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Advanced Drug Delivery Reviews (v.58, #7)
The coming of age of two-photon excitation imaging for intravital microscopy by David W. Piston (pp. 770-772).
Two-photon excitation imaging; Intravital microscopy; Cellular physiology; Organ physiology; Pathophysiology
Imaging of cortical astrocytes using 2-photon laser scanning microscopy in the intact mouse brain by Guo-Feng Tian; Takahiro Takano; Jane H.-C. Lin; Xiaohai Wang; Lane Bekar; Maiken Nedergaard (pp. 773-787).
A number of studies over the past decade have shown that astrocytes, the supportive cells of the brain, play important roles in synaptic transmission including regulating the strength of both excitatory and inhibitory synapses. A major challenge for the future is to define the role of astrocytes in complex tasks, such as functional hyperemia and sensory processing, as well as their contribution to acute and degenerative diseases of the nervous system. Multiphoton imaging approaches are ideally suited to study electrically non-excitable astrocytes. We here discuss novel in vivo studies aimed at defining the role of astrocytes in normal and pathological brain function. With a better understanding of the role astrocytes play in information processing and regulation of the brain microenvironment in vivo, and the understanding that astrocytes are heavily implicated in the pathology of many diseases such as epilepsy, Alzheimer's and Parkinson's diseases, astrocytes provide a promising target for future drug therapy approaches.
Keywords: Ca; 2+; signaling; Cerebral blood flow; Sensory stimulation; Epilepsy; Purinergic signaling; In vivo
Principles of two-photon excitation fluorescence microscopy and other nonlinear imaging approaches by Martin Oheim; Darren J. Michael; Matthias Geisbauer; Dorte Madsen; Robert H. Chow (pp. 788-808).
The aim of this article is to review the basic principles of two-photon excitation fluorescence (2PEF) microscopy and to compare the advantages and disadvantages of 2PEF imaging to other microscopy methodologies. 2PEF imaging is a nonlinear approach that generates images of optical sections and that is particularly well suited for deep-tissue and in vivo imaging of live animals. The nonlinear excitation used for 2PEF offers the advantage, too, of being able to generate contrast from second or third harmonic generation as well as coherent anti-Stokes Raman scattering. We also review the recent use of nonlinear excitation to provide image resolution beyond the diffraction limit and discuss the progress in non-scanning (planar) 2PEF microscopy, an approach that holds great potential for large-scale quantitative imaging and plate reading, e.g., in screening applications.
Keywords: Two-photon excitation fluorescence (2PEF) microscopy; Coherent anti-Stokes Raman scattering; Planar 2PEF microscopy
Pharmacophotonics: Utilizing multi-photon microscopy to quantify drug delivery and intracellular trafficking in the kidney by Bruce A. Molitoris; Ruben M. Sandoval (pp. 809-823).
The recent introduction of multi-photon microscopy coupled with advances in optics, computer sciences and the available fluorophores used to label molecules of interest have empowered investigators to study the dynamic events within the functioning kidney at cellular and subcellular levels. This emerging technique, with improved spatial and temporal resolution and sensitivity, enables investigators to follow the cell specific uptake of large and small molecules, determine the mode of cellular uptake, intracellular trafficking and drug metabolism in complex heterogeneous organs such as the kidney over time. Repeat determinations over seconds to hours to days allow for multiple observations within the same animal, thereby minimizing animal use and inter-animal variability. This can be particularly useful for preclinical studies. Furthermore, the ability to obtain volumetric data (3-D) makes quantitative 4-D (time) analysis possible. Finally, up to three fluorophores can be visualized simultaneously allowing for three different or interactive processes to be observed and resolved.
Keywords: Kidney; Multi-photon microscopy; Toxicology; Imaging; Pharmacology; Cell toxicity; Preclinical data
Imaging the renin–angiotensin system: An important target of anti-hypertensive therapy by Jung Julie Kang; Ildikó Toma; Arnold Sipos; Fiona McCulloch; János Peti-Peterdi (pp. 824-833).
Multiphoton fluorescence microscopy allows visualization, manipulation, and quantification of the structure–function relationships between pharmacological interventions and their physiological effects. The application of these methods to live animals permits direct observation of acute physical responses that lack chemically detectable signals in the blood or urine and would otherwise remain unknown. With the use of special fluorescent dyes, chemical/hormonal responses may also be detected. The delivery and site-specific effects of drugs can be monitored in real-time. The capacity to simultaneously visualize both proximal and distal segments of the nephron permits observation of the dynamic processes within the living kidney and a quantitative assessment of the various operations. Consequently, a clinically valuable and pending application for multi-photon microscopy will be to provide real-time, quantitative imaging of basic organ functions and their responses to therapeutic intervention. Imaging of the intra-renal renin content and enzymatic activity of renin in situ and in real-time is a new, more informative measure of RAS activity. Direct visualization of the molecular and cellular components of renin release signals and the interactions between the vascular endothelium, tubular epithelium, local mediators, and the renin producing cells provides great insight for drug development. Examples of how the effects of various RAS inhibitors can be visualized in the intact kidney are provided: including angiotensin converting enzyme inhibition (captopril), angiotensin II type 1 receptor blockade (olmesartan), and renin inhibition (aliskiren). The site-specific actions of diuretics, like furosemide, have also been visualized. Quantitative imaging of basic renal functions in health and disease can provide key information to assess the delivery and effects of pharmaceutical interventions.
Keywords: Multi-photon excitation; Intravital microscopy; Diabetes; Diuretics; Angiotensin converting enzyme inhibitors; Angiotensin II type 1 receptor blockers; Renin inhibitors
Intravital fluorescence microscopy in pulmonary research by Claudette M. St. Croix; Karanee Leelavanichkul; Simon C. Watkins (pp. 834-840).
Over the last several years, microscopy as a scientific tool has reinvented itself evolving from a group of principally descriptive methodologies to encompass a wide range of primary tools and techniques to investigate the molecular organization of organs, tissues and cells. Advances in microscope and camera design, fluorescent dye technology, the development of fluorescent proteins as well as the advent of inexpensive powerful computers, has led to the feasibility of simultaneous sub micron resolution and quantitation of multiple concurrent molecular markers for both protein and DNA. Confocal microscopy has allowed optical sectioning and reconstruction of tissues in three dimensions. Finally, the development of multiphoton methodologies as an extension of optical sectioning microscopy has further improved the potential utility of this technology when examining living or light scattering tissues such as the lung. In order to illustrate the utility of two-photon methods in pulmonary biology, we present the application of this approach to the study of cellular trafficking in situ and to the study of pulmonary vasoregulation in an ex vivo rodent model.
Keywords: Lung; Multiphoton microscopy; Confocal microscopy; Tumor biology
Application of two-photon microscopy to the study of cellular pharmacology of central neurons by B. Lendvai; S.I. Szabo; A.I. Barth; T. Zelles; E.S. Vizi (pp. 841-849).
Two-photon microscopy is an especially powerful tool for combining anatomical and physiological experiments in the central nervous system: the possibility of simultaneously studying physiological phenomena in well-defined anatomical compartments allows fluorescence imaging of neurons in deeper layers of the brain. In this review we summarize the most commonly used brain preparation techniques together with the methods of loading neurons with fluorescent indicators. We will focus primarily on issues of drug delivery specifically related to two-photon experiments highlighting the different ways of drug administration. Methods of chemical stimulation via caged neurotransmitters are also discussed. Finally a few specific areas of two-photon applications in drug research on neuronal tissue are highlighted.
Keywords: Pressure ejection; Bath perfusion; Brain preparation; Uncaging; Two-photon microscopy; Cellular pharmacology; Brain slice; Organotypic slice culture
Two-photon excitation imaging of exocytosis and endocytosis and determination of their spatial organization by Haruo Kasai; Takuya Kishimoto; Tomomi Nemoto; Hiroyasu Hatakeyama; Ting-Ting Liu; Noriko Takahashi (pp. 850-877).
Two-photon excitation imaging is the least invasive optical approach to study living tissues. We have established two-photon extracellular polar-tracer (TEP) imaging with which it is possible to visualize and quantify all exocytic events in the plane of focus within secretory tissues. This technology also enables estimate of the precise diameters of vesicles independently of the spatial resolution of the optical microscope, and determination of the fusion pore dynamics at nanometer resolution using TEP-imaging based quantification (TEPIQ). TEP imaging has been applied to representative secretory glands, e.g., exocrine pancreas, endocrine pancreas, adrenal medulla and a pheochromocytoma cell line (PC12), and has revealed unexpected diversity in the spatial organization of exocytosis and endocytosis crucial for the physiology and pathology of secretory tissues and neurons. TEP imaging and TEPIQ analysis are powerful tools for elucidating the molecular and cellular mechanisms of exocytosis and certain related diseases, such as diabetes mellitus, and the development of new therapeutic agents and diagnostic tools.
Keywords: Insulin secretion; Pancreatic islet; Exocrine gland; Adrenal medulla; Chromaffin cell; Synapse; Pheochromocytoma cell; Compound exocytosis
Two-photon microscopes and in vivo multiphoton tomographs — Powerful diagnostic tools for tissue engineering and drug delivery by Katja Schenke-Layland; Iris Riemann; Odile Damour; Ulrich A. Stock; Karsten König (pp. 878-896).
Near-infrared multiphoton microscopes and in vivo femtosecond laser tomographs are novel powerful diagnostic tools for intra-tissue drug screening and high-resolution structural imaging applicable to many areas of biomedical research. Deep tissue cells and extracellular matrix (ECM) compartments can be visualized in situ with submicron resolution without the need for tissue processing. In particular, the reduced fluorescent coenzyme NAD(P)H, flavoproteins, keratin, melanin, and elastin are detected by two-photon excited autofluorescence, whereas myosin, tubulin and the ECM protein collagen can be imaged additionally by second harmonic generation (SHG). Therefore, these innovative multiphoton technologies have been used to probe architecture and state of a variety of native tissues, as well as of tissue-engineered constructs, giving insights on the interaction between scaffolds and seeded cells in vitro prior implantation. Moreover, non-invasive 4-D multiphoton tomographs are employed in clinical studies to examine the diffusion behavior, the intra-tissue accumulation of topically applied cosmetic and pharmaceutical components, and their interaction with skin cells.
Keywords: Multiphoton imaging; Non-invasive microscopy; Second harmonic generation; Cardiovascular tissue engineering; Heart valve; Skin equivalent; ECM; Collagen; Elastin