Departments of 1Biochemistry and 2Urology, Weill Medical College of Cornell University, New York, New York 10021
THE PAST TWO DECADES have seen spectacular advances in the ability to directly observe physiological processes in living cells. While various forms of transmitted light microscopy (e.g., differential interference contrast microscopy) have made very significant contributions, the greatest advances have come from applications of fluorescence microscopy. This rapid progress has been made possible by parallel advances in several fields. Although optical microscopy might already have been considered a mature field 20 years ago, there have been fundamental innovations in instrumentation that have changed the field dramatically. The most prominent of these are the practical implementation of various forms of confocal microscopy (15) and the invention of multiphoton microscopy (4, 5). In addition, improvements in detectors such as charge-coupled device cameras have greatly improved the sensitivity, accuracy, and resolution of quantitative microscopic imaging. These improved detectors, along with sophisticated computational algorithms for analyzing and reconstructing three-dimensional (3-D) images, have further advanced the ability to obtain precise, high-resolution 3-D images of cells.
At the same time, there has been an ever-increasing repertoire of fluorescent labels that can report a variety of cellular processes. While fluorescent antibody reagents have been invaluable for localizing proteins in fixed cells and tissues, they have been of limited value in examining dynamic processes in living cells. For certain processes such as endocytosis, proteins with chemically conjugated fluorophores have proved useful for following membrane traffic in living cells (16, 17). In a more cumbersome method, fluorescently labeled cytoplasmic proteins (e.g., cytoskeletal proteins) can be microinjected into the cytoplasm, and many seminal findings about cytoskeletal dynamics have been made from these studies (11, 13). The advent of the use of genetically encoded fluorescent proteins (1) has made it very convenient to label almost any protein in a living cell. These genetically encoded fluorescent proteins now cover almost the entire visible spectrum, allowing multicolor imaging. While caution must always be exercised to ensure that the fluorescent protein is not modifying the behavior of the protein of interest, there are continuing improvements in the properties of the genetically encoded fluorescent proteins to reduce problems such as aggregation.
Fluorescent indicator dyes have provided the ability to monitor physiological changes in living cells. Chemical dyes have been developed to monitor pH, Ca2+, Na+, Cl, membrane electrical potential, redox levels, protease activity, and many other cellular functions (7, 8, 27, 28). These provide unique information about the spatial localization of signals and the heterogeneity of cellular responses in a population of cells. Single-cell measurements also provide dramatic improvements in temporal resolution of signaling responses, because the average response reflects the differences in timing of responses in individual cells. Recently, genetically encoded fluorescent proteins have also been used to report on signal transduction activation in cells. This often involves the use of two genetically encoded fluorescent proteins that form a fluorescence resonance energy transfer donor-acceptor pair such that the distance between the donor and the acceptor changes in response to a signal (e.g., phosphorylation of a linker sequence between the genetically encoded fluorescent proteins) (25).
All of these advances in microscopy have contributed greatly to the understanding of many aspects of cell biology. Making movies of specific proteins and their involvement in cellular processes such as mitosis or protein secretion is now almost routine. Furthermore, we are able to obtain increasingly sophisticated kinetic models to describe these processes. As valuable as these tissue culture microscopic studies are, they are of cells in a laboratory environment. We know that the milieu surrounding a cell has profound effects on almost all cellular processes, so there is great interest and value in extending these studies to cells and tissues in living organisms. Many of the recent advances in microscopy facilitate this transition. In some cases, however, microscopy is not even needed for the imaging, and the fluorescence expressed in some cancer cells can be seen by eye as a result of the very high level of the fluorescent protein expression. For example, a recent study (20) described the imaging of tumor xenografts and metastases of human melanoma cells in living mice with cells expressing trifusion reporter gene with a luciferase reporter, a monomeric red fluorescence protein, and a positron emission tomography reporter gene.
In vivo imaging itself has a long history, although the range of its applications is relatively limited. For example, optical microscopy was used more than 40 years ago to directly visualize changes in capillaries and the movement of blood cells through them in living human skin (19). For fluorescence imaging in live animals, several experimental issues become critical. One of the first is that photodamage should be minimized. This can be achieved by choosing wavelengths that have low absorption and by using the lowest amount of light that provides an adequate signal-to-noise ratio. For thin samples, 3-D deconvolution of epifluorescence images and image reconstruction may provide better images at lower light doses than conventional confocal imaging (23). Both confocal imaging and 3-D deconvolution techniques have been very useful in obtaining 3-D images through small organisms. For example, in Drosophila embryos, several rounds of cell division (24) have been observed with the use of rhodamine-labeled topoisomerase II, and the dynamics of mitotic spindle formation during cell division were monitored with the use of microinjected fluorescent tubulin (3).
Light absorption and scattering limit the depth of penetration that can be used for fluorescence microscopy in vivo. The development of multiphoton picosecond pulsed laser scanning fluorescence microscopy (4, 5) provides several advantages in this regard. The excitation of visible wavelength fluorophores by the simultaneous absorption of two or three photons allows the use of near-infrared wavelength light, which has relatively low absorption by biological tissues. The requirement for high photon densities ensures that fluorophores are excited only near the focal point, and this confines photobleaching and photodamage to the vicinity of the focal plane. Because the excitation is highly localized, there is no need for a confocal pinhole, which would reduce the efficiency of light collection. Recently, deep tissue imaging of several millimeters in an intact animal was achieved with the use of multiphoton microscopy through gradient index lenses (12).
Multiphoton microscopy provides the opportunity to visualize cellular process in their natural environment, including even tissues such as the neurons of mammals. Using two-photon scanning microscopy and time-lapse imaging of transgenic mice expressing yellow fluorescent protein, alterations of neuronal behavior from high plasticity during development to remarkable stability in the adult mice were observed, providing a potential structural basis for long-term information storage (9). These approaches were also used to investigate the molecular mechanisms underlying experience-dependent plasticity (26). Multiphoton imaging in the brains of transgenic mice as a model of Alzheimer's disease showed no detectable changes in plaque size or geometry over extended periods of time (2). This observation addresses a long-standing question about the dynamics of plaque formation. Multiphoton microscopy has also been used to release caged compounds in subfemtoliter volumes, to measure calcium transients 500 µm deep in mouse brain, and to quantify blood flow by imaging shadows of blood cells as they travel through capillaries (22, 30).
One side benefit of multiphoton imaging has been the ability to obtain detailed information from unstained samples. For example, serotonin is a weak multiphoton fluorophore, and its secretion can be observed directly (29). A distinct imaging method is based on second harmonic generation. This is a scattering process in which two photons combine during the scattering interaction, resulting in the output of a photon at one-half the input wavelength. The precise basis for determining the efficiency of second harmonic generation is not well understood, but empirically, different types of structures can vary significantly in their scattering efficiency. For example, collagen is a very strong second harmonic generator, and it can be visualized easily in animal tissues. With the use of changes in second harmonic generation, action potentials of primary Aplysia neurons in culture were recoded with 0.8-ms temporal and 0.6-µm spatial resolution (6). Recently, two-photon laser scanning microscopy has been used for phenotype screening of transgenic and gene-targeted animal models. Intracortical injections of a viral vector with EGFP gene expressing abnormalities of dendritic spine development in both length and density in mice with mutation resulted in mental retardation disorders that were directly visualized and characterized (18).
In the current article in focus (Ref. 21, see p. C517 in this issue), Sandoval et al. examine the binding and endocytic trafficking of a conjugate of folic acid, folate Texas red (FTR), in vivo. Using two-photon fluorescence and electron microscopy, the authors followed the fate of fluorescent folate after internalization by proximal tubule cells in live rats or mice by infusion of FTR by one of three methods: 1) a femoral venous line inserted into the left leg, 2) a butterfly catheter inserted into the tail vein, or 3) intraperitoneal injection. Folic acid is important for nucleic acid synthesis, and folate deficiency has been associated with developmental disorders. Previous studies in which in vitro models were used have shown that the folate receptor (FR), a glycosylphosphoinositol lipid-anchored protein, binds folate with high affinity. FR is expressed at the apical or basal lateral surface of different cell types and is internalized into cells through clathrin-coated vesicles and by nonclathrin mechanisms (10, 21). In this report, the authors demonstrate that folate conjugates were localized only at the apical surface of kidney proximal tubules in rat or mouse and that the bulk of the rapidly internalized FTR accumulated in lysosomes. However, a small fraction of the internalized FTR was also delivered by transcytosis directly from the apical surface to the basal membrane of the kidney proximal tubules. Moreover, no cytosolic release of FTR was observed. These results provide unique insights into the trafficking of folate across the proximal tubules, which otherwise would not be possible.
Molecular imaging of FTR or anti-folate antibody may provide new insight into ways to improve cancer treatments. The high affinity of FR for folic acid has led to its use as both a marker and a target for chemotherapy with folate for delivering therapeutic agents. FR has been shown to be abundant on the membranes of many malignant cells. While future work is needed to fully understand folic acid and its related disease process, FR has already been used in clinical settings as a potential delivery system for bound ligand or folate analogs to FR-positive tumor cells (14).
Currently, multiphoton microscopy, with its high resolution and narrow focal plane, is the best high-resolution, noninvasive means of imaging in living animals for direct visualization of tissue morphology, cell metabolism, and disease states (30). It offers a powerful in vivo imaging tool with unprecedented temporal and spatial resolution that will provide distinct benefits in a broad range of applications in 1) elucidating specific cellular and molecular interaction and linking in vitro cell culture observations with in vivo processes; 2) monitoring gene expressing and protein trafficking and targeting; 3) phenotype screening of transgenic and gene-targeted animal models; 4) imaging disease progression, tumor development, and physiological abnormalities with repetitive imaging; and 5) improving drug discovery and accelerating preclinical trials of clinical treatments. We can expect that continued improvements in many underlying technologies will keep fluorescent imaging at the forefront of physiological studies.
Address for reprint requests and other correspondence: F. R. Maxfield, Dept. of Biochemistry, Weill Medical College of Cornell Univ., 1300 York Ave., New York, NY 10021 (E-mail: frmaxfie{at}med.cornell.edu).
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