ADVANCES IN MOLECULAR AND CELL BIOLOGY ORGANELLES IN VIVO Series Editor: E. EDWARD BITTAR Department of Physiology University of Wisconsin Madison, Wisconsin Guest Editor: LAN BO CHEN Dana- Farber Cancer Institute Harvard Medical School Boston, Massachusetts VOLUME 8 1994 @ JAl PRESS INC. Greenwich, Connecticut Lon don, England Copyright 0 1994 by)Al PRESS INC. 55 Old Post Road, No. 2 Greenwich, Connecticut 06836 )A1 PRESS L TO. The Courtyard 28 High Street Hampton Hill, Middlesex TW12 1PD England All rights reserved. No part of this publication may be reproduced, stored on a retrieval system, or transmitted in any form or by any means, electronic, mechanical, photocopying, filming, recording, or otherwise, without prior permission in writing from the publisher. ISBN: 1-55938-636-3 Manufactured in the United States of America LIST OF CONTRIBUTORS Guojun Bu Mallinckrodt Departments of Pediatrics, Molecular Biology, and Pharmacology Washington University School of Medicine and St. Louis Children's Hospital St. Louis, Missouri Lucie H. Clapp Department of Pharmacology United Medical and Dental Schools St. Thomas's Hospital London, England Theresa A. Davies Department of Biochemistry Boston University School of Medicine Boston, Massachusetts Adolf Ellinger Institute of Micromorphology and Electron Microscopy University of Vienna Vienna, Austria Ahon M. Gurney Department of Pharmacology United Medical and Dental Schools St. Thomas's Hospital London, England Dick Hoekstra Department of Physiological Chemistry University of Groningen Groningen, The Netherlands Jan Willem Kok Department of Physiological Chemistry University of Groningen Groningen, The Netherlands Folkert Kuipers Department of Pediatrics University of Groni n gen Groningen, The Netherlands vi i ... LIST OF CONTRIBUTORS Vlll Barry R. Masters Department of Anatomy and Cell Biology Uniformed Services University of the Health Sciences Bethesda, Maryland Phillip A. Morton Division of Immunology Monsanto Company Chesterfield, Missouri Margit Pavelka Institute of Histology and Embryology University of lnnsbruck Innsbruck, Austria Jane Somsel Rodman Department of Physiology Tufts University School of Medicine Boston, Massachusetts J. L. Roti Roti Mallinckrodt Institute of Radiology Washington University School of Medicine St. Louis, Missouri Alan L. Schwartz Mallinckrodt Departments of Pediatrics, Molecular Biology, and Pharmacology Washington University School of Medicine and St. Louis Children’s Hospital St. Louis, Missouri Elizabeth R. Simons Department of Biochemistry Boston University School of Medicine Boston, Massachusetts W. 0.W right Mallinckrodt Institute of Radiology Washington University School of Medicine St. Louis, Missouri Kristien J. M. Zaal Department of Physiological Chemistry University of Groningen Croningen, The Netherlands PREFACE After a decade of dominance by recombinant DNA technology, the field of molecular and cell biology is witnessing a renewed interest in techniques and approaches that are not driven by DNA acrobatics. In hindsight, this is an inevitable outcome. Deoxyribonucleic acid is not the master; it is only a storage house. If one wishes to know how cells work, the secret is not to be found in DNA, but rather in everything outside DNA. Science based on DNA is useful but does not itself solve the problem. It is most fortunate that at the height of the DNA phenomenon, there remain scientists who continue to probe cells by non-DNA means. Suddenly, people with such expertise are in high demand. In this volume, some truly original scientists take the time to tell us their stories of innovations-some almost iconoclastic. All of these researchers have pioneered approaches that were long neglected; moreover, each is now in the fruitful phase of great harvests. It is a wonderful lesson for graduate students and postdoctorates that, although not being in the pack might be risky, the reward of such work is sweeter. As in physics, biology needs more young people who think like Richard Feynman-the ultimate iconoclast. On the surface, the eight chapters of this volume appear to be diverse, but they are not. If our purpose is to understand cells, we must stop the habit of constantly dissecting leaves. Once in a while we have to see which forest we are in. The contributions included herein cannot cover the whole cell, but they give a sufficient flavor to arouse a desire to think more globally about cells. At a time when our field is in danger of being buried by thousands of kinases and phosphatases, these chapters inform our intended audiences that there are other ways-other tech- niques, other approaches, other thinkings. and other stories. We need all of them to appreciate the holistic aspect of cells, which has been a taboo until now. Lan Bo Chen Guest Editor ix CONFOCAL REDOX IMAGING OF CELLS . Barry R Masters 1. INTRODUCTION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 A . NAD(P)H Redox Imaging . . . . . . . . . . . . . . . . . . . . . . . . . . 2 B . Flavoprotein Redox Imaging . . . . . . . . . . . . . . . . . . . . . . . . . 3 11 . MATERIAL AND MmHODS . . . . . . . . . . . . . . . . . . . . . . . . . . 5 A . Sources of Biological Material . . . . . . . . . . . . . . . . . . . . . . . . 5 B . One-Dimensional Confocal Redox Fluorometer . . . . . . . . . . . . . . . 5 C . Confocal Redox NAD(P)H Imaging . . . . . . . . . . . . . . . . . . . . . 7 D. Confocal Redox Havoprotein Imaging . . . . . . . . . . . . . . . . . . . 10 E . Volume Reconstruction . . . . . . . . . . . . . . . . . . . . . . . . . . . 12 111 . RESULTS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12 A . NAD(P)H Redox Imaging . . . . . . . . . . . . . . . . . . . . . . . . . 12 B . No-Photon NAD(P)H Redox Imaging . . . . . . . . . . . . . . . . . . 12 C . Flavoprotein Redox Imaging . . . . . . . . . . . . . . . . . . . . . . . . 13 IV. DISCUSSION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14 A . NAD(P)H Redox Imaging . . . . . . . . . . . . . . . . . . . . . . . . . 14 B . Flavoprotein Redox Imaging . . . . . . . . . . . . . . . . . . . . . . . . 15 V. CONCLUSIONS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18 ACKNOWLEDGMENT . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18 REFERENCES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18 Advances in Molecular and Cell Biology Vol~me8. 1-19 . Copyright 0 1994 by JAI Press Inc . All rights of reproduction in any form reserved ISBN: 1-55938-636-3 1 2 BARRY R. MASTERS 1. INTRODUCTION This paper demonstrates the application of confocal fluorescence microscopic imaging to noninvasively monitor cellular function across the full thickness of the in vitro cornea. The cornea is an avascular, thick (400 pm), semitransparent, living optical element in the front region of the eye. The corneal epithelium and endothe- lium comprise the limiting cell layers on the anterior and posterior surfaces of the cornea. An optical technique for two-dimensional metabolic imaging of cellular metabolism in cellular layers of the cornea is described herein. These methods are also suitable for other cells and tissues and provide a general noninvasive optical method to monitor cellular function. A. NAD(P)H Redox Imaging The fluorescence from the naturally occurring reduced pyridine nucleotides in cells is an indicator of cellular respiration. The fluorescence of reduced pyridine nucleotides is excited with light of 364 nm, and these substances show a fluores- cence emission in the range of 400-500 nm. Cellular hypoxia is associated with an increased ratio of reduced to oxidized pyridine nucleotides, and therefore an increased fluorescence intensity in the region of 400-500 nm is observed. Since the quantum efficiency, and thus the fluorescence intensity of the reduced pyridine nucleotides is significantly greater than that of the oxidized pyridine nucleotides. the fluorescence intensity monitors the degree of cellular hypoxia. This noninvasive technique is called redoxfluoromerry. We have demonstrated that the 400-500-nm fluorescence excited at 364 nm is due to the reduced pyridine nucleotides. While the fluorescence intensity of the cornea has been investigated using optically sectioning microscopes to monitor the degree of cellular hypoxia, it was not previously possible to obtain single-cell images of the reduced pyridine nucleotide fluorescence. However, two-dimensional redox imaging has been demonstrated by other investigators for isolated cardiac myocytes in tissue culture (Eng et al.. 1989). The confocal microscope serves as an optical device to observe a single focal plane of thick objects with high resolution and contrast as compared with standard microscopes (Masters and Kino, 1990). The confocal microscope used in the fluorescent mode has the excitation at one wavelength, and the fluorescent image is formed at a longer wavelength. This differs from the reflected light mode in which the confocal image is formed at the same wavelength as that of the laser illumina- tion. The depth resolution of fluorescence-mode confocal scanning optical micro- scopes is reduced as compared with the reflected imaging mode. The advantages of UV confocal microscopy include increased resolution and a reduced depth of focus as compared with visible light confocal microscopy. These advantages depend on a microscope objective that is corrected for the UV. The use of the confocal microscope to optically section the cornea has been demonstrated (Lemp et al., 1986). The fine structure of the in virro cornea has been anfbcal Redox Imaging of Cells 3 shown with both the one-sided Nipkow disk confocal microscope and with the 1-r-scanning confocal microscope (Masters and Paddock, 1990a). The three- dimensional volume feCOnStfUCtiOnf rom serial confocal optical sections of the virtu cornea has been demonstrated (Masters and Paddock, 1990b). The confocal laser-scanning microscope (Zeiss, UV confocal LSM) permits twodimensional confocal imaging of the redox fluorescence intensity of corneal endothelid cells (Kapitzaand Wilke, 1988).T hus, a two-dimensional image or map of cellular hypoxia can be obtained. The combining of both the reflected light images of cell morphology and the redox fluorescence images of cellular metabo- lism may be used to construct a multimodality three-dimensional image of cell structure and function. The fluorescence from NAD(P)H. reduced pyridine nucleotides, is an intrinsic probe to study cellular metabolism (Chance and Thorell, 1959). The fluorescence intensity from these intrinsic probes provides a noninvasive optical method to monitor cellular respiration (Chance et al., 1978). Due to the strength of its fluorescence intensity, the fluorescence from NAD(P)H has been used to study cellular metabolism in many tissues and organs. The NAD(P)H fluorescence intensity occurs in two compartments, the mitochondrial and the cytosolic; this complicates the interpretation of the fluorescence studies; however, in some tissues (e.g., rat cardiac myocytes). the NAD(P)H fluorescence is predominantly from the mitochondrial space. Tbo-dimensional images of the fluorescence intensity from NAD(P)H have been studied in brain slices and in isolated perfused hearts. At the cellular level, NAD(P)H imaging of isolated rat cardiac myocytes have been studied with a standard fluorescence microscope (Balaban and Mandel, 1990). These authors demonstrated that the fluorescence images are mainly due to mitochondrial NAD(P)H fluorescence (Eng et al., 1989). Two-dimensional imaging of the NAD(P)H fluorescence intensity of in vim corneal endothelial cells has been studied with an ultraviolet confocal laser-scanning fluorescence microscope. B. Flavoprotein Redox Imaging The fluorescence intensity from intrinsic oxidized flavoproteins present in the mitochondria of cells is a noninvasive measure of cellular respiratory function. The main advantage of measuring the fluorescence intensity from the oxidized flavo- proteins is that the fluorescence is localized in the mitochondrial space. The use of a confocal laser-scanning microscope to image the flavoprotein fluorescence in cultured cell spheroids has been reported (Weinlich and Acker. 1990).T he fluores- cence excitation line was the488-nm line of the argon ion laser, and the fluorescence emission was detected with a filter cutting-off at 5 15 nm; these results are consistent with flavoprotein fluorescence. The fluorescence intensity from oxidized flavoproteins in the corneal epithelium occurs in the region of 520-590 nm, with a broad maximum at 540 nm (Chance 4 BARRY R. MASTERS and Schoener, 1966). The light absorption of oxidized flavoproteins has a broad maximum at 460 nm and extends from 430-500 nm. The 488-nm laser line can excite flavoprotein fluorescence although it does not coincide with the optimal 450460-nm wavelength band. The fluorescence intensity of the oxidized flavo- proteins in the corneal epithelial cells is much lower than the fluorescence intensity from the reduced pyridine nucleotides. The confocal microscope was used to produce images in both backscattered light and in the fluorescence mode, in order to exploit both the superb optical sectioning properties of the confocal microscope, as well as its strong rejection of stray light. Therefore, the reflection and fluorescence images made with a confocal laser- scanning microscope could be limited to a single cell layer (superficial epithelial cells) in a tissue that is 400-pm thick. The cornea is a semitransparent, avascular tissue in the anterior portion of the eye. The rabbit cornea is about 400-pm thick and is composed of several layers: the 40ym thick epithelial layer that is directly adjacent to the tear film, the stromal portion (354 pm) or middle region of the cornea, and the endothelial cell layer (6 pm) on the posterior side of the cornea. The epithelial cell layer itself is composed of 5-6 layers of epithelial cells. Fluorescence from the intrinsic oxidized flavoproteins in the cornea has been spectroscopically characterized in rabbit corneas frozen to 77 K (Chance and Lieberman, 1978). Other evidence that the corneal epithelial cell fluorescence in both in vitro and in vivo corneas is due to oxidized flavoproteins is based on the studies of Masters (1984a, b). The distribution of mitochondria (stained with the cationic dye rhodamine 123) in the Superficial epithelial cells of the rabbit cornea has been studied with a confocal laser-scanning fluorescence microscope. The alteration of the fluorescence intensity of the oxidized flavoproteins in the corneal epithelial cells of the in vivo rabbit as a function of cellular hypoxia has been demonstrated (Masters et al., 1982a). A HeCd laser at 442 nm was used to excite the corneal epithelial cells in a living rabbit. The flavoprotein fluorescence intensity was measured in the wavelength region of 550 nm (Masters et al, 1982b). The fluorescence intensity was reduced in the presence of a flow of hydrated nitrogen (tissue hypoxia) and the effect was reversed in the presents of hydrated air. These studies are in agreement with in virro studies of flavoprotein fluorescence from corneal epithelial cells conducted in both in vitro rabbit perfused corneas at 37 "C, and in freeze-trapped rabbit cornea at 77 K. Confocal microscopy has been used to image the cornea in both the in vitro eye and the in vivo eye (Masters, 1990a, b). High-contrast images of cellular and subcellular cytoskeleton components have been visualized; for example, the basal epithelial cells, the interdigitations of the stromal fibroblasts. and the actin distri- bution around the corneal endothelial cells have been observed with this technique. The functional anatomy, which is acombination of corneal morphology and cellular function, had not been investigated prior to this study. Confocal Redox Imaging of Cells 5 This study by Masters demonstrates that the cellular respiratory function of corneal epithelial cells can be visualized, together with epithelial cellular morphol- ogy, with the use of a laser-scanning confocal microscope. Two-dimensional imaging of the superficial epithelial cell functional anatomy is demonstrated at m m t emperature in an in vim rabbit eye. The source of the functional signal is the fluorescence intensity from the mitochondrial-oxidized flavoproteins. The Source of the morphological signal is the backscattered light intensity that results from differences of refractive index in the corneal epithelium. A confocal laser- scanning microscope operating in the fluorescent mode and then in the backscat- tered reflected-light mode was used to generate the images. The images of flavoprotein fluorescence intensity is a measure of cellular metabolism and pro- vides a noninvasive optical method to study cell metabolism. The combination of confocal microscopy, with its excellent optical sectioning capabilities, and the optical imaging of cell metabolism provides a useful technique for cell biology. This is the first time that the confocal microscope has been used to image the functional anatomy of the cornea. Previous studies of two-dimensional imaging of either flavoprotein fluorescence or NAD(P)H fluorescence were made on freeze- trapped, frozen, mechanically cut sections, or mechanically milled surfaces of sections of tissue (Chance and Lieberman, 1978). This chapter demonstrates the feasibility of the confocal microscopic imaging technique to visualize functional anatomy in the living cornea. II. MATERIAL AND METHODS A. Sources of Biological Material Eyes were obtained from male New Zealand white rabbits weighing 2.5 kg. The rabbits were maintained and handled in accordance with the ARVO (Association for Research in Vision and Ophthalmology,B ethesda, MD)“ Resolution on the Use of Animals in Research.” The rabbits were anesthetized with an intramuscular injection of ketamine HCI (40 mg/kg) and xylazine (5 mg/kg). The eyes were freed of adhering tissue and were swiftly removed. The eyes were immediately placed in abeaker containing bicarbonate Ringer’s solution with glucose (5 mM) and calcium (2 mM) at 25 “C. These conditions have been previously shown to maintain the physiological state of the cornea. B. One-Dimensional Confocal Redox Fluorometer Figures 1 and 2 illustrate the mechanical and optical components of a confocal redox fluorometer. The device is a confocal microscope since it contains two slits, located in conjugate planes, for the illumination and the image plane. The confocal microscope has been used in the vertical mode for work on tissue culture and for