Imaging intracellular protein dynamics by spinning disk confocal microscopy

The palette of fluorescent proteins (FPs) has grown exponentially over the past decade, and as a result, live imaging of cells expressing fluorescently tagged proteins is becoming more and more mainstream. Spinning disk confocal (SDC) microscopy is a high-speed optical sectioning technique and a method of choice to observe and analyze intracellular FP dynamics at high spatial and temporal resolution. In an SDC system, a rapidly rotating pinhole disk generates thousands of points of light that scan the specimen simultaneously, which allows direct capture of the confocal image with low-noise scientific grade-cooled charge-coupled device cameras, and can achieve frame rates of up to 1000 frames per second. In this chapter, we describe important components of a state-of-the-art spinning disk system optimized for live cell microscopy and provide a rationale for specific design choices. We also give guidelines of how other imaging techniques such as total internal reflection microscopy or spatially controlled photoactivation can be coupled with SDC imaging and provide a short protocol on how to generate cell lines stably expressing fluorescently tagged proteins by lentivirus-mediated transduction.

Identifying weak linear features with the "coalescing shortest path image transform"

The detection of line-like features in images finds many applications in microanalysis. Actin fibers, microtubules, neurites, pilis, DNA, and other biological structures all come up as tenuous curved lines in microscopy images. A reliable tracing method that preserves the integrity and details of these structures is particularly important for quantitative analyses. We have developed a new image transform called the "Coalescing Shortest Path Image Transform" with very encouraging properties. Our scheme efficiently combines information from an extensive collection of shortest paths in the image to delineate even very weak linear features. © Copyright Microscopy Society of America 2011.

Clustering algorithms and shape factor methods to discriminate among small GTPase phenotypes using DIC image analysis.

Naïvement perçu, le processus d’évolution est une succession d’événements de duplication et de mutations graduelles dans le génome qui mènent à des changements dans les fonctions et les interactions du protéome. La famille des hydrolases de guanosine triphosphate (GTPases) similaire à Ras constitue un bon modèle de travail afin de comprendre ce phénomène fondamental, car cette famille de protéines contient un nombre limité d’éléments qui diffèrent en fonctionnalité et en interactions. Globalement, nous désirons comprendre comment les mutations singulières au niveau des GTPases affectent la morphologie des cellules ainsi que leur degré d’impact sur les populations asynchrones. Mon travail de maîtrise vise à classifier de manière significative différents phénotypes de la levure Saccaromyces cerevisiae via l’analyse de plusieurs critères morphologiques de souches exprimant des GTPases mutées et natives. Notre approche à base de microscopie et d’analyses bioinformatique des images DIC (microscopie d’interférence différentielle de contraste) permet de distinguer les phénotypes propres aux cellules natives et aux mutants. L’emploi de cette méthode a permis une détection automatisée et une caractérisation des phénotypes mutants associés à la sur-expression de GTPases constitutivement actives. Les mutants de GTPases constitutivement actifs Cdc42 Q61L, Rho5 Q91H, Ras1 Q68L et Rsr1 G12V ont été analysés avec succès. En effet, l’implémentation de différents algorithmes de partitionnement, permet d’analyser des données qui combinent les mesures morphologiques de population native et mutantes. Nos résultats démontrent que l’algorithme Fuzzy C-Means performe un partitionnement efficace des cellules natives ou mutantes, où les différents types de cellules sont classifiés en fonction de plusieurs facteurs de formes cellulaires obtenus à partir des images DIC. Cette analyse démontre que les mutations Cdc42 Q61L, Rho5 Q91H, Ras1 Q68L et Rsr1 G12V induisent respectivement des phénotypes amorphe, allongé, rond et large qui sont représentés par des vecteurs de facteurs de forme distincts. Ces distinctions sont observées avec différentes proportions (morphologie mutante / morphologie native) dans les populations de mutants. Le développement de nouvelles méthodes automatisées d’analyse morphologique des cellules natives et mutantes s’avère extrêmement utile pour l’étude de la famille des GTPases ainsi que des résidus spécifiques qui dictent leurs fonctions et réseau d’interaction. Nous pouvons maintenant envisager de produire des mutants de GTPases qui inversent leur fonction en ciblant des résidus divergents. La substitution fonctionnelle est ensuite détectée au niveau morphologique grâce à notre nouvelle stratégie quantitative. Ce type d’analyse peut également être transposé à d’autres familles de protéines et contribuer de manière significative au domaine de la biologie évolutive.

A novel method for measuring lag times in division of individual bacterial cells using image analysis

A method is presented for determining the time to first division of individual bacterial cells growing on agar media. Bacteria were inoculated onto agar-coated slides and viewed by phase-contrast microscopy. Digital images of the growing bacteria were captured at intervals and the time to first division estimated by calculating the "box area ratio". This is the area of the smallest rectangle that can be drawn around an object, divided by the area of the object itself. The box area ratios of cells were found to increase suddenly during growth at a time that correlated with cell division as estimated by visual inspection of the digital images. This was caused by a change in the orientation of the two daughter cells that occurred when sufficient flexibility arose at their point of attachment. This method was used successfully to generate lag time distributions for populations of Escherichia coli, Listeria monocytogenes and Pseudomonas aeruginosa, but did not work with the coccoid organism Staphylococcus aureus. This method provides an objective measure of the time to first cell division, whilst automation of the data processing allows a large number of cells to be examined per experiment. (c) 2005 Elsevier B.V. All rights reserved.

Alveolar bone dynamics in osteoporotic rats treated with raloxifene or alendronate: confocal microscopy analysis

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Analysis of Band 4.1B in Integrin-Mediated Cell Adhesion and Signaling

Band 4.1B is a cytoskeletal adaptor protein that regulates various cellular behavior; however, the mechanisms by which Band 4.1B contributes to intracellular signaling are unclear. This project addresses in vivo and in vitro functions for Band 4.1B in integrin-mediated cell adhesion and signaling. Band 4.1B has been shown to bind to β8 integrin, although cooperative functions of these two proteins have not been determined. Here, functional links between β8 integrin and Band 4.1B were investigated using gene knockout strategies. Ablation of β8 integrin and Band 4.1B genes resulted in impaired cardiac morphogenesis, leading to embryonic lethality by E11.5. These embryos displayed malformation of the outflow tract that was likely linked to abnormal regulation of cardiac neural crest migration. These data indicate the importance of cooperative signaling between β8 integrin and Band 4.1B in cardiac development. The involvement of Band 4.1B in integrin-mediated cell adhesion and signaling was further demonstrated by studying its functional roles in vitro. Band 4.1B is highly expressed in the brain, but its signaling in astrocytes is not understood. Here, Band 4.1B was shown to promote cell spreading likely by interacting with β1 integrin via its band 4.1, ezrin, radixin, and moesin (FERM) domain in cell adhesions. In astrocytes, both Band 4.1B and β1 integrin were expressed in cell-ECM contact sites during early cell spreading. Exogenous expression of Band 4.1B, especially its FERM domain, enhanced cell spreading on fibronectin, an ECM ligand for β1 integrin. However, the increased cell spreading was prohibited by blocking β1 integrin. These findings suggest that Band 4.1B is crucial for early adhesion assembly and/or signaling that are mediated by β1 integrin. Collectively, this study was the first to establish Band 4.1B as a modulator of integrin-mediated adhesion and signaling.

Molecular analysis of $\beta$1,4-galactosyl-transferase localization to the cell surface and participation in cell adhesion

$\beta$1,4-Galactosyltransferase (GalTase) is unusual among the glycosyltransferases in that it is found in two subcellular compartments where it performs different functions. In the trans-Golgi complex, GalTase participates in oligosaccharide biosynthesis as do other glycosyltransferases. GalTase is also found on the cell surface, where it associates with the cytoskeleton and functions as a receptor for extracellular oligosaccharide ligands. Although we know much regarding GalTase function on the cell surface, little is known about the mechanisms underlying its transport to the plasma membrane. Cloning of the GalTase gene revealed that there are two GalTase proteins (i.e., long and short) with different size cytoplasmic tails. This raises the possibility that differences in the cytoplasmic domain of GalTase may influence its subcellular distribution. The object of this study was to examine this hypothesis directly through the use of molecular, immunological, and biochemical approaches.^ To examine whether the two GalTase proteins are targeted to different subcellular compartments, F9 embryonal carcinoma cells were transfected with either long or short GalTase cDNAs and intracellular and cell surface enzyme levels measured. Cell surface GalTase activity was enriched in cells overexpressing the long, but not the form of short GalTase. Furthermore, a dominant negative mutation in cell surface GalTase was created by transfecting cells with GalTase cDNAs encoding a truncated version of long GalTase devoid of the extracellular catalytic domain. Overexpressing the complete cytoplasmic and transmembrane domains of long GalTase led to a loss of GalTase-dependent cellular adhesion by specifically displacing surface GalTase from its cytoskeletal associations. In contrast, overexpressing the analogous truncated protein of short GalTase had no effect on cell adhesion. Finally, chloramphenicol acetyltransferase (CAT) reporter proteins were used to determine directly whether the cytoplasmic domains of long and short GalTase were responsible for differential subcellular distribution. The cytoplasmic and transmembrane domains of long GalTase led to CAT expression on the ceil surface and its association with the detergent-insoluble cytoskeleton; the analogous fusion protein containing short GalTase was restricted to the Golgi compartment. These results suggest that the cytoplasmic domain unique to long GalTase is responsible for targeting a portion of this protein to the cell surface and associating it with the cytoskeleton, enabling it to function as a cell adhesion molecule. ^

A New Technique for Firn Grain-Size Measurement Using SEM Image Analysis

Firn microstructure is accurately characterized using images obtained from scanning electron microscopy (SEM). Visibly etched grain boundaries within images are used to create a skeleton outline of the microstructure. A pixel-counting utility is applied to the outline to determine grain area. Firn grain sizes calculated using the technique described here are compared to those calculated using the techniques of Cow (1969) and Gay and Weiss (1999) on samples of the same material, and are found to be substantially smaller. The differences in grain size between the techniques are attributed to sampling deficiencies (e.g. the inclusion of pore filler in the grain area) in earlier methods. The new technique offers the advantages of greater accuracy and the ability to determine individual components of the microstructure (grain and pore), which have important applications in ice-core analyses. The new method is validated by calculating activation energies of grain boundary diffusion using predicted values based on the ratio of grain-size measurements between the new and existing techniques. The resulting activation energy falls within the range of values previously reported for firn/ice.

In vivo confocal microscopy of the bulbar conjunctiva

Background: The aim of this work is to develop a more complete qualitative and quantitative understanding of the in vivo histology of the human bulbar conjunctiva. Methods: Laser scanning confocal microscopy (LSCM) was used to observe and measure morphological characteristics of the bulbar conjunctiva of 11 healthy human volunteer subjects. Results: The superficial epithelial layer of the bulbar conjunctiva is seen as a mass of small cell nuclei. Cell borders are sometimes visible. The light grey borders of basal epithelial cells are clearly visible, but nuclei can not be seen. The conjunctival stroma is comprised of a dense meshwork of white fibres, through which traverse blood vessels containing cellular elements. Orifices at the epithelial surface may represent goblet cells that have opened and expelled their contents. Goblet cells are also observed in the deeper epithelial layers, as well as conjunctival microcysts and mature forms of Langerhans cells. The bulbar conjunctiva has a mean thickness of 32.9 1.1 mm, and a superficial and basal epithelial cell density of 2212 782 and 2368 741 cells/ mm2, respectively. Overall goblet and mature Langerhans cell densities are 111 58 and 23 25 cells/mm2, respectively. Conclusions: LSCM is a powerful technique for studying the human bulbar conjunctiva in vivo and quantifying key aspects of cell morphology. The observations presented here may serve as a useful marker against which changes in conjunctival morphology due to disease, surgery, drug therapy or contact lens wear can be assessed.

Confocal microscopy of the bulbar conjunctiva in contact lens wear

Purpose: The aim of this study was to investigate the capabilities of laser scanning confocal microscopy (LSCM) for undertaking qualitative and quantitative investigations of the response of the bulbar conjunctiva to contact lens wear. Methods: LSCM was used to observe and measure morphological characteristics of the bulbar conjunctiva of 11 asymptomatic soft contact lens wearers and 11 healthy volunteer subjects (controls). Results: The appearance of the bulbar conjunctiva is consistent with known histology of this tissue based on light and electron microscopy. The thickness of the bulbar conjunctival epithelium of lens wearers (30.9 ± 1.1 μm) was less than that of controls (32.9 ± 1.1 μm) (P < 0.0001). Superficial and basal bulbar conjunctival epithelial cell densities in contact lens wearers were 91% and 79% higher, respectively, than that in controls (P < 0.0001). No difference was observed in goblet and Langerhans cell density between lens wearers and controls. Conjunctival microcysts were observed in greater numbers, and were larger in size, in lens wearers compared with controls. Conclusions: The effects of contact lens wear on the human bulbar conjunctiva can be investigated effectively at a cellular level using LSCM. The observations in this study suggest that contact lens wear can induce changes in the bulbar conjunctiva such as epithelial thinning and accelerated formation and enlargement of microcysts, increased epithelial cell density, but has no impact on goblet or Langerhans cell density.

Corneal confocal microscopy : a novel noninvasive test to diagnose and stratify the severity of human diabetic neuropathy

OBJECTIVE: The accurate quantification of human diabetic neuropathy is important to define at-risk patients, anticipate deterioration, and assess new therapies. ---------- RESEARCH DESIGN AND METHODS: A total of 101 diabetic patients and 17 age-matched control subjects underwent neurological evaluation, neurophysiology tests, quantitative sensory testing, and evaluation of corneal sensation and corneal nerve morphology using corneal confocal microscopy (CCM). ---------- RESULTS: Corneal sensation decreased significantly (P = 0.0001) with increasing neuropathic severity and correlated with the neuropathy disability score (NDS) (r = 0.441, P < 0.0001). Corneal nerve fiber density (NFD) (P < 0.0001), nerve fiber length (NFL), (P < 0.0001), and nerve branch density (NBD) (P < 0.0001) decreased significantly with increasing neuropathic severity and correlated with NDS (NFD r = −0.475, P < 0.0001; NBD r = −0.511, P < 0.0001; and NFL r = −0.581, P < 0.0001). NBD and NFL demonstrated a significant and progressive reduction with worsening heat pain thresholds (P = 0.01). Receiver operating characteristic curve analysis for the diagnosis of neuropathy (NDS >3) defined an NFD of <27.8/mm2 with a sensitivity of 0.82 (95% CI 0.68–0.92) and specificity of 0.52 (0.40–0.64) and for detecting patients at risk of foot ulceration (NDS >6) defined a NFD cutoff of <20.8/mm2 with a sensitivity of 0.71 (0.42–0.92) and specificity of 0.64 (0.54–0.74). ---------- CONCLUSIONS: CCM is a noninvasive clinical technique that may be used to detect early nerve damage and stratify diabetic patients with increasing neuropathic severity. Established diabetic neuropathy leads to pain and foot ulceration. Detecting neuropathy early may allow intervention with treatments to slow or reverse this condition (1). Recent studies suggested that small unmyelinated C-fibers are damaged early in diabetic neuropathy (2–4) but can only be detected using invasive procedures such as sural nerve biopsy (4,5) or skin-punch biopsy (6–8). Our studies have shown that corneal confocal microscopy (CCM) can identify early small nerve fiber damage and accurately quantify the severity of diabetic neuropathy (9–11). We have also shown that CCM relates to intraepidermal nerve fiber loss (12) and a reduction in corneal sensitivity (13) and detects early nerve fiber regeneration after pancreas transplantation (14). Recently we have also shown that CCM detects nerve fiber damage in patients with Fabry disease (15) and idiopathic small fiber neuropathy (16) when results of electrophysiology tests and quantitative sensory testing (QST) are normal. In this study we assessed corneal sensitivity and corneal nerve morphology using CCM in diabetic patients stratified for the severity of diabetic neuropathy using neurological evaluation, electrophysiology tests, and QST. This enabled us to compare CCM and corneal esthesiometry with established tests of diabetic neuropathy and define their sensitivity and specificity to detect diabetic patients with early neuropathy and those at risk of foot ulceration.

Utility of corneal confocal microscopy for assessing mild diabetic neuropathy: baseline findings of the LANDMark study

Background:  For those in the field of managing diabetic complications, the accurate diagnosis and monitoring of diabetic peripheral neuropathy (DPN) continues to be a challenge. Assessment of sub-basal corneal nerve morphology has recently shown promise as a novel ophthalmic marker for the detection of DPN. Methods:  Two hundred and thirty-one individuals with diabetes with predominantly mild or no neuropathy and 61 controls underwent evaluation of diabetic neuropathy symptom score, neuropathy disability score, testing with 10 g monofilament, quantitative sensory testing (warm, cold, vibration detection) and nerve conduction studies. Corneal nerve fibre length, branch density and tortuosity were measured using corneal confocal microscopy. Differences in corneal nerve morphology between individuals with and without DPN and controls were investigated using analysis of variance and correlations were determined between corneal morphology and established tests of, and risk factors for, DPN. Results:  Corneal nerve fibre length was significantly reduced in diabetic individuals with mild DPN compared with both controls (p < 0.001) and diabetic individuals without DPN (p = 0.012). Corneal nerve branch density was significantly reduced in individuals with mild DPN compared with controls (p = 0.032). Corneal nerve fibre tortuosity did not show significant differences. Corneal nerve fibre length and corneal nerve branch density showed modest correlations to most measures of neuropathy, with the strongest correlations to nerve conduction study parameters (r = 0.15 to 0.25). Corneal nerve fibre tortuosity showed only a weak correlation to the vibration detection threshold. Corneal nerve fibre length was inversely correlated to glycated haemoglobin (r = -0.24) and duration of diabetes (r = -0.20). Conclusion:  Assessment of corneal nerve morphology is a non-invasive, rapid test capable of showing differences between individuals with and without DPN. Corneal nerve fibre length shows the strongest associations with other diagnostic tests of neuropathy and with established risk factors for neuropathy.