The in vivo and in vitro interactions of elastic and rigid vesicles with human skin

Elastic vesicles are the most novel development in vesicular systems design for dermal and transdermal drug delivery. However, interactions between these vesicles and human skin are not yet fully understood. In this study, the in vivo and in vitro interactions between elastic-, rigid vesicles and micelles with human skin were investigated. Vesicle and micelle solutions were applied onto human skin in vitro and in vivo. Subsequently, a series of tape strippings were performed, which were visualised by freeze fracture electron microscopy (FFEM). The results showed no ultrastructural changes in skin treated with rigid vesicles. Skin treated with elastic vesicles, however, showed a fast partitioning of intact vesicles into the deeper layers of the stratum corneum (SC), where they accumulated in channel-like regions. Only little vesicle material was found in the deepest layers of the SC, suggesting that the partitioning of intact vesicles from the SC into the viable epidermis is unlikely to happen. Treatment with micelles resulted in rough, irregular fracture planes. Similar results were obtained in vitro and in vivo, indicating an excellent in vitro/in vivo correlation. These results support the hypothesis that elastic vesicles have superior characteristics to rigid vesicles for the interaction with human skin. Elastic vesicles and micelles demonstrated very different interactions with human skin and hence probably also have different mechanisms of action for the enhancement of drug transport.     

Multiphoton excitation fluorescence microscopy and spectroscopy of in vivo human skin

Multiphoton excitation microscopy at 730 nm and 960 nm was used to image in vivo human skin autofluorescence from the surface to a depth of approximately 200 microm. The emission spectra and fluorescence lifetime images were obtained at selected locations near the surface (0-50 microm) and at deeper depths (100-150 microm) for both excitation wavelengths. Cell borders and cell nuclei were the prominent structures observed. The spectroscopic data suggest that reduced pyridine nucleotides, NAD(P)H, are the primary source of the skin autofluorescence at 730 nm excitation. With 960 nm excitation, a two-photon fluorescence emission at 520 nm indicates the presence of a variable, position-dependent intensity component of flavoprotein. A second fluorescence emission component, which starts at 425 nm, is observed with 960-nm excitation. Such fluorescence emission at wavelengths less than half the excitation wavelength suggests an excitation process involving three or more photons. This conjecture is further confirmed by the observation of the super-quadratic dependence of the fluorescence intensity on the excitation power. Further work is required to spectroscopically identify these emitting species. This study demonstrates the use of multiphoton excitation microscopy for functional imaging of the metabolic states of in vivo human skin cells.     

Video-rate scanning two-photon excitation fluorescence microscopy and ratio imaging with cameleons

A video-rate (30 frames/s) scanning two-photon excitation microscope has been successfully tested. The microscope, based on a Nikon RCM 8000, incorporates a femtosecond pulsed laser with wavelength tunable from 690 to 1050 nm, prechirper optics for laser pulse-width compression, resonant galvanometer for video-rate point scanning, and a pair of nonconfocal detectors for fast emission ratioing. An increase in fluorescent emission of 1.75-fold is consistently obtained with the use of the prechirper optics. The nonconfocal detectors provide another 2.25-fold increase in detection efficiency. Ratio imaging and optical sectioning can therefore be performed more efficiently without confocal optics. Faster frame rates, at 60, 120, and 240 frames/s, can be achieved with proportionally reduced scan lines per frame. Useful two-photon images can be acquired at video rate with a laser power as low as 2.7 mW at specimen with the genetically modified green fluorescent proteins. Preliminary results obtained using this system confirm that the yellow "cameleons" exhibit similar optical properties as under one-photon excitation conditions. Dynamic two-photon images of cardiac myocytes and ratio images of yellow cameleon-2.1, -3.1, and -3.1nu are also presented.     

Principles of two-photon excitation microscopy and its applications to neuroscience

The brain is complex and dynamic. The spatial scales of interest to the neurobiologist range from individual synapses (approximately 1 microm) to neural circuits (centimeters); the timescales range from the flickering of channels (less than a millisecond) to long-term memory (years). Remarkably, fluorescence microscopy has the potential to revolutionize research on all of these spatial and temporal scales. Two-photon excitation (2PE) laser scanning microscopy allows high-resolution and high-sensitivity fluorescence microscopy in intact neural tissue, which is hostile to traditional forms of microscopy. Over the last 10 years, applications of 2PE, including microscopy and photostimulation, have contributed to our understanding of a broad array of neurobiological phenomena, including the dynamics of single channels in individual synapses and the functional organization of cortical maps. Here we review the principles of 2PE microscopy, highlight recent applications, discuss its limitations, and point to areas for future research and development.     

Depth penetration and detection of pH gradients in biofilms by two-photon excitation microscopy.

Deep microbial biofilms are a major problem in many industrial, environmental, and medical settings. Novel approaches are needed to understand the structure and metabolism of these biofilms. Two-photon excitation microscopy (TPE) and conventional confocal laser scanning microscopy (CLSM) were compared quantitatively for the ability to visualize bacteria within deep in vitro biofilms. pH gradients within these biofilms were determined by fluorescence lifetime imaging, together with TPE. A constant-depth film fermentor (CDFF) was inoculated for 8 h at 50 ml. h(-1) with a defined mixed culture of 10 species of bacteria grown in continuous culture. Biofilms of fixed depths were developed in the CDFF for 10 or 11 days. The microbial compositions of the biofilms were determined by using viable counts on selective and nonselective agar media; diverse mixed-culture biofilms developed, including aerobic, facultative, and anaerobic species. TPE was able to record images four times deeper than CLSM. Importantly, in contrast to CLSM images, TPE images recorded deep within the biofilm showed no loss of contrast. The pH within the biofilms was measured directly by means of fluorescence lifetime imaging; the fluorescence decay of carboxyfluorescein was correlated with biofilm pH and was used to construct a calibration curve. pH gradients were detectable, in both the lateral and axial directions, in steady-state biofilms. When biofilms were overlaid with 14 mM sucrose for 1 h, distinct pH gradients developed. Microcolonies with pH values of below pH 3.0 were visible, in some cases adjacent to areas with a much higher pH (>5.0). TPE allowed resolution of images at significantly greater depths (as deep as 140 microm) than were possible with CLSM. Fluorescence lifetime imaging allowed the in situ, real-time imaging of pH and the detection of sharp gradients of pH within microbial biofilms.     

Extracellular vesicles release by cardiac telocytes: electron microscopy and electron tomography

Telocytes have been reported to play an important role in long‐distance heterocellular communication in normal and diseased heart, both through direct contact (atypical junctions), as well as by releasing extracellular vesicles (EVs) which may act as paracrine mediators. Exosomes and ectosomes are the two main types of EVs, as classified by size and the mechanism of biogenesis. Using electron microscopy (EM) and electron tomography (ET) we have found that telocytes in culture release at least three types of EVs: exosomes (released from endosomes; 45 ± 8 nm), ectosomes (which bud directly from the plasma membrane; 128 ± 28 nm) and multivesicular cargos (MVC; 1 ± 0.4 μm), the latter containing tightly packaged endomembrane‐bound vesicles (145 ± 35 nm). Electron tomography revealed that endomembrane vesicles are released into the extracellular space as a cargo enclosed by plasma membranes (estimated area of up to 3 μm²). This new type of EV, also released by telocytes in tissue, likely represents an essential component in the paracrine secretion of telocytes and may consequently be directly involved in heart physiology and regeneration.     

Characterization of one- and two-photon excitation fluorescence resonance energy transfer microscopy

Advances in molecular biology provide various methods to define the structure and function of the individual proteins that form the component parts of subcellular structures. The ability to see the dynamic behavior of a specific protein inside the living cell became possible through the application of advanced fluorescence resonance energy transfer (FRET) microscope techniques. The fluorophore molecule used for FRET imaging has a characteristic absorption and emission spectrum that should be considered for characterizing the FRET signal. In this article we describe the system development for the image acquisition for one- and two-photon excitation FRET microscopy. We also describe the precision FRET (PFRET) data analysis algorithm that we developed to remove spectral bleed-through and variation in the fluorophore expression level (or concentration) for the donor and acceptor molecules. The acquired images have been processed using a PFRET algorithm to calculate the energy transfer efficiency and the distance between donor and acceptor molecules. We implemented the software correction to study the organization of the apical endosome in epithelial polarized MDCK cells and dimerization of the CAATT/enhancer binding protein alpha (C/EBPalpha). For these proteins, the results revealed that the extent of correction affects the conventionally calculated energy transfer efficiency (E) and the distance (r) between donor and acceptor molecules by 38 and 9%, respectively.     

High-order photobleaching of green fluorescent protein inside live cells in two-photon excitation microscopy

Combination of green fluorescent protein (GFP) and two-photon excitation fluorescence microscopy (TPE) has been used increasingly to study dynamic biochemical events within living cells, sometimes even in vivo. However, the high photon flux required in TPE may lead to higher-order photobleaching within the focal volume, which would introduce misinterpretation about the fine biochemical events. Here we first studied the high-order photobleaching rate of GFP inside live cells by measuring the dependence of the photobleaching rate on the excitation power. The photobleaching rate under one- and two-photon excitation increased with 1-power and 4-power of the incident intensity, respectively, implying the excitation photons might interact with excited fluorophore molecules and increase the probability of photobleaching. These results suggest that in applications where two-photon imaging of GFP is used to study dynamic molecular process, photobleaching may ruin the imaging results and attention should be paid in interpreting the imaging results.     

Tenascin/hexabrachion in human skin: biochemical identification and localization by light and electron microscopy

Tenascin/hexabrachion is a large glycoprotein of the extracellular matrix. Previous reports have demonstrated that tenascin is associated with epithelial-mesenchymal interfaces during embryogenesis and is prominent in the matrix of many tumors. However, the distribution of tenascin is more restricted in adult tissues. We have found tenascin to be present in normal human skin in a distribution distinct from other matrix proteins. Immunohistochemical studies showed staining of the papillary dermis immediately beneath the basal lamina. Examination of skin that had been split within the lamina lucida of the basement membrane suggested a localization of tenascin beneath the lamina lucida. In addition, there was finely localized staining within the walls of blood vessels and in the smooth muscle bundles of the arrectori pilorem. Very prominent staining was seen around the cuboidal cells that formed the basal layer of sweat gland ducts. The sweat glands themselves did not stain. The distribution of tenascin in the papillary dermis was studied at high resolution by immunoelectron microscopy. Staining was concentrated in small amorphous patches scattered amongst the collagen fibers beneath the basal lamina. These patches were not associated with cell structures, collagen, or elastic fibers. Tenascin could be partially extracted from the papillary dermis by urea, guanidine hydrochloride, or high pH solution. The extracted protein showed a 320-kD subunit similar to that purified from fibroblast or glioma cell cultures. We have developed a sensitive ELISA assay that can quantitate tenascin at concentrations as low as 5 ng/ml. Tests on extracts of the papillary dermis showed tenascin constituted about 0.02-0.05% of the protein extracted.     

Activation of dynamin II by POPC in giant unilamellar vesicles: a two-photon fluorescence microscopy study

The interaction of dynamin II with giant unilamellar vesicles was studied using two-photon fluorescence microscopy. Dynamin II, labeled with fluorescein, was injected into a microscope chamber containing giant unilamellar vesicles, which were composed of either pure 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) or a mixture of POPC and phosphatidylinositol 4,5-bisphosphate (PI(4,5)P₂). Binding of the fluorescent dynamin II to giant unilamellar vesicles, in the presence and absence of PI(4,5)P₂, was directly observed using two-photon fluorescence microscopy. This binding was also visualized using the fluorescent N-methylanthraniloyl guanosine 5-[-thio]triphosphate analogue. The membrane probe 6-dodecanoyl-2-dimethylamine-naphthalene was used to monitor the physical state of the lipid in the giant unilamellar vesicles in the absence and presence of dynamin. A surprising finding was the fact that dynamin II bound to vesicles in the absence of PI(4,5)P₂. Activation of the GTPase activity of dynamin II by pure POPC was then shown.     

Interactions of native and modified human low density lipoproteins with human skin fibroblasts.

125I-labeled low density lipoprotein (LDL) covalently bonded to Sepharose beads was not degraded by normal human fibroblasts nor did it trigger inhibition of sterol synthesis. The Sepharose beads loaded with LDL bound very tightly to the surface both of normal fibroblasts and fibroblasts from a subject with homozygous familial hypercholesterolemia; control Sepharose beads (activated sites covered with glycine) did not adhere to either cell type. LDL was extracted by a modification of the method of Gustafson (Gustafson, A. (1965) J. Lipid Res. 6, 512-517), so as to remove essentially all cholesterol, cholesterol ester and triglyceride. This modified LDL was bound, internalized and degraded as well as or better than native LDL. However, it failed to suppress sterol synthesis. These results provide additional evidence that the sterol moiety of the LDL is the key component affecting sterol synthesis. They also imply that the neutral lipids of LDL play a minor role in the binding of LDL to cell membranes and that the apoprotein rather than molecular size and shape is the critical factor.     

FtsZ condensates: An in vitro electron microscopy study

In vivo cell division protein FtsZ from E. coli forms rings and spirals which have only been observed by low resolution light microscopy. We show that these suprastructures are likely formed by molecular crowding which is a predominant factor in prokaryotic cells and enhances the weak lateral bonds between proto‐filaments. Although FtsZ assembles into single proto‐filaments in dilute aqueous buffer, with crowding agents above a critical concentration, it forms polymorphic supramolecular structures including rings and toroids (with multiple protofilaments) about 200 nm in diameter, similar in appearance to DNA toroids, and helices with pitches of several hundred nm as well as long, linear bundles. Helices resemble those observed in vivo, whereas the rings and toroids may represent a novel energy minimized state of FtsZ, at a later stage of Z‐ring constriction. We shed light on the molecular arrangement of FtsZ filaments within these suprastructures using high resolution electron microscopy. © 2009 Wiley Periodicals, Inc. Biopolymers 91: 340–350, 2009. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

Sedimentation field flow fractionation of fused unilamellar vesicles: comparison with electron microscopy and gel filtration

Sedimentation field flow fractionation (SF3), a relatively new instrumentation methodology for separating particles according to size, has been used to monitor changes in vesicle size during the formation of fused unilamellar vesicles of dipalmitoylphosphatidylcholine. The fusion of 500-A small unilamellar vesicles to 700-A large unilamellar vesicles (LUVs) and the slower conversion to 950-A LUVs at 4 degrees C was readily monitored by SF3 over the time course of a month. Changes in the physical state of these vesicles as a result of structural reorganizations were characterized by high sensitivity differential scanning calorimetry. The advantages and limitations of SF3 are discussed and correlated with electron microscopy and gel filtration.     

Interactions of phospholipid vesicles with rat hepatocytes in vitro influence of vesicle-incorporated glycolipids

We examined the interaction of glycolipid-containing phospholipid vesicles with rat hepatocytes in vitro. Incorporation of either $\text{N-}\text{lignoceroyldihydrolactocerebroside}$ or the monosialoganglioside, G_M1, enhanced liposomal lipid uptake 4–5-fold as judged by the uptake of radioactive phosphatidylcholine as a vesicle marker. Cerebroside enhanced phospholipid uptake only when incorporated into dimyristoyl, but not into egg phosphatidylcholine vesicles. The lack of cerebroside effect in egg phosphatidylcholine-containing vesicles appeared to be due to a limited exposure of the carbohydrate part of the glycolipid as suggested by the reduced agglutinability of those vesicles by Ricinus communis agglutinin.In contrast to the results with radioactive phosphatidylcholine, we observed only a 20% increase in vesicle-cell association as a result of glycolipid incorporation, when a trace amount of [¹⁴C]cholesteryloleate served as a marker of the liposomal lipids or when using the fluorescent dye, carboxyfluorescein, as a marker of the aqueous space of the vesicles. By the same token, intracellular delivery of vesicle-contents was only slightly enhanced (approx. 10%).The discrepancy between the association with the cells of phosphatidylcholine on the one hand and cholesteryoleate or entrapped marker on the other suggests different mechanisms of uptake for these markers. Our results are compatible with the notion that the main effect of incorporation of glycolipids into the vesicles is the enhancement of exchange or transfer of phospholipid molecules between vesicles and cells. Incubation of the cells with galactose or lactose, prior to addition of vesicles, suggests that this enhanced phospholipid exchange or transfer involves specific recognition of the terminal galactose residues of the glycolipid vesicles by a receptor present on the plasma membranes of hepatocytes.     

High-voltage electron microscopy of human diploid fibroblasts during ageing in vitro. Morphometric analysis of mitochondria

Since recent studies have suggested a diminished mitochondrial functional capacity in late-passage ('old') compared to early-passage ('young') normal fibroblasts and fibroblasts from the Hutchinson-Gilford (progeria) syndrome of premature ageing, we analysed whole-cell preparations on the high voltage electron microscope to look for mitochondrial and related defects. All strains examined showed considerable heterogeneity in cell size and intracellular morphology. Mitochondria were readily seen in all cells, predominantly as long slender rods with frequent branching, but occasional circular and saccular forms were also evident. Various parameters of mitochondrial mass including mean number, weight, and total length of mitochondria per cell weight tended to increase in old and progeria cells, but only the former attained statistical significance due to the heterogeneity and consequent variance. A significant finding was the decreased width of mitochondria in old and progeria cells. Cystic blebs were evident in mitochondria of some cells with an apparent increase in old and progeria fibroblasts. These blebs appeared to be due to weakening of the inner membrane, allowing dilatation of the outer membrane which otherwise appeared intact. The number of osmiophilic inclusions per cell weight, particularly lipofuscin granules and autophagic vacuoles, was significantly increased in old and progeria cells. In conclusion, despite some morphological changes, mitochondria of old and progeria cells maintain a structurally and bioenergetically adequate mass compatible with continued cellular viability.     

Special features of phosphatidylcholine vesicles as seen in cryo-transmission electron microscopy

Vesicles of egg yolk phosphatidylcholine (EYPC) were studied by cryo-transmission electron microscopy. The electron micrographs indicate that, despite the rapidity of cooling, membrane undulations are flattened and some vesicles change their shapes before the samples freeze. These artefacts are attributed to the action of the lateral tension that results from the membrane area contraction associated with the temperature drop. Other micrographs represent grainy membranes and angular vesicles. We regard them as the first direct evidence for the superstructure and optically invisible roughness which were recently postulated for these membranes.