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688 KiB

Open AccessArticle

Absorption and Fluorescence Spectroscopic Properties of 1- and 1,4-Silyl-Substituted Naphthalene Derivatives

by Hajime Maeda, Tomohiro Maeda and Kazuhiko Mizuno

Molecules 2012, 17(5), 5108-5125; https://doi.org/10.3390/molecules17055108 - 3 May 2012

Cited by 67 | Viewed by 11267

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 of [...] 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

(This article belongs to the Special Issue Fluorophores - The Fluorescent Toolbox in Biological and Biomedical Research)

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284 KiB

Open AccessCommunication

Selective Formation of Twisted Intramolecular Charge Transfer and Excimer Emissions on 2,7-bis(4-Diethylaminophenyl)-fluorenone by Choice of Solvent

by Masayuki Shigeta, Mifumi Morita and Gen-ichi Konishi

Molecules 2012, 17(4), 4452-4459; https://doi.org/10.3390/molecules17044452 - 13 Apr 2012

Cited by 28 | Viewed by 8182

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 the [...] 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

(This article belongs to the Special Issue Fluorophores - The Fluorescent Toolbox in Biological and Biomedical Research)

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1122 KiB

Open AccessArticle

Developing Fluorescent Hyaluronan Analogs for Hyaluronan Studies

by Wei Wang, Arlin G. Cameron and Shi Ke

Molecules 2012, 17(2), 1520-1534; https://doi.org/10.3390/molecules17021520 - 7 Feb 2012

Cited by 14 | Viewed by 5687

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 HA [...] 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

(This article belongs to the Special Issue Fluorophores - The Fluorescent Toolbox in Biological and Biomedical Research)

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1126 KiB

Open AccessArticle

Fluorescent Lipids: Functional Parts of Fusogenic Liposomes and Tools for Cell Membrane Labeling and Visualization

by Christian Kleusch, Nils Hersch, Bernd Hoffmann, Rudolf Merkel and Agnes Csiszár

Molecules 2012, 17(1), 1055-1073; https://doi.org/10.3390/molecules17011055 - 20 Jan 2012

Cited by 75 | Viewed by 14402

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 bilayers [...] 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

(This article belongs to the Special Issue Fluorophores - The Fluorescent Toolbox in Biological and Biomedical Research)

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402 KiB

Open AccessArticle

Li⁺ Selective Podand-Type Fluoroionophore Based on a Diphenyl Sulfoxide Derivative Bearing Two Pyrene Groups

by Yukihiro Nishimura, Tetsuo Takemura and Sadao Arai

Molecules 2011, 16(8), 6844-6857; https://doi.org/10.3390/molecules16086844 - 10 Aug 2011

Cited by 5 | Viewed by 6333

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, respectively. [...] 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

(This article belongs to the Special Issue Fluorophores - The Fluorescent Toolbox in Biological and Biomedical Research)

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529 KiB

Open AccessArticle

Fluorescent Probes Detecting the Phagocytic Phase of Apoptosis: Enzyme-Substrate Complexes of Topoisomerase and DNA

by Candace L. Minchew and Vladimir V. Didenko

Molecules 2011, 16(6), 4599-4614; https://doi.org/10.3390/molecules16064599 - 3 Jun 2011

Cited by 18 | Viewed by 7591

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 DNA [...] 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

(This article belongs to the Special Issue Fluorophores - The Fluorescent Toolbox in Biological and Biomedical Research)

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9608 KiB

Open AccessArticle

Chemically Induced Photoswitching of Fluorescent Probes—A General Concept for Super-Resolution Microscopy

by Ulrike Endesfelder, Sebastian Malkusch, Benjamin Flottmann, Justine Mondry, Piotr Liguzinski, Peter J. Verveer and Mike Heilemann

Molecules 2011, 16(4), 3106-3118; https://doi.org/10.3390/molecules16043106 - 13 Apr 2011

Cited by 87 | Viewed by 16282

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 impact [...] 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

(This article belongs to the Special Issue Fluorophores - The Fluorescent Toolbox in Biological and Biomedical Research)

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386 KiB

Open AccessArticle

Structural Relationship and Binding Mechanisms of Five Flavonoids with Bovine Serum Albumin

by E-Hu Liu, Lian-Wen Qi and Ping Li

Molecules 2010, 15(12), 9092-9103; https://doi.org/10.3390/molecules15129092 - 9 Dec 2010

Cited by 130 | Viewed by 13259

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 (BSA) [...] 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

(This article belongs to the Special Issue Fluorophores - The Fluorescent Toolbox in Biological and Biomedical Research)

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Review

Jump to: Research

1095 KiB

Open AccessReview

Fluorescent Nanoprobes Dedicated to in Vivo Imaging: From Preclinical Validations to Clinical Translation

by Juliette Mérian, Julien Gravier, Fabrice Navarro and Isabelle Texier

Molecules 2012, 17(5), 5564-5591; https://doi.org/10.3390/molecules17055564 - 10 May 2012

Cited by 151 | Viewed by 11385

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 also [...] 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

(This article belongs to the Special Issue Fluorophores - The Fluorescent Toolbox in Biological and Biomedical Research)

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5522 KiB

Open AccessReview

Advanced Fluorescence Microscopy Techniques—FRAP, FLIP, FLAP, FRET and FLIM

by Hellen C. Ishikawa-Ankerhold, Richard Ankerhold and Gregor P. C. Drummen

Molecules 2012, 17(4), 4047-4132; https://doi.org/10.3390/molecules17044047 - 2 Apr 2012

Cited by 390 | Viewed by 83819

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 which [...] 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

(This article belongs to the Special Issue Fluorophores - The Fluorescent Toolbox in Biological and Biomedical Research)

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867 KiB

Open AccessReview

Fluorescence-Based Multiplex Protein Detection Using Optically Encoded Microbeads

by Bong-Hyun Jun, Homan Kang, Yoon-Sik Lee and Dae Hong Jeong

Molecules 2012, 17(3), 2474-2490; https://doi.org/10.3390/molecules17032474 - 1 Mar 2012

Cited by 71 | Viewed by 11555

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 over [...] 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

(This article belongs to the Special Issue Fluorophores - The Fluorescent Toolbox in Biological and Biomedical Research)

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1216 KiB

Open AccessReview

Oligothiophenes as Fluorescent Markers for Biological Applications

by Massimo L. Capobianco, Giovanna Barbarella and Antonio Manetto

Molecules 2012, 17(1), 910-933; https://doi.org/10.3390/molecules17010910 - 18 Jan 2012

Cited by 45 | Viewed by 8327

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 absorbance [...] 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

(This article belongs to the Special Issue Fluorophores - The Fluorescent Toolbox in Biological and Biomedical Research)

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630 KiB

Open AccessReview

Like a Bolt from the Blue: Phthalocyanines in Biomedical Optics

by Nawal Sekkat, Hubert van den Bergh, Tebello Nyokong and Norbert Lange

Molecules 2012, 17(1), 98-144; https://doi.org/10.3390/molecules17010098 - 23 Dec 2011

Cited by 225 | Viewed by 11674

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 the [...] 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

(This article belongs to the Special Issue Fluorophores - The Fluorescent Toolbox in Biological and Biomedical Research)

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2960 KiB

Open AccessReview

Pyrene: A Probe to Study Protein Conformation and Conformational Changes

by Gursharan Bains, Arti B. Patel and Vasanthy Narayanaswami

Molecules 2011, 16(9), 7909-7935; https://doi.org/10.3390/molecules16097909 - 14 Sep 2011

Cited by 234 | Viewed by 14366

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 pyrene [...] 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

(This article belongs to the Special Issue Fluorophores - The Fluorescent Toolbox in Biological and Biomedical Research)

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751 KiB

Open AccessReview

Recent Developments in Molecular Dynamics Simulations of Fluorescent Membrane Probes

by Luís M. S. Loura and J. P. Prates Ramalho

Molecules 2011, 16(7), 5437-5452; https://doi.org/10.3390/molecules16075437 - 27 Jun 2011

Cited by 53 | Viewed by 10621

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, and [...] 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

(This article belongs to the Special Issue Fluorophores - The Fluorescent Toolbox in Biological and Biomedical Research)

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1988 KiB

Open AccessReview

Molecular Morphology of Pituitary Cells, from Conventional Immunohistochemistry to Fluorescein Imaging

by Akira Matsuno, Akiko Mizutani, Hiroko Okinaga, Koji Takano, So Yamada, Shoko M. Yamada, Hiroshi Nakaguchi, Katsumi Hoya, Mineko Murakami, Masato Takeuchi, Mutsumi Sugaya, Johbu Itoh, Susumu Takekoshi and R. Yoshiyuki Osamura

Molecules 2011, 16(5), 3618-3635; https://doi.org/10.3390/molecules16053618 - 29 Apr 2011

Cited by 8 | Viewed by 7975 | Correction

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 [...] Read more.

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

(This article belongs to the Special Issue Fluorophores - The Fluorescent Toolbox in Biological and Biomedical Research)

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