Light Microscopy
17 articles
ToupTek™ ToupView™ Image Analysis Software
ToupView™ ToupTek™ Software ToupView™, previously known as ScopePhoto™ ScopeTek™, is image processing software professionally designed for our digital cameras. This software is supplied with our cameras. As well as basic image viewing and processing, ToupView™ allows scientific analysis of the captured image through the camera or other image capture devices. Prior to capturing an image, all adjustments - colour, contrast, brightness frame-speed - may be optimised. After capture, image files are saved in the desired image-format on the computer's hard drive. Once calibrated using a stage micrometer, the software allows measurement of lengths, angles and areas across microscope images. It is also possible to include a scale marker on micrographs and to count particles. For counting select Plugin and then Count, after that set parameters and Count. The main functions of ToupView™: Image archival with data attachment (*.PTL format, other formats are also supported). Image capture through VFW and Twain interface. Image cut & paste (image to image, image to file). Basic image operations are included in the Image menu for easy adjustment of the image. Image layer operation specially designed for measurement (Layer technique is used to separate the measured results and the different results can be put on different layers). Image processing filter for noise reduction and enhancement (Image enhancement filters, edge enhancement filters, morphology operation filters, user defined kernel filter, convolution and morphological filters). Image arithmetic operation between image and image, image and digital number (Including AND, OR, XOR, NAND, NOR, NOT, Add, Subtract, Multiply, Divide, Absolute, Min, Max, Average, Invert, Logarithm, Exponential, Square Root, Square, x to the power of Y operation). Image pseudocolor, range, segmentation operations are also include in the application. Image zooming and panning operations. STARTING PARAMETERS for capturing images with a ODCM camera Open ToupView™ (ScopePhoto™ ScopeTek™) and focus the specimen in the live capture window. Select Video Source Property from the Setup menu. White Balancetab Hold a sheet of white paper under the microscope so that the light source is projected onto it. Click Auto White Balance. The colour temperature will be set to a neutral grey (~20%). Exposuretab Tick Auto Exposure and click the Default button. Colourtab Adjust the gamma, saturation, and contrast as desired. Note: The white balance will not change unless the colour temperature is changed. This can occur when increasing the intensity of a light source, changing the bulb, or using a different microscope. Using the white paper method to achieve a neutral grey will adjust the white balance to different light sources/intensities. Information: http://en.wikipedia.org/wiki/Contrast_(vision) http://en.wikipedia.org/wiki/Exposure_(photography) http://en.wikipedia.org/wiki/Image_editing http://en.wikipedia.org/wiki/Dynamic_range http://en.wikipedia.org/wiki/Gamma_correction CALIBRATION Required is a known length for calibration (e.g. a stage‐micrometer). Place this under the microscope so that it is visible in the computer's monitor. Ensure the video window is set to 100%. Select the "Define Software Power" icon. Choices are: horizontal line, vertical line and any line. For calibration purposes, horizontal line is recommended. A cross‐line will be super imposed. Using the mouse drag the cross‐line to a starting point, then again, left click, and then move to the measurement's end‐point and click again. Consider. Measurements may be more accurate if always starting and end‐points always are from the beginning of a graduation. The calibration should include as much of the calibration ruler as can fit onto the screen at the used magnification setting. The centre of the screen will be the least distorted part of the image. When the end‐point of the measurement has been selected, the "Define Software Power" window appears. Index 1 and 2 and co‐ordinates denote the location on the image where the measurement was made based on the horizontal and vertical rulers surrounding the image. Under "Actual Length" enter the length that was measured and select the unit of measurement. Then select "OK". The "Software Power" window will appear. Select "Add". Enter the total magnification (i.e. objective x eyepiece power) then click "OK". This will be used indicative, to identify a particular calibration, but is unlikely to be accurate.) This completes the calibration for this particular magnification. Click "OK". Repeat steps 1‐7 to calibrate other magnifications on your microscope. All calibrations will be stored in the system for future measurements. Note: If other microscopes are being used with the software, they will also have to be calibrated. MEASURING Before measuring, ensure draw preferences are set as desired. Select Options and then Draw. Note: Ensure correct "software power" is set (the nominal magnification on the microscope) Measuring on a live image (e.g. not a jpg) Quick measurements can be made on the live image. Click the ruler icon and select the type of measurement required (length or angle). A crosshair will appear allowing point‐to‐point length or angle measurements to be made (depending on the type of measurement selected). Note: These one‐off measurements will not be saved. Batch measurements can be made for angle and length measurements. Select Batch Measurement and the type of measurement required. Again, a crossline will appear. Make the measurement as described above. The batch measurement function is particularly useful for making the same measurement on multiple samples. When measurements are complete, click "View Results". The following window will be displayed detailing all measurements made. Click the "Process" button to export results to Excel or a text file. Batch results may also be averaged. Batch measurements will only be saved if exported to Excel or a text file. Closing ToupView will delete all batch measurements. Before starting a new batch of measurements delete any other. Measuring on a captured image Measuring on a captured image has two advantages. Measurements can be saved on the image (useful for reporting/archiving) and they can be exported to Excel (image and measurement data). A measurement made on a captured image is stored on a layer over the raw image. After captured the image, select Layer from the menu and then New. Enter a name for this new layer. Inserting a layer on a raw image will activate the "Draw" menu and icons. Select the measurement type required. Select start and end point of measurement. Further measurements can be made on the same layer or new layers can be inserted for different measurements. Multiple layers are useful if a number of measurements need to be made on the image. Layers can be made visible or hidden as required. To save the image (with layers), select Save As from the File menu. For a raw image and layers to be saved as one file, it must be saved as the ToupView file type (.sft). This will allow the image to be opened in ToupView at a future date and for layers to be manipulated or added to ‐ if necessary. Saving as any other file type at this stage will not save layers. To print an image select Print from the File menu. The saved image can also be exported to Excel. The image including data will be exported to allow insertion in spreadsheets as required. If Export to Image is selected, a new image file will appear on the screen. Use the Save As function to save the file (complete with layers) as another file type (e.g. .bmp, .jpg etc.) for easy insertion in other programs, reports etc. COUNTING Counting how many particles with an image: With the desired captured image in focus, select "Plugin" from the "Menu Bar" and click "Count" To set the parameters within this plugin, click "Setting" → "Parameter" This panel will allow you to set your desired settings. Once you have adjusted your parameters, press "M&C" → "Count" The program will allocate a set number to each particle the is found Clicking on "Show All Results In Image" Selecting "M&C" → "Check the Result" display a list of found particles shown below.
Dunn Chemotaxis Chamber
GRAHAM A. DUNN Randall Division, New Hunt's House, King's College London, London, SE1 1UL, UK Design The Dunn Chemotaxis Chamber (SVDCC100) allows the behaviour of cells subjected to a linear concentration gradient of chemoattractant to be observed directly in the light microscope. The chamber was designed to have good optical properties and long-term stability of the gradient, thus permitting time-lapse recording of cell behaviour over many hours. The chamber consists of a glass microscope slide with two concentric annular wells ground into the centre of one face to a depth of about half the thickness of the slide (see figure). The annular platform that separates the wells (the bridge) is about 1mm wide. This bridge and the central pip are optically polished to lie precisely 20 ìm below the slide's face. Thus when the wells are covered with a coverslip carrying the cells to be studied, there is a gap between coverslip and bridge of 20 ìm. If the inner well of the chamber is filled with control medium and the outer well filled with a medium containing chemoattractant, a radially directed linear diffusion gradient becomes quickly established in this gap and is subsequently maintained for several hours. Setting up the chamber Cells are seeded onto a suitably washed sterile coverslip and allowed to settle prior to assembling the chemotaxis chamber. Special thick coverslips are provided that ensure dimensional stability, though other types can be used with care. Initially, both annular wells are filled with control medium and the coverslip seeded with cells is inverted onto the chamber in an offset position in order to leave a narrow filling slit at one edge for access to the outer well (see figure). After firmly seating the coverslip on the face of the chamber and blotting up surplus medium, it is sealed in place using hot wax mixture (Vaseline : paraffin : beeswax - 1:1:1) applied with a paintbrush around all the edges except for the filling slit. Take care not to apply any pressure directly over the bridge or you will crush the cells! In order to set up a chemotactic gradient, the medium is then drained from the outer well using a syringe with a fine-bore needle and replaced with medium containing the chemoattractant. The slit is finally sealed with the hot wax mixture. If the chemoattractant is a globular protein of around 20 kDa, the gradient becomes linear within approximately 20 min and has a half-life of about 24 h (Zicha et al., 1991). Large changes in molecular weight will have only a small effect on these times. Recording cell behaviour The chamber is placed on a temperature-controlled microscope stage with suitable provision for time-lapse recording of cell migration in the gradient. It is usually positioned by locating the outer edge of the bridge to coincide with the upper margin of the recording field, so that the direction of increasing chemoattractant concentration is vertically upwards in the image. The useful recording field is limited by the width of the bridge and can have a maximum size at the specimen of about 1 mm in the vertical direction. However, a motorised X-Y stage under software control will allow cell behaviour in several different fields over the bridge to be recorded during each time-lapse interval, thus maximising the data collected in a single experiment. Cells over the central pip region are not subjected to a gradient and simultaneous recording of their behaviour may be a useful control in some experiments. The design of the chemotaxis chamber, especially the blind inner well, helps to ensure stability of the gradient but it is advisable not to handle the chamber roughly during the course of an experiment. Cell tracking and analysis of chemotaxis The method of tracking cells in the recordings in order to obtain trajectories of cell locomotion will depend on the software available and specific details cannot be given here. Chemotaxis can be evaluated by assessing directional clustering of cell migration using standard methods for the statistical analysis of directional data (Zicha et al.,1997). References Zicha, D., Dunn, G.A. and Brown, A.F. (1991) A new direct-viewing chemotaxis chamber. J. Cell Sci. 99, 769-775. Zicha D., Dunn G. and Jones G. (1997) Analyzing chemotaxis using the Dunn direct-viewing chamber. Methods Mol. Biol. 75, 449-57. April 2006
EliSpot™ Slide Procedure
Cells can be stimulated directly in the antibody coated wells (Direct) or first stimulated in 24‐well plates or flasks, harvested and then plated into the coated wells (Indirect). The method used is dependent on the type of cell assayed the expected secretion frequency number of cytokine producing cells expected. Following stimulation the detection method is the same. Pre‐incubate 16‐well ELISPOT Slides with 100µl of 70% ethanol for 10 min at room temperature. Empty wells by flicking the 16‐well ELISPOT Slide over a sink and tapping it on absorbent paper and wash three times with 100µl of PBS. Dilute capture antibody according to kit instructions and dispense 100µl into each well. Incubate 4 hours to overnight at 4°C. Empty wells and wash once with 100µl of PBS. Dispense 100µl of 2% skimmed dry milk or other blocker in PBS into wells. Cover and incubate for 1 hour at room temperature. Wash 16‐well ELISPOT Slide once with PBS. Dispense into wells, 100µl of cell suspension containing the appropriate number of cells and appropriate concentration of stimulator. Cells may have been previously in‐vitro stimulated (Indirect ELISPOT). Cover and incubate cells at 37°C in a CO₂ incubator for an appropriate length of time (15‐20 hours). During this period do not agitate or move the 16‐well ELISPOT Slide. Empty wells by flicking 16‐well ELISPOT Slide over a sink and gently tapping it on absorbent paper. Dispense 100µl of PBS‐0.1% Tween 20 in wells and let sit for 10 min at 4°C. Wash wells three times with PBS‐0.1% Tween 20. Dilute 100µl of reconstituted detection antibody into PBS containing 1% BSA. Dispense 100µl in wells, cover and incubate 30‐60 min at 37°C. Empty wells and wash three times with PBS‐0.1% Tween 20. Detection can be performed using streptavidin‐alkaline phosphatase conjugate in PBS‐1% BSA, followed by washing and addition of substrate. Rinse wells three times with distilled water. Dry wells. Read spots. Upper structures may be removed from both plastic and glass slides for storage. The sealing gasket may be removed cleanly from the plastic slide but not the glass slides. After removal of upper structures, slides can be stored at room temperature away from direct light. EliSpot Slides are for Laboratory Use Only
Fertility Semen Counting Chamber
Important: The metallised surface of the counting cell is delicate. It is composed of metallic Rhodium ‐ a few atoms thick ‐ fused into the glass surface. Cleaning: Great care should be taken not to scratch this surface either with capillaries in use or by abrasion whilst cleaning. Never rub or brush the chamber. Clean by immersion in warm water or in the most dilute solution of a neutral detergent cleanser. Sterilisation prior to cleaning may be by immersion in alcohol, if suitable, or by immersion in a 2% solution of glutaraldehyde. Method of use: Total count: Allow 15‐20 minutes for the semen sample to liquefy completely. Place 4.5µl of sample centrally on the counting chamber's metallised cell platform. This sample may be measured using a capillary pipette in conjunction with an aspirator. Dilution of the sample may be necessary if it is too viscous for pipetting and the factor of dilution; i.e. 1 in 2, 1 in 4 etc noted. If dilution is required the sample should be thoroughly mixed before pipetting. The coverslip is placed firmly over the counting chamber and the counting performed in the usual way, under a microscope typically of x 200 magnification. As the grid squares are 0.1 x 0.1mm and the depth of the cell 0.01mm, the volume of original sample corresponding to one square is 0.0001µl. It is convenient practice to count the sperm cells in 10 squares and to multiply this count by 1 million for an undiluted sample or by 1 million times the dilution factor for a diluted sample. This provides a count of cells per ml. Motility: Due to the fact that sperm cells are trapped in a mono‐layer their motility is easily assessed as follows: Count the immobile cells in a suitable number of squares and then count the moving cells in the same square. Repeat this procedure a number of times and then calculate the average percentage motility.
Howard Cell Counting Chambers
The material to be examined should be a pulp. Mix a small quantity with water until the solids of the diluted pulp are between 8.37% and 9.37%. This corresponds to an Abbe refractometer reading at 20°C of 1.3460. Spread a small drop of the well‐mixed sample with the end of a glass rod over the counting chamber. Place the cover glass on to the counting chamber and carefully press down the shoulder of the chamber until Newton's rings are visible. Prepared samples containing air bubbles beneath the cover glass or an over‐full moat should be discarded. If using a compound microscope, examine using the X10 eyepiece and the X10 objective. Systematically examine all 25 fields and note those with a presence or absence of mould filaments (hyphae). A field is regarded as positive if the aggregate length of not more than three filaments present exceed one sixth of the diameter of the field. This is a general description of how a Howard Cell is used. The results are interpreted as a percentage of positive fields observed in all the fields examined. Precise interpretation of the results is made by a statistical analysis of the sample and should be carried out in accordance with your own internal or published procedures.
Knittel Coverglass
KNITTEL Coverglass is made of a colourless borosilicate glass (D 263) with an optimum hydrolytic resistance HGB1. This ultra thin glass is produced by only two special glass companies worldwide and by patented technology working with very expensive platinum orifices for getting blister and striae-free glass. The "ultra-thin glass foils" are regularly checked by the Quality Control Systems of our supplier to ensure accurate chemical composition and glass thickness. Through our consistent developments and automatisation of the process and cutting technology, we have reached the optimum dimensional accuracy. Our coverglass is manufactured in accordance of the international standard ISO 8255/1 and is ready to use. D 263 M cover glass is a colourless borosilicate glass with a very low iron content. Its composition assures excellent resistance to chemical attack. D 263 M meets the requirements laid down in ISO 8255/1. The outstanding features of D 263 M include: Virtually colourless appearance Excellent internal glass quality with very low levels of inclusions, striae, bubbles, streaks, etc. High spectral transmission Exceptional cutting and grinding characteristics Excellent flatness Optimum resistance to chemical attack Refractive index finely adapted to microscopes D 263 M is used as coverglass in microscopy for medical, biological and research work. Spectral transmittance (d = 0.15mm) Transformation temperature is 557°C Between 0.15 und 0.40mm glass thicknesses, the light transmittance is ôVA = 91.8%. In the visible range of the spectrum D 263 M is without absorption. The excellent UV absorption properties makes D 263 M an ideal material for use in fluorescence microscopy. Optical properties: Refractive indices (directive values) ne (ë = 546.1nm): 1.5255 ± 0.0015 nD (ë = 589.3nm): 1.5230 Abbe value íe : 55 Chemical properties: Hydrolytic class: HGB 1 (DIN?ISO 719) Thermal properties: Transformation temperature Tg in °C 557 (straining point: 529°C) EC Declaration of Conformity We herewith declare that the products Coverglass, water-white glass of hydrolytic class 1, precise cutting, in accordance with ISO 8255/1 Microscope slides, soda lime glass, cleanly washed and polished, in accordance with ISO 8037/1 are in conformity with the provisions of the following EC Directive: European Guideline 98/79 EG for in vitro diagnostics dated 27th October 1998 Dimensions Other thicknesses are available upon request. All data are subject to change. The accuracy of measurement for the thickness is ±5µm For trimmed glass (without the bead), flatness is measured net width x length. The maximum deviation of the height at any point on the total surface of the glass panel is measured from an ideal plane surface. The determined maximum value is the index for the flatness deviation. It does not, however, include the nominal thickness of the glass panel.
Lab-Tek Chamber Slides and Coverglass
Chamber Slide or coverglass products allow cell culture on a microscope slide or coverglass. A plastic upper structure mounted on a slide is removable after culture. Cells can be grown, fixed and stained on the same slide. Chamber Slide products are ideal for microscopic analysis of cells in culture. Growth Surfaces: Chamber Slide products are available with a soda lime glass, Permanox® plastic or polystyrene plastic slide. Chambered Coverglass products incorporate a #1.0 borosilicate coverglass as the growth surface. Slide surfaces are not coated with any biological or chemical reagents. Both Permanox plastic and polystyrene slide surfaces are cell culture treated for optimal cell performance. The polystyrene SlideFlask™ has a surface identical to other Nunclon® products. Fluorescence: Both glass and Permanox plastic slides may be used with most fluorescent labels. The polystyrene slide of the SlideFlask can be used with fluorescein with a blocking filter to remove red autofluorescence. Fixation: Permanox plastic is compatible with many standard fixation protocols (i.e., 3:1 Methanol: Glacial Acetic Acid, 3:2 Acetone: PBS, 2:3 Acetone Ethanol, 100% Methanol).3 Note: Permanox plastic slides will float when submerged in stain solutions. Weigh the slides or use a holder to keep them submerged during staining procedures. Cover slipping: Permanox slides are not compatible with non-aqueous mounting media containing xylene or toluene. For permanent storage, Permanox slides can be cover slipped with many aqueous mounting media. Manufacturing: Gasket is medical grade silicone. Permanox plastic and polystyrene are virgin materials. All components are non-toxic. All products are carefully tested to assure quality and sterility. Permanox Plastic: Permanox plastic is a plastic material, resistant to many acids, bases and some inorganic solvents. It has good transmission in the UV range and >70%T at 400nm. Flask Styles: The Flaskette Chamber (white cap) has a glass slide sealed with a gasket. It is useful with Lowicryl K4M embedding resin, where air inhibits polymerisation of embedding resin.1 The SlideFlask (black cap) features a polystyrene slide growth surface. A special sonic weld seals the slide to the upper structure without a gasket. The upper easily snaps away after culture. Do not use 100% acetone fixation with SlideFlask. The polystyrene slide is compatible with diluted Acetone, 3:2 Acetone: PBS and 2:3 Acetone: Methanol.3 Chambered Coverglass: This is a cell culture vessel with a #1.0 borosilicate coverglass as the growth surface. The very thin glass allows high magnification microscopy with inverted microscopes. Media structure of the Chambered Coverglass is not removable. References: Bou-Gharios, G. et al. J. Microscopy, 150, pt2, 161-163, 1998. NUNC™ TechNote: Compatibility of Different Mounting Media on Permanox® Slides. NUNC A/S: NUNC SlideFlask™ Procedures, August, 1986. NUNC Brand Product, Bulletin No. 13, 'Cell Adhesion And Growth on Coated or Modified Glass or Plastic Surfaces', 1998
Makler Semen Counting Chambers
Makler semen counting chamber INSTRUCTIONS: IMPORTANT: REMEMBER TO HEAT THE MAKLER AT 37ºC BEFORE USING IT (10 MINUTES IN THE INCUBATOR OR ON HEATING STAGE). Mix the material well, taking care not to generate bubbles. Place a little drop in the centre of the disc region using a wooden rod or pipette. Take hold of the cover glass with your fingers opposite the black dots, then position it on the four pins promptly. Gently press, watching for the emergence of colour fringes. The drop will spread across the whole surface of the disc to a thickness of 10 microns. After the cover glass is in place, avoid contact, lifting, and covering the chamber again, because this may disrupt the uniform distribution of sperm within it. Place the chamber on the microscope's stage using the handles. To correctly fit it, use the chamber grip. Sperm count: The sperm heads within the squares of the grid are counted in the same way blood cells are counted in a haemocytometer. If the quantity of sperm is significant, count them in a strip of 10 squares. The value represents their concentration in millions per ml. To determine the average, repeat this count in another strip or two. To maximize the reliability of the count result, it is advised that the count be conducted from 2 or 3 different drops of the material. In the event of oligospermic specimens, it is recommended that sperm be counted across the whole grid area. The concentration in millions per mL is then calculated by adding five zeros to the amount counted. Cleaning and preparation for reuse: Do not use tap water to rinse or wet the chamber. Simply dip the brush in water or a noncorrosive antiseptic solution and wash both sides of the glasses. Then, squeeze the brush to remove any residual water. Finally, use lint-free lens paper to dry the surface. Prevent touching the pin points as much as possible. The chamber can now be reused. Makler Sperm Counting Chamber for CASA is the same as the non-Casa system, with the exception of the cover glass which has no Grid
McMaster Egg Counting Chamber
Description: Worm egg Counting Slide for use in the estimation of HELMINTHIC infections in faecal suspensions: Slide = 76 x 32mm Platforms = 4mm wide Plain Glass Top = 52mm x 17.5mm cemented on 2mm up from lower edge of slide The cell depth of 1.5mm must be accurate. The underside of top cover is to be ruled with 1 x 10mm squares and with 10 equal distant vertical lines forming a 10mm square with 10 equal vertical columns. The 10mm square is to be in the centre of each cell. The cells are to be open at top and bottom. Method of use: Under 2/3 objective of the microscope all eggs are counted which lie within the lined centimetre square of the counting chamber. Each egg counted represents '100 eggs per g' of faeces. This calculation is based on the fact that the depth of chamber is 1.5mm and consequently the volume of fluid examined is 0.15ml, which is 1/300 of the original volume of 45mls, made up of 42ml of saturated salt solution and 3g of faeces. Therefore each egg counted represents 300 per 3g of faeces, which is equivalent to 100 per g. Where two chambers are employed the total count is multiplied by 100 and divided by 2. Cleaning: Clean by immersion in warm water or in the most dilute solution of a neutral detergent cleanser. Sterilisation prior to cleaning may be by immersion in alcohol, if suitable, or by immersion in a 2% solution of glutaraldehyde.
MetroChip Microscope Calibration Target Cleaning
THIS PRODUCT HAS BEEN DISCONTINUED BY SUPPLIER Metro Chip Cleaning Procedure The cleaning procedure largely depends on the nature of the contamination and what facilities are available at your site. Large particles can be blown off the sample by nitrogen gas sprayed from a nozzle, preferably with a point-of-use particle filter. Most facilities have high purity nitrogen plumbed in. Otherwise, use "Dust-off" cans. But avoid the kind that mention "Do not use on camera lenses." in their instructions. Use CDA (clean dry air) only if you are sure how it is produced. Some facilities use compressors that cause their air to have pump oil residues. In most semiconductor fabs and equipment manufacturer facilities, this has been resolved and CDA is quite pure and is fine to use. Rinsing the sample in DI water is safe and effective for removing larger particles. For removing smaller particles, use DI water in combination with ultrasound. For cleaning fingerprints, chemical baths that remove photoresist, if available, are effective. Any resist strip used in semiconductor manufacturing is safe, including sulphuric peroxide clean. (This requires a heated bath and well ventilated area.) Facilities such as analysis labs often have small stand alone plasma cleaners for sample surface preparation. One can also use those plasma sample cleaners with pure oxygen, or mixtures of oxygen and forming gas (N2/H2) for removing fingerprints and also for cleaning hydrocarbon residues formed as a result of inspection in a scanning electron microscope. Here are things that should be avoided: Wiping the sample with a cloth. This will destroy the features. Spaying the sample with gases that leave residues. Also immersing the sample in liquids that leave residues. Using cleaning solutions that attack silicon dioxide (glass). Any HF dip will attack the oxide and should be avoided. Avoid plasma treatments that attack silicon dioxide (glass). The list includes gas mixtures containing CF4 or CHF3 or similar gases. Naturally, harsher plasma treatments, such as those using chlorinated gases, should be avoided since the polysilicon can be etched.
Neon Bulb Array
EMS97036-01 The Neon Bulb Array is a tool which helps you to locate power peaks in your microwave oven. It shows you where to place your samples in the microwave, therefore, improving the reproducibility of your results. Procedure Make two photocopies of Figure 1. Be sure the number and position of circles on your drawing match your Neon Bulb Array. You will chart illuminating patterns on these maps. Place the Neon Bulb Array on the floor of the microwave oven. Place the corner with the dot against the oven's left, rear corner. Close the oven door. Program the oven to irradiate at 100% power for 30 seconds. Press start. Observe the illumination pattern of the bulbs in the left, rear quadrant of the Neon Bulb Array. On the drawing mark an "X" on each bulb that is continuously lit. Let the bulbs cool for two minutes. Do not remove the Neon Bulb Array. Repeat steps 3 through 5 three more times, one for each of the remaining quadrants. Place a beaker with 200 ml of room-temperature water in the right, rear corner of the oven. If necessary, remove enough bulbs from the Neon Bulb Array to make room for the beaker of water to sit on the mat. Repeat steps 3 through 5 one time for each of the four quadrants of the array. Compare the illumination patterns of each map. Place the maps in your notebook. Expected Observations When the microwave oven contains only the Neon Bulb Array, most of the neon bulbs will remain continuously lit. With the beaker of water in the oven, the pattern of bulb illumination will change dramatically. Few bulbs will be lit; those that light up may flicker. Precautions Do not irradiate the neon bulbs for more than 1 minute. They will become too hot to handle. Do not place the beaker of water on top of the bulbs. The bulbs may break and the water may spill. Instead, remove some bulbs to make room for the water load. Reference Login and Dvorak. (1994) "The Microwave Tool Book, a practical guide for Microscopists". Published and distributed by: Beth Israel Hospital, Department of Pathology, 330 Brookline Avenue, Boston, MA. 0215-5491.
Phase Contrast Test Slide
Description and Manufacture The Phase Contrast Test Slide was developed by the Health and Safety Laboratory (HSL), on behalf of the Health and Safety Executive (HSE), in collaboration with ULO Optics (formerly PTR Optics) and the National Physical Laboratory (NPL). The slide is known as HSE/NPL Test Slide. Its purpose is to provide a reproducible phase object to check microscope system performance prior to counting asbestos fibre samples. The master engraving was produced by the NPL using a ruling engine. It consists of seven bands of lines, with 20 lines in each band, progressively reducing in width and depth, from 1.1μm to 0.25μm wide. The lines were produced using a V shaped inscriber with a depth to width ratio of approximately 1:10. The bands are separated by 20μm gaps. A test zone is delineated by a rectangle bounded by deep groves, which can be viewed using a microscope with x100 magnification in either dark field or phase contrast mode. Figure 1 illustrates the bands of lines on a test slide and Table 1 gives the widths of the lines within each band and the associated phase contrast induced. Figure 1: An illustration of the bands of lines that form the basis of the HSE/NPL Mark III Test Slide Table 1: The width of the lines within each band and the associated phase contrast introduced when green light (Lambda = 530nm) passes through a line. Band Numbers Line Width (um) Maximum theoretical phase change for light (lambda=530nm)passing through a test line 1 1.08 6.6° 2 0.77 4.7° 3 0.64 3.9° 4 0.53 3.2° 5 0.44 2.7° 6 0.36 2.2° 7 0.25 1.5° Each of the HSE standard test slides is an epoxy replica of the NPL Master. An impression of the lines is produced in a resin of refractive index 1.58 and is mounted on a standard glass microscope slide (76 x 25 x 1.2mm). This impression is sealed in another resin of refractive index 1.485 and covered with a standard (0.17mm thick) glass coverslip. To maintain a steady level of quality between slides HSL assesses each one. Only the test zone bound by the four sets of intense lines (Figure 1) is examined assess the contrast of the band 4, 5, 6 & 7 and inspected for dust deposits that may cause light or dark spots. The whole slide is inspected for cracks, splits and other defaults in manufacture. Although these defects cannot be eliminated, all slides are considered to be satisfactory. HSL certifies the test slides as satisfactory for use to set up phase contrast microscope and categorises them into three sets: those that have band 4 fully visible and band 5 partially visible (red documents) those that have band 5 fully visible and band 6 partially visible (green documents) those that have band 6 fully visible and band 7 partially visible (yellow documents) Each slide is allocated a unique number and this is shown on the accompanying certificate. Records and computer images of each slide inspected are kept by HSL. Anyone experiencing difficulty in using the test slide may contact the Minerals and Fibres Section of the HSL.
Poly-L-Lysine Coated German Glass Coverslips
Handling Procedure Take a package from freezer and cut a bag of 15 pieces off the package – cut along middle seal line so that both bags remain sealed after cut – then put package back into freezer. Spray bag of 15 pieces with 70% ethanol in hood and let it dry completely. Cut to open bag and pour all coverslips into a well of a 6-well plate. Cover it and vigorously shake the plate side to side (horizontally) to loosen coverslip. Invert the plate so that the coverslips sit on the lid of the plate. Let coverslips roll along edge of the lid to separate from each other. Pick them up easily with sharp forceps. Store unused coverslips at room temperature in TC hood and use them within 2 weeks. (If longer storage is desired, place one coverslip per well and store the plate at 4°C. Optional: Pre-incubate coverslips in culture medium before seeding cells. Removing Coverslip from Well after Culture Create a lifting tool by bending a needle 0.5-1mm from tip. Use one hand with the lifting tool to lift coverslip from its edge and use other hand with forceps to hold and remove coverslip from well. Note: Do not drop the coverslip and remember which side contains cultured cell. Coating Compatibility Different cells require different coating substrates. If this type of coating did not produce satisfactory results, try a different coating substrate. Important Store in Freezer – Fragile Glass Inside Air shipped at room temperature Once received, store unopened bags at -20°C Expiration: 6 months at -20°C
S7 Stage Micrometer, England Finder Counting Slide
The England Finder is a glass slide marked over the top surface in such a way that a reference position can be deduced by direct reading, the relationship between the reference pattern and the locating edges being the same in all finders. The object of the Finder is to give the microscopist an easy method of recording the position of a particular field of interest in a specimen mounted on a slide, so that the same position can be re‐located using any other England Finder on any microscope. The England Finder consists of a glass slide 3" x 1" marked with a square grid at 1mm intervals. Each square contains a centre ring bearing reference letter and number, the remainder of the square being subdivided into four segments number 1 to 4. Reference numbers run horizontally 1 to 75, and letters vertically A‐Z (omitting I). The main locating edge is the bottom of the slide which is used in conjunction with either the left or right vertical edge of the slide, according to the fixed stops of the stage of the microscope, all three locating edges being marked with arrow heads. The label on the finder should always appear visually at the bottom left corner when through most microscopes the reference image will appear correct. Method of Use Mark the specimen slide with a label on the left indicating with arrows which sides are to be used for location. Place the slide on the stage of the microscope bringing the bottom long edge in contact with the base stops of the stage and then sliding either left or right into contact with the vertical fixed stops as appropriate. It is important always to obtain the main location of the slide and finder on the base stops first. Having examined the specimen in the normal way and found a point of interest, bring this to the centre of the field of view (a crosswire in the eyepiece is useful in this respect). Taking care not to alter the position of the fixed stops of the stage, remove the slide and replace with the England Finder, again bringing the bottom edge in contact first and sliding to the appropriate vertical stop, the label of the Finder being at the bottom left corner. The reference pattern of the Finder will now be seen through the microscope (adjusting the focus if necessary). The reference number of the main square is recorded followed by an oblique stroke and the number of the segment in which the centre of the field of view lies (1 to 4 or 0 if in the centre circle). The boundary lines of the main squares are easily distinguishable as these are the only continuous straight lines of the pattern. The reverse procedure is adopted to re‐locate the point of interest, The England Finder is placed on the stage as outlined above and the stage is adjusted until the appropriate reference square and segment appear in the centre of the field of view. Remove the finder and replace with the specimen slide with label to left and appropriate vertical slide in contact with the fixed stop, when the point of interest will appear in the centre of the field of view. Notes: When using high powered objectives with fields of view less than 1mm, it is advisable to adjust the turret to a lower powered objective, relocating the point of interest, before taking the England Reference. If it is not possible, first note the segment in which the point lies and then adjust the mechanical stage until the reference number of the main square can be found, taking care only to cross a curved, but never a straight line. To facilitate re‐location a small sketch indicating the position of the point of interest in the actual segment is very helpful with high power. When there are no fixed stops on the stage of the microscope, a fixed stop glass plate should be used, which we would be pleased to supply. This consists of a glass plate approximately 4" x 2", on which four fixed stops are cemented in the form of glass discs. Two edges are brought in contact with the appropriate three fixed stops of the plate, which is then moved on the stage until the point of interest is in the centre of the field. The plate is now clamped to the stage of the microscope with the stage clips and the slide and finder are interchanged as outlined above. It should be emphasised that only when the specimen slide is identical in size and shape to the finder will the references taken from the right or left edges be identical. As this will seldom be the case, where interchange between different microscopes is likely to be required, it may be convenient to take both right and left references, and in any case the references should be clearly pre‐fixed with the word right or left as appropriate. The glass fixed stop plate mentioned above is particularly convenient for taking readings from both right and left. The successful use of the England Finder is partly dependent on the use of an efficient stage with good fixed stops, and partly on the use of well cut specimen slides. These should have good clean edges and should be cut square. If you should experience any difficulty in obtaining suitable slide glasses, we should be pleased to supply these to you in a satisfactory quality. Care of the England Finder It is most important to avoid spoiling the locating edges and to keep these clean. Finders can be conveniently cleaned by soaking in Nitric Acid or solvents normally used for glass cleaning, polishing with a soft cleaning tissue or cloth, being careful not to scratch the glass surface. If you require a replacement finder, you will obtain the same references for the appropriate locating edges.
Thermanox Coverslips
GL081, GL082, GL083, G;084, GL085 Purpose: Cell attachment and growth is equal to or better than polystyrene, plastic or glass. Resistant to all commonly used solvents (xylene, acetone, acetic acid) so you can use them with most staining techniques and with regular mounting and embedding materials. Use with amyl acetate for EM preparations. Very low vapour and gas permeability properties. Suitable for use in scintillation counting. Warning COVERSLIPS ARE PACKAGED "RIGHT SIDE UP" To assure best orientation for cell growth, keep coverslips in package until ready for use. When removing from package. USE THE SIDE FACING LABELLED TOP OF PACKAGE FOR BEST CELL GROWTH. DO NOT FLAME THERMANOX coverslips. DO NOT HANDLE WITH RUBBER GLOVES OR OTHER RUBBER PRODUCTS. Most rubber is toxic to cells and the toxic agent is transferred to the THERMANOX surface. NOT RECOMMENDED for phase contrast microscopy or techniques involving fluorescent stains. Suggestions for Use Cut to special shapes or sizes with sterile scissors. To prevent scratches, handle only by the corner or edges, preferably with sterile forceps. A pick-up tab may be formed by bending one corner to a right angle with sterile forceps. Resterilise, if necessary, with 70% isopropyl alcohol or exposure to ultraviolet light overnight. After embedding, you may peel THERMANOX coverslips off the epoxy, leaving cells or other objects in the mounting material or you may section them with the mount. Note: THERMANOX coverslips may float due to air bubbles or surface tension. Air bubbles may form when the coverslip is placed in the medium, or if the medium is below incubator temperature. Bubbles may form as the medium comes up to temperature, releasing dissolved gas. Coverslips may be pushed to the bottom of a dish or other container with sterile forceps, pipette or gentle agitation. In Leighton tubes, a gentle shake will cause the coverslip to settle to the bottom of the vessel. Permanox Plastic; Non Fluorescent: Permanox plastic is nonfluorescent and may be used with most fluorescent labels. Permanox has better chemical resistance to fixation and staining solvents than polystyrene. An aqueous based mounting medium must be used when cover slipping to prevent warping. Coverslip treatment test: A simple method to determine which side of the coverslip is treated is the "droplet" test. A drop of water or culture medium will spread on the hydrophilic, treated side. - If the droplet forms a bead, then the side of the coverslip is not treated - If the droplet spreads evenly over the coverslip, the side is treated. To prepare for light microscopy: Fix cells grown on THERMANOX coverslips in methanol, acetone or other fixatives. Stain. Dehydrate in acetone. Clean in Xylene. Cover with a mountant. Place clear glass coverslip on top. Examine the cells through the glass coverslip and mountant for improved resolution. To prepare for transmission electron microscopy: Fix cells with routine fixative (glutaraldehyde) Post fix with OsO4. Rinse and prestain if desired. Dehydrate in ethanol followed by propylene oxide. Infiltrate with epoxy resin. Invert on previously polymerised blank resin block, wipe back and polymerise. Remove coverslip by inverting on a warm hotplate and peeling off or by touching coverslip to dry ice. The coverslip will peel off leaving the cells on the resin block. References BUCKLEY, C.E. Coverslips for Use in Tissue Culture. Laboratory Equipment Digest, May 1976 DETER, R. L. Quantitative Morphological Analysis of Early Mouse Embryogenesis in Vitro. Perfusion Culture System. Tissue Preparation; Sampling. Journal Embryol.Morph. 40:91-100. 1977 PAULI, B. U., ANDERSON, S. N., MEMOLI, V. A., and KUETTNER, K.A. Development of an in Vitro and in Vivo Epithelial Tumor Model for the study of Invasion. Cancer Research, 40:4571-4580. December, 1980. SWIERENGA, S. H. H., WHITFIELD, J. F., and MORRIS, H. P. The Reduced Extracellular Calcium Requirement for Proliferation by Neoplastic Hepatocytes. In Vitro. 14 No. 6. 527-535. 1978 WERB, Z., BAINTON, D. F., and JONES, P. A., Degradation of Connective Tissue Matrices by Macrophages. Journal of Experimental Medicine. 152:1537-1553. December, 1980. ZEMBALA, M., LEMMEL, E. M., and URACZ, W. Activation of Human Monocytes For Nitroblue Tetrazolium Reduction and the Suppression of Lymphocytes Response to Mitogens. Clin. Exp. Immunol. 41:309-316. 1980
Universal Worm Egg Counting Chamber
The Universal worm egg counting chamber is particularly designed for the quantitative estimation of the number of parasite eggs per gram of faeces in cattle, horses, sheep, goats, and small animals. The overall methods which utilise this information can estimate the degree of infestation in livestock and the efficacy of treatments. The counting chamber has 4 x 0.5mL chambers. Each chamber is subdivided by guidelines to five counting strips of approximately 0.1ml. Universal worm egg counting chambers feature guidelines in each chamber for easier counting of worm eggs. The guidelines were introduced to assist users in alignment during the scanning of individual chambers. The lines should only be used as a counting aid. The considered volume of each chamber is that area bounded by the inside of the thicker lines and NOT the area between each of the glass support strips. Remember that not all microscopes are the same. This can affect individual usage. The engravings are on the under surface of the top‐piece for floatation egg counts and are opaqued for improved contrast. The chambers feature: Wide front filling zone Silicone fillets between chambers Overhanging top‐pieces for better grip Silicone bonding to absorb minor impacts and withstand autoclaving and most cleaning agents.
Very Low Light CCD Camera for Fluorescence Microscopy
User note for fluorescence highest sensitivity 1.4mp monochrome CCD camera for microscope, OCCD01400KPA-C Key Features: Super quantum efficiency up to 65% Extremely low noise, down to 4e -rms 12bit dynamic range at the hardware level and 8 bit to the PC High resolution ( 1376 x 1040pixel) Shutter / exposure times from 500ns -1000s Shutter / exposure times from 500ns -1000s Binning ( H & V) Region of interest ( ROI) 10 frames per second at full CCD resolution Free software included Areas of Application: Laser induced fluorescence Luorescence microscopy Electron microscopy Red and NIR fluorescence applications Bioluminescence / chemo luminescence Spectroscopy Gel imaging Ion imaging Low light level imaging Semiconductor quality control Imaging of bio markers (e.g. green fluorescent protein, GFP) Technical Specifications: Sensor Format 2/3" SONY CCD 1.4M hardware pixels Pixel Size 6.45µmx6.45µm Resolution 1376x1024 Binning 2x2 , 4x4 Peak Quantum Efficiency 62% Dynamic Range 70.1dB A/D 12 bit Readout Noise 4..5 e-rms @ gain high /5..6 e-rms @ gain low Pixel Scan Rate 16MHz Spectral Range 350-1000(w/o IR cut) , 350-780 (with IR cut) Exposure Time 133ns-582s Smear < 0.002% Region of Interest down to 32x32pixels Power Supply USB port Operating Temperature -20°C ~ 40°C Operating Humidity 10%~90% Optical Input Window fused silica Data Interface USB2.0 Data transfer Direct show and Twain interface OS Windows 2000 (Professional-SP4)-Windows XP (Professional-SP2)-Windows VISTA-Windows 2008 PC requirement CPU: Inter Pentium4 2.8GHz or higher 1GB RAM or more 1 Hi-speed USB 2.0Port 17" or 19" Monitor Special Function: Live capture quick measure Video scale (e.g.100 x 20µm) Video Marker (e.g. size, colour, position can be changed) Video Date Marker Video Clarity Factor Manual Fusion (make image large depth of field) Auto Fusion (Need DAF Z-axis controller) Auto Focus (Need DAF Z-axis controller) Dimensions and Weight: Body length: 60mm (H) Body Diameter: 50mm (D) Cable length: 1.5m Body with USB cable: 350g Total unit with cable weight: 600g Package Size: 16.5mm (L) x 14.5mm (W) x 8.5mm (H) Package Includes: Digital Microscope C-mount Camera CD: Driver and Software (ScopePhoto, MiniSee) High-Speed USB2.0 cable (1.5m) User manual Optional Accessories:TS-M1 or TS-M2 Calibration Slide(Stage Micrometer) System Requirements: Intel Pentium 2.0GHz processor minimum (Recommend Dual core 2.8GHz or faster) Windows 2000/XP(SP2-SP3)/ Vista DirectX 9.0 or later 1GB RAM or more CD-ROM drive 32-bit colour graphics card 21" display monitor or larger Hi-speed USB 2.0 Port Download: Basic manual (PDF format 660KB)