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Advances in fluorescence imaging: opportunities for pharmaceutical science
Advanced Drug Delivery Reviews 57 (2005) 1 – 4 www.elsevier.com/locate/addr
Preface
Advances in fluorescence imaging: opportunities for pharmaceutical science
The decision to compile this Advanced Drug Delivery Reviews theme issue came from a wish to highlight the potential of fluorescence imaging technology to contribute to many aspects of pharmaceutical research. Fluorescence imaging has, over the last 20 years or so, made a significant impact in many areas of drug delivery research, from formulation to analysis of the distribution of drug delivery vehicles and characterisation of cellular barriers to delivery. The rapid development of fluorescent probes and advanced light microscopy techniques are providing a constantly changing spectrum of opportunities to apply these technologies in all branches of biomedical sciences. As the opportunities for applying fluorescence imaging techniques in pharmaceutical science increase, it is also true that the expanding array of available technologies can be somewhat confusing to the researcher faced with the question of which imaging technique is most applicable to their particular research questions. This dproblemT was very much in mind when the theme issue was conceived. By drawing together articles from experts with a broad range of research interests, and who use a wide range of imaging techniques, the issue aims to provide a useful source of information about the available technologies to broaden the scope of fluorescence imaging in pharmaceutical research. The papers also differ in type. At one extreme, some focus primarily on theoretical and practical aspects of the available technology and indicate potential and actual applications in drug delivery research, while at the other extreme are dcase historiesT which outline how 0169-409X/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.addr.2004.08.001
fluorescence (and other) imaging techniques are being applied to address specific drug delivery questions. In between these extremes lie contributions focusing on the application of fluorescence imaging to studies of intracellular trafficking, epithelial function and on viral gene delivery systems that illustrate the capabilities of different modes of fluorescence imaging in addressing questions in cell biology and physiology research and highlight their applications in pharmaceutical research. In many cases, the authors have also considered how developments in fluorescence imaging may impact on drug delivery research in the future. Readers can scarcely have failed to notice the massive expansion in the use of fluorescence imaging in all fields of biomedical sciences over recent years. This has arisen from, on the one hand, the emergence of new fluorescent probes, including the near-ubiquitous Green Fluorescent Protein (GFP) and its derivatives, that have provided new ways of labelling almost anything of interest to the biomedical researcher and, on the other hand, technological developments in imaging systems, including new imaging modes, software, computing power and laser technology. Conventional (widefield) fluorescence microscopes are available to virtually every researcher, and more sophisticated technologies (e.g., confocal microscopy, multi-photon microscopy, deconvolution and 3D/4D image processing) have now become accessible to most. Almost certainly the same will increasingly be true of the fluorescence techniques that are currently less
2
Preface
mainstream, such as fluorescence lifetime imaging microscopy (FLIM), total internal reflection fluorescence (TIRF) microscopy and fluorescence correlation spectroscopy (FCS). The exploitation of GFP and its spectral variants has led to a huge increase in the use of live cell imaging techniques to study protein dynamics and protein interactions including fluorescence recovery after photobleaching (FRAP) and fluorescence resonant energy transfer (FRET) techniques which, as with all the methodologies mentioned above, are finding applications in pharmaceutical research. The organization of this theme issue broadly reflects the scope of imaging technologies and their relative complexities. After broad-ranging introductory papers, there are a series of articles which review, and/or provide dcase historiesT to illustrate, applications of fluorescence imaging techniques. Each of these papers focuses predominantly on applications of, in turn, conventional (wide-field) imaging, confocal microscopy, multi-photon microscopy and fluorescence correlation spectroscopy. It is important to stress, however, that it is relatively rare for one type of imaging technology to be ideally suited to all the requirements of a research project, and this is reflected here by the fact that the research discussed in these papers is rarely limited to just one imaging mode. Indeed, the relative strengths of different techniques for specific types of application are discussed in several of these papers. It is clear that some understanding of the basic concepts of fluorescence and of theoretical aspects of the available fluorescence imaging technology are required in order to appreciate how such technology can best be applied to specific research areas. With this in mind, White and Errington have provide an overview of theoretical and practical aspects of fluorescence microscopy. This serves as an introduction to the theme issue and, by comparing the types of imaging equipment available, will be a valuable starting point for those requiring further insight into the fundamental aspects of the daunting range of available imaging technologies, some of which are discussed in more detail elsewhere in the issue. Since knowledge of the intracellular distribution and trafficking of drugs and other agents is of paramount importance in pharmaceutical research,
Watson et al. discuss microscopical methods for studying intracellular trafficking. Their paper reviews practical aspects of fluorescence imaging of cells and focuses in depth on the characterisation of intracellular compartments and trafficking pathways. In addition to outlining the main trafficking pathways of relevance to intracellular drug delivery, the paper provides a fairly comprehensive list of probes used to label intracellular compartments and processes. This paper provides a valuable resource for researchers wishing to go beyond a basic description of cellular uptake to characterize intracellular trafficking and targeting in greater depth. As well as identifying cellular compartments accessed by agents and delivery vehicles, it is also important to understand the kinetics of their transport within complex biological environments, both extracellular and intracellular. In recent years, there have been significant advances in the understanding of these transport processes facilitated by time-lapse imaging and associated computational analyses. Suh et al have written an informative overview of this topic, illustrated with examples of how they are tracking and quantifying movement of viral and non-viral gene (and drug) delivery vehicles within cells, gastrointestinal mucus and cystic fibrosis sputum. The techniques discussed are revealing important information about interactions of delivery vehicles with intra- and extra-cellular components. The theme of gene delivery is also explored by Teschemacher et al. who outline the use of various viral gene delivery vectors, brain cell preparations and imaging technologies to illustrate how they can be used for high-resolution live cell imaging. The authors describe how such technology is allowing them to study aspects of central nervous system structure and function. The authors’ group predominantly applies laser-scanning confocal microscopy to image fluorescent-labelled neurons since this has major advantages over wide-field microscopy in their relatively thick specimens. The next three dcase historyT papers outline how wide-field fluorescence microscopy and/or laser-scanning confocal microscopy and other imaging techniques are being applied to study aspects of drug delivery and the epithelial barrier. Torchilin outlines how fluorescence imaging is being applied by his group to study cellular uptake and fate of micelles and
Preface
liposome vectors containing fluorescent-labelled tracers. The studies described by Torchilin predominantly utilize conventional (wide-field) microscopy on cultured cells or tissue sections, with confocal microscopy being applied in some experiments to confirm the intracellular distribution of liposomes. Two articles then outline the use of fluorescence imaging to study aspects of epithelial function where, due to the nature of the cells or tissue examined, confocal microscopy provides a major advantage over widefield imaging. Johnson provides an overview of studies from his and other groups where confocal imaging is being used to study tight junction structure (using immunofluorescence techniques) and barrier function (using fluorescent-labelled lipids and extracellular tracers). These techniques are being applied to examine the pathophysiological regulation of tight junction barrier function and the paracellular route of uptake across epithelia. Buda et al. then describe how fluorescence imaging, and especially confocal microscopy, is being applied to study specialized antigentransporting epithelial M cells. Their paper uses examples from their own research and that of other groups to highlight the advantages of confocal imaging on whole tissue preparations to characterize M cells and study the transcellular transport of fluorescent bacteria and inert particulates. In addition to clearly describing the advantages that confocal imaging has in their particular research areas, the Johnson and Buda et al. papers also discuss how other imaging techniques, such as wide-field (deconvolution) imaging, electron microscopy, ion-sensitive fluorophores and multi-photon imaging, can contribute to studies of epithelial function. The next two papers in the issue focus primarily on applications of multi-photon microscopy in drug delivery and related research. Tozer et al. describe how multi-photon microscopy can be applied to provide quantitative data on tumour angiogenesis and vascular permeability in intact animals. Using window chambers in which developing tumours can be imaged at high resolution, the capabilities of multiphoton microscopy to image deep within tissue can be fully exploited to study how vascular development and properties affect drug delivery at different levels within the tumour. As explained in this paper, and elsewhere in the issue, the greater penetration of tissue by near-infrared light, on which multi-photon micro-
3
scopy is based, has fundamental advantages for imaging intact tissues where deeper layers are of interest. The other main advantage of multi-photon microscopy is that it can avoid the photodamage that is a ubiquitous problem with shorter-wavelength excitation, particularly when imaging fluorophores in the UV region. This facet of multi-photon microscopy is being exploited in research described by Errington et al. to study the cellular interactions of DNA-binding (anti-cancer) drugs with intrinsic fluorescence, which is excited by UV light in singlephoton microscopy. The ability of multi-photon microscopy to image fluorescent compounds in living cells without damaging the cells is allowing Errington et al. to obtain quantitative single-cell pharmacodynamic data that would not be revealed by other imaging modes. In addition, these authors describe FLIM studies performed with multi-photon microscopy which reveal information about the environment in which the fluorescent drug is found in living cells. The theme of advanced light microscopy and related fluorescence techniques, including fluorescence correlation spectroscopy, are continued in the final two papers in the issue. Gfsch and Rigler have contributed an informative introduction to theoretical and applied aspects of fluorescence fluctuation (correlation) spectroscopy (FFS and FCS). Measurement of the fluorescence events in a population of fluorophore molecules within a diffraction-limited spot can provide detailed information about the mobility of the molecules and has great potential in many areas of cell biology including studying the dynamics of drug mobility and drug-receptor interactions. Gfsch and Rigler also outline in their paper how these highly sensitive techniques are being used to quantify fluorophore interactions with labelled and unlabelled cellular components. This theme is continued by De Smedt et al. in the final paper of the issue which includes discussion of how FFS and FCS are being applied to study DNA delivery. De Smedt et al. show how the mobility of DNA complexes and intracellular DNA degradation can be analysed by FCS techniques. The paper also explains how this type of technique is complemented by other advanced light microscopy techniques, including confocal microscopy, FRAP and FRET, in their studies on the extracellular and intracellular mobilities of DNA
4
Preface
complexes and nanoparticles and also to examine the mobility of fluorescent molecules within microparticulate delivery vehicles. Structured photobleaching is also taken a step further in their demonstration of techniques for encoding polystyrene particulates by photobleaching a dbar codeT within immobilized fluorophore. The papers included in this issue illustrate the enormous potential for fluorescence techniques to address key questions in drug delivery and other aspects of pharmaceutical research. With careful application, each of the techniques described here can go far beyond the use of fluorescence microscopes to generate dpretty picturesT and provide robust quantitative information on drug delivery and action in single cells and complex tissues. I am grateful to all the authors for their contributions that reveal their obvious enthusiasm for the topic. Some of the authors have highlighted technical challenges in fully exploiting the available imaging technology and the advantages to be gained from collaboration between experts in pharmaceutical sciences, cell biology and optics. It should also be stressed that continued improvements in advanced
light microscopy techniques are making these technologies increasingly accessible, user-friendly, flexible and powerful. It is clear that fluorescence imaging has enormous potential in all aspects of biomedical sciences and I hope that this theme issue will help to further promote the use of high-quality fluorescence imaging technologies by the pharmaceutical research community. Finally, I would like to thank all the reviewers for their constructive comments on the submitted manuscripts and the ADDR executive editor, Mark Gumbleton, for commissioning this issue and his invaluable support at each stage of its development. Mark A. Jepson (Theme Editor) Cell Imaging Facility and Department of Biochemistry School of Medical Sciences University of Bristol Bristol BS8 1TD UK E-mail: [email protected].
Preface
Advances in fluorescence imaging: opportunities for pharmaceutical science
The decision to compile this Advanced Drug Delivery Reviews theme issue came from a wish to highlight the potential of fluorescence imaging technology to contribute to many aspects of pharmaceutical research. Fluorescence imaging has, over the last 20 years or so, made a significant impact in many areas of drug delivery research, from formulation to analysis of the distribution of drug delivery vehicles and characterisation of cellular barriers to delivery. The rapid development of fluorescent probes and advanced light microscopy techniques are providing a constantly changing spectrum of opportunities to apply these technologies in all branches of biomedical sciences. As the opportunities for applying fluorescence imaging techniques in pharmaceutical science increase, it is also true that the expanding array of available technologies can be somewhat confusing to the researcher faced with the question of which imaging technique is most applicable to their particular research questions. This dproblemT was very much in mind when the theme issue was conceived. By drawing together articles from experts with a broad range of research interests, and who use a wide range of imaging techniques, the issue aims to provide a useful source of information about the available technologies to broaden the scope of fluorescence imaging in pharmaceutical research. The papers also differ in type. At one extreme, some focus primarily on theoretical and practical aspects of the available technology and indicate potential and actual applications in drug delivery research, while at the other extreme are dcase historiesT which outline how 0169-409X/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.addr.2004.08.001
fluorescence (and other) imaging techniques are being applied to address specific drug delivery questions. In between these extremes lie contributions focusing on the application of fluorescence imaging to studies of intracellular trafficking, epithelial function and on viral gene delivery systems that illustrate the capabilities of different modes of fluorescence imaging in addressing questions in cell biology and physiology research and highlight their applications in pharmaceutical research. In many cases, the authors have also considered how developments in fluorescence imaging may impact on drug delivery research in the future. Readers can scarcely have failed to notice the massive expansion in the use of fluorescence imaging in all fields of biomedical sciences over recent years. This has arisen from, on the one hand, the emergence of new fluorescent probes, including the near-ubiquitous Green Fluorescent Protein (GFP) and its derivatives, that have provided new ways of labelling almost anything of interest to the biomedical researcher and, on the other hand, technological developments in imaging systems, including new imaging modes, software, computing power and laser technology. Conventional (widefield) fluorescence microscopes are available to virtually every researcher, and more sophisticated technologies (e.g., confocal microscopy, multi-photon microscopy, deconvolution and 3D/4D image processing) have now become accessible to most. Almost certainly the same will increasingly be true of the fluorescence techniques that are currently less
2
Preface
mainstream, such as fluorescence lifetime imaging microscopy (FLIM), total internal reflection fluorescence (TIRF) microscopy and fluorescence correlation spectroscopy (FCS). The exploitation of GFP and its spectral variants has led to a huge increase in the use of live cell imaging techniques to study protein dynamics and protein interactions including fluorescence recovery after photobleaching (FRAP) and fluorescence resonant energy transfer (FRET) techniques which, as with all the methodologies mentioned above, are finding applications in pharmaceutical research. The organization of this theme issue broadly reflects the scope of imaging technologies and their relative complexities. After broad-ranging introductory papers, there are a series of articles which review, and/or provide dcase historiesT to illustrate, applications of fluorescence imaging techniques. Each of these papers focuses predominantly on applications of, in turn, conventional (wide-field) imaging, confocal microscopy, multi-photon microscopy and fluorescence correlation spectroscopy. It is important to stress, however, that it is relatively rare for one type of imaging technology to be ideally suited to all the requirements of a research project, and this is reflected here by the fact that the research discussed in these papers is rarely limited to just one imaging mode. Indeed, the relative strengths of different techniques for specific types of application are discussed in several of these papers. It is clear that some understanding of the basic concepts of fluorescence and of theoretical aspects of the available fluorescence imaging technology are required in order to appreciate how such technology can best be applied to specific research areas. With this in mind, White and Errington have provide an overview of theoretical and practical aspects of fluorescence microscopy. This serves as an introduction to the theme issue and, by comparing the types of imaging equipment available, will be a valuable starting point for those requiring further insight into the fundamental aspects of the daunting range of available imaging technologies, some of which are discussed in more detail elsewhere in the issue. Since knowledge of the intracellular distribution and trafficking of drugs and other agents is of paramount importance in pharmaceutical research,
Watson et al. discuss microscopical methods for studying intracellular trafficking. Their paper reviews practical aspects of fluorescence imaging of cells and focuses in depth on the characterisation of intracellular compartments and trafficking pathways. In addition to outlining the main trafficking pathways of relevance to intracellular drug delivery, the paper provides a fairly comprehensive list of probes used to label intracellular compartments and processes. This paper provides a valuable resource for researchers wishing to go beyond a basic description of cellular uptake to characterize intracellular trafficking and targeting in greater depth. As well as identifying cellular compartments accessed by agents and delivery vehicles, it is also important to understand the kinetics of their transport within complex biological environments, both extracellular and intracellular. In recent years, there have been significant advances in the understanding of these transport processes facilitated by time-lapse imaging and associated computational analyses. Suh et al have written an informative overview of this topic, illustrated with examples of how they are tracking and quantifying movement of viral and non-viral gene (and drug) delivery vehicles within cells, gastrointestinal mucus and cystic fibrosis sputum. The techniques discussed are revealing important information about interactions of delivery vehicles with intra- and extra-cellular components. The theme of gene delivery is also explored by Teschemacher et al. who outline the use of various viral gene delivery vectors, brain cell preparations and imaging technologies to illustrate how they can be used for high-resolution live cell imaging. The authors describe how such technology is allowing them to study aspects of central nervous system structure and function. The authors’ group predominantly applies laser-scanning confocal microscopy to image fluorescent-labelled neurons since this has major advantages over wide-field microscopy in their relatively thick specimens. The next three dcase historyT papers outline how wide-field fluorescence microscopy and/or laser-scanning confocal microscopy and other imaging techniques are being applied to study aspects of drug delivery and the epithelial barrier. Torchilin outlines how fluorescence imaging is being applied by his group to study cellular uptake and fate of micelles and
Preface
liposome vectors containing fluorescent-labelled tracers. The studies described by Torchilin predominantly utilize conventional (wide-field) microscopy on cultured cells or tissue sections, with confocal microscopy being applied in some experiments to confirm the intracellular distribution of liposomes. Two articles then outline the use of fluorescence imaging to study aspects of epithelial function where, due to the nature of the cells or tissue examined, confocal microscopy provides a major advantage over widefield imaging. Johnson provides an overview of studies from his and other groups where confocal imaging is being used to study tight junction structure (using immunofluorescence techniques) and barrier function (using fluorescent-labelled lipids and extracellular tracers). These techniques are being applied to examine the pathophysiological regulation of tight junction barrier function and the paracellular route of uptake across epithelia. Buda et al. then describe how fluorescence imaging, and especially confocal microscopy, is being applied to study specialized antigentransporting epithelial M cells. Their paper uses examples from their own research and that of other groups to highlight the advantages of confocal imaging on whole tissue preparations to characterize M cells and study the transcellular transport of fluorescent bacteria and inert particulates. In addition to clearly describing the advantages that confocal imaging has in their particular research areas, the Johnson and Buda et al. papers also discuss how other imaging techniques, such as wide-field (deconvolution) imaging, electron microscopy, ion-sensitive fluorophores and multi-photon imaging, can contribute to studies of epithelial function. The next two papers in the issue focus primarily on applications of multi-photon microscopy in drug delivery and related research. Tozer et al. describe how multi-photon microscopy can be applied to provide quantitative data on tumour angiogenesis and vascular permeability in intact animals. Using window chambers in which developing tumours can be imaged at high resolution, the capabilities of multiphoton microscopy to image deep within tissue can be fully exploited to study how vascular development and properties affect drug delivery at different levels within the tumour. As explained in this paper, and elsewhere in the issue, the greater penetration of tissue by near-infrared light, on which multi-photon micro-
3
scopy is based, has fundamental advantages for imaging intact tissues where deeper layers are of interest. The other main advantage of multi-photon microscopy is that it can avoid the photodamage that is a ubiquitous problem with shorter-wavelength excitation, particularly when imaging fluorophores in the UV region. This facet of multi-photon microscopy is being exploited in research described by Errington et al. to study the cellular interactions of DNA-binding (anti-cancer) drugs with intrinsic fluorescence, which is excited by UV light in singlephoton microscopy. The ability of multi-photon microscopy to image fluorescent compounds in living cells without damaging the cells is allowing Errington et al. to obtain quantitative single-cell pharmacodynamic data that would not be revealed by other imaging modes. In addition, these authors describe FLIM studies performed with multi-photon microscopy which reveal information about the environment in which the fluorescent drug is found in living cells. The theme of advanced light microscopy and related fluorescence techniques, including fluorescence correlation spectroscopy, are continued in the final two papers in the issue. Gfsch and Rigler have contributed an informative introduction to theoretical and applied aspects of fluorescence fluctuation (correlation) spectroscopy (FFS and FCS). Measurement of the fluorescence events in a population of fluorophore molecules within a diffraction-limited spot can provide detailed information about the mobility of the molecules and has great potential in many areas of cell biology including studying the dynamics of drug mobility and drug-receptor interactions. Gfsch and Rigler also outline in their paper how these highly sensitive techniques are being used to quantify fluorophore interactions with labelled and unlabelled cellular components. This theme is continued by De Smedt et al. in the final paper of the issue which includes discussion of how FFS and FCS are being applied to study DNA delivery. De Smedt et al. show how the mobility of DNA complexes and intracellular DNA degradation can be analysed by FCS techniques. The paper also explains how this type of technique is complemented by other advanced light microscopy techniques, including confocal microscopy, FRAP and FRET, in their studies on the extracellular and intracellular mobilities of DNA
4
Preface
complexes and nanoparticles and also to examine the mobility of fluorescent molecules within microparticulate delivery vehicles. Structured photobleaching is also taken a step further in their demonstration of techniques for encoding polystyrene particulates by photobleaching a dbar codeT within immobilized fluorophore. The papers included in this issue illustrate the enormous potential for fluorescence techniques to address key questions in drug delivery and other aspects of pharmaceutical research. With careful application, each of the techniques described here can go far beyond the use of fluorescence microscopes to generate dpretty picturesT and provide robust quantitative information on drug delivery and action in single cells and complex tissues. I am grateful to all the authors for their contributions that reveal their obvious enthusiasm for the topic. Some of the authors have highlighted technical challenges in fully exploiting the available imaging technology and the advantages to be gained from collaboration between experts in pharmaceutical sciences, cell biology and optics. It should also be stressed that continued improvements in advanced
light microscopy techniques are making these technologies increasingly accessible, user-friendly, flexible and powerful. It is clear that fluorescence imaging has enormous potential in all aspects of biomedical sciences and I hope that this theme issue will help to further promote the use of high-quality fluorescence imaging technologies by the pharmaceutical research community. Finally, I would like to thank all the reviewers for their constructive comments on the submitted manuscripts and the ADDR executive editor, Mark Gumbleton, for commissioning this issue and his invaluable support at each stage of its development. Mark A. Jepson (Theme Editor) Cell Imaging Facility and Department of Biochemistry School of Medical Sciences University of Bristol Bristol BS8 1TD UK E-mail: [email protected].