Fixation Artifacts Research Articles

Low temperature FESEM of the calcifying interface of a scleractinian coral

The ultrastructural nature of the calcifying interface in the scleractinian coral Galaxea fascicularis has been investigated using high-resolution, low temperature field emission scanning electron microscopy (FESEM). This technique permitted structural analyses of soft tissue and skeleton in G. fascicularis in a frozen-hydrated state, without the need for chemical fixation or decalcification. Structural comparisons are made between frozen-hydrated polyps and polyps that have undergone conventional fixation and decalcification. Vesicles expelled by the calicoblastic ectodermal cells into sub-skeletal spaces and previously suggested to play a role in calcification were commonly observed in fixed samples but were distinctly absent in frozen-hydrated preparations. We propose that these vesicles are fixation artefacts. Two distinct types of vesicles (380 and 70 nm in diameter, respectively), were predominant throughout the calicoblastic ectodermal cells of frozen-hydrated preparations, but these were never seen to be entering, or to be contained within, sub-skeletal spaces, nor did they contain any crystalline material. In frozen-hydrated preparations, membranous sheets were seen to surround and isolate portions of aboral mesogloea and to form junctional complexes with calicoblastic cells. The calicoblastic ectoderm was closely associated with the underlying skeleton, with sub-skeletal spaces significantly smaller ( P<0.0001) in frozen-hydrated polyps compared to fixed polyps. A network of organic filaments (26 nm in diameter) extended from the apical membranes of calicoblastic cells into these small sub-skeletal cavities. A thin sheath was also frequently observed adjacent to the apical membrane of calicoblastic cells.

Positively Charged DNA-Binding Proteins Cause Apparent Cell Membrane Translocation

Several positively charged DNA-binding proteins such as the human immunodeficiency virus Tat protein, the Antennapedia (Antp) homeobox protein, and the herpes simplex virus VP22 protein have been reported to translocate across cell membranes and accumulate in cell nuclei. The import occurs by a poorly understood mechanism that appears to be receptor- and energy-independent. We showed that both VP22 and the positively charged histone H1 adhered to the cell membrane of living cells and were not removed by extensive washing. However, after fixation the proteins relocated to the cell nucleus. The nuclear accumulation of VP22 and histone H1 after fixation shows that positively charged proteins may appear to translocate across the cell membrane because of a fixation artifact. The majority of studies on “membrane permeable” proteins and peptides have been performed using fixation techniques, and our study shows that influx of these proteins may occur during fixation rather than in living cells.

Superselective mesenteric embolization with microcoils in a porcine model.

There is a lack of data regarding the degree of ischemic change that may occur in small and large bowel after superselective arterial embolotherapy with platinum microcoils. The purpose of this study was to gain a clearer understanding of the ischemic complications of superselective embolization of peripheral mesenteric arteries with microcoils by gross and histologic examination of postembolectomy bowel specimens at several time intervals in an adult porcine model. Two-millimeter platinum microcoils with fibrils were superselectively deployed into six isolated mesenteric branch vessels in each of nine adult pigs. The animals were observed for two days (n = 3), one month (n = 3), or three months (n = 3) and then killed. At necropsy, intestines were examined for gross abnormalities. Sections of bowel containing microcoils were identified under fluoroscopy, resected, and evaluated histologically. A total of 54 microcoils were deployed into the distal arterial mesentery supplying the jejunum (n = 14), ileum (n = 26), and colon (n = 14) of nine adult pigs. Each animal received six microcoils. There were no clinical complications, and all pigs gained weight during their observation periods. Gross examination of the intestines did not reveal any evidence of acute or chronic ischemia. The coils were found in the distal arterial vasculature of the intestine. Histologic examination revealed mild superficial necrosis of villous tips in several samples; however, this finding is likely related to a fixation artifact rather than ischemic injury. There was no other histologic evidence suggestive of ischemic injury. Gross and histologic findings after superselective arterial embolotherapy demonstrated minimal changes in the bowel, and there were no significant clinical consequences to the animals. We conclude that transcatheter arterial embolotherapy in pigs is safe and may be applicable in the control of massive lower gastrointestinal hemorrhage in humans.

LACK OF ASSOCIATION OF PROSTATE CARCINOMA NUCLEAR GRADING WITH PROSTATE SPECIFIC ANTIGEN RECURRENCE AFTER RADICAL PROSTATECTOMY

Grading prostate cancer using the Gleason system relies only on architectural tumor growth, in contrast to other systems, such as the WHO system, which grade prostate carcinoma based on nuclear features as well as architectural patterns. The prognostic significance of nuclear grading remains controversial since most studies were performed before prostate specific antigen (PSA) screening became widely available. We evaluated the significance of nuclear grade for predicting PSA recurrence in a contemporary cohort of patients treated with radical prostatectomy for clinically localized prostate carcinoma. Nuclear grades 1 to 3 were determined in 141 consecutive radical prostatectomies in 1995. Predominant and worst nuclear grade was determined by a consensus of 3 pathologists. Statistical analysis compared nuclear grade with Gleason score using the chi-square test. The Cox proportional hazards analysis was performed to calculate the ability of nuclear grade, Gleason score and other variables to predict PSA recurrence. We identified a significant association of Gleason score with worst nuclear grade (p = 0.007). All 6 cases with a Gleason score of 8 or greater had a worst nuclear grade of 3, in contrast to 36 of 60 (60%) with a score 6 or less, in which the worst nuclear grade was 3. Of the 141 patients 31 (21.9%) had PSA recurrence at a median followup of 3.7 years. The univariate Cox model revealed significant associations of PSA recurrence with Gleason score 8 or greater (hazards ratio 5.5, p = 0.005), extraprostatic extension (hazards ratio 3.4, p = 0.001), positive surgical margin (hazards ratio 2.6, p = 0.009), seminal vesicle involvement (hazards ratio 7.3, p <0.001), preoperative serum PSA (hazards ratio 1.03, p = 0.007), tumor stage (hazards ratio 3.6, p = 0.001) and maximal tumor dimension (hazards ratio 2.4, p <0.001). However, overall and worst nuclear grade did not predict PSA recurrence (p = 0.89 and 0.13, respectively). Nuclear grade did not fit any multivariate model tested, which otherwise included Gleason score, log(PSA), surgical margin status, extraprostatic extension, seminal vesicle status, tumor size and pathological stage. By varying sample fixation time we also showed that benign prostate tissue in the same section as prostate carcinoma had grade 2 or 3 nuclear changes, that is moderate to marked anaplasia. High nuclear grade is associated with high Gleason score. However, prostate carcinoma with a Gleason score of 6 or less shows extreme variability. Nuclear grade determined by light microscopy failed to predict PSA recurrence in a contemporary series of men with clinically localized prostate cancer treated with radical prostatectomy. Nuclear morphology is subject to tissue fixation and processing artifact. Any nuclear morphometric study must consider this artifact.

Freeze-substitution of dehydrated plant tissues: artefacts of aqueous fixation revisited.

This investigation assessed the extent of rehydration of dehydrated plant tissues during aqueous fixation in comparison with the fine structure revealed by freeze-substitution. Radicles from desiccation-tolerant pea (Pisum sativum L.), desiccation-sensitive jackfruit seeds (Artocarpus heterophyllus Lamk.), and leaves of the resurrection plant Eragrostis nindensis Ficalho & Hiern. were selected for their developmentally diverse characteristics. Following freeze-substitution, electron microscopy of dehydrated cells revealed variable wall infolding. Plasmalemmas had a trilaminar appearance and were continuous and closely appressed to cell walls, while the cytoplasm was compacted but ordered. Following aqueous fixation, separation of the plasmalemma and the cell wall, membrane vesiculation and distortion of cellular substructure were evident in all material studied. The sectional area enclosed by the cell wall in cortical cells of dehydrated pea and jackfruit radicles and mesophyll of E. nindensis increased after aqueous fixation by 55, 20, and 30%, respectively. Separation of the plasmalemma and the cell wall was attributed to the characteristics of aqueous fixatives, which limited the expansion of the plasmalemma and cellular contents but not that of the cell wall. It is proposed that severed plasmodesmatal connections, plasmalemma discontinuities, and membrane vesiculation that frequently accompany separation of walls and protoplasm are artefacts of aqueous fixation and should not be interpreted as evidence of desiccation damage or membrane recycling. Evidence suggests that, unlike aqueous fixation, freeze-substitution facilitates reliable preservation of tissues in the dehydrated state and is therefore essential for ultrastructural studies of desiccation.

Procoagulant platelet balloons: evidence from cryopreparation and electron microscopy.

Visualisation of the procoagulant transformation of human platelets has recently become possible through use of an in vitro approach combined with fluorescence and phase contrast microscopy. Here, we extended these studies to the ultrastructural level by employing both rapid freezing/freeze-substitution and conventional ambient-temperature chemical fixation for transmission and scanning electron microscopy. Procoagulant transformation was only inducible by adhering platelets to collagen fibrils or to the collagen-related peptide and exposing them to physiological extracellular Ca2+ levels. Under these conditions prominent, 2- to 4-micron-wide balloon-like structures were regularly observed, regardless of the specimen fixation protocol. In strong contrast to normal platelets in their vicinity, the balloons' subcellular architecture proved remarkably poor: dilute cytoplasm, no cytoskeleton, only a few, randomly distributed organelles and/or their remnants. Cryofixed balloons displayed intact and smooth surfaces whereas conventional specimen processing caused plasma membrane perforations and shrinkage of the balloons. Our results clearly show that neither the balloons themselves, nor their simple ultrastructure reflect fixation artefacts caused by inadequate membrane stabilisation. The balloons are interpreted as to be transformed and/or fragmented procoagulant platelets. Thus, the generation of balloons represents a genuine, final stage of platelet ontogenesis, presumably occurring alternatively to aggregate formation.

Dark hair cells in the inner ear of the rainbow trout. A study of the influence of different fixation methods

AbstractThe occurrence of dark staining cells in different tissues has been suggested to be artefactual and caused during the fixation process. In inner ear sensory epithelia, dark hair cells (DHC) have been suggested to be apoptotic cells. We have examined whether dark cells represent dying cells or whether they are the results of fixation artefacts. The effects of buffer osmolarity and different fixation methods on the incidence of dark hair cells in the inner ear macula sacculi of the rainbow trout (Oncorhynchus mykiss) were investigated by light and electron microscopy. Glutaraldehyde in phosphate buffer with osmolarities of 0, 135, 225, 425, and 560 mosmol were used for fixation by immersion. For comparison, fixation by vascular perfusion as well as the effects of mechanical injury and delayed fixation were studied. DHC were found in all examined saccular maculae except for the delayed fixation protocol where almost all the sensory cells were lost. The number of DHC accounted for 2.5–12.9‰ of the sensory cells. Neither the buffer osmolarity nor the fixation method had significant effects on the frequencies of DHC. Mitotic cell division events were seen exclusively in the apical cell strata of the sensory epithelium. The DHC are suggested to be associated with apoptosis rather than fixation artefacts.

"Tubular Spitz's nevus" an artifact of fixation?

Spitz's nevi may contain numerous tubule-like structures. This phenomenon has been described as "tubular Spitz's nevus" and has been attributed to either apoptosis in the center of nests of melanocytes or tubular metaplasia of melanocytes. In order to study this peculiar phenomenon, we reviewed 31 Spitz's nevi from our teaching collection. Empty spaces vaguely resembling tubules were found in 15 Spitz's nevi, all of which were examples of the epithelioid-cell type of Spitz's nevus. Histopathologically and immunohistochemically, there was no evidence of apoptosis within or around tubule-like structures. Empty; spaces in the center of nests ranged from distinctive tubule-like structures to only slight separation of melanocytes from one another. Furthermore, empty spaces were also seen around nests, often in combination with distinctive tubule-like changes. Our findings suggest that so-called "tubular" structures in epithelioid Spitz's nevi result from retraction of tissue secondary to fixation in formalin. "Tubular Spitz's nevus" seems to represent an artifact rather than a distinctive variant of Spitz's nevus.

The application of atomic force microscopy to topographical studies and force measurements on the secreted adhesive of the green alga Enteromorpha.

Atomic force microscopy (AFM) enables the topographical structure of cells and biological materials to be resolved under natural (physiological) conditions, without fixation and dehydration artefacts associated with imaging methods in vacuo. It also provides a means of measuring interaction forces and the mechanical properties of biomaterials. In the present study, AFM has been applied for the first time to the study of the mechanical properties of a natural adhesive produced by a green plant cell. Swimming spores of the green alga Enteromorpha linza (L.) J. Ag. (7-10 microm) secrete an adhesive glycoprotein which provides firm anchorage to the substratum. Imaging of the adhesive in its hydrated state revealed a swollen gel-like pad, approximately 1 microm thick, surrounding the spore body. Force measurements revealed that freshly released adhesive has an adhesion strength of 173 +/- 1.7 mN m(-1) (mean +/- SE; n=90) with a maximum value for a single adhesion force curve of 458 mN m(-1). The adhesive had a compressibility (equivalent to Young's modulus) of 0.54 x 10(6) +/- 0.05 x 10(6) N m-2 (mean +/- SE; n=30). Within minutes of release the adhesive underwent a progressive 'curing' process with a 65% reduction in mean adhesive strength within an hour of settlement, which was also reflected in a reduction in the average length of the adhesive polymer strands (polymer extension) and a 10-fold increase in Young's modulus. Measurements on the spore surface itself revealed considerably lower adhesion-strength values but higher polymer-extension values than the adhesive pad, which may reflect the deposition of different polymers on this surface as a new cell wall is formed. The study demonstrates the value of AFM to the imaging of plant cells in the absence of fixation and dehydration artefacts and to the characterisation of the mechanical properties of plant glycoproteins that have potential utility as adhesives.

Immunohistochemical Demonstration of Hyaluronan and Its Possible Involvement in Axolotl Neural Crest Cell Migration

Hyaluronan (HA), an extracellular matrix component, is involved mainly in the control of cell proliferation, neural crest and tumor cell migration, and wound repair. We investigated the effect of hyaluronan on neural crest (NC) cell migration and its ultrastructural localization in dark (wild-type) and white mutant embryos of the Mexican axolotl (Ambystoma mexicanum, Amphibia). The axolotl system is an accepted model for studying mechanims of NC cell migration. Using a biotinylated hyaluronan binding protein (HABP), major extracellular matrix (ECM) spaces, including those of NC cell migration, reacted equally positive on cryosections through dark and white embryos. Since neural crest-derived pigment cells migrate only in subepidermal spaces of dark embryos, HA does not seem to influence crest cell migration in vivo. However, when tested on different alternating substrates in vitro, migrating NC cells in dark and white embryos prefer HA to fibronectin. In vivo, such an HA migration stimulating effect might exist as well, but be counteracted to differing degrees in dark and white embryos. The ultrastructural localization of HA was studied by means of transmission electron microscopic immunohistochemistry using HABP and different protocols of standard chemical fixation, cryofixation, embedding, and immunolabeling. The binding reaction of HA to HABP was strong and showed an equal distribution throughout ECM spaces after both standard chemical fixation/freeze substitution and cryofixation. A preference for the somite or subepidermal side was not observed. Following standard fixation/freeze substitution HABP-labeled “honeycomb”-like networks reminiscent of fixation artifacts were more prominent than labeled fibrillar or irregular net-like structures. The latter predominated in adequately frozen specimens following high-pressure freezing/freeze substitution. For this reason fibrillar or irregular net-like structures very likely represent hyaluronan in the complex subepidermal matrix of the axolotl embryo in its native arrangement.

Ultrastructure and Cytopathology of a Rickettsia-like Organism Causing Systemic Infection in the Redclaw Crayfish, Cherax quadricarinatus (Crustacea: Decapoda), in Ecuador

A studyof the ultrastructural characteristics of an intracellular bacterium infecting the redclaw crayfish, Cherax quadricarinatus, a pathogen referred to previously as a rickettsia-like organism (RLO), revealed the presence of different developmental stages. These included a rod-shaped and uniformly electron-dense elementary body (EB) and an intermediate body (IB). The length of the EB varied between 0.48 and 0.6 μm, and the diameter was 0.3 μm. The IB was 0.75 to 1.1 μm long by 0.36 to 0.44 μm in diameter. Although the EB of this bacterium has ultrastructural characteristics similar to those of Rickettsiella, no information is available regarding its genetic relationship to this genus, and the intracellular bacterium should continue to be referred to as a rickettsia-like organism. The hemocytes had different levels of infection, and the RLO proliferated inside these cells. The EB appeared to be free in the cytoplasm of infected hemocytes and other cells; however, this might be a fixation artifact. The EB was also contained in membrane-bound vacuoles along with the IB. RLO colonies were observed inside small granular cells. No large granular cells were observed in the sections examined; therefore, no data were obtained regarding infection of this type of hemocyte. The fixed phagocytes on the external side of the terminal hepatic arterioles had an activated interrupted layer containing RLO bacteria. Stem cells in the hematopoietic tissue were also infected, and some cells were apparently being released into circulation.

Mitochondria‐targeted GFP highlights the heterogeneity of mitochondrial shape, size and movement within living plant cells

Little is known concerning the heterogeneity of mitochondrial shape, size, number, cytoplasmic distribution, and motility in planta. Ultrastructural studies using the electron microscope have shown a variety of mitochondrial shapes and sizes within fixed cells, however, it is not possible to dismiss the possibility that any heterogeneity observed resulted from preparation or fixation artefacts. Unambiguous demonstration of the extent and nature of mitochondrial heterogeneity in vivo necessitates the use of a truly in vivo mitochondrial detection system. Green fluorescent protein is an excellent in vivo marker for gene expression and protein localization studies. It is particularly useful for real-time spatiotemporal analysis of intracellular protein targeting and dynamics and as such is an ideal marker for analysing mitochondria in planta. Stably transformed Arabidopsis lines have been generated with GFP targeted to the mitochondria using either of two plant mitochondrial signal sequences from the beta-ATPase subunit or the mitochondrial chaperonin CPN-60. Mitochondrially targeted GFP, which is easily detectable using an epifluorescent or confocal microscope, highlights heterogeneity of mitochondrial shape, size, position, and dynamic within living plant cells.

Mast cell derangement in salivary glands in patients with Sjögren's syndrome

Mast cells (MCs) have been implicated in many immune-inflammatory disorders. Deranged mast cell distribution and function may contribute to the local pathomechanisms in the labial salivary glands (LSG) in Sjogren's syndrome (SS). Evidence for MC presence, localization, frequency, subtype, and degree of activation was sought by using reverse transcriptase-polymerase chain reaction (RT-PCR), immunohistochemistry (IHC)/image analysis, transmission electron microscopy, Western blotting, spectrophotometric activity assay, and radioimmunoassay. The overall expression (densitometric units) of MC tryptase mRNA (1483 +/- 228 vs 1044 +/- 308) did not differ between SS and control LSGs. However, IHC disclosed an uneven distribution of MCs in SS with an absence in lymphocyte foci and increased numbers (cells/mm2 77 +/- 7 vs 38 +/- 4, P < 0.01) elsewhere. Absence of MCs in the lymphocyte foci was not a fixation artefact and was not explained by the presence of "phantom" MCs in these areas. In both SS and controls, 80% of all MCs were chymase containing, but the typical lattices/gratings characteristic for connective tissue MCs (CTMCs) were not found. Instead, MC granules had mostly a homogeneous, finely granular substructure characteristic of "new" granules subjected to a continuous, low-grade release. 32 kDa MC tryptase was found in saliva and its activity/concentration was comparable to that found in controls. However, tryptase output was low in SS (1.30 +/- 0.30 microg/min vs 3.49 +/- 0.66 microg/min, P < 0.001). Normal LSGs contain mostly CTMCs, in close contact with various resident and immigrant cells, characterized by a low-grade release of MC mediators. In SS this normal pattern is disturbed so that MCs are absent in lymphocyte foci (but increased elsewhere in the glands). The net salivary output of MC mediators is low in SS. This derangement of MCs may contribute to the pathogenesis of SS via multiple mechanisms.

Blastocystis hominis: origin and significance of vacuolar and granular forms.

Using supravital neutral red staining and light microscopy, individual Blastocystis organisms, subcultured from clinical isolates in modified monophasic Robinson's medium, were followed over various periods on glass slides. A rapid transition from uniformly stained to granular and vacuolated forms preceded the organism's death as evidenced by pale staining and Brownian motion in the cell's interior. Granular and vacuolar forms of Blastocystis may indicate degenerative changes in individual cells, fixation artifact, or both.

Green Fluorescent Protein as a Reporter for Macromolecular Localization in Bacterial Cells

Green fluorescent protein (GFP) is a highly useful fluorescent tag for studying the localization, structure, and dynamics of macromolecules in living cells, and has quickly become a primary tool for analysis of DNA and protein localization in prokaryotes. Several properties of GFP make it an attractive and versatile reporter. It is fluorescent and soluble in a wide variety of species, can be monitored noninvasively by external illumination, and needs no external substrates. Localization of GFP fusion proteins can be analyzed in live bacteria, therefore eliminating potential fixation artifacts and enabling real-time monitoring of dynamics in situ. Such real-time studies have been facilitated by brighter, more soluble GFP variants. In addition, red-shifted GFPs that can be excited by blue light have lessened the problem of UV-induced toxicity and photobleaching. The self-contained domain structure of GFP reduces the chance of major perturbations to GFP fluorescence by fused proteins and, conversely, to the activities of the proteins to which it is fused. As a result, many proteins fused to GFP retain their activities. The stability of GFP also allows detection of its fluorescence in vitro during protein purification and in cells fixed for indirect immunofluorescence and other staining protocols. Finally, the different properties of GFP variants have given rise to several technological innovations in the study of cellular physiology that should prove useful for studies in live bacteria. These include fluorescence resonance energy transfer (FRET) for studying protein–protein interactions and specially engineered GFP constructs for direct determination of cellular ion fluxes.

High Pressure Freezing has Come of Age - but is it Mature?

It should be no secret by now that ultrarapid freezing is a superior method of specimen preparation for many biological EM projects, and that high pressure freezing is the most versatile of the freezing methods. While cryopreservation is not necessary for all EM studies, it is the method of choice for high resolution work and where “fixation artifacts”, such as distorted membranes, or extraction of the cytosol is a problem. It is true that the machines are expensive, and not always immediately available, but for some questions it is the appropriate technology to use. In the U.S., there are machines available for general use in the far West (Berkeley, CA), the upper Midwest (Madision, WI and Minneapolis, MN), and in the Northeast (Albany, NY). For locations of any of the other 8 machines around the country, interested users can call Technotrade, International at (603) 622-5011.Current Status of High Pressure Freezing: A decade ago, Studer et al. wrote an article entitled: “High Pressure Freezing Comes of Age” that illustrated how high pressure freezing (HPF) had become a proven technology, useful for preserving ultrastructure with unmatched fidelity in cell types that had previously been difficult to fix well for electron microscopy (EM).

The Perineuronal Net: A Weapon for a Challenge

Theories and data do not always fit and sometimes are sources of conflicts among scientists. This is the case of a morphological structure, the perineuronal net, which was denied on the basis of an ideological conflict between two giants of neurosciences: Camillo Golgi and Santiago Ramón y Cajal. The perineuronal net is a reticular structure enveloping many neurons. Originally reported by Golgi in 1893 and 1898 and confirmed by several authors before the turn of the century, the perineuronal net was used by Golgi to support the reticular theory of the organization of the nervous system. Ramón y Cajal, the paladin of the neuronal theory who had also observed this anatomical structure, denied its existence suggesting that it was a fixation artifact. After Cajal's statements, only a few Italian scientists continued to work in this field, and after the 1930s the perineuronal net was forgotten. Only the recent advances in histochemical and immunocytochemical technology confirmed the existence of this structure opening new fields in functional neuroanatomy and neuropathology.

Live confocal analysis with fluorescently labeled proteins.

AbstractThe development of laser scanning confocal microscopy provides a powerful means to observe structures and components within the cell. Equally important advances have also occurred in the development of reagents and techniques for generating functional fluorescently tagged proteins. When these labeled proteins are used in conjunction with confocal and other advanced fluorescence microscopes, the dynamics of a given protein within the living cell can be readily analyzed in real time. Live analysis provides an appreciation of the cellular and developmental dynamics that is virtually impossible to obtain through observation of fixed samples. In addition, tracking down the primary defect and determining causal relationships in mutant and drug-mediated phenotypes often can be accomplished only through live fluorescence analysis. Finally, live analysis has been used to confirm the existence of structures in the embryo that were previously contested as possible fixation artifacts (1).KeywordsDrosophila EmbryoSuccinimidyl EsterSulfonyl ChlorideLive AnalysisInjection BufferThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

1] Membrane trafficking

Publisher Summary Immunofluorescence microscopy has played a central role in providing information concerning the localization of specific integral membrane proteins. However, immunofluorescence microscopy simply generates a “snapshot” of what is going on and fails to provide direct information concerning the movement of integral membrane proteins in living cells. Time course experiments, for example, designed to study the effects of specific hormones and drugs on intracellular morphology and membrane trafficking, are also particularly laborious to perform if based on the use of immunofluorescence microscopy. Such studies, and many others on the dynamic nature of membrane traffic within eukaryotic cells, would be much easier to perform and would not be subject to possible fixation artifacts if specific integral membrane proteins could be visualized in living cells. The expression of recombinant, green fluorescent protein (GFP)-tagged integral membrane proteins has provided us with precisely the right tools with which to perform such experiments. This chapter describes the use of a Leica [Leica Microsystems GmbH (Heidelberg, Germany)] DM IRBE inverted epifluorescence microscope attached to a Leica TCS-NT confocal laser scanning system to monitor the localization and movement of GFP-tagged integral membrane proteins in living cells; however, the general principles are applicable to a wide range of microscope systems.

Pseudolipomatosis cutis: superficial dermal vacuoles resembling fatty infiltration of the skin.

Empty spaces within the dermis of paraffin-embedded sections of skin have been attributed to fatty infiltration and postulated to originate from topical steroid administration or sebaceous gland rupture. We examined skin biopsy specimens exhibiting dermal vacuolation to determine whether this phenomenon was associated with specific skin diseases and to attempt to illuminate its etiology. Routine hematoxylin-eosin-stained sections from 26 formalin-fixed, paraffin-embedded biopsy specimens were examined. Histochemical stains for mucin and immunohistochemical staining for S100 protein and vascular markers were performed. Dermal vacuolation was characterized by empty spaces, 15-120 microm in diameter, in the superficial dermis, associated with either fibrosis/sclerosis or a lymphocytic infiltrate. There was no relationship to clinical findings, topical steroid treatment, or histologic diagnosis. There was no evidence of true adipocyte differentiation, and vascular markers were negative. Transmission electron microscopy showed nonmembrane-bound irregular spaces in the dermis. Dermal vacuoles likely represent an artifact of tissue fixation or processing and are unrelated to the underlying pathologic process. We propose the name pseudolipomatosis cutis, analogous to insufflation-induced colonic vacuolation, to distinguish this phenomenon from true dermal fatty infiltration and to emphasize its incidental, likely artifactual nature.