimens, obtained at several times after treatment with (3)H-labeled free CPT, pHPMA-Gly-Phe-Leu-Gly-CPT, or pHPMA-Gly-6-aminohexanoyl-Gly-CPT, were either imaged for time-resolved phase-shift fluorescence or subjected to autoradiography. Phase shifts of CPT conjugates were equal or longer than those of free CPT, indicating the presence of both free and polymer-bound drug in the tumor, in agreement with autoradiograms. pHPMA-Gly-Phe-Leu-Gly-CPT underwent relevant intratumor hydrolysis during the first 24 h, whereas the hydrolysis of pHPMA-Gly-6-aminohexanoyl-Gly-CPT was slow. The latter showed antitumor activity at doses from 10 to 22.5 mg/kg/day against s.c. HT-29, A2780, M14, and A549 s.c. xenografts. Moreover, inhibition of tumor growth lasted for up to 73-88 days, and cures were observed on mice with orthotopic implanted HT-29; pHPMA-Gly-Phe-Leu-Gly-CPT was 2-fold more potent than pHPMA-Gly-6-aminohexanoyl-Gly-CPT but less tolerated. Our data suggest that the efficacy of pHPMA-CPT copolymers is related to their intratumor accumulation, and in vivo properties of releasing CPT by esterolytic and proteolytic degradation.

Plant leaves possess microscopic valves, called stomata, that enable control of transpirational water loss. In case of water shortage, stomata close, resulting in decreased transpirational cooling. The ensuing temperature increase is readily visualized by thermography. Salicylic acid, a central compound in the defense of plants against pathogens, also closes stomata in several species. In previous work, thermography permitted to monitor an increase in temperature after infection of resistant tobacco by tobacco mosaic virus, before visual symptoms appeared. Furthermore, cell death was visualized with high contrast in both tobacco and Arabidopsis. In addition to transpiration, photosynthetic assimilation is a key physiological parameter. If the amount of light absorbed by chlorophyll exceeds the capacity of the photosynthetic chain, the surplus is dissipated as light of longer wavelength. This phenomenon is known as chlorophyll fluorescence. If a plant leaf is affected by stress, photosynthesis is impaired resulting in a bigger share of non-utilized light energy emitted as fluorescence. The potential of an automated imaging setup combining thermal and fluorescence imaging was shown by monitoring spontaneous cell death in tobacco. This represents a first step to multispectral characterization of a wide range of emerging stresses, which likely affect one or both key physiological parameters.© (2002) COPYRIGHT SPIE--The International Society for Optical Engineering. Downloading of the abstract is permitted for personal use only.

Chronic wound healing is very seriously hampered by a profound lack of basic understanding of the influence of bacteria and bacterial biofilms on the process. Treatment could greatly benefit from a longitudinal 3D assessment of spatial and biochemical properties of woundbed and fluid using molecular techniques. Inexpensive biosensor monitoring of a range of wound parameters and tissue cellular interactions would be beneficial, specially the profiling and early detection of multi-drug resistant organisms (MDROs).Point-Of-Care (POC) wound assessment to monitor the healing process will benefit from digitization and automation of wound shape, size and volume determinations, wound color imaging including a simultaneously imaged calibration color card for digital processing, as well as imaging of pH, tissue oxygenation, vascularization and temperature distributions.After initial haemostatis, a chronic wound develops when (a)biotic influences extend the inflammation period and hamper wound repair proliferation and remodeling. For healing to proceed the influence of the species and total number of bacteria present, the fraction of persister bacteria with reduced metabolic rates, the quantity of multiple drug resistance present in a (chronic) wound, disruption by wound dressings and the biochemistry of the woundbed - bacteria interaction eg. Extra Cellular Matrix (ECM) remodeling and bacterial motility has to be understood. To gain an understanding in chronic wound characteristics for theranostic purposes we present experiments in silico using Matlab and complement results with available open-source to simulate biofilms consisting of single and multiple species with species-specific properties under several of above described conditions. This may help to better understand the outcome of clinical trials and assist in the design of POC biosensors for monitoring and aiding chronic wound healing.

Reactive Oxygen Species (ROS) production and impairment of mitochondrial functioning is a central research topic. A recent review for Multiple Sclerosis1. Investigation of mitochondrial functioning in living cells under normal physiological conditions and metabolic inhibition provides biologically relevant information using confocal cell imaging2,3 Cytosolic calcium clearance does not necessarily lead to mitochondrial dysfunction as long as the mitochondrial matrix pH stayed acidified compared to the cytosol.2 Studying intra-mitochondrial versus cytosolic electrolyte homeostasis in living cells under metabolic stress might be crucial to understand the conditions where mitochondria become detrimental by producing ROS. Caveats are presented to properly obtain information about mitochondrial and cellular pH, structure and properties of the mitochondrial network under normal conditions and metabolic inhibition, by using optimized protocols4. The method critically depends on availability and selection of nuclear areas with lowest background fluorescence contribution while keeping experiment conditions rigorously similar. We present the influence of factors such as cell type, plating number, age of the cells, attachment surface properties, cell confluency, loading protocol of the dyes, background area selection on the values obtained. Optimization of parameters such as objective choice, detector voltage stability, fluorescence intensity saturation, illumination power and bleaching, imaging depth, point spread function (PSF) and reduction of cell motility are elucidated. Confluent Madin Darby Canine Kidney (MDCK) cells were loaded with mitotracker green and pH indicator SNARF. Images were collected with a Zeiss LSM 510 Meta CLSM. Data were analyzed with ImageJ4, and Matlab.1. Lassmann, H., and van Horssen, J., FEBS Lett. 20112. Smets, I. et al., Am. J. Physiol. Renal Physiol., 20043. Baron, Sz. et al., JASN, 20054. vandeVen, M, & Balut, C. et al. Methods Mol Biol. 2010

Author(s): Fishkin, JB; Fantini, S; vandeVen, MJ; Gratton, E | Abstract: The predictions of the frequency-domain standard diffusion equation (SDE) model for light propagation in an infinite turbid medium diverge from the more complete [Formula Presented] approximation to the linear Boltzmann transport equation at intensity modulation frequencies greater than several hundred MHz. The [Formula Presented] approximation is based on keeping only the terms l=0 and l=1 in the expansion of the angular photon density in spherical harmonics, and the nomenclature [Formula Presented] approximation is used since the spherical harmonics of order l=1 can be written in terms of the first order Legendre polynomial, which is traditionally represented by the symbol [Formula Presented]. Frequency-domain data acquired in a quasi-infinite turbid medium at modulation frequencies ranging from 0.38 to 3.2 GHz using a superheterodyning microwave detection system were analyzed using expressions derived from both the [Formula Presented] aproximation equation and the SDE. This analysis shows that the [Formula Presented] approximation provides a more accurate description of the data over this range of modulation frequencies. Some researchers have claimed that the [Formula Presented] approximation predicts that a light pulse should propagate with an average speed of c/ √3 in a thick turbid medium. However, an examination of the Green’s function that we obtained from the frequency-domain [Formula Presented] approximation model indicates that a photon density wave phase velocity of c/ √3 is only asymptotically approached in a regime where the light intensity modulation frequency aproaches infinity. The Fourier transform of this frequency-domain result shows that in the time domain, the [Formula Presented] approximation predicts that only the leading edge of the pulse (i.e., the photons arriving at the detector at the earliest time) approaches a speed of c/√3. © 1996 The American Physical Society.

Fluorescence recovery after photobleaching (FRAP) carried out on a confocal laser-scanning microscope (CLSM) performs well for photobleached disks that are large compared to the resolution of the bleaching beam. For smaller disks approaching this resolution, current FRAP models providing a closed-form solution do not allow one to extract the diffusion coefficient accurately. The new generalized disk model we present addresses this shortcoming by bringing into account the bleaching resolution and the total confocal imaging resolution. A closed-form solution is obtained under the assumption of linear photobleaching. Furthermore, simultaneous analysis of FRAP data collected at various disk sizes allows for the intrinsic determination of the instrumental resolution parameters, thereby obviating the need for an extrinsic calibration. A new method to estimate the variance of FRAP data is introduced to allow for proper weighting in this global analysis approach by nonlinear least squares. Experiments are performed on two independent CLSMs on homogeneous samples providing validation over a large range of diffusion coefficients. (C) 2011 Society of Photo-Optical Instrumentation Engineers (SPIE). [DOI: 10.1117/1.3569620]

In frequency domain fluorometry, as well as all other spectroscopic techniques, the noise ultimately limits the sensitivity of the instrument and the precision of the measurement. The analysis of the sources of noise in different instruments has revealed that the noise is due to a number of different instrumental factors rather than photon statistics. The ultimate goal is to eliminate those factors to achieve a situation in which the limit is the detector intrinsic noise. We have developed a system, based on digital signal processing, in which the influence of several spurious noise sources has been reduced. A study of the range of cross-correlation frequencies used to obtain the best signal-to-noise ratio is presented.© (1992) COPYRIGHT SPIE--The International Society for Optical Engineering. Downloading of the abstract is permitted for personal use only.

The attainable frequency range of ordinary heterodyning and super-heterodyning frequency- domain fluorescence instrumentation is limited by the response of the optical detector rather than by the harmonic content of the light source. The replacement of the photomultiplier detector by a 6 (mu) microchannel plate detector has improved the frequency response from 500 MHz to 10 GHz. A new method is developed to detect fast, excited state processes by extending to the frequency-domain the well known pump/probe (absorption) technique used in the time-domain. The upper frequency limit attainable with this method is limited only by the pulse width of the light sources. For picosecond pulse lasers this limit extends to hundreds of gigahertz. A theoretical determination of the basic equations is given, and data are shown for the excited state decay of a rhodamine 6G sample in ethylene glycol.

Light propagation in turbid media can be described by photon diffusion. In the frequency domain, sinusoidally intensity-modulated light gives rise to diffusive waves which have a coherent front. In a homogeneous medium, the wave front propagates with a constant phase velocity and the amplitude attenuates exponentially as the diffusional wave advances. We have studied the diffusion approximation to the one-speed linear transport equation with a sinusoidally intensity modulated point source of particles and performed experiments using frequency domain detection methods on homogeneous scattering and absorbing media to test the applicability of the above mentioned transport equation to photon migration in turbid media. We have used the analytical solutions of the linear transport equation in homogeneous, infinite media to determine via a simple analysis of our frequency domain data the linear scattering and absorption coefficients.© (1991) COPYRIGHT SPIE--The International Society for Optical Engineering. Downloading of the abstract is permitted for personal use only.

The basic theory of a frequency domain fluorometer has been known since the original work of Gaviola (1)• With the introduction of multifrequency capabilities and the use of ultra high speed detectors, the basic understanding of the details of the instrumentation has become essential. The ultimate time resolution and separation capabilities for complex decay species critically depends on the noise characteristics of the instrument's various components. In frequency domain fluorometry, it is customary to measure the standard deviation of the phase and modulation at each individual frequency point. In the correlated single photon counting method, it is assumed that the standard deviation is given by '[i, where N is the number of counts in a time channel. In single photon counting techniques, the fluctuation of the number of counts is assumed to be intrinsic to the statistical properties of light, therefore, the only way to decrease the standard deviation of an individual channel is to increase the light intensity at the detector. A reduction of the standard deviation results in lower integration time and better instrument resolvability. The major feature emerging from the analysis of the residues and the standard deviation in the frequency domain technique is that, although they appear randomly distributed and uncorrelated from one frequency point to the next, there is a correlation between different experiments. The pattern of the residues is often reproducible. This observation questions the basic assumption used for statistical data analysis, i.e., that there is a gaussian distribution of each data point around the mean value and that the standard deviation of each point is significant for the statistical analysis of the frequency response.

Frequency domain fluorometry provides an alternative method for the recording of fluorescence decay kinetics (1), The major advantages of this method are the time resolution and the fast measurement of lifetime values. There are several systems, where a change in lifetime occurs across the emission band. These systems include heterogeneous ground state systems, where species emit at different wavelengths; excited state reactions, with products emitting at different parts of the spectrum; and dipolar relaxation processes, where the emission spectra change with time. To characterize the decay of the systems, it is necessary to collect the emission decay at several wavelengths. Generally, this process is obtained by successive measurements using a monochromator or a series of bandpass filters. For steady-state spectra, optical multichannel analyzers (OMA) are available, which can collect the entire emission spectra at once. By gating the image intensifier, used with some of these analyzers, it is possible to obtain time windows of the order of a few nanoseconds (2), This time resolution is inadequate for most of the fluorescence substances. We have developed a new method based of frequency domain fluorometry which extends the time resolution of an OMA to the picosecond region. The entire emission decay is collected in a few seconds and the lifetime information across the entire emission band is analyzed. The same method can be applied to other array detectors such as TV and CCD cameras used in fluorescence microscopy (3).