Fluorescence microscopy is one of the most powerful tools for elucidating the cellular functions of proteins and other molecules. In many cases, the function of a molecule can be inferred from its association with specific intracellular compartments or molecular complexes, which is typically determined by comparing the distribution of a fluorescently labeled version of the molecule with that of a second, complementarily labeled probe. Although arguably the most common application of fluorescence microscopy in biomedical research, studies evaluating the "colocalization" of two probes are seldom quantified, despite a diversity of image analysis tools that have been specifically developed for that purpose. Here we provide a guide to analyzing colocalization in cell biology studies, emphasizing practical application of quantitative tools that are now widely available in commercial and free image analysis software.

Optical microscopy, when applied to living animals, provides a powerful means of studying cell biology in the most physiologically relevant setting. The ability of two-photon microscopy to collect optical sections deep into biological tissues has opened up the field of intravital microscopy to high-resolution studies of the brain, lens, skin, and tumors. Here we present examples of the way in which two-photon microscopy can be applied to intravital studies of kidney physiology. Because the kidney is easily externalized without compromising its function, microscopy can be used to evaluate various aspects of renal function in vivo. These include cell vitality and apoptosis, fluid transport, receptor-mediated endocytosis, blood flow, and leukocyte trafficking. Efficient two-photon excitation of multiple fluorophores permits comparison of multiple probes and simultaneous characterization of multiple parameters and yields spectral information that is crucial to the interpretation of images containing uncharacterized autofluorescence. The studies described here demonstrate the way in which two-photon microscopy can provide a level of resolution previously unattainable in intravital microscopy, enabling kinetic analyses and physiological studies of the organs of living animals with subcellular resolution.

Abstract. To study the fusion and separation of endocytic compartments, we have used digital image analysis to quantify the accumulation of fluorescent ligands in endosomes during continuous endocytosis for periods of 1-20 min. Fluorescently labeled transferrin (Tf) and low density lipoproteins (LDL) were used as markers of recycling receptors and lysosomally directed ligands respectively. By measuring the intensity of individual endosomes, we found that the amount of LDL per endosome increases 30-40-fold between 1 and 10 min and then plateaus. In contrast, the amount of Tf per endosome reaches a steady state within 2 min at a level that is only three to four times that at 1 min.We used pulse-chase double label methods to demonstrate that Tf cycles through the compartment in which the LDL accumulates. When both Tf and LDL are added to cells simultaneously for 2 min, nearly all endosomes contain both labels. With 2--4 min further incubation in the absence of external ligands, LDLcontaining compartments become depleted of Tf as Tf is directed to para-Golgi recycling endosomes. However, if Tf is added to the medium 2-4 min after a pulse with LDL, most of the LDL-containing endosomes become labeled with "If. The data indicate that at least 30-40 endocytic vesicles containing both Tf and LDL fuse with an endosomal compartment over a period of 5-10 min. LDL accumulates within this compartment and Tf is simultaneously removed. Simple mathematical models suggest that this type of iterative fractionation can lead to very high efficiency sorting.

Abstract. Cataract is a major ocular disease that causes blindness in many developing countries of the world. It is well established that various factors such as oxidative stress, UV, and other toxic agents can induce both in vivo and in vitro cataract formation. However, a common cellular basis for this induction has not been previously recognized. The present study of lens epithelial cell viability suggests such a general mechanism. When lens epithelial cells from a group of 20 cataract patients 12 to 94 years old were analyzed by terminal deoxynucleotidyl transferase (TdT) labeling and DNA fragmentation assays, it was found that all of these patients had apoptotic epithelial cells ranging from 4.4 to 41.8%. By contrast, in eight normal human lenses of comparable age, very few apoptotic epithelial cells were observed.We suggest that cataract patients may have deficient defense systems against factors such as oxidative stress and UV at the onset of the disease. Such stress can trigger lens epithelial cell apoptosis that then may initiate cataract development. To test this hypothesis, it is also demonstrated here that hydrogen peroxide at concentrations previously found in some cataract patients induces both lens epithelial cell apoptosis and cortical opacity. Moreover, the temporal and spatial distribution of induced apoptotic lens epithelial cells precedes development of lens opacification. These results suggest that lens epithelial cell apoptosis may be a common cellular basis for initiation of noncongenital cataract formation.

Abstract. Endocytosed proteins are sorted in early endosomes to be recycled to the plasma membrane or transported further into the degradative pathway. We studied the role of endosome acidification on the endocytic trafficking of the transferrin receptor (TfR) as a representative for the recycling pathway, the cationindependent mannose 6-phosphate receptor (MPR) as a prototype for transport to late endosomes, and fluidphase endocytosed HRP as a marker for transport to lysosomes. Toward this purpose, bafilomycin A1 (Bar), a specific inhibitor of the vacuolar proton pump, was used to inhibit acidification of the vacuolar system. Microspectrofluorometric measurement of the pH of fluorescein-rhodamine-conjugated transferrin (Tf)-conraining endocytic compartments in living cells revealed elevated endosomal pH values (pH >7.0) within 2 min after addition of Bar. Although recycling of endocytosed Tf to the plasma membrane continued in the presence of Baf, recycled Tf did not dissociate from its receptor, indicating failure of Fe 3+ release due to a neutral endosomal pH. In the presence of Baf, the rates of internalization and recycling of Tf were reduced by a factor of 1.40 +_ 0.08 and 1.57 + 0.25, respectively. Consequently, little if any change in TfR expression at the cell surface was measured during Bar treatment. Sorting between endocytosed TfR and MPR was analyzed by the HRP-catalyzed 3,3'-diaminobenzidine crosslinking technique, using transferrin conjugated to HRP to label the endocytic pathway of the TfR. In the absence of Baf, endocytosed surface 125I-labeled MPR was sorted from the TfR pathway starting at 10 min after uptake, reaching a plateau of 40% after 45 min. In the presence of Bar, sorting was initiated after 20 min of uptake, reaching ~40% after 60 min. Transport of fluid-phase endocytosed HRP to late endosomes and lysosomes was measured using cell fractionation and immunogold electron microscopy. Baf did not interfere with transport of HRP to MPR-labeled late endosomes, but nearly completely abrogated transport to cathepsin D-labeled lysosomes. From these results, we conclude that trafficking through early and late endosomes, but not to lysosomes, continued upon inactivation of the vacuolar proton pump.URING receptor-mediated endocytosis, ligand-receptor complexes are internalized and transported via clathrin-coated vesicles to endosomes (for reviews see Van Deurs et al., 1989;Courtoy, 1991). In endosomes, ligands and receptors may dissociate from each other, after which receptors and ligands are transported to their specific destination in the cell. Since endocytosed proteins are transported from endosomes to lysosomes, the trans-Golgi network (TGN) 1, or to either apical or ba-

Wound healing in adult human skin results in varying degrees of scar formation, ranging clinically from fine asymptomatic scars to problematic hypertrophic and keloid scars, which may limit function and restrict further growth. At present, no good objective method of clinically assessing scars exists, which is problematic for the evaluation of scar prevention or treatment regimens. Similarly lacking are histologic correlates of what we consider good and bad clinical scars. The objective of this study was to quantitatively assess human scarring (1) clinically, by developing a comprehensive rating scale, (2) photographically, using an image capture system and a scar assessment panel, and (3) by histologic analysis following scar excision. We assessed 69 scars, with a wide clinical range of severity, in patients who were undergoing surgery, for whatever reason, that involved removal of an old scar. Preoperatively, patients had their scars assessed, clinically using our newly developed scale and photographically using a computerized image capture system. These photographs were then sent to a panel for assessment using similar criteria to those used clinically. Assessment of scars from photographs correlated well with the clinical scar evaluation, indicating its potential utility in multicenter scar prevention/treatment trials. Following excision, scars were processed and analyzed for histology. We also found a strong correlation between the macroscopic and microscopic appearance of scars, particularly between the clinical appearance and histologic scores of features in the epidermis and papillary dermis. This suggests that our clinical scale is a sensitive instrument in scar assessment, allowing validated quantification of the severity of a wide range of scars.

Previous studies of fibroblasts have demonstrated that recycling of endocytic receptors occurs through a default mechanism of membrane-volume sorting. Epithelial cells require an additional level of polar membrane sorting, but there are conflicting models of polar sorting, some suggesting that it occurs in early endosomes, others suggesting it occurs in a specialized apical recycling endosome (ARE). The relationship between endocytic sorting to the lysosomal, recycling and transcytotic pathways in polarized cells was addressed by characterizing the endocytic itineraries of LDL, transferrin (Tf) and IgA, respectively, in polarized Madin-Darby canine kidney (MDCK) cells. Quantitative analyses of 3-dimensional images of living and fixed polarized cells demonstrate that endocytic sorting occurs sequentially. Initially internalized into lateral sorting endosomes, Tf and IgA are jointly sorted from LDL into apical and medial recycling endosomes, in a manner consistent with default sorting of membrane from volume. While Tf is recycled to the basolateral membrane from recycling endosomes, IgA is sorted to the ARE prior to apical delivery. Quantifications of the efficiency of sorting of IgA from Tf between the recycling endosomes and the ARE match biochemical measurements of transepithelial protein transport, indicating that all polar sorting occurs in this step. Unlike fibroblasts, rab11 is not associated with Tf recycling compartments in either polarized or glass-grown MDCK cells, rather it is associated with the compartments to which IgA is directed after sorting from Tf. These results complicate a suggested homology between the ARE and the fibroblast perinuclear recycling compartment and provide a framework that justifies previous conflicting models of polarized sorting.Key words: Endocytosis, endosome, epithelia, low density lipoprotein, MDCK, polarity, polymeric Ig receptor, transcytosis, transferrin Received 9 August 1999, revised and accepted for publication 26 October 1999The transport functions of an epithelium are determined by the distinct compositions of the apical and basolateral plasma membrane domains. This membrane polarity is maintained despite significant endocytic turnover, which in MadinDarby canine kidney (MDCK) cells can amount to 40% of the plasma membrane internalized per hour (1). Whereas early studies indicated that the apical and basolateral endocytic recycling pathways of MDCK cells are distinct (2,3), recent evidence indicates that the two pathways are interconnected (4 -6). With this continuous intermixing of apical and basolateral membranes, it is clear that endocytic sorting is crucial to maintaining the plasma membrane polarity of epithelial cells.

To examine the relationship between endosome acidification and receptor trafficking, transferrin receptor trafficking was characterized in Chinese hamster ovary cells in which endosome acidification was blocked by treatment with the specific inhibitor of the vacuolar H+-ATPase, bafilomycin A1. Elevating endosome pH slowed the receptor externalization rate to approximately one-half of control but did not affect receptor internalization kinetics. The slowed receptor externalization required the receptor's cytoplasmic domain and was largely eliminated by substitutions replacing either of two aromatic amino acids within the receptor's cytoplasmic YTRF internalization motif. These results confirm, using a specific inhibitor of the vacuolar proton pump, that proper endosome acidification is necessary to maintain rapid recycling of intracellular receptors back to the plasma membrane. Moreover, receptor return to the plasma membrane is slowed in the absence of proper endosome acidification by a signal-dependent mechanism involving the receptor's cytoplasmic tyrosine-containing internalization motif. These results, in conjunction with results from other studies, suggest that the mechanism for clustering receptors in plasma membrane clathrin-coated pits may be an example of a more general mechanism that determines the dynamic distribution of membrane proteins among various compartments with luminal acidification playing a crucial role in this process.