Spatial-temporal imaging of bacterial infection and antibiotic response in intact animals.

We describe imaging the luminance of green fluorescent protein (GFP)-expressing bacteria from outside intact infected animals. This simple, nonintrusive technique can show in great detail the spatial-temporal behavior of the infectious process. The bacteria, expressing the GFP, are sufficiently bright as to be clearly visible from outside the infected animal and recorded with simple equipment. Introduced bacteria were observed in several mouse organs including the peritoneal cavity, stomach, small intestine, and colon. Instantaneous real-time images of the infectious process were acquired by using a color charge-coupled device video camera by simply illuminating mice at 490 nm. Most techniques for imaging the interior of intact animals may require the administration of exogenous substrates, anesthesia, or contrasting substances and require very long data collection times. In contrast, the whole-body fluorescence imaging described here is fast and requires no extraneous agents. The progress of Escherichia coli-GFP through the mouse gastrointestinal tract after gavage was followed in real-time by whole-body imaging. Bacteria, seen first in the stomach, migrated into the small intestine and subsequently into the colon, an observation confirmed by intravital direct imaging. An i.p. infection was established by i.p. injection of E. coli-GFP. The development of infection over 6 h and its regression after kanamycin treatment were visualized by whole-body imaging. This imaging technology affords a powerful approach to visualizing the infection process, determining the tissue specificity of infection, and the spatial migration of the infectious agents.

Whole-body and intravital optical imaging of angiogenesis in orthotopically implanted tumors.

The development of drugs for the control of tumor angiogenesis requires a simple, accurate, and economical assay for tumor-induced vascularization. We have adapted the orthotopic implantation model to angiogenesis measurement by using human tumors labeled with Aequorea victoria green fluorescent protein for grafting into nude mice. The nonluminous induced capillaries are clearly visible against the very bright tumor fluorescence examined either intravitally or by whole-body luminance in real time. The orthotopic implantation model of human cancer has been well characterized, and fluorescence shadowing replaces the laborious histological techniques for determining blood vessel density. Intravital images of orthotopically implanted human pancreatic tumors clearly show angiogenic capillaries at both primary and metastatic sites. A quantitative time course of angiogenesis was determined for an orthotopically growing human prostate tumor periodically imaged intravitally in a single nude mouse over a 19-day period. Whole-body optical imaging of tumor angiogenesis was demonstrated by injecting fluorescent Lewis lung carcinoma cells into the s.c. site of the footpad of nude mice. The footpad is relatively transparent, with comparatively few resident blood vessels, allowing quantitative imaging of tumor angiogenesis in the intact animal. Capillary density increased linearly over a 10-day period as determined by whole-body imaging. Similarly, the green fluorescent protein-expressing human breast tumor MDA-MB-435 was orthotopically transplanted to the mouse fat pad, where whole-body optical imaging showed that blood vessel density increased linearly over a 20-week period. These powerful and clinically relevant angiogenesis mouse models can be used for real-time in vivo evaluation of agents inhibiting or promoting tumor angiogenesis in physiological microenvironments.

Visualizing gene expression by whole-body fluorescence imaging.

Transgene expression in intact animals now can be visualized by noninvasive techniques. However, the instruments and protocols developed so far have been formidable and expensive. We describe here a system for rapidly visualizing transgene expression in major organs of intact live mice that is simple, rapid, and eminently affordable. Green fluorescent protein (GFP) is expressed in the cells of brain, liver, pancreas, prostate, and bone, and its fluorescence is encoded in whole-body optical images. For low-magnification images, animals are illuminated atop a fluorescence light box and directly viewed with a thermoelectrically cooled color charge-coupled device camera. Higher-magnification images are made with the camera focused through an epi-fluorescence dissecting microscope. Both nude and normal mice were labeled by directly injecting 8 x 10(10) plaque-forming units/ml of adenoviral GFP in 20-100 microl PBS and 10% glycerol into either the brain, liver, pancreas, prostate, or bone marrow. Within 5-8 h after adenoviral GFP injection, the fluorescence of the expressed GFP in brain and liver became visible, and whole-body images were recorded at video rates. The GFP fluorescence continued to increase for at least 12 h and remained detectable in liver for up to 4 months. The system's rapidity of image acquisition makes it capable of real-time recording. It requires neither exogenous contrast agents, radioactive substrates, nor long processing times. The method requires only that the expressed gene or promoter be fused or operatively linked to GFP. A comparatively modest investment allows the study of the therapeutic and diagnostic potential of suitably tagged genes in relatively opaque organisms.

Whole-body optical imaging of green fluorescent protein-expressing tumors and metastases.

We have imaged, in real time, fluorescent tumors growing and metastasizing in live mice. The whole-body optical imaging system is external and noninvasive. It affords unprecedented continuous visual monitoring of malignant growth and spread within intact animals. We have established new human and rodent tumors that stably express very high levels of the Aequorea victoria green fluorescent protein (GFP) and transplanted these to appropriate animals. B16F0-GFP mouse melanoma cells were injected into the tail vein or portal vein of 6-week-old C57BL/6 and nude mice. Whole-body optical images showed metastatic lesions in the brain, liver, and bone of B16F0-GFP that were used for real time, quantitative measurement of tumor growth in each of these organs. The AC3488-GFP human colon cancer was surgically implanted orthotopically into nude mice. Whole-body optical images showed, in real time, growth of the primary colon tumor and its metastatic lesions in the liver and skeleton. Imaging was with either a trans-illuminated epifluorescence microscope or a fluorescence light box and thermoelectrically cooled color charge-coupled device camera. The depth to which metastasis and micrometastasis could be imaged depended on their size. A 60-microm diameter tumor was detectable at a depth of 0.5 mm whereas a 1, 800-microm tumor could be visualized at 2.2-mm depth. The simple, noninvasive, and highly selective imaging of growing tumors, made possible by strong GFP fluorescence, enables the detailed imaging of tumor growth and metastasis formation. This should facilitate studies of modulators of cancer growth including inhibition by potential chemotherapeutic agents.

The nuclear matrix prepared by amine modification.

The nucleus is spatially ordered by attachments to a nonchromatin nuclear structure, the nuclear matrix. The nuclear matrix and chromatin are intimately connected and integrated structures, and so a major technical challenge in nuclear matrix research has been to remove chromatin while retaining a native nuclear matrix. Most methods for removing chromatin require first a nuclease digestion and then a salt extraction to remove cut chromatin. We have hypothesized that cut chromatin is held in place by charge interactions involving nucleosomal amino groups. We have tested this hypothesis by chemically modifying amino groups after nuclease digestion. By using this protocol, chromatin could be effectively removed at physiological ionic strength. We compared the ultrastructure and composition of this nuclear matrix preparation with the traditional high-salt nuclear matrix and with the third nuclear matrix preparation that we have developed from which chromatin is removed after extensive crosslinking. All three matrix preparations reveal internal nuclear matrix structures that are built on a network of branched filaments of about 10 nm diameter. That such different chromatin-removal protocols reveal similar principles of nuclear matrix construction increases our confidence that we are observing important architectural elements of the native structure in the living cell.

Developmental association of the beta-galactoside-binding protein galectin-1 with the nuclear matrix of rat calvarial osteoblasts.

The protein composition of the nuclear matrix changes significantly as the osteoblast matures from a proliferating pre-osteoblast to an osteocyte embedded in a mineralized matrix. These matrix protein are the result of developmental stage-specific gene expression during osteoblast differentiation. To isolate nuclear matrix proteins unique to the bone phenotype we analyzed nuclear matrix preparations from cultures of rat calvarial osteoblasts by high resolution two-dimensional gel electrophoresis at two different stages: proliferation (day 3) and differentiation (day 18, mineralized). We characterized one protein (14 kDa; pI 5.0), that was detectable only in the nuclear matrix of differentiated osteoblasts. By mass spectrometry and microsequencing, this protein was identified as the beta -galactoside-binding protein galectin-1. Both immunofluorescence staining of nuclear matrix preparations with the galectin-1 antibody and western blot analysis of subcellular fractions confirmed that galectin-1 is only associated with the nuclear matrix in differentiated osteoblasts as the result of differential retention. Galectin-1 protein and mRNA are present throughout osteoblast differentiation. Galectin-1 is present in the cytoplasmic and nuclear fractions in both proliferating and differentiated osteoblasts. However, its only stable binding is to the nuclear matrix of the differentiated osteoblast; but, in proliferating osteoblasts, galectin-1 is not retained in the nuclear matrix. Taken together, our results suggest that developmental association of galectin-1 with the nuclear matrix reflects differential subnuclear binding of galectin-1 during osteoblast differentiation.

TGF-beta1 modifications in nuclear matrix proteins of osteoblasts during differentiation.

Nuclear matrix protein (NMP) composition of osteoblasts shows distinct two-dimensional gel electrophoretic profiles of labeled proteins as a function of stages of cellular differentiation. Because NMPs are involved in the control of gene expression, we examined modifications in the representation of NMPs induced by TGF-beta1 treatment of osteoblasts to gain insight into the effects of TGF-beta on development of the osteoblast phenotype. Exposure of proliferating fetal rat calvarial derived primary cells in culture to TGF-beta1 for 48 h (day 4-6) modifies osteoblast cell morphology and proliferation and blocks subsequent formation of mineralized nodules. Nuclear matrix protein profiles were very similar between control and TGF-beta-treated cultures until day 14, but subsequently differences in nuclear matrix proteins were apparent in TGF-beta-treated cultures. These findings support the concept that TGF-beta1 modifies the final stage of osteoblast mineralization and alters the composition of the osteoblast nuclear matrix as reflected by selective and TGF-beta-dependent modifications in the levels of specific nuclear matrix proteins. The specific changes induced by TGF-beta in nuclear matrix associated proteins may reflect specialized mechanisms by which TGF-beta signalling mediates the alterations in cell organization and nodule formation and/or the consequential block in extracellular mineralization.

Intranuclear targeting of AML/CBFalpha regulatory factors to nuclear matrix-associated transcriptional domains.

The AML/CBFalpha runt transcription factors are key regulators of hematopoietic and bone tissue-specific gene expression. These factors contain a 31-amino acid nuclear matrix targeting signal that supports association with the nuclear matrix. We determined that the AML/CBFalpha factors must bind to the nuclear matrix to exert control of transcription. Fusing the nuclear matrix targeting signal to the GAL4 DNA binding domain transactivates a genomically integrated GAL4 responsive reporter gene. These data suggest that AML/CBFalpha must associate with the nuclear matrix to effect transcription. We used fluorescence labeling of epitope-tagged AML-1B (CBFA2) to show it colocalizes with a subset of hyperphosphorylated RNA polymerase II molecules concentrated in foci and linked to the nuclear matrix. This association of AML-1B with RNA polymerase II requires active transcription and a functional DNA binding domain. The nuclear matrix domains that contain AML-1B are distinct from SC35 RNA processing domains. Our results suggest two of the requirements for AML-dependent transcription initiation by RNA polymerase II are association of AML-1B with the nuclear matrix together with specific binding of AML to gene promoters.

Targeting of the YY1 transcription factor to the nucleolus and the nuclear matrix in situ: the C-terminus is a principal determinant for nuclear trafficking.

The multifunctional transcription factor YY1 is associated with the nuclear matrix. In osteoblasts, the interaction of several nuclear matrix-associated transcription factors with the bone specific osteocalcin gene contributes to tissue-specific and steroid hormone-mediated transcription. A canonical nuclear matrix targeting signal (NMTS) is present in all members of the AML/CBFbeta transcription factor family, but not in other transcription factors. Therefore, we defined sequences that direct YY1 (414 amino acids) to the nuclear matrix. A series of epitope tagged deletion constructs were expressed in HeLa S3 and in human Saos-2 osteosarcoma cells. Subcellular distribution was determined in whole cells and nuclear matrices in situ by immunofluorescence. We demonstrated that amino acids 257-341 in the C-terminal domain of YY1 are necessary for nuclear matrix association. We also observed that sequences within the N-terminal domain of YY1 permit weak nuclear matrix binding. Our data further suggest that the Gal4 epitope tag contains sequences that affect subcellular localization, but not targeting to the nuclear matrix. The targeted association of YY1 with the nuclear matrix provides an additional level of functional regulation for this transcription factor that can exhibit positive and negative control.

Governing step of metastasis visualized in vitro.

Metastasis is the ultimate life-threatening stage of cancer. The lack of accurate model systems thwarted studies of the metastatic cell's basic biology. To follow continuously the succeeding stages of metastatic colony growth, we heritably labeled cells from the human lung adenocarcinoma cell line ANIP 973 with green fluorescent protein (GFP) by transfection with GFP cDNA. Labeled cells were then injected intravenously into nude mice, where, by 7 days, they formed brilliantly fluorescing metastatic colonies on mouse lung [Chishima, T., Miyagi, Y., Wang, X., Yang, M., Tan, Y., Shimada, H., Moossa, A. R. & Hoffman, R. M. (1997) Clin. Exp. Metastasis 15, 547-552]. The seeded lung tissue was then excised and incubated in the three-dimensional sponge-gel-matrix-supported histoculture that maintained the critical features of progressive in vivo tumor colonization while allowing continuous access for measurement and manipulation. Tumor progression was continuously visualized by GFP fluorescence in the same individual cultures over a 52-day period, during which the tumors spread throughout the lung. Histoculture tumor colonization was selective for lung cancer cells to grow on lung tissue, because no growth occurred on histocultured mouse liver tissue, which was also observed in vivo. The ability to support selective organ colonization in histoculture and visualize tumor progression by GFP fluorescence allows the in vitro study of the governing processes of metastasis [Kuo, T.-H., Kubota, T., Watanbe, M., Furukawa, T., Teramoto, T., Ishibiki, K., Kitajima, M., Moossa, A. R., Penman, S. & Hoffman, R. M. (1995) Proc. Natl. Acad. Sci. USA 92, 12085-12089]. The results presented here provide significant, new opportunities to understand and to develop treatments that prevent and possibly reverse metastasis.

Structural protein 4.1 is located in mammalian centrosomes.

Structural protein 4.1 was first characterized as an important 80-kDa protein in the mature red cell membrane skeleton. It is now known to be a member of a family of protein isoforms detected at diverse intracellular sites in many nucleated mammalian cells. We recently reported that protein 4.1 isoforms are present at interphase in nuclear matrix and are rearranged during the cell cycle. Here we report that protein 4.1 epitopes are present in centrosomes of human and murine cells and are detected by using affinity-purified antibodies specific for 80-kDa red cell 4.1 and for 4.1 peptides. Immunofluorescence, by both conventional and confocal microscopy, showed that protein 4.1 epitopes localized in the pericentriolar region. Protein 4.1 epitopes remained in centrosomes after extraction of cells with detergent, salt, and DNase. Higher resolution electron microscopy of detergent-extracted cell whole mounts showed centrosomal protein 4.1 epitopes distributed along centriolar cylinders and on pericentriolar fibers, at least some of which constitute the filamentous network surrounding each centriole. Double-label electron microscopy showed that protein 4.1 epitopes were predominately localized in regions also occupied by epitopes for centrosome-specific autoimmune serum 5051 but were not found on microtubules. Our results suggest that protein 4.1 is an integral component of centrosome structure, in which it may play an important role in centrosome function during cell division and organization of cellular architecture.

Identification of a nuclear matrix targeting signal in the leukemia and bone-related AML/CBF-alpha transcription factors.

Transcription factors of the AML (core binding factor-alpha/polyoma enhancer binding protein 2) class are key transactivators of tissue-specific genes of the hematopoietic and bone lineages. Alternative splicing of the AML-1 gene results in two major AML variants, AML-1 and AML-1B. We show here that the transcriptionally active AML-1B binds to the nuclear matrix, and the inactive AML-1 does not. The association of AML-1B with the nuclear matrix is independent of DNA binding and requires a nuclear matrix targeting signal (NMTS), a 31 amino acid segment near the C terminus that is distinct from nuclear localization signals. A similar NMTS is present in AML-2 and the bone-related AML-3 transcription factors. Fusion of the AML-1B NMTS to the heterologous GAL4-(1-147) protein directs GAL4 to the nuclear matrix. Thus, the NMTS is necessary and sufficient to target the transcriptionally active AML-1B to the nuclear matrix. The loss of the C-terminal domain of AML-1B is a frequent consequence of the leukemia-related t(8;21) and t(3;21) translocations. Our results suggest this loss may be functionally linked to the modified interrelationships between nuclear structure and gene expression characteristic of cancer cells.

Resinless section electron microscopy reveals the yeast cytoskeleton.

The cytoskeleton of Saccharomyces cerevisiae is essentially invisible using conventional microscopy techniques. A similar problem was solved for the mammalian cell cytoskeleton using resinless section electron microscopy, a technique applied here to yeast. In the resinless image, soluble proteins are no longer cloaked by embedding medium and must be removed by selective detergent extraction. In yeast, this requires breaching the cell wall by digesting with Zymolyase sufficiently to allow detergent extraction of the plasma membrane lipids. Gel electropherograms show that the extracted or "soluble" proteins are distinct from the retained or "structural" proteins that presumably comprise the cytoskeleton. These putative cytoskeleton proteins include the major portions of a 43-kDa protein, which is presumably actin, and of proteins in a band appearing at 55 kDa, as well as numerous less abundant, nonactin proteins. Resinless section electron micrographs show a dense, three-dimensional web of anastomosing, polymorphic filaments bounded by the remnant cell wall. Although the filament network is very heterogenous, there appear to be two principal classes of filament diameters-5 nm and 15-20 nm-which may correspond to actin and intermediate filaments, respectively. A large oval region of lower filament density probably corresponds to the vacuole, and an electron dense spheroidal body, 300-500 nm in diameter, is likely the nucleus. The techniques detailed in this report afford new approaches to the study of yeast cytoarchitecture.

Cell cycle independent interaction of CDC2 with the centrosome, which is associated with the nuclear matrix-intermediate filament scaffold.

The cell cycle regulating Cdc2 protein kinase helps orchestrate cell cycle dependent changes in cell structure and function. This report shows that Cdc2 is localized to the centrosome region and is tightly bound to the nuclear matrix-intermediate filament scaffold. Antibodies to Cdc2 and to the centrosome-specific protein, pericentrin, label the centrosome in an apparently cell cycle independent manner. Isolated centrosomes also label similarly with both antibodies. Essentially, all cells show Cdc2 labeling of the centrosomes, implying an independence of the stage in the cell cycle, a conclusion supported by studies of synchronized cells. In contrast to the labeling of every cell with the Cdc2 monoclonal antibody, fewer centrosomes were labeled with an antibody to the PSTAIRE domain of Cdc2. Embedment-free, immunogold electron micrographs of extracted cell whole mounts show the centrioles and a pericentriolar network of filaments. Both Cdc2 and pericentrin antibodies decorate the amorphous pericentriolar material, while the Cdc2 antibodies also decorate the centrioles themselves. The constitutive presence of Cdc2 at the centrosome suggests a continuing role in the dynamics of centrosome function throughout the cell cycle.

Structural protein 4.1 in the nucleus of human cells: dynamic rearrangements during cell division.

Structural protein 4.1, first identified as a crucial 80-kD protein in the mature red cell membrane skeleton, is now known to be a diverse family of protein isoforms generated by complex alternative mRNA splicing, variable usage of translation initiation sites, and posttranslational modification. Protein 4.1 epitopes are detected at multiple intracellular sites in nucleated mammalian cells. We report here investigations of protein 4.1 in the nucleus. Reconstructions of optical sections of human diploid fibroblast nuclei using antibodies specific for 80-kD red cell 4.1 and for 4.1 peptides showed 4.1 immunofluorescent signals were intranuclear and distributed throughout the volume of the nucleus. After sequential extractions of cells in situ, 4.1 epitopes were detected in nuclear matrix both by immunofluorescence light microscopy and resinless section immunoelectron microscopy. Western blot analysis of fibroblast nuclear matrix protein fractions, isolated under identical extraction conditions as those for microscopy, revealed several polypeptide bands reactive to multiple 4.1 antibodies against different domains. Epitope-tagged protein 4.1 was detected in fibroblast nuclei after transient transfections using a construct encoding red cell 80-kD 4.1 fused to an epitope tag. Endogenous protein 4.1 epitopes were detected throughout the cell cycle but underwent dynamic spatial rearrangements during cell division. Protein 4.1 was observed in nucleoplasm and centrosomes at interphase, in the mitotic spindle during mitosis, in perichromatin during telophase, as well as in the midbody during cytokinesis. These results suggest that multiple protein 4.1 isoforms may contribute significantly to nuclear architecture and ultimately to nuclear function.

The nuclear matrix revealed by eluting chromatin from a cross-linked nucleus.

The nucleus is an intricately structured integration of many functional domains whose complex spatial organization is maintained by a nonchromatin scaffolding, the nuclear matrix. We report here a method for preparing the nuclear matrix with improved preservation of ultrastructure. After the removal of soluble proteins, the structures of the nucleus were extensively cross-linked with formaldehyde. Surprisingly, the chromatin could be efficiently removed by DNase I digestion leaving a well preserved nuclear matrix. The nuclear matrix uncovered by this procedure consisted of highly structured fibers, connected to the nuclear lamina and built on an underlying network of branched 10-nm core filaments. The relative ease with which chromatin and the nuclear matrix could be separated despite extensive prior cross-linking suggests that there are few attachment points between the two structures other than the connections at the bases of chromatin loops. This is an important clue for understanding chromatin organization in the nucleus.

Comparison of caudal bupivacaine and diamorphine with caudal bupivacaine alone for repair of hypospadias.

Forty-five boys undergoing repair of hypospadias were allocated randomly to one of two groups. After induction of anaesthesia, 22 patients received 0.25% caudal bupivacaine 0.5 ml kg-1 and diamorphine 30 micrograms kg-1 and the remaining 23 patients received 0.25% caudal bupivacaine 0.5 ml kg-1 alone. Pain scores (Children's Hospital of Eastern Ontario Pain Scale), sedation scores, ventilatory frequency, analgesic requirements and associated side effects were recorded for the first 24 h after operation. The two groups were indistinguishable in age, weight and duration of surgery. There was a statistically significant reduction in early pain scores. There was also a statistically significant increase in the time to first passage of urine in those boys in the diamorphine group who were not catheterized during operation.

Liver colonization competence governs colon cancer metastasis.

Tumors that metastasize do so to preferred target organs. To explain this apparent specificity, Paget, > 100 years ago, formulated his seed and soil hypothesis; i.e., the cells from a given tumor would "seed'' only favorable "soil'' offered by certain groups. The hypothesis implies that cancer cells must find a suitable "soil'' in a target organ--i.e., one that supports colonization--for metastasis to occur. We demonstrate in this report that ability of human colon cancer cells to colonize liver tissue governs whether a particular colon cancer is metastatic. In the model used in this study, human colon tumors are transplanted into the nude mouse colon as intact tissue blocks by surgical orthotopic implantation. These implanted tumors closely simulate the metastatic behavior of the original human patient tumor and are clearly metastatic or nonmetastatic to the liver. Both classes of tumors were equally invasive locally into tissues and blood vessels. However, the cells from each class of tumor behave very differently when directly injected into nude mouse livers. Only cells from metastasizing tumors are competent to colonize after direct intrahepatic injection. Also, tissue blocks from metastatic tumors af fixed directly to the liver resulted in colonization, whereas no colonization resulted from nonmetastatic tumor tissue blocks even though some growth occurred within the tissue block itself. Thus, local invasion (injection) and even adhesion to the metastatic target organ (blocks) are not sufficient for metastasis. The results suggest that the ability to colonize the liver is the governing step in the metastasis of human colon cancer.

The nuclear matrix protein NMP-1 is the transcription factor YY1.

NMP-1 was initially identified as a nuclear matrix-associated DNA-binding factor that exhibits sequence-specific recognition for the site IV regulatory element of a histone H4 gene. This distal promoter domain is a nuclear matrix interaction site. In the present study, we show that NMP-1 is the multifunctional transcription factor YY1. Gel-shift and Western blot analyses demonstrate that NMP-1 is immunoreactive with YY1 antibody. Furthermore, purified YY1 protein specifically recognizes site IV and reconstitutes the NMP-1 complex. Western blot and gel-shift analyses indicate that YY1 is present within the nuclear matrix. In situ immunofluorescence studies show that a significant fraction of YY1 is localized in the nuclear matrix, principally but not exclusively associated with residual nucleoli. Our results confirm that NMP-1/YY1 is a ubiquitous protein that is present in both human cells and in rat osteosarcoma ROS 17/2.8 cells. The finding that NMP-1 is identical to YY1 suggests that this transcriptional regulator may mediate gene-matrix interactions. Our results are consistent with the concept that the nuclear matrix may functionally compartmentalize the eukaryotic nucleus to support regulation of gene expression.

Rethinking cell structure.

Cell structure, emerging from behind the veil of conventional electron microscopy, appears far more complex than formerly realized. The standard plastic-embedded, ultrathin section can image only what is on the section surface and masks the elaborate networks of the cytoplasm and nucleus. Embedment-free electron microscopy gives clear, high-contrast micrographs of cell structure when combined with removal of obscuring material such as soluble proteins. The resinless ultrathin section is the technique of choice; it is simple and inexpensive, and it uses ordinary electron microscopes. The resulting pictures reveal a world of complex cell structure and function. These images necessarily change our conception of the cytoskeleton, nuclear matrix, mitosis, and the relation of membranes to cytostructure.