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Special Issue "Fluorophores - The Fluorescent Toolbox in Biological and Biomedical Research"

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A special issue of Molecules (ISSN 1420-3049).

Deadline for manuscript submissions: closed (29 February 2012)

Special Issue Editor

Guest Editor
Dr. Gregor Drummen

Cellular Stress and Ageing Program, Bionanoscience and Bioimaging Program, BNS, 33647 Bielefeld, Germany
Interests: quantum dots; bionanotechnology; two-photon fluorescence imaging; cellular imaging; fluorescence microscopy; cancer; cell signaling; oxidative stress; lipids and biomembranes; lipid peroxidation; antioxidants; renal pathobiology

Special Issue Information

Dear Colleagues,

Ever since the invention of lenses, man has not only looked into the skies, but also into the realm of the invisible microscopic world of tissues, cells, and micro-organisms. The use of fluorescent labels and the development of various forms of optical microscopy, in particular confocal laser scanning microscopy (CLSM), have significantly advanced our knowledge about the basic mechanisms underpinning biology and the pathophysiological processes that lead to disease. Furthermore, fluorescence-based assays have largely replayed radioactive assays in the lab.

It is intended that this special issue of “Molecules” will consider fundamental physicochemical properties, synthesis and modification, biomedical, imaging and assay applications of fluorophores, from organic dyes to fluorescent nanoparticles and fluorescent proteins. In this respect it should be stressed that the fluorescent molecule always takes centre stage. Previously unpublished experimental, theoretical, prospective, historical, and review papers are solicited on the following and related topics:

  • Synthesis and modification of fluorophores
  • Physico-chemical and fluorescent properties
  • Biocompatibility and cytotoxicity
  • Live-cell tracking and imaging
  • Whole animal imaging
  • FRET, FLIM, FRAP, FLIP et al.
  • Applications in cell biology and (bio)medicine
  • Fluorescence-based assays and biosensors

Dr. Gregor Drummen
Guest Editor

Keywords

  • fluorophores
  • synthesis
  • imaging
  • fluorescence microscopy
  • BODIPY
  • FLUORESCEIN
  • ALEXA
  • fluorescence sensors
  • toxicity
  • FRET
  • FLIM
  • FRAP
  • correlation spectroscopy
  • CLSM
  • fluorescence
  • quantum dots
  • nanoparticles
  • super resolution microscopy

Published Papers (16 papers)

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Research

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Open AccessArticle Absorption and Fluorescence Spectroscopic Properties of 1- and 1,4-Silyl-Substituted Naphthalene Derivatives
Molecules 2012, 17(5), 5108-5125; doi:10.3390/molecules17055108
Received: 13 February 2012 / Revised: 21 April 2012 / Accepted: 23 April 2012 / Published: 3 May 2012
Cited by 24 | PDF Full-text (688 KB)
Abstract
Silyl-substituted naphthalene derivatives at the 1- and 1,4-positions were synthesized and their UV absorption, fluorescence spectroscopic properties, and fluorescence lifetimes were determined. Analysis of the results shows that the introduction of silyl groups at these positions of the naphthalene chromophore/fluorophore causes shifts [...] Read more.
Silyl-substituted naphthalene derivatives at the 1- and 1,4-positions were synthesized and their UV absorption, fluorescence spectroscopic properties, and fluorescence lifetimes were determined. Analysis of the results shows that the introduction of silyl groups at these positions of the naphthalene chromophore/fluorophore causes shifts of the absorption maxima to longer wavelengths and increases in fluorescence intensities. Bathochromic shifts of the absorption maxima and increases in fluorescence intensities are also promoted by the introduction of methoxy and cyano groups at the naphthalene 4- and 5-positions. In addition, the fluorescence of 9,10-dicyanoanthracene is efficiently quenched by these naphthalene derivatives with Stern-Volmer plot calculated rate constants that depend on the steric bulk of the silyl groups. Full article
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Open AccessCommunication Selective Formation of Twisted Intramolecular Charge Transfer and Excimer Emissions on 2,7-bis(4-Diethylaminophenyl)-fluorenone by Choice of Solvent
Molecules 2012, 17(4), 4452-4459; doi:10.3390/molecules17044452
Received: 1 March 2012 / Revised: 30 March 2012 / Accepted: 6 April 2012 / Published: 13 April 2012
Cited by 13 | PDF Full-text (284 KB) | Supplementary Files
Abstract
We designed and synthesized a donor-acceptor-donor dye consisting of a 2,7-disubstituted fluorenone with diethylaminophenyl moieties present as strong electron donating groups. Switching between twisted intramolecular charge transfer (TICT) emission and excimer emission was achieved, with no ground state changes, by simply changing [...] Read more.
We designed and synthesized a donor-acceptor-donor dye consisting of a 2,7-disubstituted fluorenone with diethylaminophenyl moieties present as strong electron donating groups. Switching between twisted intramolecular charge transfer (TICT) emission and excimer emission was achieved, with no ground state changes, by simply changing the solvent used. In a nonpolar solvent, excimer emission was observed; with increasing polarity, the emission gradually disappeared, and the TICT emission appeared. Full article
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Open AccessArticle Developing Fluorescent Hyaluronan Analogs for Hyaluronan Studies
Molecules 2012, 17(2), 1520-1534; doi:10.3390/molecules17021520
Received: 22 November 2011 / Revised: 2 February 2012 / Accepted: 3 February 2012 / Published: 7 February 2012
Cited by 7 | PDF Full-text (1122 KB)
Abstract
Two kinds of fluorescent hyaluronan (HA) analogs, one serving as normal imaging agent and the other used as a biosensitive contrast agent, were developed for the investigation of HA uptake and degradation. Our approach of developing HA imaging agents depends on labeling [...] Read more.
Two kinds of fluorescent hyaluronan (HA) analogs, one serving as normal imaging agent and the other used as a biosensitive contrast agent, were developed for the investigation of HA uptake and degradation. Our approach of developing HA imaging agents depends on labeling HA with varying molar percentages of a near-infrared (NIR) dye. At low labeling ratios, the hyaluronan uptake can be directly imaged while at high labeling ratios, the fluorescent signal is quenched and signal generation occurs only after degradation. It is found that the conjugate containing 1%–2% NIR dye can be used as a normal optical imaging agent, while bioactivable imaging agents are formed at 6% to 17% dye loading. It was determined that the conjugation of dye to HA with different loading percentages does not impact HA biodegradation by hyaluronidase (Hyal). The feasibility of using these two NIR fluorescent hyaluronan analogs for HA investigation was evaluated in vivo with optical imaging. The data demonstrates that the 1% dye loaded fluorescent HA can be used to monitor the behavior of HA and its fragments, whereas bioactivatable HA imaging agent (17% dye in HA) is more suitable for detecting HA fragments. Full article
Open AccessArticle Fluorescent Lipids: Functional Parts of Fusogenic Liposomes and Tools for Cell Membrane Labeling and Visualization
Molecules 2012, 17(1), 1055-1073; doi:10.3390/molecules17011055
Received: 30 November 2011 / Revised: 10 January 2012 / Accepted: 16 January 2012 / Published: 20 January 2012
Cited by 16 | PDF Full-text (1126 KB) | Supplementary Files
Abstract
In this paper a rapid and highly efficient method for controlled incorporation of fluorescent lipids into living mammalian cells is introduced. Here, the fluorescent molecules have two consecutive functions: First, they trigger rapid membrane fusion between cellular plasma membranes and the lipid [...] Read more.
In this paper a rapid and highly efficient method for controlled incorporation of fluorescent lipids into living mammalian cells is introduced. Here, the fluorescent molecules have two consecutive functions: First, they trigger rapid membrane fusion between cellular plasma membranes and the lipid bilayers of their carrier particles, so called fusogenic liposomes, and second, after insertion into cellular membranes these molecules enable fluorescence imaging of cell membranes and membrane traffic processes. We tested the fluorescent derivatives of the following essential membrane lipids for membrane fusion: Ceramide, sphingomyelin, phosphocholine, phosphatidylinositol-bisphosphate, ganglioside, cholesterol, and cholesteryl ester. Our results show that all probed lipids could more efficiently be incorporated into the plasma membrane of living cells than by using other methods. Moreover, labeling occurred in a gentle manner under classical cell culture conditions reducing cellular stress responses. Staining procedures were monitored by fluorescence microscopy and it was observed that sphingolipids and cholesterol containing free hydroxyl groups exhibit a decreased distribution velocity as well as a longer persistence in the plasma membrane compared to lipids without hydroxyl groups like phospholipids or other artificial lipid analogs. After membrane staining, the fluorescent molecules were sorted into membranes of cell organelles according to their chemical properties and biological functions without any influence of the delivery system. Full article
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Open AccessArticle Li+ Selective Podand-Type Fluoroionophore Based on a Diphenyl Sulfoxide Derivative Bearing Two Pyrene Groups
Molecules 2011, 16(8), 6844-6857; doi:10.3390/molecules16086844
Received: 11 July 2011 / Revised: 28 July 2011 / Accepted: 8 August 2011 / Published: 10 August 2011
Cited by 3 | PDF Full-text (402 KB)
Abstract
New podand-type fluoroionophores having two pyrene moieties: 2,2´-bis(1-pyrenylacetyloxy)diphenyl sulfide (3), 2,2´-bis(1-pyrenylacetyloxy)diphenyl sulfoxide (4), and 2,2´-bis(1-pyrenylacetyloxy)diphenyl sulfone (5), have been synthesized by connecting two 1-pyrenecarbonylmethyl groups with the two hydroxy groups of 2,2´-dihydroxydiphenyl sulfide, sulfoxide, and sulfone, [...] Read more.
New podand-type fluoroionophores having two pyrene moieties: 2,2´-bis(1-pyrenylacetyloxy)diphenyl sulfide (3), 2,2´-bis(1-pyrenylacetyloxy)diphenyl sulfoxide (4), and 2,2´-bis(1-pyrenylacetyloxy)diphenyl sulfone (5), have been synthesized by connecting two 1-pyrenecarbonylmethyl groups with the two hydroxy groups of 2,2´-dihydroxydiphenyl sulfide, sulfoxide, and sulfone, respectively. Their complexation behavior toward alkali metal ions was examined by fluorescence spectroscopy. Among these fluoroionophores, compound 4, having a sulfinyl group, showed high selectivity toward Li+. Full article
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Open AccessArticle Fluorescent Probes Detecting the Phagocytic Phase of Apoptosis: Enzyme-Substrate Complexes of Topoisomerase and DNA
Molecules 2011, 16(6), 4599-4614; doi:10.3390/molecules16064599
Received: 28 April 2011 / Revised: 20 May 2011 / Accepted: 24 May 2011 / Published: 3 June 2011
Cited by 9 | PDF Full-text (529 KB)
Abstract
In apoptosis, the initial self-driven suicide phase generates cellular corpses which are digested in the phagolysosomes of professional and amateur phagocytes during the subsequent waste-management phase. This ensures the complete elimination of the genetic material which often contains pathological, viral or cancerous [...] Read more.
In apoptosis, the initial self-driven suicide phase generates cellular corpses which are digested in the phagolysosomes of professional and amateur phagocytes during the subsequent waste-management phase. This ensures the complete elimination of the genetic material which often contains pathological, viral or cancerous DNA sequences. Although the phagocytic phase is critical for the efficient execution of apoptosis, there are currently few methods specifically adapted for its detailed visualization in the fixed tissue section format. To resolve this we developed new fluorescent probes for in situ research. The probes selectively visualize active phagocytic cells of any lineage (professional, amateur phagocytes or surrounding tissue cells) which engulf and digest apoptotic cell DNA. These fluorescent probes are the covalently-bound enzyme-DNA intermediates produced in a topoisomerase reaction with specific “starting” oligonucleotides. They detect a specific marker of DNase II cleavage activity, which occurs exclusively in phagolysosomes of the cells that engulfed apoptotic nuclei. The probes provide snap-shot images of the digestion process occurring in cellular organelles responsible for the actual execution of phagocytic degradation of apoptotic cell corpses. We applied the probes for visualization of the phagocytic reaction in tissue sections of normal thymus and in several human lymphomas. We also discuss the nature, stability and properties of DNase II-type breaks as a marker of phagocytic activity. This development provides a useful fluorescent tool for studies of pathologies where clearance of dying cells is essential, such as cancers, inflammation, infection and auto-immune disorders. Full article
Open AccessArticle Chemically Induced Photoswitching of Fluorescent Probes—A General Concept for Super-Resolution Microscopy
Molecules 2011, 16(4), 3106-3118; doi:10.3390/molecules16043106
Received: 18 March 2011 / Revised: 8 April 2011 / Accepted: 12 April 2011 / Published: 13 April 2011
Cited by 49 | PDF Full-text (9608 KB) | Supplementary Files
Abstract
We review fluorescent probes that can be photoswitched or photoactivated and are suited for single-molecule localization based super-resolution microscopy. We exploit the underlying photochemical mechanisms that allow photoswitching of many synthetic organic fluorophores in the presence of reducing agents, and study the [...] Read more.
We review fluorescent probes that can be photoswitched or photoactivated and are suited for single-molecule localization based super-resolution microscopy. We exploit the underlying photochemical mechanisms that allow photoswitching of many synthetic organic fluorophores in the presence of reducing agents, and study the impact of these on the photoswitching properties of various photoactivatable or photoconvertible fluorescent proteins. We have identified mEos2 as a fluorescent protein that exhibits reversible photoswitching under various imaging buffer conditions and present strategies to characterize reversible photoswitching. Finally, we discuss opportunities to combine fluorescent proteins with organic fluorophores for dual-color photoswitching microscopy. Full article
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Open AccessArticle Structural Relationship and Binding Mechanisms of Five Flavonoids with Bovine Serum Albumin
Molecules 2010, 15(12), 9092-9103; doi:10.3390/molecules15129092
Received: 9 October 2010 / Revised: 1 December 2010 / Accepted: 6 December 2010 / Published: 9 December 2010
Cited by 49 | PDF Full-text (386 KB)
Abstract
Flavonoids are structurally diverse and the most ubiquitous groups of dietary polyphenols distributed in various fruits and vegetables. In this study, the interaction between five flavonoids, namely formononetin-7-O-β-D-glucoside, calycosin- 7-O-β-D-glucoside, calycosin, rutin, and quercetin, and bovine serum albumin [...] Read more.
Flavonoids are structurally diverse and the most ubiquitous groups of dietary polyphenols distributed in various fruits and vegetables. In this study, the interaction between five flavonoids, namely formononetin-7-O-β-D-glucoside, calycosin- 7-O-β-D-glucoside, calycosin, rutin, and quercetin, and bovine serum albumin (BSA) was investigated by fluorescence and UV-vis absorbance spectroscopy. In the discussion, it was proved that the fluorescence quenching of BSA by flavonoids was a result of the formation of a flavonoid-BSA complex. Fluorescence quenching constants were determined using the Stern-Volmer and Lineweaver-Burk equations to provide a measure of the binding affinity between the flavonoids and BSA. The binding constants ranked in the order quercetin > rutin > calycosin > calycosin-7-O-β-D-glucoside ≈ formononetin-7-O-β-D-glucoside. The results of thermodynamic parameters ΔG, ΔH, and ΔS at different temperatures indicated that the hydrophobic interaction played a major role in flavonoid-BSA association. The distance r between BSA and acceptor flavonoids was also obtained according to Förster’s theory of non-radiative energy transfer. Full article

Review

Jump to: Research

Open AccessReview Fluorescent Nanoprobes Dedicated to in Vivo Imaging: From Preclinical Validations to Clinical Translation
Molecules 2012, 17(5), 5564-5591; doi:10.3390/molecules17055564
Received: 5 April 2012 / Revised: 6 May 2012 / Accepted: 7 May 2012 / Published: 10 May 2012
Cited by 56 | PDF Full-text (1095 KB)
Abstract
With the fast development, in the last ten years, of a large choice of set-ups dedicated to routine in vivo measurements in rodents, fluorescence imaging techniques are becoming essential tools in preclinical studies. Human clinical uses for diagnostic and image-guided surgery are [...] Read more.
With the fast development, in the last ten years, of a large choice of set-ups dedicated to routine in vivo measurements in rodents, fluorescence imaging techniques are becoming essential tools in preclinical studies. Human clinical uses for diagnostic and image-guided surgery are also emerging. In comparison to low-molecular weight organic dyes, the use of fluorescent nanoprobes can improve both the signal sensitivity (better in vivo optical properties) and the fluorescence biodistribution (passive “nano” uptake in tumours for instance). A wide range of fluorescent nanoprobes have been designed and tested in preclinical studies for the last few years. They will be reviewed and discussed considering the obstacles that need to be overcome for their potential everyday use in clinics. The conjugation of fluorescence imaging with the benefits of nanotechnology should open the way to new medical applications in the near future. Full article
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Open AccessReview Advanced Fluorescence Microscopy Techniques—FRAP, FLIP, FLAP, FRET and FLIM
Molecules 2012, 17(4), 4047-4132; doi:10.3390/molecules17044047
Received: 14 March 2012 / Revised: 21 March 2012 / Accepted: 21 March 2012 / Published: 2 April 2012
Cited by 102 | PDF Full-text (5522 KB) | HTML Full-text | XML Full-text
Abstract
Fluorescence microscopy provides an efficient and unique approach to study fixed and living cells because of its versatility, specificity, and high sensitivity. Fluorescence microscopes can both detect the fluorescence emitted from labeled molecules in biological samples as images or photometric data from [...] Read more.
Fluorescence microscopy provides an efficient and unique approach to study fixed and living cells because of its versatility, specificity, and high sensitivity. Fluorescence microscopes can both detect the fluorescence emitted from labeled molecules in biological samples as images or photometric data from which intensities and emission spectra can be deduced. By exploiting the characteristics of fluorescence, various techniques have been developed that enable the visualization and analysis of complex dynamic events in cells, organelles, and sub-organelle components within the biological specimen. The techniques described here are fluorescence recovery after photobleaching (FRAP), the related fluorescence loss in photobleaching (FLIP), fluorescence localization after photobleaching (FLAP), Förster or fluorescence resonance energy transfer (FRET) and the different ways how to measure FRET, such as acceptor bleaching, sensitized emission, polarization anisotropy, and fluorescence lifetime imaging microscopy (FLIM). First, a brief introduction into the mechanisms underlying fluorescence as a physical phenomenon and fluorescence, confocal, and multiphoton microscopy is given. Subsequently, these advanced microscopy techniques are introduced in more detail, with a description of how these techniques are performed, what needs to be considered, and what practical advantages they can bring to cell biological research. Full article
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Open AccessReview Fluorescence-Based Multiplex Protein Detection Using Optically Encoded Microbeads
Molecules 2012, 17(3), 2474-2490; doi:10.3390/molecules17032474
Received: 22 December 2011 / Revised: 24 February 2012 / Accepted: 24 February 2012 / Published: 1 March 2012
Cited by 23 | PDF Full-text (867 KB)
Abstract
Potential utilization of proteins for early detection and diagnosis of various diseases has drawn considerable interest in the development of protein-based multiplex detection techniques. Among the various techniques for high-throughput protein screening, optically-encoded beads combined with fluorescence-based target monitoring have great advantages [...] Read more.
Potential utilization of proteins for early detection and diagnosis of various diseases has drawn considerable interest in the development of protein-based multiplex detection techniques. Among the various techniques for high-throughput protein screening, optically-encoded beads combined with fluorescence-based target monitoring have great advantages over the planar array-based multiplexing assays. This review discusses recent developments of analytical methods of screening protein molecules on microbead-based platforms. These include various strategies such as barcoded microbeads, molecular beacon-based techniques, and surface-enhanced Raman scattering-based techniques. Their applications for label-free protein detection are also addressed. Especially, the optically-encoded beads such as multilayer fluorescence beads and SERS-encoded beads are successful for generating a large number of coding. Full article
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Open AccessReview Oligothiophenes as Fluorescent Markers for Biological Applications
Molecules 2012, 17(1), 910-933; doi:10.3390/molecules17010910
Received: 2 December 2011 / Revised: 4 January 2012 / Accepted: 9 January 2012 / Published: 18 January 2012
Cited by 19 | PDF Full-text (1216 KB)
Abstract
This paper summarizes some of our results on the application of oligothiophenes as fluorescent markers for biological studies. The oligomers of thiophene, widely known for their semiconductor properties in organic electronics, are also fluorescent compounds characterized by chemical and optical stability, high [...] Read more.
This paper summarizes some of our results on the application of oligothiophenes as fluorescent markers for biological studies. The oligomers of thiophene, widely known for their semiconductor properties in organic electronics, are also fluorescent compounds characterized by chemical and optical stability, high absorbance and quantum yield. Their fluorescent emission can be easily modulated via organic synthesis by changing the number of thiophene rings and the nature of side-chains. This review shows how oligothiophenes can be derivatized with active groups such as phosphoramidite, N-hydroxysuccinimidyl and 4-sulfotetrafluorophenyl esters, isothiocyanate and azide by which the (bio)molecules of interest can be covalently bound. This paper also describes how molecules such as oligonucleotides, proteins and even nanoparticles, tagged with oligothiophenes, can be used in experiments ranging from hybridization studies to imaging of fixed and living cells. Finally, a few multilabeling experiments are described. Full article
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Open AccessReview Like a Bolt from the Blue: Phthalocyanines in Biomedical Optics
Molecules 2012, 17(1), 98-144; doi:10.3390/molecules17010098
Received: 4 November 2011 / Revised: 5 December 2011 / Accepted: 14 December 2011 / Published: 23 December 2011
Cited by 75 | PDF Full-text (630 KB)
Abstract
The purpose of this review is to compile preclinical and clinical results on phthalocyanines (Pcs) as photosensitizers (PS) for Photodynamic Therapy (PDT) and contrast agents for fluorescence imaging. Indeed, Pcs are excellent candidates in these fields due to their strong absorbance in [...] Read more.
The purpose of this review is to compile preclinical and clinical results on phthalocyanines (Pcs) as photosensitizers (PS) for Photodynamic Therapy (PDT) and contrast agents for fluorescence imaging. Indeed, Pcs are excellent candidates in these fields due to their strong absorbance in the NIR region and high chemical and photo-stability. In particular, this is mostly relevant for their in vivo activation in deeper tissular regions. However, most Pcs present two major limitations, i.e., a strong tendency to aggregate and a low water-solubility. In order to overcome these issues, both chemical tuning and pharmaceutical formulation combined with tumor targeting strategies were applied. These aspects will be developed in this review for the most extensively studied Pcs during the last 25 years, i.e., aluminium-, zinc- and silicon-based Pcs. Full article
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Open AccessReview Pyrene: A Probe to Study Protein Conformation and Conformational Changes
Molecules 2011, 16(9), 7909-7935; doi:10.3390/molecules16097909
Received: 1 August 2011 / Revised: 4 September 2011 / Accepted: 6 September 2011 / Published: 14 September 2011
Cited by 71 | PDF Full-text (2960 KB)
Abstract
The review focuses on the unique spectral features of pyrene that can be utilized to investigate protein structure and conformation. Pyrene is a fluorescent probe that can be attached covalently to protein side chains, such as sulfhydryl groups. The spectral features of [...] Read more.
The review focuses on the unique spectral features of pyrene that can be utilized to investigate protein structure and conformation. Pyrene is a fluorescent probe that can be attached covalently to protein side chains, such as sulfhydryl groups. The spectral features of pyrene are exquisitely sensitive to the microenvironment of the probe: it exhibits an ensemble of monomer fluorescence emission peaks that report on the polarity of the probe microenvironment, and an additional band at longer wavelengths, the appearance of which reflects the presence of another pyrene molecule in spatial proximity (~10 Å). Its high extinction coefficient allows us to study labeled proteins in solution at physiologically relevant concentrations. The environmentally- and spatially-sensitive features of pyrene allow monitoring protein conformation, conformational changes, protein folding and unfolding, protein-protein, protein-lipid and protein-membrane interactions. Full article
Open AccessReview Recent Developments in Molecular Dynamics Simulations of Fluorescent Membrane Probes
Molecules 2011, 16(7), 5437-5452; doi:10.3390/molecules16075437
Received: 29 March 2011 / Revised: 21 June 2011 / Accepted: 22 June 2011 / Published: 27 June 2011
Cited by 34 | PDF Full-text (751 KB)
Abstract
Due to their sensitivity and versatility, the use of fluorescence techniques in membrane biophysics is widespread. Because membrane lipids are non-fluorescent, extrinsic membrane probes are widely used. However, the behaviour of these probes when inserted in the bilayer is often poorly understood, [...] Read more.
Due to their sensitivity and versatility, the use of fluorescence techniques in membrane biophysics is widespread. Because membrane lipids are non-fluorescent, extrinsic membrane probes are widely used. However, the behaviour of these probes when inserted in the bilayer is often poorly understood, and it can be hard to distinguish between legitimate membrane properties and perturbation resulting from probe incorporation. Atomistic molecular dynamics simulations present a convenient way to address these issues and have been increasingly used in recent years in this context. This article reviews the application of molecular dynamics to the study of fluorescent membrane probes, focusing on recent work with complex design fluorophores and ordered bilayer systems. Full article
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Open AccessReview Molecular Morphology of Pituitary Cells, from Conventional Immunohistochemistry to Fluorescein Imaging
Molecules 2011, 16(5), 3618-3635; doi:10.3390/molecules16053618
Received: 9 March 2011 / Revised: 25 April 2011 / Accepted: 26 April 2011 / Published: 29 April 2011
Cited by 3 | PDF Full-text (1988 KB) | Correction | Supplementary Files
Abstract
In situ hybridization (ISH) at the electron microscopic (EM) level is essential for elucidating the intracellular distribution and role of mRNA in protein synthesis. EM-ISH is considered to be an important tool for clarifying the intracellular localization of mRNA and the exact site of pituitary hormone synthesis on the rough endoplasmic reticulum. A combined ISH and immunohistochemistry (IHC) under EM (EM-ISH&IHC) approach has sufficient ultrastructural resolution, and provides two-dimensional images of the subcellular localization of pituitary hormone and its mRNA in a pituitary cell. The advantages of semiconductor nanocrystals (quantum dots, Qdots) and confocal laser scanning microscopy (CLSM) enable us to obtain three-dimensional images of the subcellular localization of pituitary hormone and its mRNA. Both EM-ISH&IHC and ISH & IHC using Qdots and CLSM are useful for understanding the relationships between protein and mRNA simultaneously in two or three dimensions. CLSM observation of rab3B and SNARE proteins such as SNAP-25 and syntaxin has revealed that both rab3B and SNARE system proteins play important roles and work together as the exocytotic machinery in anterior pituitary cells. Another important issue is the intracellular transport and secretion of pituitary hormone. We have developed an experimental pituitary cell line, GH3 cell, which has growth hormone (GH) linked to enhanced yellow fluorescein protein (EYFP). This stable GH3 cell secretes GH linked to EYFP upon stimulation by Ca2+ influx or Ca2+ release from storage. This GH3 cell line is useful for the real-time visualization of the intracellular transport and secretion of GH. These three methods from conventional immunohistochemistry and fluorescein imaging allow us to consecutively visualize the process of transcription, translation, transport and secretion of anterior pituitary hormone. Full article

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