Three-dimensional resolution doubling in wide-field fluorescence microscopy by structured illumination.
AuthorsGustafsson MG, Shao L, Carlton PM, Wang CJ, Golubovskaya IN, Cande WZ, Agard DA, Sedat JW,
JournalBiophys J
PubMed ID18326650
'Structured illumination microscopy is a method that can increase the spatial resolution of wide-field fluorescence microscopy beyond its classical limit by using spatially structured illumination light. Here we describe how this method can be applied in three dimensions to double the axial as well as the lateral resolution, with true ... More
The power of single and multibeam two-photon microscopy for high-resolution and high-speed deep tissue and intravital imaging.
AuthorsNiesner R, Andresen V, Neumann J, Spiecker H, Gunzer M,
JournalBiophys J
PubMed ID17557785
'Two-photon microscopy is indispensable for deep tissue and intravital imaging. However, current technology based on single-beam point scanning has reached sensitivity and speed limits because higher performance requires higher laser power leading to sample degradation. We utilize a multifocal scanhead splitting a laser beam into a line of 64 foci, ... More
Role of glycocalyx in leukocyte-endothelial cell adhesion.
AuthorsMulivor AW, Lipowsky HH
JournalAm J Physiol Heart Circ Physiol
PubMed ID12234777
'The binding of fluorescently labeled microspheres (FLMs, 0.1-microm diameter) coated with antibody (1a29) to ICAM-1 was studied in postcapillary venules during topical application of the chemoattractant N-formylmethionyl-leucyl-phenylalanine (fMLP). FLM adhesion to endothelial cells (ECs) increased dramatically from 50 to 150 spheres per 100-microm length of venule after superfusion of the ... More
a2 Adrenergic receptor-mediated inhibition of thermogenesis.
AuthorsMadden CJ, Tupone D, Cano G, Morrison SF,
JournalJ Neurosci
PubMed ID23365239
'a2 adrenergic receptor (a2-AR) agonists have been used as antihypertensive agents, in the management of drug withdrawal, and as sedative analgesics. Since a2-AR agonists also influence the regulation of body temperature, we explored their potential as antipyretic agents. This study delineates the central neural substrate for the inhibition of rat ... More
Glucoprivation in the ventrolateral medulla decreases brown adipose tissue sympathetic nerve activity by decreasing the activity of neurons in raphe pallidus.
AuthorsMadden CJ,
JournalAm J Physiol Regul Integr Comp Physiol
PubMed ID22071154
'In urethane/a-chloralose anesthetized rats, cold exposure increased brown adipose tissue sympathetic nerve activity (BAT SNA: +699 ± 104% control). Intravenous administration of 2-deoxy-D-glucose (2-DG; 200 mg·ml(-1)·kg(-1)) reversed the cold-evoked activation of BAT SNA (nadir: 139 ± 36% of control) and decreased BAT temperature (-1.1 ± 0.2°C), expired CO(2) (-0.4 ± ... More
Why molecules move along a temperature gradient.
AuthorsDuhr S, Braun D
JournalProc Natl Acad Sci U S A
PubMed ID17164337
'Molecules drift along temperature gradients, an effect called thermophoresis, the Soret effect, or thermodiffusion. In liquids, its theoretical foundation is the subject of a long-standing debate. By using an all-optical microfluidic fluorescence method, we present experimental results for DNA and polystyrene beads over a large range of particle sizes, salt ... More
Rapid transport of large polymeric nanoparticles in fresh undiluted human mucus.
AuthorsLai SK, O'Hanlon DE, Harrold S, Man ST, Wang YY, Cone R, Hanes J
JournalProc Natl Acad Sci U S A
PubMed ID17244708
'Nanoparticles larger than the reported mesh-pore size range (10-200 nm) in mucus have been thought to be much too large to undergo rapid diffusional transport through mucus barriers. However, large nanoparticles are preferred for higher drug encapsulation efficiency and the ability to provide sustained delivery of a wider array of ... More
Analysis of ryanodine receptor clusters in rat and human cardiac myocytes.
Single rat ventricular myocytes and human ventricle tissue sections were labeled with antibodies against the ryanodine receptor (RyR) and alpha-actinin to examine the 3D distribution of RyRs with confocal microscopy. Image contrast was maximized by refractive index matching and deconvolution. The RyR label formed discrete puncta representing clusters of RyRs ... More
I5S: wide-field light microscopy with 100-nm-scale resolution in three dimensions.
AuthorsShao L, Isaac B, Uzawa S, Agard DA, Sedat JW, Gustafsson MG,
JournalBiophys J
PubMed ID18326649
A new type of wide-field fluorescence microscopy is described, which produces 100-nm-scale spatial resolution in all three dimensions, by using structured illumination in a microscope that has two opposing objective lenses. Illumination light is split by a grating and a beam splitter into six mutually coherent beams, three of which ... More
A nanoparticle-based model delivery system to guide the rational design of gene delivery to the liver. 1. Synthesis and characterization.
AuthorsPopielarski SR, Pun SH, Davis ME,
JournalBioconjug Chem
PubMed ID16173781
Nonviral gene delivery systems are amenable to forming colloidal particles with a wide range of physicochemical properties that include size, surface charge, and density and type of ligand presented. However, it is not known how to best design these particles without having a set of physicochemical design constraints that have ... More
Inhibition of brown adipose tissue thermogenesis by neurons in the ventrolateral medulla and in the nucleus tractus solitarius.
AuthorsCao WH, Madden CJ, Morrison SF,
JournalAm J Physiol Regul Integr Comp Physiol
PubMed ID20410479
Neurons in the ventrolateral medulla (VLM) and in the nucleus tractus solitarius (NTS) play important roles in the regulation of cardiovascular and other autonomic functions. In the present study, we demonstrate an inhibition of brown adipose tissue (BAT) thermogenesis evoked by activation of neurons in the VLM, as well as ... More
Endogenous activation of spinal 5-hydroxytryptamine (5-HT) receptors contributes to the thermoregulatory activation of brown adipose tissue.
AuthorsMadden CJ, Morrison SF,
JournalAm J Physiol Regul Integr Comp Physiol
PubMed ID20071609
Neurons in the rostral raphe pallidus (RPa) play an essential role in the regulation of sympathetically mediated metabolism and thermogenesis in brown adipose tissue (BAT). The presence of serotonergic neurons in the RPa that are retrogradely labeled following pseudorabies virus injections into BAT suggests that these neurons play a role ... More
Enhanced viscoelasticity of human cystic fibrotic sputum correlates with increasing microheterogeneity in particle transport.
AuthorsDawson M, Wirtz D, Hanes J,
JournalJ Biol Chem
PubMed ID13679362
Current biochemical characterizations of cystic fibrosis (CF) sputum do not address the high degree of microheterogeneity in the rheological properties of the mucosal matrix and only provide bulk-average particle diffusion coefficients. The viscoelasticity of CF sputum greatly reduces the diffusion rates of colloidal particles, limiting the effectiveness of gene delivery ... More
Determination of absolute protein numbers in single synapses by a GFP-based calibration technique.
AuthorsSugiyama Y, Kawabata I, Sobue K, Okabe S,
JournalNat Methods
PubMed ID16118638
To build a quantitative model of molecular organization of neurons, it is essential to have information about the number of protein molecules at individual synapses. Here we developed a method to estimate absolute numbers of individual proteins at actual excitatory synapses by calibrating the fluorescence intensity of microspheres with single ... More
Characterization of the sensitivity of side scatter in a flow-stream waveguide flow cytometer.
AuthorsMariella RP, Huang Z, Langlois RG
JournalCytometry
PubMed ID10486529
BACKGROUND: We previously reported a new optical configuration, in which both the side scatter and the fluorescence are collected using the index-guided, total internal reflection of a flow stream in air (the flow-stream waveguide). METHODS: Using a mixture of 0.202-microm and 0.093-microm diameter polystyrene beads, we have characterized the side ... More
Detection of changes occurring during recovery from the dauer stage in Heterorhabditis bacteriophora.
AuthorsDolan KM, Jones JT, Burnell AM
JournalParasitology
PubMed ID12166523
Nematodes of the genus Heterorhabditis are insect parasites that are widely used as biological control agents. When conditions are unfavourable for reproduction in H. bacteriophora, a long-lived, non-feeding, survival and dispersal stage, the dauer juvenile (DJ), is formed. This DJ stage is also adapted for host finding and infection. When ... More
Measurement of monomer-oligomer distributions via fluorescence moment image analysis.
AuthorsSergeev M, Costantino S, Wiseman PW
JournalBiophys J
PubMed ID16935950
We present higher-order moment analysis of fluorescence intensity fluctuations from individual laser scanning microscopy images applied to study monomer-oligomer distributions. We demonstrate that the number densities and brightness ratios of a mixed population of monomers and oligomers can be determined by analyzing higher-order moments of the fluorescence intensity fluctuations from ... More
k-Space image correlation spectroscopy: a method for accurate transport measurements independent of fluorophore photophysics.
AuthorsKolin DL, Ronis D, Wiseman PW
JournalBiophys J
PubMed ID16861272
We present the theory and application of reciprocal space image correlation spectroscopy (kICS). This technique measures the number density, diffusion coefficient, and velocity of fluorescently labeled macromolecules in a cell membrane imaged on a confocal, two-photon, or total internal reflection fluorescence microscope. In contrast to r-space correlation techniques, we show ... More
Measuring near plasma membrane and global intracellular calcium dynamics in astrocytes.
AuthorsShigetomi E, Khakh BS,
JournalJ Vis Exp
PubMed ID19396060
The brain contains glial cells. Astrocytes, a type of glial cell, have long been known to provide a passive supportive role to neurons. However, increasing evidence suggests that astrocytes may also actively participate in brain function through functional interactions with neurons. However, many fundamental aspects of astrocyte biology remain controversial, ... More
Nucleolar assembly of the rRNA processing machinery in living cells.
AuthorsSavino TM, Gébrane-Younès J, De Mey J, Sibarita JB, Hernandez-Verdun D
JournalJ Cell Biol
PubMed ID11381093
To understand how nuclear machineries are targeted to accurate locations during nuclear assembly, we investigated the pathway of the ribosomal RNA (rRNA) processing machinery towards ribosomal genes (nucleolar organizer regions [NORs]) at exit of mitosis. To follow in living cells two permanently transfected green fluorescence protein-tagged nucleolar proteins, fibrillarin and ... More
Performance comparison between the high-speed Yokogawa spinning disc confocal system and single-point scanning confocal systems.
AuthorsWang E, Babbey CM, Dunn KW
JournalJ Microsc
PubMed ID15857376
Fluorescence microscopy of the dynamics of living cells presents a special challenge to a microscope imaging system, simultaneously requiring both high spatial resolution and high temporal resolution, but with illumination levels low enough to prevent fluorophore damage and cytotoxicity. We have compared the high-speed Yokogawa CSU10 spinning disc confocal system ... More
Spatially correlated fluorescence/AFM of individual nanosized particles and biomolecules.
AuthorsKolodny LA, Willard DM, Carillo LL, Nelson MW, Van Orden A
JournalAnal Chem
PubMed ID11354476
Individual fluorescent polystyrene nanospheres (<10-100-nm diameter) and individual fluorescently labeled DNA molecules were dispersed on mica and analyzed using time-resolved fluorescence spectroscopy and atomic force microscopy (AFM). Spatial correlation of the fluorescence and AFM measurements was accomplished by (1) positioning a single fluorescent particle into the near diffraction-limited confocal excitation ... More