Measuring Serotonin Distribution in Live Cells with Three-Photon Excitation

Maiti, Sudipta; Shear, Jason B.; Williams, Rebecca M.; Zipfel, Warren R.; Webb, Watt W.

doi:10.1126/science.275.5299.530

Cited by 405 publications

(304 citation statements)

References 19 publications

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“…While the nature of the water soluble component facilitating formation of the side reaction ergosterol D isomer was not identified, it has been reported that small amounts of nitric acid or mercuric nitrate catalyze the formation of the tetraene DHE as well as a side product, i.e., mercurated triene, and so such impurities may be possible contaminants in the original ergosterol (64). Recently, real-time multiphoton imaging of the naturally-occurring fluorescent sterol DHE in the plasma membrane of living cells (70,82,203,204) was used to examine sterol distribution directly with high optical sectioning capability (206)(207)(208)(209). While subcellular distribution (plasma membrane, lysosome, lipid droplet, etc) of DHE was highly dependent on the method of delivery (microcrystals, LUV, MβCD), DHE at the plasma membrane was found to be distributed non-randomly into sterol-rich and sterol-poor regions in plasma membranes of living cells with the size range of sterol-rich clustering domains estimated to be from 200 nm (limit of optical microscopy) to 565 nm (70,71,82,203).…”

Section: Summary and Discussionmentioning

confidence: 99%

Fluorescence Techniques Using Dehydroergosterol to Study Cholesterol Trafficking

McIntosh

Atshaves

Huang

et al. 2008

Lipids

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Cholesterol itself has very few structural/chemical features suitable for real-time imaging in living cells. Thus, the advent of dehydroergosterol [ergosta-5,7,9(11),22-tetraen-3β-ol, DHE] the fluorescent sterol most structurally and functionally similar to cholesterol to date, has proven to be a major asset for real-time probing/elucidating the sterol environment and intracellular sterol trafficking in living organisms. DHE is a naturally-occurring, fluorescent sterol analog that faithfully mimics many of the properties of cholesterol. Because these properties are very sensitive to sterol structure and degradation, such studies require the use of extremely pure (>98%) quantities of fluorescent sterol. DHE is readily bound by cholesterol-binding proteins, is incorporated into lipoproteins (from the diet of animals or by exchange in vitro), and for real-time imaging studies is easily incorporated into cultured cells where it co-distributes with endogenous sterol. Incorporation from an ethanolic stock solution to cell culture media is effective, but this process forms an aqueous dispersion of dehydroergosterol crystals which can result in endocytic cellular uptake and distribution into lysosomes which is problematic in imaging DHE at the plasma membrane of living cells. In contrast, monomeric DHE can be incorporated from unilamellar vesicles by exchange/fusion with the plasma membrane or from DHE-methyl-β-cyclodextrin (DHE-MβCD) complexes by exchange with the plasma membrane. Both of the latter techniques can deliver large quantities of monomeric dehydroergosterol with significant distribution into the plasma membrane. The properties and behavior of DHE in protein-binding, lipoproteins, model membranes, biological membranes, lipid rafts/caveolae, and real-time imaging in living cells indicate that this naturally-occurring fluorescent sterol is a useful mimic for probing the properties of cholesterol in these systems. Keywordsdehydroergosterol; cholesterol; ergosterol; fluorescent sterols; microscopy Historical PerspectiveIn order to resolve the dynamics and factors governing cholesterol trafficking within cells, it is important to recognize that the cholesterol molecule has very few polar constituents (Fig. 1A). Consequently, cholesterol is poorly soluble in aqueous environments such as cytosol as evidenced by critical micellar concentrations of cholesterol and other sterols being very low, *To whom correspondence should be addressed: Department of Physiology and Pharmacology, Texas A&M University, TVMC, College Station, TX 862-1433, FAX: (979) NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript near 20-30 nM (1-5). The physiological impact of cholesterol's low aqueous solubility is that spontaneous cholesterol desorption from the cytofacial leaflet of the plasma membrane or lysosomal membrane into the cytosol for transfer to intracellular sites for esterification, oxidation, or secretion is extremely slow (t 1/2 3 hours-days) (6-9). Therefore, it is important to resolve factors...

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Section: Summary and Discussionmentioning

confidence: 99%

Fluorescence Techniques Using Dehydroergosterol to Study Cholesterol Trafficking

McIntosh

Atshaves

Huang

et al. 2008

Lipids

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“…Based on this technique, visualization of the intracellular mitochondria (Barzda et al, 2005) and quantification of intracellular serotonin (Maiti et al, 1997) and NAD(P)H (Patterson et al, 2000) have been reported. It makes high-resolution imaging of endogenous fluorophores possible and is well suited to intrinsic molecular imaging in biological specimens.…”

Section: Corneal 2-photon Excitation Imagingmentioning

confidence: 99%

Two‐photon microscopy of deep intravital tissues and its merits in clinical research

Wang

König

2010

Journal of Microscopy

174

131

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SummaryMultiphoton excitation laser scanning microscopy, relying on the simultaneous absorption of two or more photons by a molecule, is one of the most exciting recent developments in biomedical imaging. Thanks to its superior imaging capability of deeper tissue penetration and efficient light detection, this system becomes more and more an inspiring tool for intravital bulk tissue imaging. Two-photon excitation microscopy including 2-photon fluorescence and second harmonic generated signal microscopy is the most common multiphoton microscopic application. In the present review we take diverse ocular tissues as intravital samples to demonstrate the advantages of this approach. Experiments with registration of intracellular 2-photon fluorescence and extracellular collagen second harmonic generated signal microscopy in native ocular tissues are focused. Data show that the in-tandem combination of 2-photon fluorescence and second harmonic generated signal microscopy as two-modality microscopy allows for in situ co-localization imaging of various microstructural components in the whole-mount deep intravital tissues. New applications and recent developments of this high technology in clinical studies such as 2-photon-controlled drug release, in vivo drug screening and administration in skin and kidney, as well as its uses in tumourous tissues such as melanoma and glioma, in diseased lung, brain and heart are additionally reviewed. Intrinsic emission two-modal 2-photon microscopy/tomography, acting as an efficient and sensitive non-injurious imaging approach featured by high contrast and subcellular spatial resolution, has been proved to be a promising tool for intravital deep tissue imaging and clinical studies. Given the level of its performance, we believe Correspondence to B.-G. Wang. Tel: +49 3641 938496; fax: +49 3641 938552; e-mail: Baogui.Wang@mti.uni-jena.de that the non-linear optical imaging technique has tremendous potentials to find more applications in biomedical fundamental and clinical research in the near future.

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“…Fourth, an isoelectric focusing gel of the unlabeled lysozyme showed only one band, indicating that the lysozyme preparation was very pure in terms of the charge of its constituent proteins, reducing the likelihood that impurities or lysozyme isoforms are the source of the observed overshoot. Finally, to eliminate fluorescent probes completely, we conducted experiments using multiphoton excitation to obtain uptake images from the intrinsic fluorescence of the tryptophan residues in lysozyme (19). Excitation was with a mode-locked titanium:sapphire laser (Coherent Radiation, Palo Alto, CA) with ϭ 718 nm and a pulse frequency of Ϸ80 MHz.…”

Section: Figmentioning

confidence: 99%

Nondiffusive mechanisms enhance protein uptake rates in ion exchange particles

Dziennik

Belcher²,

Barker³

et al. 2003

Proc. Natl. Acad. Sci. U.S.A.

125

145

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Scanning confocal fluorescence microscopy and multiphoton fluorescence microscopy were used to image the uptake of the protein lysozyme into individual ion exchange chromatography particles in a packed bed in real time. Self-sharpening concentration fronts penetrating into the particles were observed at low salt concentrations in all of the adsorbents studied, but persisted to 100 mM ionic strength only in some materials. In other adsorbents, diffuse profiles were seen at these higher salt concentrations, with the transition region exhibiting a pronounced fluorescence peak at the front at intermediate salt concentrations. These patterns in the uptake profiles are accompanied by significant increases in protein uptake rates that are also seen macroscopically in batch uptake experiments. The fluorescence peak appears to be a concentration overshoot that may develop, in part, from an electrokinetic contribution to transport that also enhances the uptake rate. Further evidence for an electrokinetic origin is that the effect is correlated with high adsorbent surface charge densities. Predictions of a mathematical model incorporating the electrokinetic effect are in qualitative agreement with the observations. These findings indicate that mechanisms other than diffusion contribute to protein transport in oppositely charged porous materials and may be exploited to achieve rapid uptake in process chromatography.T he dramatic growth of the biotechnology industry in recent years has depended critically on preparative separation techniques for protein products. Ion exchange chromatography, introduced for protein separations in the 1950s, remains one of the most widely used preparative-scale purification and separation methods (1). Ion exchange adsorbents have been designed to offer a range of mechanical and chemical properties for chromatographic separation and purification (2), but a fundamental understanding of transport mechanisms in these materials remains elusive. For most materials, simple diffusive mechanisms are thought to control intraparticle transport, but studies using batch or column techniques (3-7) yield model-dependent transport coefficients, and they cannot definitively determine the physical transport mechanism. With the projected acceleration in the discovery and development of protein therapeutics as a result of the human genome project, the demand for improved chromatographic materials and methods for optimizing protein separation and purification will increase. Therefore, a more fundamental understanding of the transport mechanisms in these materials is required to develop better heuristics for process development as well as methodologies for the design of new adsorbents.A key complication in the interpretation of batch or column studies is that the transport is coupled to adsorption, making additional assumptions regarding the adsorption behavior necessary to characterize the transport. Microscopic techniques have a greater potential to resolve the resulting ambiguities regarding transport mechanisms...

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Measuring Serotonin Distribution in Live Cells with Three-Photon Excitation

Cited by 405 publications

References 19 publications

Fluorescence Techniques Using Dehydroergosterol to Study Cholesterol Trafficking

Fluorescence Techniques Using Dehydroergosterol to Study Cholesterol Trafficking

Two‐photon microscopy of deep intravital tissues and its merits in clinical research

Nondiffusive mechanisms enhance protein uptake rates in ion exchange particles

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