text:
Correlative Video-Light-Electron Microscopy
A Detailed Protocol with Useful Tricks
- Figure 1. Identification of the cell of interest. (A). Growing of cells on the cover slip with gridded cover slip. (B) Growing of cells on the cover slip without coordinated grid. It is possible to use the peculiar pattern of cell position for the identification of our cell. For instance in the upper-right-upper right position there are two elongated cells (grey elongated profiles) and the cell of interest (dark profile) could be identified between them. (C, D) Growing of cells on the cover slip without coordinated grid. To label the cell of interest after fixation one should take a wooden stick and make a ring (between two ovals) without cells by their scrapping around our cell (grey asterisk). After scrapping the cell of interest (arrow in D) is easily identified
- Figure 2. Orientation of samples for sectioning. (A) The sample map after embedding. The cell of interest (grey arrow) is located at the position B3. Two black arrows indicate the marks that should be done by the steel needle. (B) Orientation of the sample surface along the vertical direction. Arc shows the trajectory of the knife related to the sample. Arrows indicate the limits of movement of the holder. If the orientation of the sample is vertical, the ends of the blocks should be equally cut by the knife (triangle on the left) whereas the central zone of the sample where the cell is situated will not be trimmed. If the orientation of block surface is not vertical (showed by grey dashed parallelogram), the upper end of the sample will be trimmed first and more intensively. (C) The surface of the sample should be parallel to the glass knife that is used for trimming. In order to measure the verticality one should measure the thickness of the shadow (grey area below the cell of interest: long arrow). This shadow should be wider when the cell of interest and marks of the needle are situated al the level of the edge of the knife and equally narrower when the marks and the cell are upper or lower than the edge of the knife. Hatching and arrows indicate the ends of the samples already cut by the glass knife. The position of the cell is shown by long black arrow. The distances between the position of the cell and the beginnings of the sectioned zones (horizontal lines bordering the hatching zones) should be equal.
- Figure 3. Picking up of serial sections with the donor slot grid from the water. (A) Left. Serial sections in the glass bath. Centre. Approaching of the donor grid. Right. Orientation of the slot grid related to the position of serial sections within the surface of the bath. (B) Correct positioning of the grid and serial sections. (C) Touching of the water with the empty slot grid leads to the capturing of sections together with the droplet of water by the transfer grid. The acceptor slot-grid with carbon coated formvar film should be placed on the scotch holders (the parallel holders covered with scotch). (D) In order to avoid the hit of dirt on the sections it is useful to place a very small droplet of distilled water on the acceptor grid that covers the formvar film. (E, F). After placement of the transfer grid with sections and droplet of water on the acceptor grid covered by a small droplet of water as well the dirt appeared on the lateral surface of the droplet and after elimination of water with a filter paper sections appeared being attached to the formvar (G). (H) The transfer grid should be eliminated. (I) The acceptor grid should detached form the scotch holder.
- Figure 4. In vivo dynamics and ultrastructure of individual ER-to-Golgi carriers studied using correlative video-light-electron microscopy. NRK cells transfected with the temperature sensitive G protein of vesicular stomatitis virus that is tagged with green FP were placed at 40 °C for 16 h to block folding of this protein and, thus, to prevent its exit from the ER. Then, cells were shifted to the permissive temperature 32 °C and examined under the confocal microscope in 10 min after the shift. Video recording was performed during 3 min and then, cells were fixed and prepared for immuno EM as described in [3]. The G protein was labeled with antibody against folded protein and then with monovalent Fab fragments of antibody against the primary antibody that were conjugated with nano-gold. Next cells were underwent gold enhancement and embedded into Epon. Panels A-D show the consecutive frames of the video-film examining the behavior of the ER-to-Golgi carrier (white arrow) that moved below the nucleus. Thick white arrow in D shows the second immobile carrier that is visible also in panel K.
Microscopical studies in biology have relied on two complementary microscope technologies - light (fluorescence) microscopy and electron microscopy. Light microscopy is used to study phenomena at a global scale and to look for unique or rare events, and it also provides an opportunity for live imaging, while the forte of electron microscopy is the high resolution. Observation of living cells under EM is still impossible. Traditionally light and electron microscopy (EM) observations are carried out in different populations of cells/tissues. The advent of true correlative light-electron microscopy has allowed high resolution imaging by EM of the same structure observed by light microscopy. Thus a rare event captured by low resolution imaging of a population or transient events captured by live imaging can now also be studied at high resolution by electron microscopy.
This chapter describes correlative light-electron microscopy with details and useful tricks, including the way to localize the same cell after its transfection with a protein fused with a fluorescent tag, examination under the microscope in living condition, fixation, immunolabelling, embedding, and observation under EM. We also illustrate here the kinds of questions that the correlative video-light-EM (CVLEM) approach was designed to address, as well as the particular know-how that is important for the successful application of this technique. The potential and difficulties of this approach, along with the most impressive breakthroughs obtained by these methods, are discussed.
1. Introduction
Although advanced optical microscopy techniques can push resolution to 50-100 nm and even below, it is still much less than the resolution of EM and far from the resolution needed for the study of, for instance, the organization of assemblies of proteins and lipids in biological specimens. Often, the analysis of immuno-fluorescently-labeled structures needs a better-than-light-microscopy resolution. On the other hand, although the spatial resolution of EM is superior, its field of view is limited: a resolution of 1 nm can only be realized when small (2x2 µm2) areas are imaged.
Consequently, the study of rarely occurring events in cells or tissues is extremely tedious and time consuming. This limitation has motivated researchers to embark on the development of imaging methods that combine, for example, LM and EM - correlative microscopy (CM). CM uses a combination of microscopy methods for the study of rare cellular events of unique samples.
Now CVLEM is a rather complex procedure with the possibility to use several techniques for the identification of the organelle of interest subsequently under LM and then under EM. The CLEM procedure includes several stages: 1) observation of the structures labeled with fluorescent protein (FP, i.e., green FP) or other fluorescent markers in living cells; 2) immobilization (fixation or freezing); 3) immuno- or other type of labeling with gold or other markers suitable for EM; 4) embedding; 5) identification of the just examined cell in the resin block or within the frozen sample; 6) sectioning of thin or thick serial sections and identification of the cell on the resin block and cutting of thin or thick serial sections; 7) EM analysis and structure identification [1]. Each of these steps could be performed by different ways and all techniques have their own advantages and disadvantages (reviewed in [2]). Here, we are presenting an example of only one from many existing methods of CVLEM. We describe only those protocols that are indispensable for the presented type of CVLEM. Protocols of transfection, observation under a confocal microscope, EM tomography, the reader can find in corresponding protocol books.
2. Procedure
2.1. Observation of living cells and fixation.
2.1.1. Grow i.e. HeLa cells in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum and 2 mM L-glutamine at 37°C and 5% CO2.
2.1.2. Suspend HeLa cells using standard procedures and plate cells for CVLEM on a MatTek Petri dish with the CELLocate cover slip attached to its bottom. The CELLocate cover slip contains an etched grid with coordinates that allow the localization of the cell of interest at any step in the preparation.
2.1.3. Transfect HeLa cells with cDNA of the GFP fusion protein using any standard method of transfection or microinjection of cDNA into the nucleus.
2.1.4. By 6 h to 48 h (depending of the fusion protein and method of transfection used) after transfection place the dish under an inverted fluorescence microscope or laser scanning confocal microscope.
2.1.5. Select the transfected cell of interest, and identify its position related to the coordinates of the CELLocate grid.
2.1.6. Draw (or photograph) the position of the cell on the map of the CELLocate grid. For instance, the cell could be near the cross of horizontal line A and vertical line 3 (see arrow in Fig. 1A, 2A, see Note 2). Another way to map the cell of interest is to scrap cells around the cell of interest using wood stick and then cells remaining in the centre of ring could be visible (Fig. 1C, D), Finally, the pattern of cell culture could be used for the labeling of the cell position (Fig. 1B)
2.1.7. Observe the dynamics of the GFP-labeled structures in the selected living cell using a multiphoton-, a laser scanning confocal-, or a digitalized fluorescent-inverted microscope, which allows the grabbing of a time-lapse series of images by a computer.
2.1.8. At the moment of interest, add fixative A to the cell culture medium while still grabbing images (fixative A: medium volume ratio is 1:1). Fixation usually induces the fast fading of GFP fluorescence and blocks the motion of labeled structures in the cell.
2.1.9. Stop grabbing time-lapse images and keep the cells in fixative for 5-10 min (during this time it is useful to grab a Z-series of images of the cell).
2.1.10. Replace the mixture with the fixative 1 and keep the cells in the fixative 1 for 5 min.
2.1.11. Replace fixative 1 with fixative 2 and keep cells there for 30 min.
2.1.12. Wash with 0.2 M HEPES (pH 7.2-7.3) for 10 min (see Note 3).
2.2. Immunolabelling for EM with Nanogold
2.2.1. Wash the fixed cells for 3 x 5 min with 0.2 M HEPES (pH 7.2-7.3).
2.2.2. Incubate the cells with the blocking solution for 30 min, and then with the primary antibodies diluted in blocking solution, overnight.
2.2.3. Wash the cells for 6 x 2 min with 0.2 M HEPES (PH 7.2-7.3).
2.2.4. Dilute the Nanogold-conjugated Fab fragments of the secondary antibodies x50 times in the blocking solution and add this to the cells; incubate for 2 h.
2.2.5. Wash the cells again for 6 x 2 min with 0.2 M HEPES (PH 7.2-7.3).
2.2.6. Fix the cells with 1% glutaraldehyde in 0.2 M HEPES buffer (pH 7.2) for 5 min.
2.2.7. Wash the cells for 3 x 5 min with 0.2 M HEPES (PH 7.2-7.3), and then for 3 x 5 min in distilled water.
2.2.8. Incubate the cells with the gold-enhancement mixture for 6-10 min according to instruction of the manufacturer. The cells will become violet-grey in color if the gold enhancement is successful.
2.2.9. Wash cells for 3 x 5 min with distilled water.
2.3. Enhancement of Sample Contrast, Sample Locating, and Embedding (see Note 4)
2.3.1. Wash cells 3 times with 0.2M Cacodylate Buffer (pH 6.9).
2.3.2. Treat cells with the reduced osmium for 1 h on ice in the darkness.
2.3.3. Wash cells with 0.2 M Cacodylate Buffer (pH 6.9) 3 x 5 min.
2.3.4. Treat samples with 0.3% Thiocarbohydrazide in 0.2 M Cacodylate Buffer (pH 6.9) for 10 min.
2.3.5. Wash cells 3 times with 0.2 M Cacodylate Buffer (pH 6,9).
2.3.6. Treat cells with 1% OsO4 in 0.2 M Cacodylate buffer (pH 6,9) for 30 min.
(To further enhance contrast, one can add three additional steps between steps number 6 and 2.3.7. Rinse samples with 0.1 M sodium cacodylate (pH 6.9) buffer until all traces of the yellow osmium fixative have been removed.
2.3.8. Stain sample with a 1% solution of tannic acid in 0.05 M sodium cacodylate buffer for 45 min.
2.3.9. Replace the tannic acid with a 1% solution of anhydrous sodium sulfate in 0.05 M sodium cacodylate buffer and leave for 5 min.)
2.3.10. Rinse sample in de-ionized water and then dehydrate stepwise with 70%, 90%, and 100% ethanol (3 x 5 min for each) for the dehydration of the specimens.
2.3.11. Prepare EPON solution as described in materials.
2.3.10. Prepare a 1:1 EPON/100% ethanol mixture (acetone and propylene oxide could dissolve plastic Petri dishes). Vortex until thoroughly mixed and stores all solutions in the fume hood.
2.3.11. Treat sample with a 1:1 EPON/100% ethanol mixture for 1 hr.
2.3.12. Replace the 1:1 EPON/100% ethanol mixture with EPON alone for 4 h with one exchange of EPON after 2 h.
2.3.13. Keep the cells in EPON for 1-2 h at room temperature, and then leave them in an oven at 60 °C for 12 h.
2.3.14. After 12 h of polymerization of the EPON, place a small droplet of a fresh resin on the site where the examined cell is located, and insert a resin cylinder (prepared before by polymerization of the resin in a cylindrical mold) with a flat lower surface; leave the samples for an additional 18 h in the oven at 60 °C. The resin cylinder should be placed on the semi polymerized EPON just over the cell of interest (Note 5).
2.3.15. Polymerize for 24 h hours at 60 °C.
2.3.16. Carefully pick up the resin from the Petri dish and glass; this is easy to do by gentle bending of the resin cylinder to and fro. The resin block and the empty MatTek Petri dish after block detachment could be separated easily. If the cover glass with a coordinated grid cannot be detached from the cells included into the resin, the latter should be placed into commercially available hydrofluoric acid (do not use glassware for this) for 30-60 min.
2.3.17. Removed the glass bottom of tissue culture dishes by hydrofluoric acid for 30-60 min.
2.3.18. Control the completeness of the glass dissolution under a stereomicroscope.
2.3.19. Wash the samples in water after the complete removal of the glass.
2.3.20. Leave the samples in 0.2 M HEPES buffer (pH 7.3) for 60 min to neutralize the hydrofluoric acid.
2.3.21. Wash the samples in water and allow them to dry. The round basement of the sample with diameter of about 1 cm or more should not be cut.
2.4. Identification of the cell of interest on Epon blocks (Fig. 1).
2.4.1. Place the embedded sample under a stereomicroscope using a fiber optic illuminator.
2.4.2. If the gridded cover slip was used the furrows on the cover slips are filled with Epon and after polymerization and dissolution of the glass or its detachment the furrows appeared as combs because Epon forms a replica from the gridded glass. The relief of grid is visible under a stereomicroscope. Using numbers and letters on the grid it is easy to find the position on the surface of the sample where the cells are situated.
2.4.3. Use the composite of bright field DIC images or manual drawings of the region of interest to locate the region of interest on the Epon block.
2.5. Sample Orientation and EM Sectioning from the Very First Section (see Note 6)
2.5.1. Place the sample under the transmission light microscope.
2.5.2. Find the cell of interest among the cells within the sample according to the coordinated grid (Fig. 1A) or pattern of the cell layer (Fig. 1B).
2.5.3. Put the resin block into the holder of an ultratome and examine it under a stereomicroscope.
2.5.4. Place the holder with the sample under the stereomicroscope and using a steel needle and rotating the sample in the holder, make two small cavities in such a way that they form a horizontal line (broken line in Fig. 2A and C) with our cell appearing in the center of the sample.
2.5.5. Introduce the holder into the ultratome in such a way that the segment arc of the ultratome is in the vertical position and the two cavities form a horizontal line.
2.5.6. By rotating the glass knife stage align the bottom edge of the pyramid parallel to the knife-edge. Using the segment arc, orient the plane of the sample vertically.
2.5.7. Bring the sample as close as possible towards the glass knife.
2.5.8. Adjust the gap (which is visible as a bright band if all three of the lamps of an ultratome are switched on) between the knife-edge and the surface of the sample. The gap has to be identical in width between the most upper and lower edges of the sample during the up and down movement of the resin block. This ascertains that every point of the sample surface containing the cell of interest is at the same distance from the knife-edge.
2.5.9. Slowly moving the sample up and down, continue its approach until the knife begins to cut one of the edges of the sample. The sectioning begins from either the upper or the lower part of the sample, the middle part of the sample where the cell of interest is situated will be unaffected because the length of the radius passing through the cell is shorter than the radii passing through the upper and the lower edges of the sample.
2.5.10. If the sectioning is to begin from the upper part of the sample, tilt the segment arc to approach the lower edge towards the knife. If the sectioning is to begin from the lower edge of the sample, tilt the segment arc and approach the upper edge of the sample towards the knife. A vertically oriented sample should produce equal sections from both the upper and lower edges of the samples (Fig. 2C).
2.5.11. Note down precisely all of parameters relating to the position of the sample in the ultratome, i.e. the degree of rotation of the sample in the holder, the degree of tilting of the segment arc, and the degree of rotation of the knife in its stage. Do not take the sample from the holder and do not rotate the sample inside the holder.
2.5.12. Take the sample and trim it to provide a narrow horizontal pyramid. The pyramid should be as narrow as possible, and the cell of interest should be at the centre of the pyramid. The length of the pyramid should be smaller than 0.9 mm and its height smaller than 0.1 mm. In this case you could place on the slot 18 serial sections. An experienced person can trim a pyramid directly with a razor blade.
2.5.13. Introduce the sample back into the ultratome, and lock it in exactly the same position as before (preserving all of the parameters of sample positioning; this is very important).
2.5.14. Replace the glass knife with the diamond one, and position the latter towards the pyramid. If the sample is not parallel to the knife, adjust the angle of the diamond knife by rotating the knife stage to make its edge parallel to the plane of the pyramid. Do not change any other parameters of the sample position.
2.5.15. Approach the sample towards the edge of the knife until the gap is extremely narrow. Using a 200-nm approaching step, begin the sectioning. Take serial sections according to the instructions with the ultratome. It is enough to take only 10 sections to pass 2 µm from the bottom of the cell.
2.5.16. Identify the position of the organelle of interest within the EM sample according to the Z-stacking, and select those thick sections that should correspond to this position. For instance, if the organelle of interest is situated at 500 nm from the surface of the sample (bottom of the cell), it is enough to collect only the first three 200-nm serial sections (or ten 50-nm sections). If the position is at 1 µm in height, it will be necessary to collect from the fourth to the eighth serial 200-nm sections (or twenty 50-nm sections).
2.6. Picking up the Serial Sections with the Empty Slot Grid (Fig. 3, see Note 7)
2.6.1. Clean the empty (not covered) slot grid (the donor transfer grid) and one slot grid covered with formvar-carbon film (the acceptor grid.
2.6.2. When the normal (not bent) slot grid is taken by the tweezer it is not convenient to take sections from the water, because the axes of grid cannot be oriented paralleling to the plane of water surface. One needs a holder on the grid. The holder on the slot grid could be done by bending just the small part of the grid from horizontal position. Using a tweezer bend just the small part of the edge of the empty slot grid from horizontal position. This bent rim will serve as a holder for the tweezer.
2.6.3. Take the slot grid with supporting film from a container by the self-closing tweezer and place it film-down on parallel holders covered with scotch tape (Fig. 3C, below).
2.6.4. Take the empty slot grid using the just made holder and place it over the band of serial sections not touching the water and in such a way that the slot is projected on the band of serial section.
2.6.5. Touch water with the donor grid in such a way that sections should be inside the slot and move the slot grid away. The droplet of water and sections will be inside the slot (Fig. 3A-C).
2.6.6. To avoid dirt on the sections it is better to place a small droplet of glass handled distilled water on the acceptor slot grid. Using stereomicroscope put a small droplet of water on the supporting film of the acceptor grid (Fig. 3D).
2.6.7. Put the donor grid with serial sections on acceptor grid as it is shown in Fig. 3E, F.
2.6.8. Using sharp and very narrow filter paper orient slot in parallel to each other and eliminate the excess of water from the space between slot grids (Fig. 3G).
2.6.9. Dry grids during at least 20 min.
2.6.10. Carefully eliminate the donor grid (Fig. 3H).
2.6.11. Take the dried acceptor grid and check whether sections are in correct position (Fig. 3I).
2.7. EM Analysis
2.7.1. Using stereomicroscope (to avoid mistakes) place the slot grid into the holder of EM and introduce it into EM.
2.7.2. Find the very first serial EM sections using the traces (usually letters) of the coordinated grid filled with the resin. These are visible mostly on the first two sections. If the central position of the cell of interest within the pyramid is used as a marker, identify the central cell within the sections.
2.7.3. Take consecutive photographs (or grab the images with a computer using a video camera) of the serial sections beginning from the very first section since the organelle of interest (just observed under the LSCM) appears and until is no longer seen. Having images from FL is useful to identify the cell of interest.
2.7.4. If EM and FM images are reflective it is necessary to change the reflection position of FM images (see Note 8)
3. Materials
3.1. Observation of Living Cells and Fixation
3.1.1. Cells of interest, i.e. HeLa cells (No. CCL 185; American Type Culture Collection, Rockville, MD), cDNA and transfection reagents;
3.1.2. Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal calf serum (FCS) and 2 mM L-glutamine (GIBCO BRL, Life technologies)
3.1.3. MatTek Petri dish with CELLocate (MatTek, Ashland, MA).
3.1.4. A digitalized fluorescent-inverted microscope or laser scanning confocal microscope (i.e. from Leica Microsystems Spa, Milan, Italy, www.leica-microsystems.com).
3.1.5. HEPES buffer (0.2 M). Dissolve 4.77 g HEPES in 100 ml distilled water and add of 1 N HCl droplet by droplet to provide a pH of about 7.2-7.3
3.1.6. Fixative 1. 0.05% glutaraldehyde plus 4% formaldehyde in 0.15 M HEPES (pH 7.2-7.3). Dissolve 8 g paraformaldehyde powder in 50 ml 0.2 M HEPES buffer, stirring and heating the solution to 60 °C. Add drops of 1 N NaOH to clarify the solution. Add 1.25 ml 8% glutaraldehyde and 50 ml 0.2 M HEPES buffer. Dilute twice before use.
3.1.7. Fixative 2. 4% formaldehyde in 0.15 M HEPES (pH 7.2-7.3). Dissolve 4 g paraformaldehyde powder in 100 ml HEPES buffer, stirring and heating the solution to 60 °C. Add drops of 1 N NaOH to clarify the solution.
3.2. Immunolabelling for EM with Nanogold
3.2.1. See item 3.1.4.
3.2.2. Blocking solution. Dissolve 0.50 g BSA, 0.10 g saponin, 0.27 g NH4Cl in 100 ml of 0.2 M HEPES (PH 7.2-7.3).
3.2.3. Nanogold-conjugated monovalent Fab fragments (Nanoprobes, Incorporated: 95 Horseblock Road, Unit 1, Yaphank, NY, USA).
3.2.3. Gold-enhance mixture. Use a gold-enhance kit (Nanoprobes, Incorporated: 95 Horseblock Road, Unit 1, Yaphank, NY, USA). Using equal amounts of the four components (Solutions A, B, C and D), prepare about 200 µl of reagent per Petri dish (a convenient method is to use an equal number of drops from each bottle). First mix Solution A (enhancer; green cap) and Solution B (activator; yellow cap). Wait for 5 min, and then add Solution C (initiator; purple cap), and finally Solution D (buffer; white cap). Mix well.
3.2.4. 1% glutaraldehyde in 0.2 M HEPES buffer (pH 7.2-7.3). Mix 1 ml of 50% glutaraldehyde (EM grade, Electron Microscopy Sciences, Hatfield PA) with 49 ml of 0.2 M HEPES (pH 7.2-7.3). Store at 0-4 oC.
3.3. Enhancement of Sample Contrast, Sample Locating, and Embedding
3.3.1. 0.2 M Cacodylate buffer. Dissolve 2.12 g sodium cacodylate (Electron Microscopy Sciences, Hatfield PA) in 100 ml of distilled water. Add 1 N HCl to provide a pH of about 6.9.
3.3.2. Reduced OsO4. Mix 2% OsO4 in water 3% Potassium Ferrocyanide in 0.2 M cacodylate buffer (pH 7.2) 1:1.
3.3.4. 0.3% Thiocarbohydrazide. Dissolve 0.1 g of Thiocarbohydrazide in 33.3 ml 0.2 M Cacodylate Buffer (pH 6.9). Use immediately after preparation.
3.3.5. 1% OsO4. Mix 2 ml of 4% OsO4 ((Electron Microscopy Sciences, Hatfield PA) with 6 ml of 0.2 M Cacodylate buffer (pH 6,9). Use immediately after preparation.
3.3.6. 1% Tannic Acid. Dissolve 0.1 g Tannic Acid (TAAB Laboratories Equipment Ltd) in 0.05 M Sodium Cacodylate buffer (pH 6.9) prepared by dilution of one volume of 0.2 M Cacodylate buffer (pH 6.9) in three volumes of distilled or de-ionized water.
3.3.7. Ethanol. To prepare N% Ethanol mix N ml of 100% Ethanol (Electron Microscopy Sciences, Hatfield PA) with 100-N ml of de-ionized water.
3.3.8. EPON mixture (TAAB 812, DDSA, MNA, DMP30 in a ratio of 24:9.5:16.5:1, respectively). Put 20.0 g EPON, 13.0 g Dodecenyl succinic anhydride (DDSA) and 11.5 g Methyl nadic anhydride (MNA) into the same test tube. Heat the tube in the oven for 2-3 min at 60 °C and then vortex it well. Add 0.9 g tri- Dimethylaminomethyl phenol (DMP-30, all from Electron Microscopy Sciences, Fort Washington, PA) and immediately vortex the tube again. It is possible to freeze the EPON in aliquots and to store it for a long time at 20 °C before use.
3.3.9. 1:1 EPON/100% ethanol mixture. Mix 10 ml of 100% ethanol with 10 ml of EPON mixture.
3.4. Identification of the cell of interest on Epon blocks
3.4.1. A stereomicroscope. I.e. (Leica Microsystems Spa, Milan, Italy, www.leica-microsystems.com) equipped with a fiber optic illuminator.
3.5. Sample Orientation and EM Sectioning from the Very First Section
3.5.1. Ultramicrotome Leica EM FC6 (Leica Microsystems Spa, Milan, Italy, www.leica-microsystems.com).
3.6. Picking up the Serial Sections with the Empty Slot Grid
3.6.1. Anti-capillary self-closing tweezers (Ted Pella Inc., Redding, CA)
3.6.2. Diamond knife (Electron Microscopy Sciences, Hatfield PA).
3.6.3. Eyelash with handle (Ted Pella Inc., Redding, CA).
3.6.4. Chloroform
3.6.5. 1x2 mm slot-grids (Electron Microscopy Sciences, Hatfield PA).
3.6.6. 1x2 mm slot-grids covered with formvar and carbon (Electron Microscopy Sciences, Hatfield PA).
3.6.7. Parallel holders covered with scotch tape is not commercially available (Note 1).
3.6.8. Whatman №1 filter paper (Fisher Scientific, Pittsburgh, PA).
3.7. EM analysis
3.7.1. A stereomicroscope (see item 2.4.1.).
3.7.1. An electron microscope with a goniometer (i.e. FEI, Eindhoven, The Netherlands).
4. Notes
4.1. Take a list of thick paper and covered it from both side with a double covered scotch tape. Then cut with scissor two narrow (3 mm in width) peaces and attach them to the glass with the distance of 2 mm between them.
4.2. Then the figure will be visible after polymerization of Epon as the replica of the roof on the cover slip.
4.3. For subsequent immuno labelling, cells should be treated in the culture dish without removing the cover glass.
4.4. If you plan to use EM tomography is could be helpful to avoid the use of lead citrate staining (see corresponding Chapter in this book) that often gives not equal staining of the thick sections. For this purpose one could use Thiocarbohydrazide staining after fixation with aldehyde.
4.5. The central position of the cylinder is important to ensure the possibility of use the transmission light for the examination of EPON samples. If the wall of the cylinder is situated near the cell of interest, the light will be scattered.
4.6. This method is applicable for CLEM even if cells were looked at as CVLEM. If you use EM tomography, the thickness of EM sections should be 200-250 nm. If you use routine serial sections the thickness could be from 40 to 80 nm. Thinner sections demand more grids necessary for the picking up of serial section. Thus, one should find a compromise between the difficulty, cost, and demands.
4.7. Standard method to pick up the serial sections for routine EM is not sufficient for CVLEM due to significant rate of possible mistakes during manipulations with sections. CVLEM the demands for the reproducibility of this step are much higher. The method should give almost 100% of success. Here, we described a specific method that after some training gives such a result.
4.8. To achieve success the researcher should make all procedures with the reproducibility of about 99%. Therefore we recommend performing all manipulations with grids under stereomicroscope. During manipulations with sections and samples it is better to use self-closing tweezers and slot grids that are previously tested under the stereomicroscope.
We thank Dr. C.P. Berrie for critical reading of the manuscript and Telethon Italia for financial support.
[1] Polishchuk, R.S., Polishchuk, E. V., Marra P., Alberti, S., Buccione, R., Luini, A., and Mironov, A.A. (2000) Correlative light-electron microscopy reveals the tubular-saccular ultrastructure of carriers operating between Golgi apparatus and plasma membrane J. Cell Biol 148, 45-58.
[2] Mironov, A.A., and Beznoussenko, G.V. (2009) Correlative microscopy: a potent tool for the study of rare or unique cellular and tissue events J. Microscopy. In press.
[3] Mironov A.A., Mironov A.A. Jr., Beznoussenko, G.V., Trucco, A., Lupetti, P., Smith, J.D., Geerts, W.J., Koster, A.J., Burger, K.N., Martone, M.E., Deerinck, T.J., Ellisman, M.H., Luini, A. (2003) ER-to-Golgi carriers arise through direct en bloc protrusion and multistage maturation of specialized ER exit domains. Dev. Cell. 5, 583-594.


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So you need something different for that man in your life. Forego that boring necktie or gift card and give him something special this year.
We found some unique items, One-of-a-Kind Finds, just for him. But even if these featured gifts aren't quite right, we still recommend checking these stores out as we didn't have enough room in our Gift Guide to profile everything we saw.
Repurposed Light Fixtures
The title alone may sound boring, but we truly were floored by the incredible creativity and ingenuity of these light fixtures created by local entrepreneur, Rich Armstrong, that we found in the back of Old Bob's, 10896 E. U.S. 36 in Avon.
Rich must see light in just about every old item he finds. His collection truly came from the unlikeliest sources, an old bass drum pedal, a vintage postal scale, hollowed out books, old hubcaps and the list goes on. He even does custom work if you have an old item you would like him to turn into a light. Though during the holiday season, his already completed light fixtures at Old Bob's are 15 percent off.
He also had rustic vanity lights for sale made from various woods and other items. I was told much of the wood was salvaged from old barns. Prices begin around $45 and go up from there with the vanities costing around $150. We also found some repurposed light fixtures at Outta the Shed, an antique store on the Hendricks County Courthouse Square in Danville.
Metal Wall & Table Art
Whenever we go out scouting each year for our holiday Gift Guide, I come away amazed by the creativity we find in Hendricks and Morgan counties like I described in the light fixtures above.
We found another example of that creativity in the form of metal wall and table art at two locations, one in Hendricks County and the other in Morgan County.
Both locations feature different artists but the end products are similar and would be a welcome addition to any man's desk or wall. We found art featuring the state of Indiana and even using old car engine parts.
Prices ranged based on the location and the art itself, but we would encourage you to check out the pieces at Gizmos Galleria, 1012 E. Main St. in Brownsburg and The Sterling Butterfly, 190 E. Morgan St., Martinsville.
Painted Cigar Storage Box
At one of our Rural Routes to Main Street Cultural Trail locations, the Art Sanctuary, 190 N. Sycamore St. in Martinsville, we found these unique painted cigar boxes made by artist Nancy Maxwell.
They provide a stylish storage solution as these attractive boxes are perfect for storing personal effects atop a dresser or table. Nancy often specializes in landscapes and rustic scenes, but the boxes can be custom made for the special man in your life if he has specific interests you'd like her to focus on.
Already-painted boxes are available at Nancy's studio inside the Art Sanctuary and also at The Sterling Butterfly and cost $40-$50 depending on the box.
Magnetic Bottle Opener
Speaking of rustic, we found these great magnetic bottle openers in an unlikely place, Artistic Designs Gallery, 1038 E. Main St. in Brownsburg. It just goes to show you that you really need to walk through the doors of all our locally owned shops as you never know what you might find.
These handmade creations sell for $65 and would be the perfect gift for a man cave or garage for that man in your life who still likes his soda pop in a bottle or of course the occasional beer.
Honey Farm Experience
I personally experienced this tour at Hunter's Honey Farm in Martinsville and loved it. One of the stops on the Rural Routes to Main Street Cultural Trail, this family-owned business offers a tour that is both interesting and educational.
You can tour their warehouse where they store and process the honey. You can experience a staff member inside a see-through, netted tent interacting with the hive and the bees, showing you the different stages of the process and if you're lucky you may get to see the queen.
Fill your own personalized bottle of honey and peruse their small shop filled with every kind of honey-related item you can imagine from honey barbecue sauce to honey lip balm and so much more.
Tours range from under $5 to $22 per person depending on the content of the tour, take home items and number of people involved.
Personalized Money Clip
Seize the Night Designs on the square in Danville had a lot of options for the man in your life, but we honed in on their money clips.
The stylish money clips can be personalized with anything you can imagine on them from sayings to names and the list goes on. Priced at $16, they are affordable, too.
Framed Racing Photography
During our visit to Old Bob's in Avon, we also came across these framed racing photos that at first glance appeared to be paintings.
However, on closer inspection, they actually were photographs taken simultaneously at different exposures at the Indianapolis Motor Speedway of primarily vintage race cars giving the appearance of a painting and actually showing some action.
These truly amazing pieces also show another aspect of that store's framing capabilities. The already framed pieces sell for about $135 though they do have unframed photographs as well.
Volkswagen Mailbox
Any VW lover in your life would want to own these truly original mailboxes that look like vintage Volkswagen buses. We found these fun mailboxes in a booth at Gizmos Galleria in Brownsburg.
They come in different colors and cost $50 each.

subset: commoncrawl


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We had a great year here at Infinio. I asked our team members what accomplishments they were most proud of, and here's what everyone said:
Topics: News
One of the benefits of our developing Infinio Accelerator on VMware's VAIO platform is that we can offer support for new versions of vSphere nearly immediately. With the recent introduction of vSphere 6.5, we are proud to announce that we are now listed in VMware's compatibility guide.
This release supports all types of datastores, including VMFS, vSAN, and vVols, on this latest version of vSphere. In fact, we remain the only caching vendor to support all these datastore types on any version of vSphere.
Topics: Talking Tech, News
Today we announced a North American agreement with worldwide technology distribution leader, Tech Data. This is a landmark moment in our company history as we move forward with a partnership to bring our server-side cache technology to partners and customers.
Topics: News, Announcement
Topics: News
Every year, CRN's annual Top 100 list "salutes the key players who continue to shape the way channel does business." The publication calls its elite 100 "influencers," "leaders," "innovators" and "disruptors." These are the people "who are changing the way business gets done in the IT channel" and Infinio CEO and Co-Founder Arun Agarwal appears on this year's list amid its top 25 innovators.
Topics: News
We are very excited to announce that after a successful beta program, Infinio Accelerator v2.0 is now in general availability. This milestone product announcement comes at a time of a strong market momentum for Infinio, including continued customer success and market adoption, and an accelerated channel recruitment campaign following the recently-launched partner program.
Topics: News
Gartner is well known for its Magic Quadrants, highly influential and eagerly anticipated research reports that provide a qualitative analysis into a market and its direction, maturity and participants. In recent years, Gartner has introduced a new series of "Cool Vendors" reports, which feature smaller organizations with future potential. These reports profile providers of innovative, original technologies that are making a difference in their respective markets. And this year's Gartner Cool Vendors in Storage Technologies (1) report included Infinio as one of five vendors.
Topics: News
Running a virtualized data center or a VDI environment can be challenging. But Minara Resources, an Australian mining company, runs both in the harsh conditions of a 24/7 mining operation.
Minara supports all of its business systems and desktops using a virtualization and VDI environment across the home office in Perth, and its Murrin Murrin mine operation 425 miles away. When workers started to experience slow response times, Minara deployed Infinio to improve performance.
Budd Van Lines uses a fully virtualized data center to manage operations for its shipping and logistics business. The company, which specializes in executive relocations, supports its office employees and field staff using VMware vSphere for a mixed enterprise workload as well as XenApp/XenDesktop VMs for VDI.
This year we are again giving away a free pass to VMworld 2014! The conference pass is part of our vIP prize package, which also includes access to the VMunderground party and lots more.
Update: The contest is now over. Thank you to everyone who participated.
Topics: News


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The Cleveland Cavaliers have reached an agreement in principle to send first overall draft pick Andrew Wiggins and former top pick Anthony Bennett to the Minnesota Timberwolves in exchange for All-Star forward Kevin Love, according to a report.
Cleveland will also send a protected 2015 first-round pick to Minnesota as part of a trade that was reported on the National Basketball Association's website on Thursday and cited league sources from an earlier Yahoo! Sports report.
The addition of Love, who would team up with recently-signed four-time league most valuable player LeBron James, would further solidify the Cavaliers as a favourite to represent the Eastern Conference in the NBA Finals.
The deal cannot be finalised until August 24th, NZT, because Wiggins, the centerpiece of the package for Minnesota who was taken first overall by Cleveland in June, cannot be traded until one month following the signing of his rookie contract, the report said.
Bennett, the first overall pick in the 2013 NBA draft, had a rough start to his rookie campaign with Cleveland but improved as the season progressed. He averaged 4.2 points and 3.0 rebounds in 52 games.
According to the report, the Cavaliers are making the deal with Minnesota with a firm agreement that Love will opt out of his contract in 2015 and re-sign with the Cavaliers on a five-year, $120 million-plus contract extension.
Love averaged 26.1 points per game, 12.5 rebounds and 4.4 assists last season for a Minnesota team that went 40-42 and was nine games back of the eighth and final playoff spot in the Western Conference.


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AVS/Express is a comprehensive and versatile data visualization tool for both non-programmers and experienced developers. Rapid data analysis and rich visualization techniques combined with an intuitive, graphical application development environment make AVS/Express the best choice for challenging problems in a vast range of fields including science, engineering, medicine, telecommunications and environmental research.
For information and trial download links please contact email@example.com.
What's New in AVS/Express 8.1
Platform updates
- Support for Visual Studio 2012 on Windows Vista and 7 (VS2012 is not available on Windows XP, use the VS2010 port).
- Support for Mac OS X 10.8 (Mountain Lion) in addition to continued support for 10.6 and 10.7.
- The following platforms have been dropped for this release: Linux EL4, Visual Studio 2005 and Visual Studio 2008.
New animation macros
Two new macros, fly_through and fly_object, have been added to the animation kit. These macro respectively allow the user to move the camera or a set of objects along a timeline by setting up checkpoints on a base plane. fly_through allows the camera position, speed and direction to be controlled and fly_object allows one set of objects to be moved through a scene while the remaining objects are static. Please refer to the example applications in the "What's New" library, each of which contains a sample timeline which can be read in.
Fullscreen viewer mode
On Windows, a viewer window can now be made to fill the entire screen. In the View Editor, Options panel is a toggle to turn on the fullscreen mode. Press the escape key to return to the standard size. If for some reason AVS/Express is unable to correctly determine the screen size (for example when using a multiple monitor setup), the fullscreen width and height can be manually set in the UI.
New macros
Please see the "What's New" library for example applications demonstrating these new macros.
- Cone – new cone geometry
- crop_view – crop a field based on the current view window, with an optional margin
- curvature – calculate the curvature of a surface
- downsize_scat – downsize scattered data by superimposing a uniform mesh
- FOrthoPlane – plane geometry fixed to the X, Y or Z axis
- FPlane_Data – enhanced version of the FPlane geometry where a node data component representing the X position, Y position, radius or angle can be added
- GISReadShape – read ESRI shape files
- interp_data_orthoplane – interpolate node data using an orthoplane
- local_rotate – animate rotation of a scene around the X, Y or Z axis
- make_volume – map non-uniform data onto a uniform grid with a single "short" data component suitable for volume rendering
- PickCenter – pick the center point about which an object is rotated
- point_render – draw an unstructured field as illuminated points
- project_vector – calculate either an axial or tangential projection of a vector component
- Read_UCD_Cache – speed up reading of large time dependent UCD files by caching some timesteps in memory
- Read_WRF – read Weather Research and Forecasting Model data
- select_material – select particular cell sets based on their material id
- stream_color – add color to streamlines based on various properties of the lines
- tensor_val – calculate various stress values from an input tensor
- tensor_glyph – create line or diamond glyphs representing stress values
- Write_KML_Image – write an image embedded in a .kml file suitable for viewing in Google Maps
- ZoomBox – zoom into a view by drawing a box
Updates to multithreaded macros
All multithreaded macros have been updated so that when instanced they use the same number of threads as the number of detected cores on the system, rather than requiring the developer to specify each one. If this behavior is not desired, the default number of threads when a macro is instanced can be set using the environment variable or config file setting XP_NUM_THREADS.
New multithreaded versions of existing macros
- mt_advector
- mt_advector_point
- mt_interp_data
- mt_isosurface_nest
- mt_slice
- mt_slice_arbitplane
- mt_slice_plane
Updated macros
- advector – add tailed lines at a particular release interval and length
- crop_area_box – now supports structured fields
- crop_cylinder – now supports structured field
- crop_sphere – now supports structured fields
- interp_data – the null value can now be specified
- Read_UCD – retain the node/cell id values from the UCD file


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Greetings Wizards,
Home Tree, well my version ;) Home tree is the place where the people of Pandora live. I did my best to recreate this wizard101 style version.This tree can hold a whole tribe of the people. I showed everyone my version of the world, now I hope you enjoy home tree just as much. The life school house has these inner root system that works perfect for hammocks the people sleep in, these hammocks hang from the the tree, so this are will represent that.
Now we move up the twisted spiral of tree columns , you will see animals plant life and Yes I do have actual gardens inside and outside of this house. First area I have stinkweed , it was kinda hard to hide the furniture item stinkweed likes litter, there is also in one part Evil Snowpeas with a Snowflake Obelisk, I really did not hide this as it went with the decor . Multi levels of this house are shown in this next picture. As we go up and through the tower we come to where they hold their weapons and shields. I planted bears here as the sand worked perfect for where they held their weapons racks. Next in line is the food circle this is where food was made and eaten.
Last on our tour I created a area I hope you enjoy as much as I do, in the movie there is this scene where the stars are swimming in a florescent pool area, water is pouring down and everything is lit up around them. I have laugh-o-dils and baby carrots for plants as they both like the fish fountains I used to recreate the water falls. bright colors to make the fluorescents even added a spinyfish to give creature to the area. I hope you enjoyed my version of my favorite movie for 2010 , Wizard101 Style ;). Until next time ,happy house decorating.
Your friend,
Paige MoonShade
Tuesday, January 4, 2011
Inside Home tree,cont Pandora wizard101
Posted by Paige MoonShade at 1:18 PM
6 comments:
- AnonymousJanuary 4, 2011 at 6:46 PM
No one has mastered housing more then you, Paige.ReplyDelete
Great Housing Paige!ReplyDelete
- AnonymousJanuary 4, 2011 at 9:11 PM
I think we know who is going to win the housing contest lol.ReplyDelete
Great job!
Woah. Thats a lot different from when i was there. Nice job!ReplyDelete
Wow, Thanks everyone. HonoredReplyDelete
- AnonymousJanuary 5, 2011 at 9:07 AM
Hey Paige just to let you know I love it and I will definetly be making another post about this later but would you mind adding necroamancernation.blogspot to your blogroll thanks so muchReplyDelete
-Duncan Dragonblood
Please make sure to have your Pirate or Wizard name attached to your question. I will answer these question as soon as possible.
Sorry any players who stays anonymous or unknown will no longer be posted or answered. Your wizard or pirate's name is required.
Thank you.
Paige MoonShade
Subscribe to: Post Comments (Atom)


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Our Sixth Form sets an example to the rest of the school so be a responsible part of our learning community by adhering to the following policies.
When you go to a lesson take off your coat, turn off your phone, take out your earphones, get your books, folders and planner ready … Be ready to learn
When you come into school wear your ID, set an example to the other students…Be a responsible part of our learning community
When you are in a study area (SF5, SF6 or the library) work quietly so that others can concentrate…Be ready to study
When you are in a social area (SFCR or Castle Green) keep your feet off the chairs and tables, put your rubbish in the bin, don't shout or swear… Be kind to each other
Please make sure that you are following all the expectations and procedures:
Mobile phones should not be visible in the main school.
You can use your phone in the sixth form corridor, SFCR and in Castle Green foyer.
You may not use your phone in lessons, in main street or any other corridors.
If a member of staff asks for your phone because you are using it in the wrong place, give it to them. They will take it to the main office. Politely find out the teachers' name if you don't know it. See Mrs Wren, Miss Campbell or Mrs Evans to get the phone back.
Be Aware:
Year 12 students must be working at a D grade or Merit in order to be able to continue into Yr 13. Everyone needs to be recommended to go into Yr 13 by their teachers.
We are working hard on supporting high attendance so that students do as well as they can. There are several strategies in use.
Students must contact school using the absence line on every day of absence. On their return to school students must supply a note to their tutor explaining their absence. There are slips for this purpose in the student planners. Students who receive Bursary are not eligible for their weekly payment unless they follow these procedures.
Tutors are using milestone assessment data and their own knowledge of the students to allow students who are successful independent learners the choice of studying at home if they do not have a timetabled lesson. Most year 13 students already have this privilege.
Students who need more support during their study periods are required to be in school instead of going home if they do not have a lesson. Support during study periods will be available if required. Our sixth form student support co-ordinator will be working with students and there is other study support available too.
If we are concerned about a student we will use an attendance card to monitor their attendance. If concerns continue we will contact parents and discuss further support.
We hope that these strategies will show that we are supporting students who need help and allowing mature students to take more responsibility for their learning. We also want to make it clear that attendance is a key factor in success.
The buses between Sixth Forms run during each break, the timetable for these buses can be found by clicking here.
Reminder that students who are unable to make school must make sure they phone into the sick line (0208 724 1531) on the days they are sick and bring in a letter to confirm their absence on their first day back. Should the student have a medical appointment that is unable to be made outside of school hours the appointment card must be brought to Miss Campbell, Mrs Evans or Mrs Yeaman BEFORE the appointment. Emergency appointments which have no prior notice must have a card given to Miss Campbell, Mrs Evans or Mrs Yeaman on the students return to school.
Should the above guidelines not be followed then students will be marked as unverified absence and this may jeopardise their sixth form place.
To improve punctuality in the morning we are using this policy:
If you are late in the mornings your tutor will give you a 20 minute detention. This will be recorded with a cfc.
If you miss registration altogether or you hit 3 lates, you will have a 20 minute detention with Miss Campbell/ Mrs Evans. This will be recorded with a cfc. After 1 detention with Miss Campbell/Mrs Evans any more will result in 1 hour. After this a conversation about whether sixth form is a suitable place for you will be had with yourself and parents.
Detentions and cfcs with letters home are not usually used by sixth form tutors as students are usually able to take mature, responsibility for their learning.
There will be dress-code checks at random times
All students should be wearing clothes suitable for a working environment.
Do not smoke outside the school. You have to move away from the building (or quit!).
Mr Smith will have a formal meeting with any student who is seen smoking outside the school.
By 4.15 students who are still at school should be
All students have to have left the site by 5:30pm
ALL students must complete an induction for the gym
All students must swipe in before their session at Castle Green Reception and must be out of the gym, with equipment away neatly by 5.30pm


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So there I was psyching myself up for my Medication Management meeting at 3pm, when at 11am I get a phone call from the clinic saying that the Nurse who was to be sitting in on the meeting wasn't available today so they would have to cancel the meeting and rearrange it for next week. As the call went on I felt all my energy slip away from me. It was like a big vacuum cleaner stuck up my rear end and sucking my will to live out of me.
I wasn't particularly looking forward to the meeting… I had been told there would be a lot of talking and I just wasn't in the right head space to be revealing my inner soul today. I have been down now for about 2 weeks and it doesn't seem to be getting any better. I am putting on a face for Mum and Linda but the cracks are starting to show. I don't want to be chirpy and chipper, I want to take to my bed and never come out.
My CPN is coming Thursday, so no doubt I will get the usual I should go out more and take the Lorazapam as much as you need. You would think she was trying to get me hooked on the stuff. Maybe they get points for referrals to different sections. 10 Pts for a Bipolar referral 20Pts fpr a Lorazapam addiction 30 Pts for a schizophrenic meltdown. first one to 100 and you get a set of steak knives. Sorry that was cynical and I know she only has my best intrests at heart but I am sick of being dependant on drugs to get through my day. I would never be allowed to get rid of the drugs by my family, I would drive them nuts inside of a week. Hell, missing one day is cause to my have my balls chopped off just to remind me that I should take them every day.
I really shouldn't complain as I don't know where I would be with the pair of them nagging at me to get things done. Dead or on the streets at the very least. Maybe even dead out on the streets. My mortality is become oh so real to me lately. There is a race going on in my body. It's between my Mind and my Pancreas. They are duelling over who will get the final blow in. My diabetes is out of control due to the medications I take for Bipolar and my Bipolar is casuing me to freak out about my diabetes. I actually think the stress of worrying about whats gonna kill me first will actually kill me first.
I am not going to keep writing tonight or else I will convince myself that it's a good idea to do something stupid. I am just going to surf around looking for picture of cute kittens and puppy dogs just to make myself feel better.
Until Next Time…


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Regardless of the incredible development of cloud computing and the expansion of information that accompanies it, there are as yet numerous myths about the cloud that hold on even right up 'til the present time. In case you're planning moving to the cloud or are now there, there are a few convictions you should realize that may make you reconsider your cloud technique.
Here are 5 basic cloud computing myths:
- Moving to the cloud is a one-man or one-team choice.
As opposed to what you may think, adopting cloud technique is not a one man business to run. It takes a group to decide the best move for your business. What's more, since cloud innovation is an IT-related idea does not imply that it's exclusively up to the IT group to settle on the choice. There are money related and official administration choices to be considered, as well. On the off chance that a cloud technique is grasped by the top level of management as well as the IT group, the appropriation procedure is a great deal more prone to go easily, and workers will probably get the training they require.
- Putting as much as you can in the cloud will make your life much easy.
Not each solution is a cloud-fits-all. There are a few sections of your production site that might be more qualified to physical environment than virtual. On the off chance that you put everything without exception in the cloud, you could be making more work for yourself without knowing it.
- The only motivation to utilize cloud is to save cash.
There are different motivations to move to the cloud other than saving cash. Convenience, technology and accessibity overhauls are only a couple (reasons) to move to the cloud. Also, at times, the cost saving may not be as much as you think. Make a point to examine your particular needs and discover how your TCO will be influenced thus.
- Only a private cloud can deal with your critical applications.
It relies on upon what basic intends to your business. On the off chance that your critical frameworks include applications that oblige you to scale in an exceptionally reliable manner, at that point maybe a private cloud is the best answer for you. In any case, if critical only implies having enough storage room or a working email server, you could be more suited to a hybrid cloud or public.
- If you aren't in the cloud, you're falling flat at life.
There are many benefits of utilizing the cloud, however that doesn't mean it's ideal for everybody. Because you don't have a cloud solution doesn't mean your present solution is bad. Regardless of the possibility that most organizations are utilizing the cloud to an ever increasing extent, you should take a good look at the options at what's right for your business. That may mean you have a colocation or committed solutions that is more qualified for your applications. What's more, that is OK! The critical thing is to enable your business to deal with your growing information requests the best it can.
There are numerous a bigger number of myths to cloud strategy than what's listed previously. In case you're thinking about moving to the cloud or are as of now there, investigate your alternatives and ensure the cloud solution you embrace or received is genuinely the best fit for your business.


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What is a Macro? Macro is one of the objects of Microsoft Access database that is used to create actions that run sequentially i.e. in the order in which those actions are listed and they are of two types: Embedded and Standalone macros. Macro is used to add logic to your database and to automate repetitive tasks and to create more usable interfaces like the switchboard and other numerous tasks. Macros work hand in hand with Visual Basic and modules. In MS Access 2013 and above, you don't really need an intense knowledge of Visual Basic coding to create a simple macro. In this tutorial part, I will give you the step by step guide of creating the various types of macros (embedded and standalone) and also show you how to run and debug them.This is part 1 of chapter 8 of the Free Online Access Tutorial Series at Microsoft Tutorial website. In this part, I will explicitly explain how you can create and run macro in any version of MS Office Access.
In the previous chapter (Chapter 7), I explained how to create professional reports, charts and mailing labels in any versions of MS Access in detail. Also see the full tutorial course content of this Microsoft Access Tutorial Series.
The term macro is often used to refer to standalone macro objects (that is, the objects that you see under Macros in the Navigation Pane), but in reality, one macro object can contain multiple macros. In that case, it is referred to as a macro group.
A macro group is displayed in the Navigation Pane as a single macro object, but a macro group actually contains more than one macro. Of course, it is possible to create each macro in a separate macro object, but often it makes sense to group several related macros into a single macro object. The name in the Macro Name column identifies each macro.
A macro consists of individual macro actions. Most actions require one or more arguments. In addition, you can assign names to each macro in a macro group, and you can add conditions to control how each action is run. The following sections discuss each of these features in more detail.
Macro Names:
If your macro object contains only one macro, macro names are unnecessary. You can just refer to the macro by the name of the macro object. However, in the case of a macro group, you must assign a unique name to each macro. If the Macro Name column is not visible in the Macro Builder, click Macro Names in the Show/Hide group on the Design tab.
An argument is a value that provides information to the action, such as what string to display in a message box, which control to operate on, and so on. Some arguments are required, and some others are optional. Arguments are visible in the Action Arguments pane at the bottom of the Macro Builder.
The Arguments column was introduced into Access 2007, which allows you to view (but not edit) an action's arguments on the same line as the action. This makes it a little easier to read your macro because you no longer need to select each action to display its arguments. To display the Arguments column, click Arguments in the Show/Hide group on the Design tab.
A condition specifies certain criteria that must be met before an action will be performed. You can use any expression that evaluates to True/False or Yes/No. The action will not be executed if the expression evaluates to False, No, or 0 (zero). If the expression evaluates to any other value, the action will be run.
You can have one condition control more than one action by typing an ellipsis (...) in the Condition column for each subsequent action that you want the condition to apply to. If the expression evaluates to False, No, or 0 (zero), none of the actions are performed. If the condition evaluates to any other value, all of the actions are performed.
Macro Actions:
Actions are the basic building blocks of macros. Access provides a large number of actions from which to choose, enabling a wide variety of commands. For example, some of the more commonly used actions can open a report, find a record, display a message box, or apply a filter to a form or report.
Temporary Variables
These three macro actions (SetTempVar, RemoveTempVar, and RemoveAllTempVars) allow you to create and use temporary variables in your macros. You can use these in conditional expressions to control running macros, or to pass data to and from reports or forms, or for any other purpose that requires a temporary storage place for a value. These temporary variables are also accessible in VBA, so you can also use them to communicate data to and from VBA modules.
Embedded Macros:
This gives you the ability to embed macros in any of the events provided by a form, report, or control. An embedded macro is not visible in the Navigation Pane; it becomes part of the form, report, or control in which it was created. If you create a copy of a form, report, or control that contains embedded macros, the macros are also present in the copy.
Standalone Macros:
From MS Access 2007 and later versions, a macro or macro group can be contained in a macro object (sometimes called a standalone macro), or a macro can be embedded into any event property of a form, report, or control. Embedded macros become part of the object or control in which they are embedded. Standalone macros are visible in the Navigation Pane, under Macros; embedded macros are not.
To Create a Simple Standalone Macro in MS Access:
1. Click Macro located in the Macros & Code group the Create tab. This opens up the Macro Builder as shown below.
2. Add an action to the macro. For example, as a sales Manager, you might want to create a macro that will automatically open a table in MS Access and then automatically exports the table to MS Excel for you. To build a macro carry out these function for you:
a. Click the Add New Action drop down arrow.
b. Type the desired action or click the arrow to select from the list of available actions. For the above example, select the OpenTable action. Then click the Table Name fields and select the desired table. Next, ensure that the View and Data Mode is set to Datasheet and Edit respectively as shown below.
The OpenTable action above will open the table once the macro is run. Note that after selecting an action, users must specify arguments for the action, if any are required.
c. Optionally, type a comment for the action by selecting Comment under Program Flow.
3. Next is to add an action that will export the table from MS Access to MS Excel. Click the Add New Action drop down arrow and select the ExportWithFormatting action from the drop down options.
4. In the Object Type textbox or drop down menu, type or select Table or any other object of MS Access data base you wish to export.
Next, select the particular table or type the object name in the Object Name dropdown menu. For example, the name of the table I want to export is workers payroll system.
Next, select your desired output file format. You have many options available here like: Excel file format (".xls", ".xlsb", ".xlsx"), HTML file format (".htm", ".html"), PDF file format (".pdf"), Rich Text Format (".rtf"), Text File format (".txt"), XPS file format (".xps"). For example, since I want to export from MS Access to the most current version of MS Excel, I will select the Excel Workbook (*.xlsx) option.
Next, select the Yes option from the Auto Start drop down menu. This means that the ExportWithFormatting action will start as soon as you run the macro. If you don't want the action to start automatically, then select the No option.
Next, for the Output Quality drop down menu, you have two options to choose from: the Print option and the Screen option. The Print option exports the file with a very high quality suitable for both viewing on screen and for printout while the Screen option is only suitable for viewing on screen. So make your choice. But I recommend the Print option.
The Output file, Template file and the Encoding options or arguments are optional. So leave them blank for now as shown below.
5. Please note that when you run the macro, Access carries out the actions in the order in which you list them.
6. To add more actions, select an action from the Add New Action drop down menu and provide the needed arguments for that action if any. Then save tour macro with a unique file name.
To Run or Edit a Standalone Macro:
1. Right click the desired macro you wish to run from the Navigations Pane and select !Run. To edit a standalone macro in design view, right click the macro name from the Navigation Pane and select Design view as shown below.
To learn more about standalone and embedded macros in MS Access 2007 and previous versions, See this article on Microsoft Office website on macro.
This is the end of part 1 of chapter 8 of this FREE online MS Access tutorial training series.
In the next part (part 2), I will give you the step by step guide on how to create switchboards and how to password- protect MS Access Database.
Recommended MS Access Textbook
Get this book (Kindle format): Designing Professional Database Management Systems Using Microsoft Access 2013 and 2016 and MySQL.Click Here to know more about the book.
In the next part (part 2), I will give you the step by step guide on how to create switchboards and how to password- protect MS Access Database.
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Remember when I said omeprazole was ordered so hopefully that wouldn't be a long time coming? Definitely jinxed it. It's been on back order; hopefully it will be here on Friday.
I say hopefully because the last three or so rides with Joker have been… interesting. We settled into a groove in late November as long as he wasn't the only horse being ridden indoors. At the beginning of December, we went on our first trail ride with a group from the barn and he did super. You could tell he really loved being ridden out as opposed to indoors. We went about 3 1/2 miles in an hour (there were people in the group who were only comfortable with walking) but it was heavenly to me.
Last week, I rode him three days – two nights in the indoor plus one mini trail ride. Each ride was progressively more tense. I had a half-day at work last Friday so another lady and I decided to take advantage and go for a trail ride in the same area as before. It took me a solid five minutes to get Joker off the property – he just planted his feet and said "No, thanks." I tried an opening rein, pony club kick, circling, zig zagging, letting the other horse go first. Nothing. Finally, I think he just got tired of me and moved off. He wasn't up or tense when we set out but when we got to the field across the way, he checked out. The trail ride only lasted half an hour because his behavior was frightening the other lady. To me, he wasn't being a nightmare (I spent almost a decade riding green beans). But to be fair, he was jigging and being looky and pulled some hard stops every once in a while. I told her I was fine and that I would definitely let her know if I felt like we were in any danger. This was this lady's third trail ride I later learned so that explained her apprehension. Joker was pretty lathered when we got back; note that he didn't break a sweat on our first trail ride and it was twice as long.
I gave Joker the next day off and went back out on Sunday with another woman from the barn who agreed to trail ride with me. It's been 60 degrees in December (Northern Indiana) which is ridiculous so I wanted to take advantage of it. We went a different route and Joker was slightly jumpy but much better although he spent most of the ride pretty up though. We got caught in the rain so headed back to the barn to finish our ride. Joker was tense and bracey inside so I tried to do some suppling exercises and used some trot poles. He improved after a while so I called it quits.
He got Monday off which brings us to Tuesday night's ride. He was in the cross ties alone again which seems to be happening a lot lately. He pawed and weaved but didn't rear. He was pretty squirmy to tack up and gave me some hard looks when I was slowly tightening his girth (anyone else hearing the screaming ULCERS yet).
Someone else was just leaving the arena (with a super cute Oldenburg gelding) when we got in and Joker was none too pleased. I worked with him on the ground a little first on the lunge and then yielding various parts of his body. He was a little up but listening.
So I got on. And sigh. And ow.
He was a freight train. He was yanking my arms out of their sockets for the whole 30 minutes. He acted like he wanted to be a run away train so I decided, why not, I'll just let him go. So up into a canter we went (it's really too small to gallop) and I pushed him out and while he grunted and stayed stiff, he didn't let any squirrels out. So we trotted some then cantered some and I tried to work in a lot of transitions to get his mind and body unwound.
Nothing. I could hear him grinding his teeth (in a hackamore) and his back was like a two by four it was so stiff. He was spooking at things that have been in there since he's been at this barn and tried to outright bolt twice. I've never heard a horse grunt almost the entire ride. And grind their teeth without a bit in their mouth. When I could finally walk him a lap without him trying to boot scoot, I called it a night. And immediately texted Elise, "Please tell me the omeprazole is off back order soon."
It sounds really rough but in reality I've been having so much fun. We've had some pretty bad days but we've had some good ones sprinkled in too. Being back in the saddle has been like coming home. Even when I'm having to get after him or preparing for sillies, I'm having a blast. My body somehow remembers all of this. Sure, I'm no equitation queen and may never be again, but I know I'm not riding like a complete sack of potatoes and I'm really proud of that. My husband has come out once or twice and taken video and I kind of do a double take. Is that my stable lower leg? Wait, my toes aren't turned out at the canter? Hold on, he looks that nice going around? It's really reaffirming.
I'm not a personal trainer so take this with a grain of salt but I'm attributing my relatively quick acclimation to riding again to the hard, hard work I've put in at the gym over the past two years. While I haven't been able to ride much before Joker since college, I've been hitting the gym hard. Five days a week at 5AM. I lift weights and I run and I do some exercises my physical therapist gave me to strengthen my hip flexors for riding (she rides too). I'm no body builder but I've spent two years methodically lifting and conditioning myself.
And damn, I gotta say, I'm not as weak as I thought I would be in the saddle. It was mainly about getting the balance right and the minute feeling of where to put/move/keep your body in different gaits, to ask for leg yields, etc. I still have a long ways to go to be technically correct again and that's ok.
If the omeprazole comes on Friday like it's supposed to, we'll start Joker on it over the weekend. I'm going to give him a week off (the weather is finally getting back to season normal 30's) from riding but go out and spend some down time with him. Hopefully after a week, when I go to ride him next, his tummy will be feeling a lot better. Fingers crossed because I hate fighting him, especially when I'm 99% sure he's in some degree of belly pain.
On a much happier note, husband agreed that we could drive up so I could ride Nibbles this weekend. I haven't seen her in person since the show in September and I haven't ridden her since July (!?!?!!). I'm out of my mind excited. First of all, I feel completely prepared because I've been riding several days a week. I know I can do this. Second, I get to ride my horse. Third, I've never cantered her before (when I backed her, we only made it through walk and trot before she went to Elise) and I'm super, super excited to feel it. Fourth, I get to ride my horse. Fifth, MY HORSE.


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Women Only Classes 27.08.17
Women only classes will run tomorrow as long as we have minimum numbers. YOU MUST book in by text 07743596420 by 12 noon.
2-3 Thai Circuit
3-4 Thai Padwork
2 Responses to "Women Only Classes 27.08.17"
Jade lee on
August 26th, 2017 2:36pm
Hi. I am interested with tomorrow class.
What is the difference between Thai circuit and Thai padwork.
trix on
September 18th, 2017 9:29pm
Thanks for your enquiry.
There are 2 classes for women only every Sunday.
The 2-3pm class is a Thai Circuit class, great for improving general fitness, building strength, stamina and flexibilty.
The 3-4pm class is a traditional Thai Padwork class, working with a partner on the pads learning Thai Boxing techniques. This class is good for developing Thai Boxing skills and improving stamina and conditioning and a really good stress buster.
You do need to book in every week by 12 noon on the Sunday via text on 07743596420 after checking our website (wickercamp.co.uk) to confirm classes are on.
Any further information please give me a call.
Feel free to leave a comment on this post...


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Background music: Good Life by OneRepublic. It's the anthem of my time abroad, the best way to convey how I feel about this past semester (and lif...Read More
This city. It's so underrated, but because of that it wasn't crowded with tourists. Half of the city was on a cliff, the other half in the valley,...Read More
Luxembourg City, Luxembourg
We were on the furthest western corner of Germany, so pretty far from Rome… and yet, the city was covered with former Roman ruins of buildings, wall...Read More
Trier, Deutschland
We took an overnight bus, only to arrive to unrelenting rain at 7 am, and remained cold and wet until the next morning…when the sun came out, and th...Read More
Zürich, Switzerland
If there's one thing I've learned from living in Germany in the springtime, it's that there's one thing that hasn't made it into the list of...Read More
It's been a while since I've written anything, but between travelling every weekend, the never-ending cycle of taking and editing photos, and scho...Read More
What Actually Matters?
I've been in Marburg for over a week now (10 days, to be exact), and it already feels like move-in day was months ago. Last night was the first ...Read More
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I still can't believe it, but my whole life I'd dreamed of skiing in the Alps, and this February, Christina and I made it happen. Despite ...Read More
Chamonix Mont-Blanc, France
...Read More
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This is the first of what I hope will become a larger series of videos. My goal is to combine photography, filmmaking, and travel, by giving a glimpse...Read More


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There are many areas of volunteer opportunities at Skylands, the largest and most active is that of gardening volunteers. Other volunteer activities include record entry, file and library maintenance, development of educational programs and materials, grant writing and fund raising, Geographic Information System mapping, carpentry, graphic arts and many areas of visitor interaction including seasonal public events. Volunteers who are interested in working in an area in which they are not experienced will receive training whenever possible. We provide training for all tour guides, including manor docents, gardens, wildflower, and family woodland hike leaders.
There are weekend gardening opportunities the third Sunday of each month during gardening season. Weekend garden volunteers meet on the third Sunday at the Carriage house at 1:30 PM. Please note that it is an Association rule that, for safety reasons, volunteers never work alone.
Click here to download the volunteer application. You may return your completed application by email, fax, or mail (see form).
Volunteers assist in the upkeep of the many beautiful gardens here at Skylands. Learn while you help in planting, weeding, deadheading, and pruning, as well as checking for plant labeling and garden problems, including insects and plant diseases. Area leaders are available on volunteer days to provide guidance and answer questions.
This is the only garden at Skylands where the plantings are mainly annuals. The eye catching displays change not only through the season but from year to year.
A photographer's delight, the perennial border consists of eight beds planted with perennials of every kind including bulbs, shrubs, roses, groundcovers, vines and trees. The colorful floral display changes from season to season attracting hummingbirds, butterflies and other pollinators. Join the enthusiastic team effort to regularly weed, deadhead, prune and plant as volunteers enlarge and enhance Mr. Lewis' original cutting garden.
The Rhododendron garden gives us a brilliant display of flowers from spring through summer. The garden also hosts a large collection of hosta that range in size from miniature to gargantuan. They sport a variety of foliage in shades of blue, green and yellow, along with white and gold variations. Volunteers work to identify and map the many cultivars, as well as maintain and mulch this spectacular garden.
New Jersey is home to many moraines, rocky deposits left by melting glaciers in the last ice age. Mr. Lewis created this interesting garden to reflect that landscape type, planting it with ground hugging alpine species that thrive on rocky slopes, with water seeping beneath. Volunteers working in this garden maintain the streams, weed and fill in plants as needed.
The rock garden surrounding the Octagonal Pool was designed so that the many dwarf plants are at optimal viewing height in waist high raised stone wall beds. The garden has been carefully restored and the tiny plants are nicely displayed. Volunteers ensure these little jewels are maintained and the garden remains weed free.
This pleasant and relaxing garden holds many wonders, as well as an opportunity to "Look, Listen and Reflect". As a volunteer maintaining this delightful area, you will see flowers and plants pop through the natural woodland gardens as the seasons progress: ferns, delicate wild flowers which change by season, including many spring blooming Japanese primroses and colorful flowering shrubs that offer berries to birds in the fall. The garden includes a frog/turtle/dragonfly friendly bog loved by children and volunteers alike.
More than 200 lilacs, planted in the 1920's grace one of Skylands favorite gardens and the pride of NJBG. Join the team effort to regularly weed, deadhead, prune and mulch the beds. Volunteers may be found in the Lilac garden every Wednesday morning from April through October. There are four Lilac Saturdays, when a Master Gardener gives lessons in Lilac care followed by hands on experience in plant maintenance. Adopt and care for a bed that will be "yours" to maintain and love. Peak bloom is Mother's Day weekend, when there is a special Lilac day and volunteers have the opportunity to greet visitors and brag about "their" fragrant beauties.
This lovely little garden was originally the site of a rose garden planted by Mr. Lewis. Over time the roses were replaced by day lilies, which put on a colorful show during summer months. Summer annuals, varying from year to year, complement the daylilies and you can count on a cheerful display of daffodils in the spring. Volunteers weed to maintain this garden's beauty.
This unique garden is planted with annual flowers grown from seeds provided by the All American Seed Company. Planted by state workers, it is maintained by volunteers. An informative August program describing the planting is given for the public.
Volunteers maintain plant inventory, propagate and divide plants, pot, label prune, deadhead plants to be sold at the annual spring plant sale. There is volunteer work in the greenhouse year round.
Trained docents lead tours of Skylands manor. Tours occur on selected Sundays and are approximately 45 minutes long. Volunteers interested in becoming docents will receive specialized training.
Interested volunteers assist the Fund Raising/Grant Committee to research, develop and write fund raising programs.
The GIS system helps define spaces in the gardens. The plants within these spaces are identified and precisely located. The plant and location information is entered into a database, which is used to generate garden maps. Volunteers map the location of gardens, trees and plants to assist the GIS team as needed. Special training is provided.
This is where the most visitor contact takes place. Volunteers work as sales people and/or greeters. They stock, price, organize and maintain stock inventory in the store. The Gift Shop is open Sundays and during special events from April through December.
The Association maintains an extensive gardening library. Volunteers file library materials, shelve and inventory books, magazines, photographs and other library materials.
Volunteers assist the Volunteer Coordinator as needed with membership mailings, record keeping and file maintenance. Computer skills are helpful for this area.
Volunteers with experience in these areas record and take photographs of the gardens and special events.
Volunteers assist with regular monthly and special programs by greeting guests, collecting tickets, serving refreshments and distributing literature during the programs. Volunteers also help with pre-program planning, set up and post program clean up. Some of our recurring seasonal events include:
When weather permits, Skylands' volunteers offer hour-long garden walks every Sunday at 2 PM, May through October. Wildflower walks are given on five designated Saturdays and family woodland hikes are given the third Sunday of the month, May through September. Skylands' First Day hike, honored throughout the US at all National Parks is on January 1st. Volunteers greet guests at the Carriage House before walks or hikes. Special training is provided to hike and walk guides.
Volunteering at Skylands, the New Jersey State Botanical Garden, gives anyone who loves gardening or history an opportunity to pursue their passion in a beautiful setting while helping preserve one of New Jersey's most opulent botanical gems.
Click here to download the NJBG/Skylands Volunteer Handbook.
Other ways to help NJBG
Visiting: Hours & Admission | Directions | Accessibility | Tours
Discover: Specialty Gardens | Skylands Manor | Tours & Family Woodlands Walks
Events: Events
Get Involved: Support | Membership
About: About | Press Releases | Contact Us | Links
New Jersey Botanical Garden, P.O. Box 302, Ringwood, NJ 07456-0302 · (973) 962-9534
Copyright © 2003-2019 NJBG/Skylands Association, Inc.


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5th Floor-bwg - 439 University Ave, Toronto, ON
DesksOffice$109.00/moHot Desk
YES, YES, YES!!! Flexible co-working at 109$/month. Come and see us today and start working immediately. One price all included. High speed internet, refreshments, coffee, tea, etc. Click now!!! Regus gives you the opportunity to grow your professional network while reducing your costs and increasing your productivity. The Business World membership gives you access to several co-working spaces in the city at the best addresses, and it allows you to work flexible during office hours. All INCLUSIVE space includes: Mail handling, access to admin support, kitchen, all utilities and cleaning, Business Lounge, access to printer, scanner & copier, receptionist, office furniture, access to Meeting rooms, and business grade internet and WiFi. We have more than 100 centres in Canada starting from 5sqm to 50 sqm or more. All sizes, all budgets. Regus is the world's largest provider of flexible workspace, able to meet the needs of large corporates and small businesses alike. We are currently serving more than 1 million clients every day providing convenient, high-quality, fully serviced spaces for people to work, whether for a few minutes or a few years. Enquire now!!! Terms & Conditions: The total space includes shared space in the Business Lounge of the centre with high quality furniture and common facilities such as kitchen and reception. Pricing is indicative and subject to availability.
The appeal of its street address just south of Dundas in downtown Toronto is fully reflected in the beautiful granite lobby of 439 University Avenue, a highly superior Class B office building. Retrofitted with state of the art services, including an environmentally responsible HVAC system, high speed telecommunications infrastructure, modernized elevators and an advanced security system, 439 University, throughout its 23 storeys meets the expectations of today's organizations. 439 University is conveniently located steps from the subway, Ontario Court House, Toronto City Hall, Toronto's Eaton Centre and Queen's Park. Convenience and service is extended with retail services including dry cleaning and a restaurant. 439 University is an affordable downtown location that delivers much more than affordability. Office layout, configurations and square footage may vary, custom build to suit options may also be available. Please contact us for details


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By Larry Fine
NEW YORK (Reuters) - Seattle Seahawks fullback Derrick Coleman has triumphed already, regardless of the outcome of Sunday's Super Bowl against the Denver Broncos.
Coleman, the third deaf player to compete in the National Football League, has beaten the odds and his story has inspired the hearing impaired and others battling adversity around the globe.
An ad he did for the Duracell batteries that power his hearing aids went viral on YouTube with more than 13 million views and led to his becoming a champion to the hearing impaired, especially among the young.
"They told me it couldn't be done, but I've been deaf since I was three, so I didn't listen," Coleman says in the ad.
Coleman, a backup fullback who also plays on special teams, has drawn crowds of reporters during media sessions ahead of the NFL title game.
"We wanted to inspire others. We wanted to let them know that whatever accomplishments you want to achieve, regardless of whatever obstacles you have to overcome, you can always endure," he said about the ads.
"Just trust the power within and do what you want to do. That's basically what I'm doing."
Coleman, who rates his ability to hear without his hearing aids as two on a scale to 10, always enjoyed playing sports as a boy and did not take up football until he was 13.
"It was the last sport that I played. When I was younger I used to play basketball, tried soccer, tried baseball, even tried tennis. I was always a sports junkie," he said.
His parents encouraged him.
"'Just go out there and be you. Don't worry about anybody else. If people start making fun of you, just walk away or tell me,'" my mom would say.
"'You only want to surround yourself with people that want to see you succeed.
"'The ones that don't, and want to pull you down to their level, walk away from them. Ignore them'. It kind of stuck, because now I'm really good at ignoring things."
Asked if anybody ever told him he could not succeed, the 6-foot, 233-pound Coleman said: "Maybe they have, but they didn't say it to my face. Maybe they did, but I didn't know it because I didn't really listen to them."
Coleman showed his grit in his first high school varsity game in Fullerton, California, bouncing back from three fumbles in the first half to score three touchdowns in the second half.
He went to college at UCLA and rushed for 1,700 yards and 19 touchdowns but went undrafted in 2012.
The Vikings signed Coleman as a free agent, but he was waived in training camp. The Seahawks signed him in December 2012, making him the NFL's first legally deaf offensive player.
Coleman is able to communicate verbally with help of hearing aids and lip reading and said he has had no trouble.
"The hardest thing about being in the deaf community is getting over wall one," he said.
The fullback experienced a feel good moment after the NFC title game victory over San Francisco from a new fan.
Nine-year-old New Jersey girl Riley Kovalcik, who like her twin sister Erin wears hearing aids, wrote a letter to Coleman, which her father took a picture of and tweeted to the player.
"It was one of those things where she's not asking for anything," Coleman said.
"She's just saying, 'I have faith in you. You're my inspiration.' That touched my heart. It made me feel warm."
Coleman wrote the girls back, telling them "even though we wear hearing aids, we can still accomplish our goals and dreams!"
"The guy's a hero, plain and simple," said father Jake Kovalcik.
"He's not just a hero to my girls, but every kid out there who has struggled with something."
(Editing by Greg Stutchbury)


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Blistering quad-core performance. Gorgeous display. Sleek design. Strong battery performance.
Disappointing camera. Runs hot. Plenty of bloatware.
Bottom Line
The LG Optimus G for AT&T is a quad-core beast, but a disappointing camera and heating issues hold it back from smartphone perfection.
LG has modified Android quite a bit here—more so than on the Sprint model. You get three home screens to start, but you can add up to four more. There's a lot of bloatware, including nine AT&T-branded apps, and unfortunately, none if it can be deleted.
LG has baked in some really cool features, like QSlide, which allows you to show a transparent video over your regular display. That means you can look up an actor on IMDb while watching a movie, or even take notes on what you're seeing without having to stop the video to load up another app. A sliding scale at the top of the screen allows you to adjust the level of transparency to your liking.
Live Zoom lets you to pinch the screen to seamlessly zoom in up to five times while watching video. SmartShare allows you to share music, photos, and video on your HDTV or monitor via DLNA, and Dual Screen Dual Play allows you to display different content on each screen. For example, a slide presentation can be displayed on the monitor while the Optimus G shows your accompanying speaking notes. That's pretty cool. You can also connect to phone to your HDTV using an MHL adapter.
LG Tag lets you store device preferences on an NFC tag. If you want to use your phone while driving, you can create a tag for your car that shuts down your apps and Wi-Fi, but turns on data and GPS, and sets your volume to high for spoken directions. FileShare lets you send documents, music, pictures, and video to other compatible devices.
Of course, this phone is also powerful enough to run all of the 500,000+ apps and games in the Google Play Store without a problem.
Camera, Multimedia, and Conclusions
Whereas Sprint's model boasts a 13-megapixel camera, the AT&T Optimus G has a more standard 8-megapixel shooter. That's not a huge deal, because as we saw with the Sprint phone, those extra megapixels don't compensate for a sensor that's merely average. Fortunately, the camera doesn't fare much worse here.
Compared with the Sprint model, this camera captures less detail. Still, our tests confirmed that even the Sprint phone was still a long way off from capturing a truly sharp image. In the realm of phone cameras, this one falls somewhere in the middle. Compared with the iPhone 5 and the Galaxy S III, images taken with the Optimus G look a little washed out, and details aren't as fine. They're still good enough for Facebook, but they're nothing to write home about. The camera records fine 1080p video at 30 frames per second both indoors and out. There's also a 1.3-megapixel, 720p front-facing camera for video chats.
You also get some fun features, like LG's silly-sounding Cheese Shutter, which triggers the camera to snap a photo when you say "cheese." It's great for setting the phone up on a tripod to take a group shot, but be warned that it may also go off when you say "knees" or "please."
The Optimus G's beautiful display makes it a good device for media playback. It comes with 11.08GB of internal storage, along with a side-mounted microSD slot, which worked with out 64GB SanDisk card. I was able to play all of our music test files except for FLAC, and all of our test videos up to 1080p. Audio was great through wired earbuds, though for video it was slightly out of sync while using Altec Lansing Backbeat Bluetooth headphones.
The LG Optimus G is the fastest phone on AT&T, but an average camera and overheating issues keep it from being the best. If you're a speed freak who can look past those issues, the Optimus G will make you very happy. There aren't enough differences between this phone and Sprint model to justify switching carriers. And if you aren't already committed to a carrier, I'd say to choose one based on carrier performance where you live rather than on the phone.
Otherwise, the Samsung Galaxy S III offers you excellent (though slightly less) performance, a microSD card slot, and even better call quality. Or, if you're willing to look past Android, the Apple iPhone 5 is also fast and elegant, with an unsurpassed app ecosystem.
More Cell Phone Reviews:
LG G8X ThinQ Dual Screen
Oppo Reno 2
OnePlus 7T
Punkt MP02
Google Pixel 4


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How to Use Kids in Playgrounds to Generate Power
Harvesting the energy kids use on playgrounds could power a new generation of interactive outdoor toys. Ben Rooney reports. Photo: Getty Images.
This transcript has been automatically generated and may not be 100% accurate.
I ... don't get some of the Senate she said one of the list and use it to something else you looking at a playground spring toying with a generation site that converts the energy that gets blown into life ... in cartoon to play in goal and longer ... or we will we will click with its uploaded to iOS Dykes but Psalm ... why Fi you repeat ... but why Fi connection or been made about the data to reach itself to take ... what we can do is also connected devices and ... between them so ... all we go ... although ... devices for the parents mobile phones and tablets ... or even weekly ... connected devices to the center also where ... we opposed to date the fall ... in a jeep with years By Kate and ... to organize a petition from a global scale ... that didn't ... maybe most synonymous ... with with just the foam on the reach ... of fish and game designers can do whatever they want ... Fossett Evin AG