Equipment
12 articles
Dry Nitrogen Operation of Desiccators and Incubators
Gas operation for desiccators or incubators Incubators may be trickle fed with CO 2 for maintaining pH levels in tissue cultures. Desiccators use the same physical arrangement with industrial dry nitrogen for a dust-free and very low humidity environment within the chamber. Incubators and desiccators are available with these gas fittings as an optional extra. The inlet is a tube fitting and the outlet a ball valve. This allows for trickling of gas through the chamber. Our catalogue item EDCG is required for this factory installation which must be specified when ordering an incubator or any one of the EDC drying cabinets. Connection recommendations: An industrial dry nitrogen (or CO 2 ) cylinder should be equipped with a two-stage regulator, with the second stage adjusted so that an outlet tube (immersed in water) would just run a continuous stream of gas. Rather than a needle valve, we recommend the use of a small additional regulator - regulator EM060, without gauge. This regulator should be adjusted to expel about one bubble per second or less. That flow rate to the cabinet (or incubator) is sufficient for maintaining pH or dry atmosphere, depending on use. A 'G' size gas cylinder may last for many months. It may also be desirable to install a by-pass around the second regulator. This would consist of a two-way valve to supply gas either to the small single stage valve or bypassing that and re-connecting to the line after the small single stage regulator, with a simple "Y" or "T" tube fitting. This bypass would allow purging the cabinet, when required, with a larger dose of gas. Leak testing: The system operates under very low pressure and leaks may be a problem only because the cylinder remains open. Over months some gas may be lost if the system is not tight between the two-stage regulator and the outlet of the additional small regulator. To test for leaks, make up a few ml of a 10% detergent solution. Work the solution with a small paintbrush to form a bubble solution. Paint the solution onto suspect connections and small leaks will become obvious.
Film Thickness Monitor Crystals (Sputter Coating)
Film Thickness Monitor (FTM) quartz crystals are used to determine the thickness of material deposited and may be used to terminate coating at a pre‐set thickness. Quorum Film Thickness Monitors (FTM) use a 5MHz RC-cut quartz crystal to measure mass deposition in real-time. The quartz crystal is placed in the vacuum work chamber. One face of the crystal is exposed towards the deposition source, such that, as material is deposited it will coat the crystal. The system functions as an oscillator, whose output is controlled by the frequency of crystal oscillation. As material is deposited on the crystal, so its frequency is modified. If coating without using the FTM, place a cover over the crystal to keep it clean. A straight edge scalpel blade may be used to very gently scrape off previously deposited coatings from the centre part of the crystal. Some coatings can be removed using a cloth with a little alcohol or with some very fine polish. Only the central area needs to be clean. It is essential to leave the gold contact on the outer edge, as this connects the crystal to the oscillator. A faulty contact area could be replaced with a new gold coating, but if both sides of the crystal are shorted, then it cannot work. If a crystal fails it may not be beyond use, but may require improving of the contacts. It is useful to have a new crystal on hand, if only to check that the system is operating properly. Replacement FTMs available for current and Q series coaters or for K series coaters .
Introduction to Critical Point Drying
Critical Point Drying is so named as it includes, as part of its process, the occurrence known as the continuity of state for which there is no apparent difference between the liquid and gas state of a medium, the surface tension between this interface reducing to zero. This occurs at a specific temperature and pressure with resulting density, and is known as the Critical Point. This condition of zero surface tension can be used to dry Biological Specimens, avoiding the damaging effects of surface tension. In biological specimens we are mainly concerned with the removal of water. The critical point of water of +347°C and 3212psi. is inconvenient, and would cause heat damage to the specimen. The most common and convenient transitional medium for critical point drying is Carbon Dioxide CO 2 which has its critical point at 31°C and 1072 p.s.i. However, it is not miscible with water, and therefore we have to involve a third medium, commonly Ethanol and Acetone, which is termed the intermediate fluid. We can now convert our transitional fluid, typically CO 2 from liquid to gas without surface tension at the critical point. The Critical Point Drier is designed for use with CO 2 , having first replaced any water in the specimen by a series of dehydration steps, often in a fluid such as Acetone, which will also be the intermediate fluid. Typical Dehydration Steps (Wet Specimen) Fixation Ethanol, Acetone - 10% - 100% CO 2 C.P.D. Dry Specimen ready for coating for SEM. The specimens for critical point drying are located in the pressure chamber of the Critical Point Dryer. The chamber is pre-cooled to allow it to be readily filled with liquid CO 2 from a gas cylinder. The chamber is then heated to just above the critical temperature with subsequent critical pressure being achieved. The CO 2 gas is vented through a needle valve, to avoid specimen distortion.
Labglass Water Stills Installation and Cleaning
Most water stills use mains water for single distillation and for cooling to condense the water vapour. Some stills allow for feeding in pre‐treated or tank water for distillation; water for cooling is also required. The two basic still designs are the CASCADE, which produces 3L/h of single distilled water and the DELTA which produces 3 or 6L/h single distilled water or 3L/h double distilled water. The output is user selectable at any time by changing around some of the tubing. The CASCADE requires a standard 10 amp power outlet. The DELTA requires a 20 amp power outlet. All water still models require a mains water tap with a ¾ inch garden hose type screw connection, to which a supplied filter connection is attached. This is for the cooling water on all models. On standard water stills this cooling water then passes into the boiler for distillation. The water supply must be available at mains pressure. A built‐in water flow regulator adjusts the water flowing into the still to the rate required for correct operation. The still has a valve to stop water flow. Hoses and fittings must be able to reliably withstand full mains pressure. The Dual Feed models require a second water supply, fitted with a tap to regulate the flow to the still. The still is connected to this water supply using 10mm ID braided tubing, which is supplied. Water for dual feed systems is pre‐treated or has other desirable properties; it is trickle fed into the still to obtain very high purity water. All models must have a drain or sink situated close to, and below, the water still. Unless the room containing the still is a 'wet room' with a floor drain, in which collecting vessels (carboys) can be on the floor, the still should be shelf‐ or wall‐mounted. The most common arrangement is to mount a strong shelf to hold the carboy above a sink, and the distillation unit needs to be a little higher than the top of the carboys. There are key holes in the rear of the distillation unit to allow hanging on the wall if required. A reinforced water hose needs to be run to the two inlets, which are positioned near the bottom left of the still, using a Y or T piece to connect to both inlets. This would produce single distilled water only. Refer to instructions if the system is to be used to produce double distilled water or when using a dual feed system. The waste water tubing from the stills must never be submerged in water in the sink or drain. Water flow to waste, and also water flow to the collecting vessels, must always be lower than the still. Any non‐pressurised tubing must be installed on a decline, if the tubing is level or runs uphill, back‐pressure will cause boilers and/or condensers to overflow. It is convenient if the cooling (and treated feed) water supply valve is within reach, but not far from the still's inlets. Any container may be used as a reservoir to collect the distilled water. The most common are 2 large (50L carboys) plastic drums with a tap on the bottom. The stills are supplied with Reservoir Monitor Tubes, which must be placed through a hole in the top of the container to be used as a reservoir. The depth must be sufficient to allow the bottom of the Reservoir Monitor Tube to be located 150mm below the level at which the water in the reservoir is to stop rising. The top of the reservoir must also have a hole drilled for the distillate delivery tube, which is supplied, to pass through the top to allow distilled water to flow into the container. Another hole must be drilled to allow the container to breathe so that it is not airtight, otherwise the distilled water will not flow into the container. This hole should be loosely plugged with gauze or cotton wool to exclude dirt but still allow gaseous exchange. The water stills have water supply monitors which, in the event of water supply failure, turn off the heating elements. The reservoir is monitored and when full, the stills are turned off, along with the water supply. If water is used from the reservoir, the still automatically restarts to maintain a full reservoir. Thermal cut‐out devices are fitted to the boilers, which turn off the water supply and heating elements in the event of an over temperature situation being detected in the boilers. CLEANING YOUR WATER STILL: A weak acid such as a 10% solution of formic acid, or vinegar, or a commercial kettle de-scaler diluted as recommended by the manufacturer. Turn off the power and remove the lid from the distiller boiling chamber. Add 2 to 3 tablespoons of either Sulfamic Acid or white vinegar to the boiling chamber. Vinegar is milder than Sulfamic Acid to work with. Agitate the solution by swirling the chamber for approximately 10 to 15 seconds. Leave the solution sitting in the chamber overnight to allow it to continue cleaning on its own. Empty the descaling solution from the boiling chamber the next morning. Fill the chamber with clean water, swirling it around to rinse it out, then emptying it. Repeat to ensure all descaling residue has been removed. Wipe the interior of the boiling chamber with a cloth to remove any last traces of solution residue then turn the power back on and watch to be sure the chamber fills and everything appears to be working properly. FAQs Does the still have a purity monitor? Not built in. We sell water purity meters separately, however, if the feed water is of reasonable quality, double distilled water is always of very high purity. Can the feed water temperature handle tap water? Yes, but warm water requires a higher flow rate of cooling water. Does the still have overflow prevention? Yes. Does the still have a stand‐by mode? Yes. Is the water suitable for PCR and DNA studies? Yes. Is the distilled water DNase and RNase free? Yes. Is the distilled water free of bacteria? When freshly distilled, yes. We suggest high wall mounting of system, best above a sink, then water can accumulate in 50L carboys, one for single, and a second for double distilled water. Replace lids with thick wad of cotton wool and pass through cotton wool the tube that controls shut‐off when carboy is filled. Large carboys allow the system to run overnight.
Laboratory Oven Types
Gravity Convection Gravity Convection Air is circulated by natural convection. Since hot air moves up, a gentle movement keeps temperatures fairly uniform within a chamber and quite uniform in any particular location. Mechanical Convection (Forced Convection) Mechanical Convection is a gravity convection oven assisted by a re‐circulating fan providing forced air circulation through the heating chamber. This system gives very rapid heat up and recovery times, combined with particularly low temperature variations within the working chamber. Adjustable vents and semi‐forced exhaust make it a good sample‐drying oven. Forced Exhaust Ovens In these ovens, air is forced through the working chamber by a fan, and dispersed through an adjustable vent. This type of oven is particularly useful in applications where the heating process produces gases or fumes that need to be quickly and continuously removed from the working chamber. All of the forced air ovens exhaust at a greater rate than a convection oven. However, much larger forced exhaust rates can be achieved by including an air inlet and an adjustable outlet. This modification has an additional cost and is only possible with forced convection ovens. Side Draught Ovens These ovens have air circulation from one side to the other i.e. left to right. Rapid heat up and recovery time make this type of oven ideal for preheating plastic sheets (hospitals, etc.) or any work where flat sheets or trays are used. Even air flow and heating is ensured. Vacuum Ovens A vacuum oven removes moisture or solvents at lower temperatures by reducing the air pressure inside the chamber. This lowered pressure decreases the boiling point of liquids, allowing materials to dry quickly without the thermal or oxidative damage that occurs under normal atmospheric conditions.
Laboratory Vacuum Microwave Processing
Introduction Microwave technology has reduced the times required for sample processing by over 90%, when compared to routine processing protocols. Water recirculation, the temperature probe and variable wattage have made protocol development possible. The addition of a microwave vacuum chamber has improved sample preservation and reduced times required for resin infiltration by >80%, over previously established microwave protocols. (Giberson et al., 1997, J. Vet. Diag. Invest. 9:61-67) Protocol:1. Microwave Oven Calibration Figure 1 indicates the placement of the finder grid mat, water loads and the vacuum chamber during VMP. The combination of water loads shown in Figure 1 creates an excellent cold spot during the fixation and buffer rinse steps. The heating rate for the microcentrifuge tubes should be 8-10°C (when 600µl of water is used - 40 seconds at 100% power under vacuum - 20"Hg). It is best to determine the actual heating rate with the buffer and fixative combination being used. Osmium will tend to heat less than the aldehydes over the same time interval. Fig. 1. The finder mat grid is taped to the oven cavity, approximately 1" from the front of the microwave cavity and centred side to side. An 800ml or 1 L plastic beaker (glass is all right but it creates more heat in the oven cavity) is filled with 500-700ml of tap water and the water is recirculated and cooled to under 35°C using the Load Cooler. The vacuum chamber is placed, as shown, in the microwave cavity. It has 2 each 100ml water loads inside which are always oriented as shown. The holder for the sample tubes (A) is placed in front to the two water loads, so that one of the sample tubes is directly below the temperature probe, which is inserted through the O-ring in the top of the chamber (see Fig. 3). Protocol 2. Aldehyde Fixation Microcentrifuge tubes which contain the specimens and 600µl of fixative are placed in the microcentrifuge tube holder (Fig. 2) which is positioned in the vacuum chamber as shown in Fig.1. A vacuum (20" Hg; 500 torr) is drawn. The microwave is then programmed for the following time sequences (these three time/power intervals are programmed sequentially on one numbered key pad): 1 minute at 0% power 40 seconds at 100% power 3 minutes at 0% power The starting temperature of the fixative should be ≤20°C. Fig. 2 The microcentrifuge tube holder is made of PTFE. The volume of fixative in the microcentrifuge tube should be 600µl ±100µl. Variations greater than 100µl will change the heating rate of the fixative solution. Fig 3. is a side view of the vacuum chamber. The temperature probe is to be positioned about 3mm above the surface of the fixative in one of the sample tubes. At the end of the 40 seconds at 100% power the microwave door is opened and the temperature probe is pushed down into the fixative to record the temperature after microwave irradiation. The temperature change should be >8°C. After noting the temperature, close the microwave door and push the start button. The final 3 minutes of 0% power will count down. The temperature probe (C) is positioned ~3mm above the fixative level in one of the microcentrifuge tubes. Beaker "A" is a standard disposable 100ml polypropylene beaker. The 3-way vacuum valve (D) is positioned toward the front of the microwave, the vacuum hose attached and a vacuum drawn (20" Hg; 500 torr). Close the valve. When the temperature probe is pushed down into the fixative to record the final temperature, after microwave irradiation, the vacuum will remain intact. Protocol 3. Buffer Rinse At the end of the 3 minute 0% power sequence, break the vacuum and remove the holder and microcentrifuge tubes. Remove the fixative and replace with buffer. Immediately remove the buffer and add 600µl of fresh buffer. Place the holder with tubes back in the vacuum chamber, draw a vacuum (20" Hg; 500 torr) and microwave for the following intervals: 1 minute at 0% power 40 seconds at 100% power Protocol 4. Osmium Fixation Remove the buffer and add 600µl of osmium fixative. It is best if the fixative temperature is <20°C before starting. Repeat the steps for aldehyde fixation (1 min. 0% power / 40 Sec. 100% power / 3 min. 0% power). At the end of the sequence, remove the osmium from the samples (under the fume hood) and rinse the tissue with water. Make sure to note the temperature after the 100% power cycle. In our experience the osmium step will heat less than the aldehyde under vacuum. NOTE: Replace the two 100ml water loads with fresh tap water prior to starting osmium fixation. Protocol 5. Water Rinse Quickly rinse the samples with tap water prior to transferring them to the flow-through baskets for dehydration (see Giberson, et al., 1997) and resin infiltration. Protocol 6. Dehydration: Fig 4. Typically use acetone in the following concentrations: 1 x 50%; 1 x 70%; 1 x 90%; 2 x 100%. Ethanol can be used in place of acetone if desired. A temperature restriction (37°C) is used during the dehydration steps which entail 40 seconds at 100% power for each step. The flow-through baskets are placed in polypropylene petri dishes and approximately 15-18ml of solution is used for each step. The Petri dish with baskets, shown in figure 4, is placed in front of the water load (about 4") serviced by the load cooler and a second water load of about 400ml is place to the right of the petri dish. The temperature probe is placed in the probe stand. Protocol 7. Vacuum Resin Infiltration The second water load, added for dehydration, is removed as are the two 100ml water loads from the vacuum chamber. The petri dish with baskets is placed in the vacuum chamber. The lid to the vacuum chamber, with the temperature probe inserted, is placed on the top of the chamber (Fig. 3). Make sure the tip of the temperature probe is in the resin in the petri dish. Position the vacuum chamber in the same location that was used for fixation. Set the temperature restriction to 43°C and draw a vacuum (20" Hg; 500 torr). Three two minute vacuum infiltration steps are done in the microwave (100% resin is used for each infiltration step). Use fresh resin for each step. After the last two minute run the tissue is ready for embedding in capsules and polymerisation. Protocol 8. Polymerisation The embedding capsules are polymerised under water in the microwave (see Giberson, et al., 1997) (Fig. 5). Since the publication of Giberson, et al., 1997, we have found that the use of the temperature probe during polymerisation improves the shape and overall block quality after polymerisation. Fig 5. a 1000ml, rectangular polypropylene dish is used during polymerisation. The large water load (serviced by the load cooler is left in the microwave. Water should be added as needed to maintain a level above that of the embedding capsules. The following polymerisation schedules are recommended for the resins listed: Epoxies: 10 min. at 60°C; 10 min. at 70°C; 10 min. at 80°C; 45 min. at 100°C LR White: 10 min. at 60°C; 10 min. at 70°C; 25 min. at 80°C Histocryl: 10 min. at 60°C; 10 min. at 70°C; 10 min. at 80°C; ~30 min. at 90°C Table 1. The Effect of Microwaves/Vacuum on the Sample Processing Times for Electron Microscopy Process Steps Process 1 . Routine Microwave Process 2. Vacuum Microwave Routine Process 1. Primary Fixation (aldehyde) 10 mins 6 mins 60 mins 2. Buffer Rinse 6 mins 4 mins 30 mins 3. Secondary Fixation (osmium) 10 mins 6 mins 60 mins 4. Dehydration (acetone/ethanol) 7 mins 7 mins 120 mins 5. Resin Infiltration 50 mins 8 mins 1080 mins (18 hrs) 6. Tissue to Embedding Capsules 15 mins 15 mins 15 mins 7. Resin Polymerisation 45-75 mins 45-75 mins 1080 mins (18 hrs) TOTALS ~170 mins ~120 mins ~2,400 mins Microwave‐assisted processing of human colon endoscopic biopsy after normal 10% NFB fix 1 x 7 minutes 100% Ethanol @ 350W TR of 67°C 1 x 5 minutes ACS reagent Grade Isopropanol @ 350W, TR of 74°C1 x 18 minutes Paraffin @ 650W, TR of 80°C (400x approximate) H&E. Liver stained with Masson's Trichrome stain using the PELCO HistoWave® (20x). Courtesy of Rick Giberson, Ted Pella, Inc. and Bruce Calkins, Pathology Sciences, Chico. Microwave Procedure Step Container Reagent Wattage Setting Temperature Restriction Time 1 50ml Coplin jar, xylene 165W None 4 min. 2 50ml Coplin jar, 100% ETOH 165W None 1 min. 3 Wash in tap water to clear BENCH STEP 4 50ml Coplin Jar, Bouin's solution 315W TR 60°C 6 min. 5 Wash in tap water to clear yellow BENCH STEP 6 50ml Coplin Jar, Gill #2 haematoxylin 315W TR 40°C 1 min. 20 sec. 7 50ml Coplin jar wash in tap water to blue BENCH STEP 8 50ml Coplin jar, Biebrich scarlet ‐ acid fuchsin 315W TR 40°C 40 sec. 9 Rinse in DI water, 3 changes BENCH STEP 10 50ml Coplin jar, phosphotungstic ‐ phosphomolybdic solution 315W TR 40°C 1 min. 11 50ml Coplin Jar, analine blue solution 165W TR 40°C 40 sec. 12 Rinse in tap then DI water BENCH STEP 13 50ml Coplin Jar, 1% acetic acid solution BENCH STEP 30 sec. 14 Dehydrate through ETOH's clear and mount BENCH STEP Intestine, cut at 4µm, stained with Mucin Alcian Blue pH2.5 using the PELCO HistoWave® (20x). Courtesy of Rick Giberson, Ted Pella, Inc. and Bruce Calkins, Pathology Sciences, Chico. Microwave Procedure Step Container Reagent Wattage Setting Temperature Restriction Time 1 50ml Coplin jar, xylene 165W None 4 min. 2 50ml Coplin jar, 95‐100% ETOH 165W None 1 min. 3 Wash in tap water to clear BENCH STEP 4 50ml Coplin jar, 3% acetic acid 165W TR 45°C 30 sec. 5 50ml Coplin jar, alcian blue solution 165W TR 45°C 1 min. 30 sec. 6 Rinse in DI water, 3 changes BENCH STEP 7 50ml Coplin jar, nuclear fast red 165W TR 45°C 1 min. 30 sec. 8 Rinse in DI water, 3 changes BENCH STEP 9 Dehydrate through ETOH's clear and mount BENCH STEP Intestinal cross‐section, cut at 4µm, stained with PAS using the PELCO HistoWave® (20x). Courtesy of Rick Giberson, Ted Pella, Inc. and Bruce Calkins, Pathology Sciences, Chico. Microwave Procedure Step Container Reagent Wattage Setting Temperature Restriction Time 1 50ml Coplin jar, xylene 165W None 4 min. 2 50ml Coplin jar, 95‐100% ETOH 165W None 1 min. 3 Wash in tap water to clear BENCH STEP 4 50ml Coplin jar, 0.5% periodic acid 315W TR 60°C 2 min. 30 sec. 5 Rinse in DI water, 3 changes BENCH STEP 6 50ml Coplin jar, Schiff's reagent 165W TR 45°C 2 min. 7 50ml Coplin jar warm tap Water 165W TR 45°C 4 min. 8 Wash in tap water BENCH STEP 9 50ml Coplin jar, Gill's #1 haematoxylin 165W TR 45°C 40 sec. 10 Wash in DI water BENCH STEP 11 Blue in Scott's water BENCH STEP 12 Wash in tap water BENCH STEP 13 Dehydrate through ETOH's clear and mount BENCH STEP Microwave Calibration The Microwave Calibration Slide Set is a set of glass slides with liquid crystal temperature strips permanently affixed to them. The slides help you predict the temperature of the staining solution around a tissue section during and after microwave irradiation. You will have two sets of calibration slides for microwave staining. Those labelled Calibration Slide #1 are for low‐temperature staining (35°C to 45°C). Those labelled Calibration Slide #2 are for high‐temperature staining (50°C to 60°C). PRECAUTION: Do not heat solutions containing the Calibration Slides above 65°C. The warm solution will melt the adhesive on the LCT strip, and the LCT strip will fall off the slides. To avoid pressure build‐up in the glass or plastic staining jars, do not cover the staining jars. If your staining protocol calls for bringing the solution to a boil or for steam at pressure, use a microwave pressure cooker. The unit is designed to safely handle 10lb/in² within a few minutes of heating in a microwave oven. Do not use your Calibration slide Set in the pressure cooker: the high temperature will melt the adhesive on the LCT strip. Do not irradiate the neon bulbs longer than 1 minute. They will become too hot to handle and could be damaged. Let the Neon Bulb Array cool for 2 minutes before re‐irradiation. Procedure The Calibrating Your Oven For Batch Microwave Staining: This procedure will allow you to predict, with confidence, the temperature of the staining solution around the microwave‐irradiated tissue sections. Materials Needed: Distilled water, 150ml 3 plastic staining jars, 30ml size. Notebook. Red marker. Thermal mitts. Microwave finder map, or alpha‐numeric oven tray. Neon Bulb Array and diagram of Neon Bulb Array. Microwave Calibration Slide Set. Microwave Tool Book. Calibrating Procedure: Make sure the alpha‐numeric Grid is in the left, rear corner of the microwave oven. Warm up the oven electronics by placing a beaker filled with 250mL of water in the right, rear corner of the oven. Program the oven to irradiate for 2 minutes at 100% power. Press start. Begin the next step within 2 minutes after the oven shuts off, or you must repeat this step. Place the Neon Bulb Array on top of the alpha‐numeric Grid. Align it so the corner with the large dot is at the left rear of the alpha‐numeric Grid and oven. Close the oven door. Programme the oven to irradiate for 30 seconds at 100% power. Press start. Observe the neon bulbs. Look for clusters of three to four bulbs that are continuously lit. Mark the alpha‐numeric Grid co‐ordinates corresponding to these bulbs on the diagram of your Neon Bulb Array. When the oven turns off, let the bulbs cool for 2 minutes. Meanwhile, fill a staining jar with 50ml or room‐temperature distilled water. Remove enough bulbs from one cluster that was lit so that you can place the staining jar between the bulbs at that co‐ordinate. Close the oven door. Programme the oven to irradiate at 100% power for 20 seconds. Press start. See if bulbs around the staining jar are continuously lit. If bulbs around the jar do not light up, refill the staining jar with 50ml of room temperature distilled water and reposition the jar on the Neon Bulb Array at another maximum power cluster (from step v.) Repeat steps 2 to 9 until you have identified a position where the bulbs remain continuously lit around the staining jar. Record your observations in your notebook. With the red marker, mark the alpha‐numeric grid at the location identified in step 9, drawing a red circle around the base of the jar. This is the optimal place to put samples for staining procedures. It is an area of maximum power with your loaded staining jar. Remove the Neon Bulb Array. Refill the staining jar with 50ml of room temperature distilled water and place it on the area marked in red on the alpha‐numeric grid. Place one calibration slide (from set #1 or set #2, as appropriate for your staining protocol) in the centre slot of the staining jar. Programme the oven to irradiate at 100% power for 5 seconds. Press start. As soon as the oven shuts off, open the oven door and observe the colour of the liquid crystals on the slide. Repeat steps 12 to 15 with the same slide, but adjust the irradiation time until the liquid crystal turns bright green for the target temperature you will use in your staining protocol. In your notebook, record the optimal alpha‐numeric grid co‐ordinates, irradiation time, and temperature for the calibration slides in the staining jar. Optional step: If your protocol requires simultaneous use of two to three staining jars, repeat steps 5 to 17 using three staining jars, each filled with 50ml water. NOTE: Some ovens do not produce enough power to reproducibly heat three jars at once. Procedure for standardised protocol for batch microwave staining : Using a standardised protocol for each microwave staining series improves reproducibility. We recommend using this protocol as a guide for adapting published staining protocols for your microwave oven. Batch Microwave Staining Procedure: Warm up the oven electronics for 2 minutes. Use the neon bulb array to identify the best location in the oven for microwave staining. Use alpha‐numeric grid for reproducible placement of the staining jars in the oven. Use calibration slides to check irradiation conditions in the loaded microwave cavity. Use standardised staining jars. Be sure all surfaces in the oven are dry Make sure the alpha‐numeric grid is in the left, rear corner of the oven. Warm up the oven electronics by placing the beaker with 250ml of water in the right, rear corner of the oven. Programme to oven to irradiate for 2 minutes at 100% power. Press start. Begin the next step with 2 minutes after the oven shuts off, or repeat this step. Select the calibration slide set that corresponds to the final temperature range you want to achieve. Place the slide in your staining jar. Place 50ml of the solution you will use for staining into the staining jar. Place the staining jar(s) on the alpha‐numeric grid on the co‐ordinated recorded from the result 17 above. Programme the oven for the power and time conditions described in your microwave staining protocol. Press start. After the microwave oven stops, the calibration slides should show the expected endpoint temperature for the staining protocol. If they do not, check the following conditions against your calibration procedure (above): staining jar location on the alpha‐numeric grid, staining solution type, volume, initial temperature, and selected irradiation time and power settings. Immerse slides containing tissue sections in 50ml of fresh, room‐temperature staining solution in the staining jar(s). Irradiate the tissue sections at the alpha‐numeric grid co‐ordinated for the power and time condition that resulted in the most even heating of the calibration slide set (from step 7). Press start. Complete the staining protocol by following the published procedure of your choice. Reference: The Microwave Tool Book ‐ A Practical Guide for Microscopists. Login and Dvorak. Beth Israel Hospital Boston. ISBN 0‐9642675‐0‐0
LED vs LCD Displays
All LED displays are technically LCDs. The key difference is their backlighting: standard LCDs use fluorescent tubes, while LED displays use light-emitting diodes. LEDs offer better contrast, deeper blacks, slimmer profiles for display, and greater energy efficiency, whereas traditional LCDs are generally more affordable. Specification Comparisons: LED LCD Operating temperature ‐40 to 85°C ‐20 to 70°C for industrial quality Storage temperature ‐40 to 100°C ‐30 to 85°C Average life 100,000 hours 50,000 hours, dependant on crystal. Backlight variable Comparable readability to 7m to 3m with backlighting Viewing angle to 60° 45°, but some high contrast versions offer 60°
National Pipe Thread Taper
American National Standard Pipe Thread standards, often called national pipe thread standards for short, are United States national technical standards for screw threads used on threaded pipes and pipe fittings. They include both tapered and straight thread series for various purposes, including rigidity, pressure-tight sealing, or both. The types are named with a full name and an abbreviation, such as NPT, NPS, NPTF, or NPSC. NPT ‐ National Pipe Thread Taper ‐ ANSI B1.20.1 Pipe Size Threads/Inch Approx. Length of Thread Approx. Number of Threads to be cut Nominal Outside Pipe Diameter Tap Drill 1/8" 27 3/8" 10 0.405" 1/4" 18 5/8" 11 0.540" 7/16" 3/8" 18 5/8" 11 0.675" 37/64" 1/2" 14 3/4" 10 0.840" 23/32" 3/4" 14 3/4" 10 1.050" 59/64" 1" 11‐1/2 7/8" 10 1.315" 1‐5/32" 1‐1/4" 11‐1/2 1" 11 1.660" 1‐1/2" 1‐1/2" 11‐1/2 1" 11 1.900" 1‐47/64" 2" 11‐1/2 1" 11 2.375" 2‐7/32" 2‐1/2" 8 1‐1/2" 12 2.875" 2‐5/8"
Quorum Technologies Glow Discharge
Quorum Technologies offer two glow discharge systems, MiniQ GD and the GloQube Plus . Preparation of TEM Grids using the GloQube Plus Transmission Electron Microscopy (TEM) is a high resolution imaging technique that provides details of internal structure and morphology of a wide range of specimens. Figure 1. Native ferritin on in-air glow discharged carbon TEM support, courtesy of Paul Simpson (Imperial College London). In TEM, a beam of electrons is focused by a condenser lens into a thin, coherent beam onto a sample. Electrons that are able to transmit through the sample are focussed by an objective lens into an image on a fluorescent screen. Areas of the sample where more electrons are able to transmit are observed as light areas, and those where few electrons are able to transmit are observed as dark areas on micrographs.1 TEM samples are prepared on specialised TEM grids, which support the specimen during imaging while allowing the electron beam to pass through. TEM grids are typically made of copper, nickel or gold, and coated with a carbon support film. Due to the hydrophobic nature of carbon, adhesion of water-based samples to the grid can be challenging. This application note explains the reasons for using glow discharge in the preparation of TEM samples and highlights the differences between glow discharge techniques. What is Glow Discharge? A glow discharge plasma is a partially ionised low-pressure gas. It is produced by application of a high voltage across two electrodes in a low pressure chamber (10-1 to 10-2 mbar). Electrons are accelerated by the electric field and inelastically collide with neutral gas molecules, causing excitation and ionisation. This process generates free radicals and/or ions. The characteristic glow that is observed arises from photons emitted as excited species return to their ground states. The plasma is sustained through the attraction of positive ions to the cathode, where they bombard the surface, ejecting more electrons which continue the ionisation process.2 Figure 2. Illustration of in air glow discharge plasma. Why do we need to glow discharge TEM grids? 1. Remove contaminants Even freshly prepared carbon layers for TEM grids will have unwanted adsorbates like water and low molecular weight material (LMWM) on the surface, typically adsorbed from the air (Figure 3). These contaminants need to be removed by a glow discharge before using the grids to ensure optimal sample spreading. 2. Improve hydrophilicity and enhance sample adhesion The deposited carbon layer on the TEM grid has a variably charged surface that is usually hydrophobic, thus even spreading of water-based sample suspensions is difficult (Figure 4). Glow discharge can be used to increase the hydrophilic nature of TEM grids to aid sample adhesion. 3. Improve sample contrast Observation of biomolecules in ambient TEM conditions also involves staining the sample with heavy metals. TEM supports that have not been treated with a glow discharge will result in uneven staining and cause poor contrast in the image. 4. Prepare grids for cryo-TEM Glow discharge of TEM prior to vitrification in cryo-TEM is an essential step. Not only will glow discharge reduce contamination and promote uniform sample spreading as mentioned previously, it also helps to promote thin vitrified ice formation and prevent particle clumping.3 5. Orient molecules to reveal areas of interest Chemical vapours can be used to tailor the TEM grid surface and influence the orientation of molecules on the surface. For example, alkylamines such as amylamine or hexylamine, can be used to functionalise the carbon support with amine-containing groups. These positively charged amine groups will attract negatively charged areas of the sample, and result in a preferential orientation. Other examples include the use of ammonia to create hydrophilic and mildly basic surfaces, and the use of organic thiols to introduce sulfhydryl groups to the grids surface. Figure 3. A typical TEM grid with carbon film and representation of surface adsorbates. Figure 4. A: Carbon support TEM grid before glow discharge with a droplet of water showing its hydrophobicity and corresponding contact angle shown below. B: Carbon support TEM grid after glow discharge with a droplet of water showing its hydrophilicity and corresponding contact angle shown below. Figure 5. Left: In-air glow discharge of 20S proteasome, showing mostly top-view orientation. Centre: TEM grid carbon support modified by blotting paper method with amylamine and used for 20s proteasome sample application. Right: In-amylamine glow discharged grid and application of 20S proteasome, using an automated valve system (GloQube Plus). Desired side-view orientation of the protein is achieved (89%). By using an automated valve system to introduce chemical vapour into the chamber, as used in the GloQube Plus, the in-chemical glow discharge can be achieved in a consistent and controlled manner (Figure 5). Glow Discharge Techniques To ensure even spreading of a sample across the TEM grid, it is crucial that an appropriate glow discharge method is selected. Depending on the application, glow discharge can either be completed in air or in chemical vapour. Table 1 shows examples of suggested surface modifications for specific key applications. Table 1. Key examples of glow discharge techniques used for different applications. Application Atmosphere Surface Type Surface Charge Advantages TEM grids Air Hydrophilic (-) No aggregation of particles on the grid square boundaries Nucleic Acids (TEM grids, Mica, HOPG*) Air Hydrophilic** (+) Improved binding of nucleic acids to surface Positively charged proteins (TEM grids) Hydrocarbons Hydrophobic (-) Covalent binding to the grid surface for positively charged molecules Negatively charged proteins, antibodies + nucleic acids (TEM grids) Alkylamines Hydrophobic (+) Covalent binding to the grid surface negatively charged molecules *Highly Oriented Pyrolytic Graphite ** Followed by treatment with 5mM magnesium acetate or 0.1% w/v poly-L-lysine Case Study: Ferritin Ferritin is the primary intracellular protein for iron storage and transport in most living organisms. Its unique nanocage structure can store up to 5000 iron atoms, which are transferred to and from the core through hydrophilic 3-fold channels.⁴ TEM is a useful technique to elucidate the structure of ferritin, as well as study the mechanisms by which it functions. Due to the hydrophilic nature of ferritin, glow discharge is essential for TEM imaging (Figure 7). In this case study, the use of in-air and inmethanol glow discharge is used to demonstrate the necessity of glow discharge for TEM imaging of ferritin. Figure 6. Human H Ferritin structure (without Fe inside core) solved using X-ray diffraction. Structure was downloaded from PDB (1FHA) and visualised using Pymol.⁴ Figure 7. The effect of non- glow discharged carbon support TEM grid on retention, spread and staining quality of native ferritin sample solution. Low (6x10-4 μg/mL)and high (6x10-2μg/mL) concentrations of the protein were used. When used in low concentrations without glow discharge, ferritin is not retained on TEM surfaces (Figure 7A). Although increased concentrations of ferritin result in retention on the TEM grid, the formation of aggregates is observed (Figure 7B). Uneven charge on the grid resulting from no glow-discharge can also hinder effective staining with uranyl acetate which results in ‘light patches’ between the proteins (7C). As demonstrated, the use of glow discharge prior to sample loading allows retention of low concentration of ferritin, and promotes uniform staining of the sample to produce clear, accurate imaging. In-chemical vapour glow discharge can also be used to further study native ferritin. All ferritin molecules are made of 24 identical peptide subunits that fold into a spherical shell with a water filled cavity inside. This cavity is connected to the outside through channels with threefold and fourfold symmetry and is thought to provide permeation pathways for iron ions and protons, essential for proper functioning of ferritin as an iron depository. Apoferritin (an empty shell of ferritin) is also used as an ion cage for templated synthesis of nanoparticles- ZnSe or CdSe. Imaging of the iron ‘load’ stored in the ferritin nanocage plays a significant role in studying the uptake of iron and other metals. During the process of in-air glow discharge, highly reactive oxygen radicals (O• and OH•) are formed. The radicals result in oxidative damage of organic molecules, which results in the abstraction of Fe3+ ions from the ferritin nanocage. In-methanol glow discharge produces less reactive radical species (CH₃• and H•), and can therefore be used to prevent loss of iron load from the nanocage (Figure 8). Figure 8. TEM images of ferritin protein complex from horse spleen (Sigma Aldrich) applied to in-air and in methanol vapor glow discharged carbon support TEM grids. Case Study: 20s Proteasome Proteasomes are enzymatic proteins responsible for the proteolytic degradation of misfolded and short-lived regulatory proteins within eukaryotic cells. This process occurs through the ubiquitin proteasome system (UPS), whereby target proteins are tagged through bonding with ubiquitin and are subsequently identified by 26S proteasome.⁵ 26S proteasome comprises of two main parts: the 20S core particle and the 19S regulatory particle. The study of the catalytic core (20S proteasome, see Figure 9) is key to understanding diseases like cancer and neurogenerative disorders. Figure 9. Left: Top-view of 20s proteasome structure. Right: Side-view of 20s proteasome structure. Structure was downloaded from PDB (6RGQ) and visualised using Pymol.⁶ In-alkylamine glow discharge can be used to produce positively charged, hydrophobic films on carbon support grids. This technique can be used to attract negatively charged areas of interest on proteins and result in the preferential orientation of proteins. ⁷ In this case study, carbon support TEM grids were modified in a GloQube Plus using an amylamine vapour glow discharge process to achieve hydrophobic and positively charged surfaces that are functionalised with amines to retain side-views of 20s proteasome complex. The effect of altering the surface charge of carbon support film on the orientation of the protein complex can be seen in Figure 10. Figure 10. TEM images of 20s human proteasome complex showing the effect of altering the surface charge of the carbon support film on the orientation of the protein molecules. Carbon film of 2.5nm thickness on Quantifoil 1.2/1.3 400 mesh was used as a support for the sample.⁸ The 20s human proteasome complex sample in TRIS buffer solution (3x10-2μg/mL) was applied on three types of carbon support TEM grids: no-glow discharge, glow-discharge in air; and glow discharge in amylamine vapour. Where no glow discharge was applied (Figure 10A), some side-views were observed due to the fact that freshly prepared carbon surface is non-uniformly charged and hydrophobic. After treating the grids with in-air glow discharge (Figure 10B), only top views could be seen as this treatment makes carbon films negatively charged and hydrophilic. This attracts the positively charged top/bottom part of the proteasome complex resulting in biased top-views orientation. When in-amylamine vapour glow discharge was used (Figure 10C), the majority of the 20s proteasome complex molecules were observed in the side-view orientation. REFERENCES: 1. De Graef, M., 2003. Introduction to conventional transmission electron microscopy. Cambridge university press. 2. Bogaerts, A. The glow discharge: an exciting plasma! J Anal At Spectrom 14, 1375–1384 (1999). 3. Wang, L. & Zimanyi, C. M. Cryo-EM sample preparation for high-resolution structure studies. Acta Crystallographica Section F 80, 74–81 (2024). 4. Lawson, D. M. et al. Solving the structure of human H ferritin by genetically engineering intermolecular crystal contacts. Nature 349, 541–544 (1991). 5. Unno, M. et al. The structure of the mammalian 20S proteasome at 2.75 Å resolution. Structure 10, 609–618 (2002). 6. Toste Rêgo, A. & da Fonseca, P. C. A. Characterization of Fully Recombinant Human 20S and 20S-PA200 Proteasome Complexes. Mol Cell 76, 138-147.e5 (2019). 7. Dubochet, J., Ducommun, M., Zollinger, M. & Kellenberger, E. A new preparation method for dark-field electron microscopy of biomacromolecules. J Ultrastruct Res 35, 147–167 (1971). 8. Morris, E. P. & Da Fonseca, P. C. A. High-resolution cryo-EM proteasome structures in drug development. Acta Crystallogr D Struct Biol 73, 522–533 (2017).
Sputter Coating in Electron Microscopy
Physical vapour deposition is a process that has been in use for over a hundred years and is used widely within current technology and manufacturing. The process involves feeding a substrate into a vacuum chamber containing two powerful magnets where a controlled gas is added and the magnets pull atoms from the substrate. The atoms collide with each other in their gaseous state before condensing into a plasma and drying into a thin film on the substrate. The final product is a thin but durable coating which is very useful across a variety of applications. The choice of target material to coat depends on the application. Applications include Semiconductors - electronics incorporate essential components which have been produced with tantalum sputtering targets. These include microchips, memory chips, print heads, flat panel displays as well as others. Glass Coating - Sputtering targets are used to produce low-radiation coated glass (Low-E glass) which is commonly used in building construction with its ability to save energy, control light, and aesthetics. Solar Cell Coating - Third generation, thin-film solar cells are prepared using sputter coating technology. Metals: Many metals are good electrical conductors but may have other unsuitable properties for the intended application. Headlight housings and CDs, aluminium is preferred for high optical reflection. SEM requires a good electrical conductivity using a very thin coating, a coating material that will not tarnish and possess a very fine grain structure. Greater resolution is possible with higher atomic number metals. Oxidisation: Most oxidising metals are not well regarded for SEM coatings except for chromium, which gives possibly the finest deposit of any metal. The target and coated specimens should be kept in a non-oxidising atmosphere for long term storage. This could be under vacuum or, more cost efficiently for large specimen collections, in a dry nitrogen gas desiccator. Coating thickness: Thick specimen coatings can hide small structures and very thin layers can form islets which may eventually merge. These islets are the graininess that may be seen at very high magnifications. The finest coatings are achieved by the simultaneous evaporation of carbon platinum; or tungsten (requires an electron gun). The finest coating using a sputterer is with chromium, followed by iridium, platinum, gold/palladium amalgam and gold. Carbon is very fine but has too low an atomic number (soft) for high resolution. Mounting: Most targets are held by a cover or are crimped around the edges, if not, it must be glued. The 'glue' also must be electrically conductive. ‘Professional mounting’ uses a few slivers of indium wire on the support plate, cover with the target and then either place in an oven with a flat weight on top of the target, or use a smoothing iron to heat the gold and melt the indium. A piece of lens tissue on the target will protect it. A temperature a little higher than melting point of indium is required (156.6°C) for a short time. Alternatively silver conductive paint, preferably a few small drops of paste is workable. Conductive paste can also be made with silver powder and commercial epoxy. Only spot gluing is required but keep weight on the target while drying/curing. Sputter coating for SEM When a target is bombarded with fast heavy particles, erosion of the target material occurs, this is termed sputtering. The arrangements of the systems are such that some of the sputtered atoms will condense on the surface of the specimen to be coated. The above process occurs under conditions of a gaseous glow discharge between an anode and cathode. It can be enhanced by the choice of a suitable gas and target material, which together with other developments of the technique, allows the deposition of a suitable coating to increase the electrical conductivity and increase "Z" number for greater resolution. These are common and important requirement for Scanning Electron Microscopy. The development of sputter coating systems embodies significant empirical design features, however, an understanding of terms such as "glow discharge characteristics" are important to these systems and may assist in the comparison of differing systems. If an inert gas such as argon is included in a cathode gas tube, the free ions and electrons are attracted to opposite electrodes and a small current is produced. As the voltage is increased some ionisation is produced by collision of electrons with gas atoms, the 'Townsend' discharge. When the voltage across the tube exceeds the breakdown potential, a self sustaining glow discharge occurs, characterised by a luminous glow. The current density and voltage drop remains relatively constant, the increase in total current being satisfied by the area of the glow increasing. Increasing the supply voltage increases current density and voltage drop, this is the abnormal glow region. Further increase in supply voltage concentrates the glow into a cathode spot and arc discharge is apparent. The operating parameters of sputter coaters are within the glow discharge regions of the characteristic described. Glow Discharge Once the condition for a sustained discharge is met, the tube exhibits the characteristic glow discharge, so called because of the associated luminous glow. It has been established that free ions and electrons are attracted to opposite electrodes producing a discharge; however, for a discharge to be self-sustaining requires regeneration of the electrons by the positive ion bombardment of the cathode. This produces secondary electrons and enhances ionisation. The resulting positive ion excess creates a positive space charge near the cathode. The voltage drop experienced is termed the cathode fall. If the discharge is established in a long narrow tube it exhibits the characteristics indicated. The positive ion density in the Crookes dark space is very high, as a result a significant voltage drop is experienced between it and the cathode. The resulting electric field accelerates the positive ions which produce secondary electron emission from the cathode. These electrons are accelerated in the direction of the anode and cause ionisation, generating positive ions to sustain a discharge. Subsequently, excitation of the gas results in intense illumination in the negative glow region. From this stage the electrons have insufficient exciting or ionising energy, resulting in the Faraday dark space. Towards the anode, a small accelerating field can produce ionisation and excitation, the gas again becoming luminous. Sputter Coating It has been indicated that under conditions of glow discharge, ion bombardment of the cathode will occur, this results in the erosion of the cathode material and is termed plasma sputtering, the subsequent omni-directional deposition of the sputtered atoms forming coatings of the cathode material. This process is enhanced in sputter coaters for use in SEM. For these applications the objective is the provision of an electrically conductive thin film representative of the surface topography of the specimen. Such films inhibit 'charging', reduce thermal damage, and enhance secondary electron emission. The most common arrangement for a D.C. (Direct Current) sputter coater is to make the negative cathode the target material to be sputtered (typically gold) and the location of the specimens the anode (which is usually 'earthed' to the system, so the specimens are effectively at 'ground' potential). The desired operating pressure (relative vacuum) is obtained usually a two stage rotary pump. An inert gas, such as argon, is admitted to the chamber by a fine control valve. Ultimate spatial resolution attainable in an SEM depends on several factors, and most importantly the average atomic number of the specimen. As a guide, the atomic number of the coating element is averaged with that of the specimen. For example, carbon (evaporated) onto a biological sample may have an average atomic number eight. A biological specimen coated with gold (79) could be given an average atomic number of 43. It is not practical to evaporate uranium which is toxic and would also oxidise during the process. The most popular target metals for SEM are from the platinum group of elements, palladium, iridium and platinum. Gold is most used for sputter targets in conventional SEM. Finer thickness deposits are only required when using magnifications over ~40k; for that usually a FESEM or a TEM is required. Gold and gold/palladium sputter well using base model sputter coaters running on a rotary vane pump only. Some other metals can also be sputtered from these simple instruments. However, oxidising metals (chromium especially) require a high vacuum pumped system and several of the heavier metals are much better sputtered by a larger instrument with a more generous power supply. Iridium is a very brittle metal and these targets are generally 0.3mm thick - which makes them expensive. Quorum Q300T Sputter Coating Workflow OPERATING CHARACTERISTICS The glow discharge in sputtering is significantly dependent on the work function of the target material and pressure of the environmental gas. A range of target materials are used including gold, gold-palladium, platinum and silver, although gold is the most common having the most effective electrical conductivity characteristics. The sputter head and sputter power supply should be effective over a range of anticipated target materials. The deposition rate is current dependant and when operated in the correct glow region of the characteristic previously described, several fold changes in current should be available for a relatively small change in sputtering voltage. The deposition rate should not be sensitive to small changes in pressure which may be experienced in the system. If an efficient sputter head design, operating on low voltage and as a result low energy input, is achieve, then radiant heating from the target and high energy electrons, (potentially the most significant sources of damage to delicate specimens) should be considerably reduced. There is evidence that such a sputter head system may also produce finer grain size for a given target material. The presence of an inert gas which will not decompose in the glow discharge is obviously desirable. Argon, having a relatively high atomic weight, provides a suitable source of ions for effective bombardment of the target material. The effectiveness is also dependent on the mean. The Micrograph (shown on right) is 3-day old concrete, freshly fractured. This is a typically difficult sample as the surface is highly granular and uneven and therefore susceptible to charging during SEM. However, after coating in the K550 such problems were not encountered. (Coating conditions: Gold, 20mA, 2 minutes, 0.1 torr, coating thickness 11nm). It is, of course, possible to satisfy very precise parameters by the selection of target material, voltage deposition current and vacuum. Under these conditions, it is possible to achieve thin films to 10nm with grain sizes better than 2nm and temperature rises of less than 1°C. The application of sputter coating has been well established. However, the improved performance of conventional Scanning Electron Microscopes requires the enhanced capabilities of EMITECH series of sputter coaters. Cathode target material is commonly gold, however, to achieve finer grain size, and thinner continuous coatings for Field-emission SEM (FSEM), it is advantageous to use cathode target materials such as chromium. To achieve sputtering with this target material requires vacuums better than those achievable with a Rotary Vacuum Pump and best provided by a turbo pump system. For chromium coating these systems require further refinements and they are offered as "Chromium Sputter Coaters". Please email PST with any questions - inquire via support@proscitech.com Full collection of Sputter Coaters Full collection of Sputter Targets
Sputter Target Materials and Mounting
ProSciTech is one of the best suppliers of targets ‐ anywhere. We supply high purity sputtering targets, and these are well produced and modestly priced. Available are a wide range of unmounted disks or washer (annular) type sputtering targets, as well as targets mounted on backing plates, replacing the original target holders. Often targets are mounted to a backing plate using a conductive glue. Alternatively, if the target is over‐sized, the edge may be formed around the edge of the backing plate ‐ if the target material is soft. If that is your preferred method, please order the target with at least 2mm greater diameter than the backing plate is. Additionally we can source many other sputtering targets in a variety of materials including ceramics, rare earth elements and metal oxides. What diameter target do I need? Manufacturer Models Ø60mm Ø57mm Ø54mm Ø50mm Ø19mm Agar Manual / Automatic X Anatech/Technics Hummer I / II / III / Jr X Bal-Tec SCD 005 / 040 / 050 / 500 X Bal-Tec MED 010 / MED 020 Bio-Rad SC5200 / SC502 X Cressington 108manual / 108auto X Cressington 108auto/SE X Cressington 208HR X Denton Desk II / II / IV / V X Edwards 150B / Scancoat X Emitech SC7620 / SC7610 X Emitech K500X / K550X / K650X X Emitech K575XD / K675X / K675XD X Emitech K575X / Q150 X EMS 150R / 300R / SC7620 X Emscope SC502 X Fullam EffaCoater / EMS-76 X Gatan 681 Ion Beam Coater X Gatan 682 PECS Ion Beam Coater X Gatan 685 PECS II Ion Beam Coater X ISI ISI 5400 X JEOL JFC-1200 / 1300 / 1400 / 1600 X JEOL JFC-2300HR X Leica EM SCD 005 / 050 / 500 X Leica EM MED 020 X Leica ACE 200 / ACE 600 X Pelco SC4 / SC5 / SC6 / SC7 X Polaron / Bio-Rad E5000 / E5200 / E5400 / E5800 X Polaron / Bio-Rad PS 3 X Polaron / Quorum SC7620 / SC7610 X Quorum Q150 Series X Quorum RotaQ/TurboQ X Quorum Q300 Series X Safematic CCU-010 LV / CCU-010 HV X SPI SPI-Module X SPI SPI Super X Sputter target materials that we can supply: W, Ag, Ti, Ce, ZnS, Au/Pd 60:40, Au/Pd 80:20, Zirconium oxide with 10‐15%wt Y2O3, Cr, Au, Al, Pt, Ir, Cu, Ag/Cu 90:10, Fe, Si micro‐crystalline, Si polycrystalline, Si n‐type, Si p‐type, Si undoped. Metal targets unless otherwise stated are 4N pure ‐ which is 99.99%. Note: All listed targets made from Au/Pd are in the ratio 60:40. Choosing your target material: Your choice of target material depends on the application. High optical reflection is preferred for headlight housings and CDs, aluminium. Most SEM users require (a) good electrical conductivity using a very thin coating, (b) the coating material should not tarnish and (c) possess a very fine grain structure. Greater resolution is possible with higher atomic number metals. Many metals are good electrical conductors (including Cu, Ag, Al), but they may not have other suitable properties. A thick specimen coating would hide small structures in the same way an increasingly thick layer of snow will hide landscape features. Very thin layers of sputtered (or evaporated) metals form islets which eventually merge. These islets are the graininess that may be seen at very high magnifications. Most oxidising metals are not well regarded for SEM coatings; the exception is Cr, which gives possibly the finest deposit of any metal. The target and coated specimens should be kept in a non-oxidising atmosphere for long term storage. This could be under vacuum or, more cost efficiently for large specimen collections, in a dry nitrogen gas desiccator ( see our vacuum desiccators ). Ultimate spatial resolution attainable in an SEM depends on several factors, and especially the average atomic number of the specimen. As a guide, the atomic number of the coating element is averaged with those of the specimen. So carbon (evaporated) onto a biological sample may at best reach the average atomic number eight. A biological specimen coated with gold (79) could, somewhat arbitrarily, rate an average atomic number of 43. It is not practical to evaporate uranium which is toxic and would oxidise. The most popular target metals for SEM are from the platinum group of elements (Pd, Ir, Pt). The finest coatings are achieved by the simultaneous evaporation of carbon platinum; or tungsten (requires an electron gun). The finest coating using a sputterer is with Cr, followed by Ir, Pt, Au/Pd amalgam and Au. Carbon is very fine, but too 'soft' (low atomic number) for high resolution. Gold is most used for conventional SEM and is perfectly satisfactory. Finer deposits are only required when using magnifications over ~40k; for that usually a FESEM or a TEM is required. Gold and gold/palladium sputter well using base model sputter coaters running on a rotary vane pump only. Some other metals can also be sputtered from these simple instruments. However, oxidising metals (Cr especially) require a high vacuum pumped system and several of the heavier metals are much better sputtered by a larger instrument with a more generous power‐supply. Iridium is a very brittle metal and we can only produce these targets at least 0.3mm thick ‐ which makes them expensive. Amalgams are more expensive to produce. So if an amalgam is sought to attain a finer coating, then it may be better to purchase Pt in lieu of Au/Pd, or Ir in lieu of Pt/Pd. Most used for metal coatings in electron microscopy is gold. Evaporated or sputtered gold until it's a fairly thick coating forms islets and these give gold coating at high powers (> x30k) a granular appearance. Comparative sputter data for Iridium and other materials on K575X sputter coater Samples were coated using an Emitech K575X Sputter Coater and were examined using a Hitachi S-5200 Field Emission SEM. Pictures are courtesy of Linda Dailey, Emitech Products Inc. Gold Magnification 15,000X 100,000X 300,000X Coating Time: 10 seconds 10 seconds 10 seconds Current Used: 20 mA 20 mA 20 mA Gold/Palladium Magnification 15,000X 100,000X 300,000X Coating Time: 10 seconds 10 seconds 10 seconds Current Used: 20 mA 20 mA 20 mA Chromium Magnification 15,000X 100,000X 300,000X Coating Time: 30 seconds 30 seconds 30 seconds Current Used: 100 mA 100 mA 100 mA Iridium Magnification 15,000X 100,000X 300,000X Coating Time: 10 seconds 10 seconds 10 seconds Current Used: 20 mA 20 mA 20 mA No Coating Magnification 15,000X 100,000X 300,000X Coating Time: N/A N/A N/A Current Used: N/A N/A N/A Platinum Magnification 15,000X 100,000X 300,000X Coating Time: N/A N/A N/A Current Used: N/A N/A N/A Magnifications provided are indicative and will vary with reproduction and monitor size used. Comparative sputtered films using the Q150T Turbo-Pumped Sputter Coater/Carbon Coater (Au, Pt, Ir and Cr) Professional Mounting Most targets are held by a cover or are crimped around the edge ‐ if yours is not that type it must be glued. The 'glue' used must be electrically conducting. Use a few slivers of indium wire on the support plate. Cover with the target and then either a) place in an oven with a flat weight on top of the target, or b) use a smoothing iron to heat the gold and hence melt the indium. Place something, like a piece of lens tissue, onto the target to protect it. Note: Use a temperature only a little higher than the melting point of indium (m.p. 156.6°C) for a short time. 4. Alternatively use a bit of silver conducting paint, preferably a few small drops of paste at the bottom of the jar. 5. You could also make a paste using our silver powder and a little commercial epoxy glue ‐ the method can be found here . Only spot-gluing is required, but keep a weight on the target while drying/ curing.
Testing Your Ultrasonic Cleaner
Testing procedure: Cut a section of aluminium foil the length and depth of the ultrasonic tank. Immerse section of aluminium foil into the liquid in a vertical hold. The foil must not make contact with tank bottom and be left for a duration of 30 seconds. A consistent pattern of perforation should result with hole sizes varying from 1 to 4mm in diameter. Foil can be purchased from any supermarket. Note: In larger ultrasonic cleaners more than one section of foil may be immersed. If the foil test is to be done at the beginning of the day, the fluid should be removed from the tank prior to the cleaning of instruments, as the solution left is foil contaminated.