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Imaging cell biology and physiology in vivo using intravital microscopy
Methods 128 (2017) 1–2
Contents lists available at ScienceDirect
Methods journal homepage: www.elsevier.com/locate/ymeth
Guest Editor’s Introduction
Imaging cell biology and physiology in vivo using intravital microscopy
The beginnings of the field of cell biology can be traced to the 17th century, when Robert Hooke used one of the first microscopes to identify the cellular components of cork. However, the application of microscopy to mammalian cell biology languished until the 1950s when methods for continuous cell culture were developed, providing researchers with the ability to observe and experimentally manipulate isolated cells. From the beginning, researchers have appreciated that cultured cells represent a compromised experimental system for understanding the function of cells in their natural environment in the body. Cultured cell systems lack the structural, biochemical, and cellular context of the intact organism that is necessary to address the most important questions in cell biology and physiology. Intravital microscopy, the microscopic examination of intact, living organisms, had been sporadically used since the 19th century to study the behaviors of cells in vivo. However, the power of intravital microscopy as a research tool in cell biology wasn’t realized until the development of confocal and multiphoton excitation microscope systems during the 1990s. The evolution of these microscope systems now provides the ability to collect high-resolution images tens-to-hundreds of microns into living biological tissues, giving biologists access to the behaviors of cells in the relevant context of the intact organism. However, realizing the full potential of intravital microscopy as a tool in cell biology has required solving a variety of new issues encountered when applying high resolution imaging to the intact organism. Over the past twenty years, these emergent problems have been addressed by an active community of researchers, whose concerted efforts have provided us with a unique window into the behaviors of cells in vivo. This volume presents an overview of some of these methods. Among the earliest applications of multiphoton intravital microscopy was to study the behavior of neurons in the intact brain (reviewed by Svoboda and Yasuda, 2006 [1]). High resolution imaging of the brain is simplified by the fact that the cranium can be completely immobilized, and neurons in the relatively transparent tissue can be imaged either through a thinned portion of the bone or an implanted window. Imaging of other organ systems has required development of specialized methods for tissue presentation, immobilization and labeling. In this volume, Amornphimoltham et al. [2] describe a novel system for intravital microscopy of the tongue and demonstrate how the unique advantages of this system can be used to characterize the cell biology of tumors in animal models of oral cancer. Surewaard and Kubes [3] then describe how they exploit the speed and reduced photo-toxicity of spinning-disk confocal microscopy for intravital studies of http://dx.doi.org/10.1016/j.ymeth.2017.08.020 1046-2023/Ó 2017 Published by Elsevier Inc.
mouse liver to characterize the in vivo interactions between bacteria and phagocytic cells that underlie bacterial pathogenesis. Next, Sandoval and Molitoris [4] describe how they use intravital multiphoton microscopy of the rat kidney to characterize renal function at the level of individual cells within single nephrons. This is followed by the work of Hato et al. [5] describing how similar methods can be applied to studies of the mouse kidney, with a particular emphasis on the challenges that they have encountered in their studies of acute kidney injury. Then Dunn and Ryan [6] describe an approach in which intravital multiphoton microscopy of the rat liver has been combined with methods of digital image analysis to dissect hepatocyte transport in vivo. Most intravital microscopy studies in living organisms are conducted immediately following acute surgical exposure of the organ of interest, an approach that essentially precludes repeated imaging of the same tissues over time. An alternative approach involves the surgical implantation of an imaging window, supporting repeated imaging of an organ. This ‘‘longitudinal” approach supports unique studies of events occurring over periods of days to months, and has been used to characterize neural plasticity and neurodegenerative disease in studies of the brain and to characterize cellular proliferation and migration in tumor models (reviewed by Alieva et al., 2014 [7]). In this volume Prunier et al. [8] review different types of imaging windows that have been used for intravital microscopy, and provide detailed descriptions of the methods that they have developed for studying cellular proliferation and migration in vivo. One of the shortcomings of high-resolution intravital microscopy is that, unlike other in vivo imaging techniques that image entire organs or even entire organisms, intravital microscopy produces images of regions that are typically only a few hundred microns across. Fields of this size are frequently too small to accurately represent the overall function of an organ or to fully capture all the salient features of a biological structure, such as a tumor. Here, Entenberg et al. [9] describe techniques that they have developed to collect millimeter-scale image volumes at a resolution sufficient to resolve filapodia extending from a macrophage to a tumor cell in vivo. The development of fluorescent proteins profoundly extended the capabilities of microscopy to inform studies in cell biology (reviewed by Day and Davidson 2014 [10]). The cloning and refinement of fluorescent proteins made it possible to create fluorescent fusion proteins that investigators could use to observe the behavior of nearly any protein in living cells. Fluorescent protein biosensors engineered for environmental sensitivity provided investigators with the ability to assay a wide range of physiological and bio-
2
Guest Editor’s Introduction / Methods 128 (2017) 1–2
chemical processes in real time. In this volume, Conway et al. [11] provide a review of the use of fluorescent protein biosensors in intravital microscopy, and describe methods for their use in studies of tumor physiology. Winfree et al. [12] then describe the unique challenges presented by fluorescent biosensors whose responses are based upon Forster Resonance Energy Transfer (FRET), and describe methods that they have developed for measuring FRET in living animals, based upon ratiometric measurements of fluorescence intensity or measurements of fluorescence lifetime. Although fluorescence represents the primary form of contrast used in most intravital microscopy studies, investigators are increasingly exploring additional non-linear imaging modalities that support optical sectioning in biological tissues. In this volume, Mazumder et al. [13] describe how polarimetry can be used to extend the power of second harmonic imaging to characterize molecular structure in vivo. Lee and Cheng [14] present a review of another non-linear imaging technique, Stimulated Raman Scattering (SRS) microscopy, which is capable of characterizing the molecular constitution of unlabeled biological tissues based upon their molecular vibrational fingerprint, and show how this technique can be used to image metabolism in vivo. The value of intravital microscopy critically depends upon the degree to which the physiology of the animal subject is maintained in the face of what are frequently highly invasive procedures. Here, George Rhodes [15] provides a review of the procedures used to present abdominal organs for intravital microscopy, and the methods used to guarantee animal physiology during imaging. Finally, Veelken et al. [16] describe a unique in vitro 3D tumor model system that they have developed as a tool for evaluating cancer therapeutics. This system capitalizes on many of the insights provided by the intravital microscopy studies of tumor biology conducted by the Friedl laboratory, while exploiting the enhanced throughput of an in vitro system. References [1] K. Svoboda, R. Yasuda, Principles of two-photon excitation microscopy and its applications to neuroscience, Neuron 50 (2006) 823–839. [2] P. Amornphimoltham et al, Non-invasive intravital imaging of head and neck squamous cell carcinomas in live mice, Methods 128 (2017) 3–11.
[3] B.G.J. Surewaard, P. Kubes, Measurement of bacterial capture and phagosome maturation of Kupffer cells by intravital microscopy, Methods 128 (2017) 12–19. [4] R.M. Sandoval, B.A. Molitoris, Intravital microscopy as a tool for studying kidney physiology and pathophysiology, Methods 128 (2017) 20–32. [5] T. Hato et al, Intravital imaging of the kidney, Methods 128 (2017) 33–39. [6] K.W. Dunn, J.C. Ryan, Using quantitative intravital multiphoton microscopy to dissect hepatic transport in rats, Methods 128 (2017) 40–51. [7] M. Alieva, L. Ritsma, R.J. Giedt, R. Weissleder, J. van Rheenen, Imaging windows for long-term intravital imaging: general overview and technical insights, Intravital 3 (2014) e29917, http://dx.doi.org/10.4161/intv.29917. [8] C. Prunier et al, Procedures and applications of long-term intravital microscopy, Methods 128 (2017) 52–64. [9] D. Entenberg et al, Time-lapsed, large-volume, high-resolution intravital imaging for tissue-wide analysis of single cell dynamics, Methods 128 (2017) 65–77. [10] R.N. Day, M.W. Davidson, The Fluorescent Protein Revolution, Taylor & Francis, Boca Raton, FL, 2014. [11] J.R.W. Conway et al, Context-dependent intravital imaging of therapeutic response using intramolecular FRET biosensors, Methods 128 (2017) 78–94. [12] S. Winfree et al, Intravital microscopy of biosensor activities and intrinsic metabolic states, Methods 128 (2017) 95–104. [13] N. Mazumder et al, Polarization resolved second harmonic microscopy, Methods 128 (2017) 105–118. [14] H.J. Lee, J.-X. Cheng, Imaging chemistry inside living cells by stimulated raman scattering microscopy, Methods 128 (2017) 119–128. [15] G.J. Rhodes, Surgical preparation of rats and mice for intravital microscopic imaging of abdominal organs, Methods 128 (2017) 129–138. [16] C. Veelken et al, Single cell-based automated quantification of therapy response of invasive cancer spheroids in organotypic 3D culture, Methods 128 (2017) 139–149.
Kenneth W. Dunn Departments of Medicine and Biochemistry, Research II, Suite E202, 950 W Walnut St, Indianapolis, United States Richard N. Day Department of Cellular and Integrative Physiology, Indiana University School of Medicine, 635 Barnhill Drive, MS 333, Indianapolis, IN 46202, USA
Contents lists available at ScienceDirect
Methods journal homepage: www.elsevier.com/locate/ymeth
Guest Editor’s Introduction
Imaging cell biology and physiology in vivo using intravital microscopy
The beginnings of the field of cell biology can be traced to the 17th century, when Robert Hooke used one of the first microscopes to identify the cellular components of cork. However, the application of microscopy to mammalian cell biology languished until the 1950s when methods for continuous cell culture were developed, providing researchers with the ability to observe and experimentally manipulate isolated cells. From the beginning, researchers have appreciated that cultured cells represent a compromised experimental system for understanding the function of cells in their natural environment in the body. Cultured cell systems lack the structural, biochemical, and cellular context of the intact organism that is necessary to address the most important questions in cell biology and physiology. Intravital microscopy, the microscopic examination of intact, living organisms, had been sporadically used since the 19th century to study the behaviors of cells in vivo. However, the power of intravital microscopy as a research tool in cell biology wasn’t realized until the development of confocal and multiphoton excitation microscope systems during the 1990s. The evolution of these microscope systems now provides the ability to collect high-resolution images tens-to-hundreds of microns into living biological tissues, giving biologists access to the behaviors of cells in the relevant context of the intact organism. However, realizing the full potential of intravital microscopy as a tool in cell biology has required solving a variety of new issues encountered when applying high resolution imaging to the intact organism. Over the past twenty years, these emergent problems have been addressed by an active community of researchers, whose concerted efforts have provided us with a unique window into the behaviors of cells in vivo. This volume presents an overview of some of these methods. Among the earliest applications of multiphoton intravital microscopy was to study the behavior of neurons in the intact brain (reviewed by Svoboda and Yasuda, 2006 [1]). High resolution imaging of the brain is simplified by the fact that the cranium can be completely immobilized, and neurons in the relatively transparent tissue can be imaged either through a thinned portion of the bone or an implanted window. Imaging of other organ systems has required development of specialized methods for tissue presentation, immobilization and labeling. In this volume, Amornphimoltham et al. [2] describe a novel system for intravital microscopy of the tongue and demonstrate how the unique advantages of this system can be used to characterize the cell biology of tumors in animal models of oral cancer. Surewaard and Kubes [3] then describe how they exploit the speed and reduced photo-toxicity of spinning-disk confocal microscopy for intravital studies of http://dx.doi.org/10.1016/j.ymeth.2017.08.020 1046-2023/Ó 2017 Published by Elsevier Inc.
mouse liver to characterize the in vivo interactions between bacteria and phagocytic cells that underlie bacterial pathogenesis. Next, Sandoval and Molitoris [4] describe how they use intravital multiphoton microscopy of the rat kidney to characterize renal function at the level of individual cells within single nephrons. This is followed by the work of Hato et al. [5] describing how similar methods can be applied to studies of the mouse kidney, with a particular emphasis on the challenges that they have encountered in their studies of acute kidney injury. Then Dunn and Ryan [6] describe an approach in which intravital multiphoton microscopy of the rat liver has been combined with methods of digital image analysis to dissect hepatocyte transport in vivo. Most intravital microscopy studies in living organisms are conducted immediately following acute surgical exposure of the organ of interest, an approach that essentially precludes repeated imaging of the same tissues over time. An alternative approach involves the surgical implantation of an imaging window, supporting repeated imaging of an organ. This ‘‘longitudinal” approach supports unique studies of events occurring over periods of days to months, and has been used to characterize neural plasticity and neurodegenerative disease in studies of the brain and to characterize cellular proliferation and migration in tumor models (reviewed by Alieva et al., 2014 [7]). In this volume Prunier et al. [8] review different types of imaging windows that have been used for intravital microscopy, and provide detailed descriptions of the methods that they have developed for studying cellular proliferation and migration in vivo. One of the shortcomings of high-resolution intravital microscopy is that, unlike other in vivo imaging techniques that image entire organs or even entire organisms, intravital microscopy produces images of regions that are typically only a few hundred microns across. Fields of this size are frequently too small to accurately represent the overall function of an organ or to fully capture all the salient features of a biological structure, such as a tumor. Here, Entenberg et al. [9] describe techniques that they have developed to collect millimeter-scale image volumes at a resolution sufficient to resolve filapodia extending from a macrophage to a tumor cell in vivo. The development of fluorescent proteins profoundly extended the capabilities of microscopy to inform studies in cell biology (reviewed by Day and Davidson 2014 [10]). The cloning and refinement of fluorescent proteins made it possible to create fluorescent fusion proteins that investigators could use to observe the behavior of nearly any protein in living cells. Fluorescent protein biosensors engineered for environmental sensitivity provided investigators with the ability to assay a wide range of physiological and bio-
2
Guest Editor’s Introduction / Methods 128 (2017) 1–2
chemical processes in real time. In this volume, Conway et al. [11] provide a review of the use of fluorescent protein biosensors in intravital microscopy, and describe methods for their use in studies of tumor physiology. Winfree et al. [12] then describe the unique challenges presented by fluorescent biosensors whose responses are based upon Forster Resonance Energy Transfer (FRET), and describe methods that they have developed for measuring FRET in living animals, based upon ratiometric measurements of fluorescence intensity or measurements of fluorescence lifetime. Although fluorescence represents the primary form of contrast used in most intravital microscopy studies, investigators are increasingly exploring additional non-linear imaging modalities that support optical sectioning in biological tissues. In this volume, Mazumder et al. [13] describe how polarimetry can be used to extend the power of second harmonic imaging to characterize molecular structure in vivo. Lee and Cheng [14] present a review of another non-linear imaging technique, Stimulated Raman Scattering (SRS) microscopy, which is capable of characterizing the molecular constitution of unlabeled biological tissues based upon their molecular vibrational fingerprint, and show how this technique can be used to image metabolism in vivo. The value of intravital microscopy critically depends upon the degree to which the physiology of the animal subject is maintained in the face of what are frequently highly invasive procedures. Here, George Rhodes [15] provides a review of the procedures used to present abdominal organs for intravital microscopy, and the methods used to guarantee animal physiology during imaging. Finally, Veelken et al. [16] describe a unique in vitro 3D tumor model system that they have developed as a tool for evaluating cancer therapeutics. This system capitalizes on many of the insights provided by the intravital microscopy studies of tumor biology conducted by the Friedl laboratory, while exploiting the enhanced throughput of an in vitro system. References [1] K. Svoboda, R. Yasuda, Principles of two-photon excitation microscopy and its applications to neuroscience, Neuron 50 (2006) 823–839. [2] P. Amornphimoltham et al, Non-invasive intravital imaging of head and neck squamous cell carcinomas in live mice, Methods 128 (2017) 3–11.
[3] B.G.J. Surewaard, P. Kubes, Measurement of bacterial capture and phagosome maturation of Kupffer cells by intravital microscopy, Methods 128 (2017) 12–19. [4] R.M. Sandoval, B.A. Molitoris, Intravital microscopy as a tool for studying kidney physiology and pathophysiology, Methods 128 (2017) 20–32. [5] T. Hato et al, Intravital imaging of the kidney, Methods 128 (2017) 33–39. [6] K.W. Dunn, J.C. Ryan, Using quantitative intravital multiphoton microscopy to dissect hepatic transport in rats, Methods 128 (2017) 40–51. [7] M. Alieva, L. Ritsma, R.J. Giedt, R. Weissleder, J. van Rheenen, Imaging windows for long-term intravital imaging: general overview and technical insights, Intravital 3 (2014) e29917, http://dx.doi.org/10.4161/intv.29917. [8] C. Prunier et al, Procedures and applications of long-term intravital microscopy, Methods 128 (2017) 52–64. [9] D. Entenberg et al, Time-lapsed, large-volume, high-resolution intravital imaging for tissue-wide analysis of single cell dynamics, Methods 128 (2017) 65–77. [10] R.N. Day, M.W. Davidson, The Fluorescent Protein Revolution, Taylor & Francis, Boca Raton, FL, 2014. [11] J.R.W. Conway et al, Context-dependent intravital imaging of therapeutic response using intramolecular FRET biosensors, Methods 128 (2017) 78–94. [12] S. Winfree et al, Intravital microscopy of biosensor activities and intrinsic metabolic states, Methods 128 (2017) 95–104. [13] N. Mazumder et al, Polarization resolved second harmonic microscopy, Methods 128 (2017) 105–118. [14] H.J. Lee, J.-X. Cheng, Imaging chemistry inside living cells by stimulated raman scattering microscopy, Methods 128 (2017) 119–128. [15] G.J. Rhodes, Surgical preparation of rats and mice for intravital microscopic imaging of abdominal organs, Methods 128 (2017) 129–138. [16] C. Veelken et al, Single cell-based automated quantification of therapy response of invasive cancer spheroids in organotypic 3D culture, Methods 128 (2017) 139–149.
Kenneth W. Dunn Departments of Medicine and Biochemistry, Research II, Suite E202, 950 W Walnut St, Indianapolis, United States Richard N. Day Department of Cellular and Integrative Physiology, Indiana University School of Medicine, 635 Barnhill Drive, MS 333, Indianapolis, IN 46202, USA