PMP22 duplication dysregulates lipid homeostasis and plasma membrane organization in developing human Schwann cells.

Charcot-Marie-Tooth disease type 1A (CMT1A) is the most common inherited peripheral neuropathy caused by a 1.5 Mb tandem duplication of chromosome 17 harbouring the PMP22 gene. This dose-dependent overexpression of PMP22 results in disrupted Schwann cell myelination of peripheral nerves.

Dopamine-mediated striatal activity and function is enhanced in GlyRα2 knockout animals.

The glycine receptor alpha 2 (GlyRα2) is a ligand-gated ion channel which upon activation induces a chloride conductance. Here, we investigated the role of GlyRα2 in dopamine-stimulated striatal cell activity and behavior. We show that depletion of GlyRα2 enhances dopamine-induced increases in the activity of putative dopamine D1 receptor-expressing striatal projection neurons, but does not alter midbrain dopamine neuron activity.

Image Correlation Spectroscopy with Second Harmonic Generating Nanoparticles in Suspension and in Cells.

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.

Dynamics of the phospholipid shell of microbubbles: a fluorescence photoselection and spectral phasor approach.

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.

Rapid and label-free optical detection of individual carbon air pollutant nanoparticulates in biomedical samples.

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.

Biocompatible Label-Free Detection of Carbon Black Particles by Femtosecond Pulsed Laser Microscopy.

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.

Coherent intensity fluctuation model for autocorrelation imaging spectroscopy with higher harmonic generating point scatterers-a comprehensive theoretical study.

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.

Fluorescence recovery after photobleaching on the confocal laser-scanning microscope: generalized model without restriction on the size of the photobleached disk.

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.

Simvastatin interferes with process outgrowth and branching of oligodendrocytes.

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.

Analysis of mitochondrial pH and ion concentrations.

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.

Measuring diffusion of lipid-like probes in artificial and natural membranes by raster image correlation spectroscopy (RICS): use of a commercial laser-scanning microscope with analog detection.

The heterogeneity in composition and interaction within the cellular membrane translates into a wide range of diffusion coefficients of its constituents. Therefore, several complementary microfluorimetric techniques such as fluorescence correlation spectroscopy (FCS), fluorescence recovery after photobleaching (FRAP) and single-particle tracking (SPT) have to be applied to explore the dynamics of membrane components. The recently introduced raster image correlation spectroscopy (RICS) offers a much wider dynamic range than each of these methods separately and allows for spatial mapping of the dynamic properties.

Structural and optical properties of DNA layers covalently attached to diamond surfaces.

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.

Diffusion of myelin oligodendrocyte glycoprotein in living OLN-93 cells investigated by raster-scanning image correlation spectroscopy (RICS).

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.

A highly reliable and budget-friendly Peltier-cooled camera for biological fluorescence imaging microscopy.

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).

Fluorescence lifetime standards for time and frequency domain fluorescence spectroscopy.

A series of fluorophores with single-exponential fluorescence decays in liquid solution at 20 degrees C were measured independently by nine laboratories using single-photon timing and multifrequency 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, water). These lifetime standards are anthracene, 9-cyanoanthracene, 9,10-diphenylanthracene, N-methylcarbazole, coumarin 153, erythrosin B, N-acetyl-l-tryptophanamide, 1,4-bis(5-phenyloxazol-2-yl)benzene, 2,5-diphenyloxazole, rhodamine B, rubrene, N-(3-sulfopropyl)acridinium, and 1,4-diphenylbenzene.

Imaging viral infection: studies on Nicotiana benthamiana plants infected with the pepper mild mottle tobamovirus.

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.

Rafts in oligodendrocytes: evidence and structure-function relationship.

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.

Probing diffusion laws within cellular membranes by Z-scan fluorescence correlation spectroscopy.

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.

Diffusion of sphingomyelin and myelin oligodendrocyte glycoprotein in the membrane of OLN-93 oligodendroglial cells studied by fluorescence correlation spectroscopy.

Evidence has been accumulated that the plasma membrane of various mammalian cell types is heterogeneous in structure and may contain lipid microdomains (lipid rafts). This study focuses on the membrane organization of living oligodendrocytes, which are the myelin-producing cells of the central nervous system. Fluorescence correlation spectroscopy (FCS) was used to monitor the lateral diffusion of a lipid and of a protein in the oligodendroglial cell line OLN-93.

Pitfalls and their remedies in time-resolved fluorescence spectroscopy and microscopy.

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.

Ca2+ uptake in mitochondria occurs via the reverse action of the Na+/Ca2+ exchanger in metabolically inhibited MDCK cells.

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+]i and [Ca2+]m were monitored via, respectively, fura 2 and rhod 2 measurements.

Camptothecin poly[n-(2-hydroxypropyl) methacrylamide] copolymers in antitopoisomerase-I tumor therapy: intratumor release and antitumor efficacy.

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.