Light Microscopy
17 articles
Adapting A Camera To Your Microscope
Connecting a camera to the microscope: digital cameras for microscopy are usually connected to a microscope by one of two means: Cameras with an integral eyepiece are inserted into the phototube of a trinocular microscope (ready to receive an eyepiece), or they can be inserted into one of the viewing eyepiece tubes after an eyepiece is removed. Alternatively the microscope has a C-mount adapter in lieu of the phototube. The C-mount camera has a thread which connects to the top of the C-mount adapter. Photo magnification is affected not only by the microscope and C-mount/ camera eyepiece, but also by the camera chip and the size of the computer screen. Most microscopists would aim for a magnification which is larger, but less than double the viewing magnification. Compared with the settings for the viewed magnifications, our cameras, using a 0.5x C-mount/ eyepiece and displayed on a 22" screen, average magnifications 1.9x greater in full screen mode and 1.4x in working mode. Particularly using macro photography or stereoscopes, a 1x C-mount is another means to obtain higher magnifications. A 1x C-mount used with a compound microscope and oil-immersion, results in several thousand times magnifications. Conventional microscopy is limited by the wavelength of light; magnifications above 1000x are increasingly blurred, and magnifications above 2000x have no merit. A 0.3x C-mount obviously results in lower magnifications, which can be useful, but in some systems may cause vignetting. Mounting a C-Mount Camera to your microscope This is what a c-mount camera looks like: You may be able to see the c-mount thread on this inside of the tube. This is the eyepiece to c-mount adapter that will allow any c-mount camera to fix the eyepiece tube microscope: Eyepiece sleeves are supplied with your camera to adapt to 30 or 30.5mm eyepiece diameters: You may need an adapter to mount a digital camera onto your microscope, or if you have a c-mount adapter already, then all you need is the camera. ProSciTech cameras fit onto the industry standard c-mount thread (Ø 25.4mm or 1 inch). If your trinocular microscope's port takes a standard eyepiece (Ø 23.2mm), you will need an eyepiece to c-mount adapter. To mount the camera on your scope, place the eyepiece to c-mount adapter into your third port and then screw the c-mount camera on top of this adapter. Using a monitor to view your specimen live Both a point and shoot and a DSLR cameras have a smaller screen for focusing the image - modern cameras usually have ports to attach a larger monitor via a cable. Alternatively you can use the camera’s WiFi or an Eye-Fi SD card. You can either plug the microscope camera into a computer and use a TV as a monitor - easy - or you use an DSLR camera that supports TV output. The latter option is more expensive and more difficult. Mounting the SLR camera to the microscope can be costly. We supply eyepiece adapters to the industry standard c-mount thread. This allows users to mount a camera onto binocular or trinocular scope where the third port does not have a c-mount. The biggest problem with point and shoot or DSLRs is mounting such a camera to a scope. Mounting an DSLR Camera to your microscope Digital cameras will take a decent image through an eyepiece, and if the eyepiece is large enough to avoid vignetting and if the camera can be held perfectly still in relation to the eyepiece, and eliminated stray light, then you can obtain great images. In Google you may find various ways of doing this. http://www.microbehunter.com/connecting-a-camera-to-a-microscope/ or http://www.truetex.com/micad.htm Basically you can either buy an expensive adapter from the camera’s manufacture or make a low-cost, DIY mount. Selecting the eyepiece to C-Mount adapter (Magnification and lens) The adjustable adapters allow you to change the distance between the camera and the specimen. The advantage of the adjustable adapter is that you can adjust the height of the camera to suit the focus of your eyepieces – par-focus. This way you will not need to readjust the focus when swapping between using the camera and the eyepieces. If you are very lucky, the third port on your scope would be at the correct height to have par-focus with a fixed adapter but this is unlikely and it’s best to use an adjustable adapter if you require par-focus. We normally recommend the 0.5x adjustable adapter (ODCMZ-EAA-5) because this way you can achieve par-focus. The 0.37x would give you lower power, which you may find advantageous, but it may (or may not) cause vignetting in the corners. Standard 10x eyepieces (oculars) are supplied with all ProSciTech microscopes. Other magnifications are available. 15x and 20x eyepieces are commonly used in stereo microscopes, these increase magnification. The objective is the more important lens group; eyepieces can only enlarge existing information projected by the objective. Final magnification in microscopy depends on several factors. In stereo or macro systems the magnification at the eye is the magnification of the objective multiplied by that of the eyepiece. If an additional lens is added to the front of the objective, both, photo and viewing magnifications, whereas the use of different eyepieces changes viewed magnifications only. Photo magnification is subject to other, additional factors. Determining the MP you require The more enlargement you need, or the greater the size of your screen, determine what MP camera is required. If you are not enlarging or printing the photographs, then a 3-5MP camera will be suitable. However, if you occasionally need to pick a central area of an images and crop a good deal or print to poster format, then you may wish for a larger format camera. The ODCM0510C is the most commonly sold camera. However, you may want to read the user note "Which camera: magnification versus resolution (Image DPI)", which will help you select the correct size camera you require. Lighting If you require top light on your stereo microscope, consider using a dimmable LED quadrant control ringlight. This ringlight has many advantages over other light sources. Software ProSciTech cameras come with a CD which includes image capturing processing software and drivers - more elaborate image analysis software is available. The provided software works with Windows (XP and later), Mac OSX, and Linux. The image processing software that comes with the cameras transmits a live image to a computer, and is capable of capturing still images and video. USB3.0 cameras are currently more expensive but offers faster transfer rates. This matters only with the very largest format cameras or if you require HD movie sequences. But please be aware that they require a USB3.0 port and are not backwards compatible with USB2.0 at this point in time. ProSciTech also have CCD cameras available. If you require to measure with your camera then you will also need a micrometer to calibrate.
Cleaning Optical Components and Instruments
Cleaning of plastic and glass lenses requires care and suitable supplies. The desired result is a clean and fibre‐free lens surface without scratching the lens surface or any coatings. To remove immersion‐oil from an objective lens Kimwipes are efficient and convenient. They absorb oil and they are non‐linting when used without rubbing. A drop of xylene applied to the Kimwipe cleans the remaining oil‐film on a lens. Kimwipes and lens tissues in particular are useful to protect delicate components. Lens tissues are low absorbent and less useful to remove immersion oil. When lenses require rubbing to remove unwanted dirt films or particles it is wise to initially blow‐off any dust particles which could scratch a fine surface; Puffers are suitable for this, blowing by mouth tends to add spittle to compound the problem. After removing lose particles, a glass, plastic or lens surface may require wiping ‐ without leaving dust and fibre particles. The most suitable, lint‐free materials are microfibre cloths. These are the most effective cleaning cloth for lenses, glass, computer screens and instrument bodies and for many applications they work impressively without any solvent or other chemical cleaning agents. For final cleaning of important optical components we recommend 3M Novec Contact Cleaner. This material was originally developed to clean electrical contacts, particularly in low voltage connection. It is now used as the best final cleaner for fibre optical joints as it dissolves many contaminants, it is non‐flammable and does not harm most plastics. Best of all is leaves absolutely no trace on optical surfaces, which makes it superbly suited for cleaning high voltage insulators, the ends of light conducting rods and fibres, lenses, prisms and front coated optical mirrors.
Correlative Microscopy Coverslips Hints and Tips
EMS66108-01, EMS66108-02, EMS66108-03, G493-01, G493-05, G493-10 Physical and Chemical Characteristics Resistant to normal chemicals used in electron microscopy No oxygen retention and compatible resin LR White Good optical quality in brightfield & UV fluorescence Excellent transparency Does not deform at temperatures (positive 100°C and negative liquid N2) Rigid, does not float in the middle of culture Easy to handle and cut with a knife or micro-punch Simple sterilisation using alcohol or UV Detaches easily from resin after polymerisation Low cost How to use the Correlative Microscopy Coverslips Sterilise the coverslip with alcohol, then dry and add the culture. Ensure that the grid is positioned correctly so that the text is readable. Observe your cell culture using light microscopy (transmitted and/or fluorescence) and identify the area of interest (Fig. 1 and 2). Record the images needed, and note the co-ordinates of the squares where there are cells of interest (Fig. 1 and 2 show co-ordinate 8C). Fix, dehydrate and embed with resin for examination by transmission electron microscopy. At the end of the embedding procedure, invert a BEEM type capsule filled with resin onto the coverslip covering the selected cells of interest (Fig. 3). Cure and detach the coverslip (Fig. 4), the footprint of the grid (Fig. 5) allows location of the position. Trim the block (Fig. 6) in the selected area then make cuts using an ultra microtome. Technical Advice 1. Use with Fluorescent Microscopes These new correlative microscopy coverslips (CMC's) are not designed to replace the glass or quartz coverslips, which have superior optical properties, and so are far better for fluorescent microscopy. The CMC's are designed to satisfy the needs of correlative microscopy where an initial, general analysis is performed using light or fluorescent microscopy and then the specimen is further processed for analysis by SEM, TEM or cryofixation. If you need to perform detailed analysis using fluorescent microscopy, then the CMC's are not the solution. If, however, you want to perform a comprehensive analysis using different analysis techniques, then the CMC's are the only solution. The key advantage of the CMC's is that they are made on special film that can be cut or punched (for cryo applications), something that is not possible with glass coverslips. The grid image is also transferred to any embedded specimen making cell location far easier. 2. Best Types of Resin to Use Because this is new technology, it often requires a change in the customer's preparation method. The method that the customer has traditionally used for glass coverslips may not be suitable for these film ones. The key thing to successful use of these CMC's seems to be the type of resin and the preparation. We have already mentioned about the need to ensure a hermetic seal between the Beem capsule and the CMC film. The preparation works perfectly with low viscosity epoxy resins (e.g. Spurr, EPON). Sufficient polymerization is obtained in 24 hours at 60°C – it does not normally require 48 hours. 3. Always clean the coverslips before applying the specimen This can be done either with UV light or alcohol. After using alcohol it is best to immerse the coverslip in polysyline to give better adhesion (just using alcohol can leave the surface 'too clean and too smooth'). 4. Film Type The CMC's are made on a special polyester base film which is 0.18mm thick. There is no absolute tolerance specified on the thickness of the film.
Counting Chambers
What is a counting chamber and what is it used for? A counting chamber is a precision measuring instrument made of special optical glass. It is used to count cells or other particles in suspensions under a microscope. Counting chambers are mainly used for blood analysis (counting leucocytes, erythrocytes and thrombozytes) and to count cells of liquor. Counting chambers are also used to count bacteria and fungus spores. Design principle All counting chambers have the same basic design principle. (Pic. 1) There are four longitudinal grooves in the central third of a rectangular and thick base plate made of special optical glass. The grooves are parallel to the short sides of the base plate and the central third has the same size as the cover glass used with the counting chamber. The two larger external surfaces are unfinished and are used for marking purposes. The central support and the two external supports are smooth and highly polished. The surface of the central support is deeper than that of the two external supports. The counting nets are engraved in the central support (chamber base). If a cover glass is placed on the external supports, a capillary gap is produced between the underside of this cover glass and the central support of the counting chamber. Designs and identification of counting chambers Two types of Counting Chambers: Single grid ruling: middle support without division (one counting grid) - Pic. 2 Double grid ruling: middle support with one division (two counting grids) - Pic. 3 Furthermore there are two different designs of grids: standard: the counting grid is directly engraved in the glass. bright-lined: the chamber base is initially coated with rhodium and the counter net is then etched into the rhodium coating. By shifting the contrast, colour inversion under the microscope is possible so that the counter net can be viewed either in light or dark field illumination. The bright lined chamber is preferred, since viewing is less strained. Identification: The following details are printed on both unworked surfaces of the counting chamber: counting net system chamber depth in mm area of the smallest square in mm² Production and quality descriptions Counting chambers are precision instruments. They are predominantly used in medical laboratories. All counting chambers are manufactured in compliance with the relevant Calibration Ordinance and DIN standard. Production: The production of counting chambers includes several individual processes, each of these is followed by stringent checks. The internal support (chamber base) as well as the two external supports are ground and polished. The flatness and the accuracy are the most important requirements. They are described in the standard DIN 12874. The central support (chamber base) is ground to requirements, for example the depth of the Neubauer must be 0.1 mm. Besides the standard depth there are special depths available (f.e. 0.2 mm / 0.5 mm). After these processes the counting net is engraved in the chamber base which is followed by the lettering on the un - worked surfaces and by a heat treatment. The production of the counting chamber is now completed with the final check which ensures that the counting chamber is in accordance with the DIN standards for weights and measures. Requirements on quality controls: The maximum deviations allowed according DIN 12847 are as follows: for the chamber depth in the area of a counting net ± 2% of the nominal value for distances of less than 0.4 mm between any net lines ± 2µm for distances of 0.4 mm or more between any net lines ± 0.5% of the desired value for the angle of the net division ± 1° the width of the division marks must not be greater than 5 µm The flatness tolerance acc. to DIN 7184 Part 1 is as follows: for the chamber base near the counting net 2 µm for the support areas 2 µm for the cover glasses 3 µm (according to DIN 58 884) Filling the counting chamber Sliding on the cover glass The external supports are to be moistened with distilled water and the cover glass then is gently pushed onto the counting chamber from the front. Important: The cover glass is fragile! The formation of interference lines (Newton rings) between the external support and the cover glass shows that the cover glass is correctly positioned. Feeding: Take a well mixed pipette from the shaker and dispose off the first few drops. Wipe the pipette dry on the outside and then hold it at an angle until a small drop has arisen at the tip of the pipette. This drop is then to be placed between the cover glass and the counting chamber. As a result of the capillary effect the gap between the cover glass and the chamber base fills up. Before the thinned blood solution can overflow at the edges of the chamber section, the tip of the pipette must be removed. If any air bubbles are visible or if the liquid has overflowed over the edges and into the grooves, the chamber must be cleaned and feeding must be repeated. (Pic.5). Blood Mixing pipettes to be used: a.) Erythrocyte pipette (red bulb) b.) Leukocyte pipette (white bulb) Counting the particles Counting technique: Counting assumes precise knowledge of the limit lines of the counting chambers used. These are shown in the illustration. To ensure that cells which are on or along the limit lines are not counted twice or are not missed during the count, certain rules have to be observed (see illustration Pic. 6.). The count should be started at the top left-hand corner and follow the direction shown by the arrow (Pic 6.1). Counting may be enhanced with the microscopes illumination reduced. Notes on counting: Use reduced microscope illumination for all chambers. The difference of the counter cells in the large squares and the group squares must not exceed 10 cells. Double checks must be performed for all cell counts. After counting the two counting nets the bottom counting net is to be counted in the same way as a check. When doing this it is to be ensured that the chamber has not dried out. This can be prevented by filling the bottom chamber only shortly before the count and the counting after the sedimentation time. The difference between the totals of the counts for the two counting nets must not exceed 10 cells. The average value of the counts is then used in the calculation formula or multiplied by the corresponding factor. Calculation Formula: counted cells x dilution factor ____________________________________ = cells per ml of blood mm2 of counted area x chamber depths Chamber: Neubauer improved Leucocytes Counted cells 156 leucocytes Counted area: four squares (= 4 x 1mm²)= 4mm² Chamber depth 0.1mm Dilution factor 1:20 156 x 20 ___________ = 7800 leucocytes /ml of blood 4 x 0.1 Erythrocytes Counted cells 528 erythrocytes Counted area: five squares (= 5 x 0.04 mm²)= 0.2mm² Chamber depth 0.1mm Dilution 1:200 528 x 200 ___________ = 5.28 million erythrocytes /ml blood 0.2 x 0.1 Cleaning the counting chamber Immediately after completing the count, remove the cover glass. The counting chamber and cover glass has to be cleaned with water or, if necessary, with a mild cleaning solution. Then dry the chamber with a soft cloth or Kimwipe lint free wiper. Chambers may be dried with acetone.
Coverslip and Slide Trace Impurities
This is a listing of trace impurities in some of our coverslips and slides. Trace Impurities Element Electrically Fused Tubing & Rod Electrically Fused Ingot Flame Fused Ingot Fused Silica Flame Fused Ag — — — <2ppm Al 14ppm 14ppm 15ppm <25ppm As <.002ppm <.002ppm — <15ppm Au — — — <1ppm B <0.2ppm <0.2ppm — <10ppm Ba — — — <1ppm Be — — — <0.5ppm Bi — — — <0ppm Ca 0.4ppm 0.4ppm 0.5ppm <15ppm Cd <0.01ppm <0.01ppm — <0.3ppm Co — — — <0.5ppm Cr <0.05ppm 0.05ppm <0.01 <1ppm Cu <0.05ppm <0.05ppm <0.01 <2ppm Fe 0.2ppm 0.2ppm 0.1ppm <20ppm Ga — — — <0.5ppm K 0.6ppm 0.6ppm 0.2ppm <5ppm Li 0.6ppm 0.6ppm 0.2ppm <7.2ppm Mg 0.1ppm 0.1ppm — <5ppm Mn <0.05ppm <0.05ppm 0.01ppm <1ppm Mo — — — <1ppm Na 0.7ppm 0.7ppm 0.1ppm <187ppm Nd — — 0.1ppm — Ni <0.1ppm <0.1ppm — <5ppm P <0.2ppm <0.2ppm — <50ppm Sb <0.003ppm <0.003ppm — <0.5ppm Sr — — — <0.5ppm Th — — — <0.5ppm Ti 1.1ppm 1.1ppm 1.3ppm <10ppm U — — — <1ppm V — — — <1ppm Y — — <0.1ppm — Zn — — — <5ppm Zr 0.8ppm 0.8 1.3ppm <5ppm OH <5ppm <5ppm 170ppm <1000ppm
Eyepiece Reticles, Stage Micrometers & Calibration Factors
The terms ocular and eyepiece mean the same thing. The terms reticle, reticule and graticule are used interchangeably by various organisation and countries, they all are referring to the same thing. These are discs marked with scales or patterns, which fit into the eyepiece at the plane of the intermediate image. This is the position of the specimen image produced by the objective lens of the microscope. Therefore, the scale or pattern is viewed superimposed on this image. In Huygenian eyepieces of older design the reticle is placed on the field aperture at the internal plane of focus. With modern Huygenian designs the reticle is held firmly within a recessed cell by a retaining ring, all within the body of the eyepiece. It should be noted however, that some eyepieces of Huygenian design need the reticle to be fitted by a qualified technician. In such cases contact the equipment manufacturer or distributor. In Kellner or Ramsden eyepieces the reticle is held by a retaining ring at the external plane of focus. We can supply most reticle in 16, 19 and 21mm diameters within two weeks, other diameters are normally supplied within 4 weeks. When ordering please state the diameter of reticle required. Nominal glass thickness is 1.5mm. Image reading correct through the glass. It is not normal to have a calibration certificate for an eyepiece reticle. The reason for this is that the reticle is installed in the eyepiece and all of the optics on the microscope can effect the size measured by the reticle. It is normal practice to use a stage micrometer to calibrate the complete microscope. EYEPIECE RETICLES When sending details to us for a special eyepiece reticle it is essential that you include the following information noting, however, that the most common 8 to 10x eyepieces are the default and only the diameter is absolutely required: External diameter of the reticle disc. Calibration of microscope. It is essential that you give calibration details of the objective magnification you intend to use on your microscope. See following section for information on calibration. e.g. If you have a x2 objective magnification and you want each division of the reticle to represent 1mm in terms of the specimen, then each division will have to be 1mm x 2 (size of objective) = 2mm. Line thickness. This will vary depending on the power of your eyepiece and we will be happy to offer advice. As a guideline it is suggested that, with a x10 eyepiece, lines should be from 10 to 12µ. MICROSCOPE STAGE MICROMETERS The terms stage micrometer and object micrometer are the same thing. Stage micrometers are used to calibrate eyepiece reticles or imaging systems like c‐mount digital cameras. They can be supplied with a certificate of calibration for customers who need traceability of measurement (For ISO or quality purposes). There are stage micrometers for transmitted brightfield light and reflected (incident) light. Transmitted light products are normally positive images (opaque lines on a clear background) and reflected light products are normally negative images (clear lines on an opaque background). You will note that Pyser offer both "S" and "PS" patterns. This is probably a good time to clarify the difference between the two. The "S" pattern stage micrometers are comprised of 16mm glass discs with the graticule pattern on them, placed in the centre of a 75mm x 25mm black, anodised aluminium slide, and is presented in a plastic box. This slide does not have an identifying serial number on it. For this reason, we do not recommend this part if the customer requires any form of calibration certificate. The "PS" patterns again have a 16mm glass disc, but this time it is centred on a 75mm x 25mm stainless steel slide, and is presented in a wooden box. The main difference from the "S" pattern though is that it has a uniquely engraved serial number on the slide. This makes it ideal for calibration and should always be used when a certificate is required. All brightfield stage micrometers have a coverglass fitted over the image so that it replicates the normal specimen stage conditions. It is always best to use a stage micrometer with a scale length that is similar to the size of the measurements you are making. The higher the objective magnification of your microscope the smaller the scale you need for calibration. Size Conversions 1mm = 1000µm, 0.1mm = 100µm, 0.01mm = 10µm, 0.001mm = 1µm 1inch = 1000thou, 0.1inch = 100thou, 0.01inch = 10thou 1mm = 39.4thou 1thou= 0.0254mm (25.4µm) CALIBRATION OF MICROSCOPES Calibration Factor Calculation In general, the total magnification of your microscope can be determined by simply multiplying the objective power by that of the eyepiece. Objective Eyepiece Total Magnification x20 x10 200 For most work, especially with modern optics, this is sufficient. However, where actual size is critical it is often necessary to modify the dimensions, so when ordering eyepiece reticle the following information is needed: A calibration factor. Objective magnification. Eyepiece magnification. Reticle diameter. Make and model of microscope. To calibrate the instrument, fit the microscope with an eyepiece scale and appropriate stage micrometer. Compare the length of the stage micrometer with the eyepiece scale. An exact calibration factor can then be calculated. Example Using a 40x objective, a circle in the eyepiece requires a diameter of 4000µm (4mm) to coincide with or read a 100µm circle on the stage. The factor is therefore defined as 4. With the stage micrometer and eyepiece reticle in place, the microscope is focused in the normal way. The eyepiece scale now becomes superimposed upon the enlarged image of the stage micrometer. Move the stage micrometer until the zeros on each scale are coincident. Further along another coincident point will be found. The relationship between the two points can now be seen and calculated. a) Number of units = Number of stage micrometer divisions Number of eyepiece scale divisions On scale shown a) there are 24 stage micrometer divisions which align with 8 eyepiece scale divisions. Therefore each eyepiece division is:- 24/8 = 3 units b) In this case the 17 divisions on the stage micrometer line up with divisions 1 to 8 on the eyepiece scale. Thus 17/7 = 2.42857 units. If the unit of the stage micrometer is 10µm, then each division = 24.2857µm. In practical terms this figure may be rounded to 24 or 24.3 c) It is possible that there is no second point of coincidence. Then on microscopes with an adjustable tube length coincidence can be obtained by lengthening or shortening the tube. Where there is no tube length adjustment, all measurements will be approximate. Size conversions and equivalents 1mm = 1000µm 1inch = 1000thou 1mm = 39.4thou 0.1mm = 100µm 0.1inch = 100thou 1thou = 0.0254mm (25.4µm) 0.01mm = 10µm 0.01inch = 10thou 0.001mm = 1µm It is far better to increase the objective magnification than use a reticle with finer details. A recent example had a customer that wanted to have a reticle with a horizontal scale to measure spores of 2‐5µm in diameter. They said they normally used a 10x objective. We answered as follows; 'In this case, our NE1, which has a 10mm scale subdivided into 0.1mm divisions will measure 0‐1mm when used with a 10x objective lens. Each division will represent 0.01mm (10um). This will not be good enough to measure spores in the 2um ‐ 5um region. If you use a reticle with a smaller scale in the eyepiece it is very difficult to read so the answer is for the customer to use the 40x objective. Now the 10mm scale will measure 0‐0.25mm and each division will represent 2.5um. If the customer is able to use the 100x objective then the measurements can be even more accurate as each division will represent 1um'. The diameter of the reticle is important to ensure it fits the eyepiece you have. When we select an eyepiece reticle we are only concerned with the objective magnification. The mathematics of microscope magnification is very simple ‐ all you do is divide the size of the feature on the reticle by the objective magnification to get the size that it will actually measure at the stage. Eg. a 10mm length scale when used with a 10x objective will measure 1mm at the stage. The same 10mm length reticle when used with a 40x objective will measure 0.25mm (250µm at the stage). If you want to look at another way, if you have a 10µm at the stage and are using a 40x objective lens this will be magnified to 400µm (0.4mm) at the reticle image plan. The next section gives more sizing information.The eyepiece merely magnifies the image on the reticle. Table Relating Stage Size and Objective Magnification with Size at Reticle Image Plane. The two columns on the left hand side show the size of the specimen or feature at the stage. Along the top are typical objective magnifications. So, if you want to measure something that is about 50µm using a 40x objective lens this will actually be 2mm at the reticle image plane. A reticle with a 10mm scale in 0.1mm divisions (Our NE1) or one with a 5mm scale in 0.05mm divisions (Our NE5) may be suitable. Measurement at Stage Measurement at Stage 1X 1.25X 2X 5X 10X 20X 40X 100X 1m 0.001mm 1m 1.25m 2m 5m 10m 20m 40m 100m 5m 0.005mm 5m 6m 10m 25m 50m 100m 200m 500m 10m 0.010mm 10m 12.5m 20m 50m 100m 200m 400m 1mm 20m 0.020mm 20m 25m 40m 100m 200m 400m 800m 2mm 50m 0.050mm 50m 60m 100m 250m 500m 1mm 2mm 5mm 100m 0.1mm 100m 125m 200m 500m 1mm 2mm 4mm 10mm 200m 0.2mm 200m 250m 400m 1000m 2mm 4mm 8mm 20mm 500m 0.5mm 500m 600m 1mm 2.5mm 5mm 10mm 20mm 50mm 1000m 1mm 1mm 1.25mm 2mm 5mm 10mm 20mm 40mm 100mm 10 000m 10mm 10mm 12.5mm 20mm 50mm 100mm 200mm 400mm 1000mm Colour of reticle lines does not matter in a microscope eyepiece. All lines, whatever their colour, block the light from the specimen/stage so the pattern will always appear dark against a brighter background. RETICLE DIAMETERS FOR COMMON MICROSCOPES Pyser‐SGI has compiled a list of eyepieces and microscope types from the major microscope manufacturers and listed alongside them the reticle sizes that fit them. Most of these eyepieces have some means of securing the reticle in position ‐ always best to just check first before ordering the reticle. Pyser offer a supply and fit service if you have any doubts. Brand Eyepiece Reticle Diameter Model NIKON C‐W 10xB/22 25mm C‐W 15x/16 19mm C‐W 20x 19mm CFI 10x CFI 12.5x CFI 15 27mm CFI UW 10x 27mm CFUW10 27mm CFW 10x CFW 15x 21mm CFWN 10x CFWN 15x 21mm E1‐CFI 10x E2‐CFI 10x E2‐CFI 15x 27mm L‐W10x ESD (FOV 22) 25mm Eclipse LV150L SME 10x /23 27mm SME 15x ~ SME 20x 16mm SME 10x /21 27mm SMZ‐10 SMZ U UW10x A/24 25mm SMZ‐U SMZ U UW15x 25mm SMZ‐U SMZ U UW20x 21mm SMZ‐U SMZ U UW30x n/p SMZ‐U SMZ 10 E10xA 24mm SMZ‐10 SMZ 10 UW15x /17 27mm SMZ‐10 SMZ 10 UW20x n/p SMZ‐10 Brand Eyepiece Reticle Diameter Model OLYMPUS WHN10X / WHN10X ‐H 24mm BX61 / BX51 / BX41 WH10X / WH10X ‐H / WH15X 24mm BX50 / BX40 WHS10X‐H / WHS15X‐H 24mm SZX7 / SZX9 / SZX12 WHSZ10X‐H / WHSZ15X‐H 24mm SZ61 WHSZ20X‐H / WHSZ30X‐H 24mm SZ61 CWH10X / CWH10X‐H 24mm CX41 GSWH10X / GSWH10X‐H 24mm SZ60 / SZ40 / SZ11 / SD / SF GSWH15X / GSWH15X‐H 24mm SZ60 / SZ40 / SZ11 / SD / SF GW10X / GWH10X 24mm SZ60 / SZ40 / SZ11 / SD / SF WH12.5X / WH12.5X ‐H 24mm GWH10X‐D 26mm SZH / SZH10 SWHK10X 28mm VANOX, and other G10X / G15X 22mm WHK10X / WHK15X / NWHK10X 20.4mm 20mm loose fitting alternative (*WHK10X‐H canonot be installed) NFK 2.5X/ 3.3X/ 5X/ 6.7X 20.4mm 20mm loose fitting alternative PE 2X/2.5X/ 3.3X/ 4X/ 5X 20.4mm 20mm loose fitting alternative GX‐SLM Scale Slider 25.5mm Special 3.8mm thick CWHK10X / NCWHK10X /G20X 19mm CK40 / CK30 / CH40 / CH30 / CH2 Brand Eyepiece Reticle Diameter Model LEICA 11507808 (S10x/25Br.M) 26mm DMR, DMIR, DML 11507807 (S10x/22B.M) 26mm DMR, DMIR, DML 11507801 (10x/20Br.M) 26mm DMR, DMIR, DML 11507802 (10x/20Br.M) 26mm DMR, DMIR, DML 11506515 (12.5x/16Br.M) 26mm DMR, DMIR, DML 10447160 (10x/21B) 23mm MX series / Z series 10445301 (16x/14B) 23mm MX series / Z series 10445302 (25x/9.5B) 23mm MX series / Z series 10445303 (40x/6B) 23mm MX series / Z series 10450023 (10x/23B) 24.5mm M205C / M165C 10450024 (16x /15B) 24.5mm M205C / M165C 10450025 (25x/9.5B) 24.5mm M205C / M165C 10450026 (40x/6B) 24.5mm M205C / M165C 10 446 333 (10x/23,adjustable) 24.5mm DM750 / S6 Series / S8APO 10 446 355 (16x/16,adjustable) 24.5mm S6 Series / S8APO 10 446 357 (20x/12,adjustable) 24.5mm S6 Series / S8APO 10 446 329 (10x/23B,adjust.f/eyeglasses) 24.5mm S6 Series / S8APO 10 447 131 (10x/23,adjustable) 24.5mm S6 Series / S8APO 10 447 133 (16x/16,adjustable) 24.5mm S6 Series / S8APO / EZ Series 10 447 135 (20x/126,adjustable) 24.5mm S6 Series / S8APO / EZ Series 10 447 137 (for eyeglasses10x/23B,adjustable) 24.5mm S6 Series?/S8APO 10 447 139 (for eyeglasses16x/15B,adjustable) 24.5mm S6 Series?/S8APO 10 445 302 (25x/9.5B,adjust.f/eyeglasses) 24.5mm S6 Series?/S8APO 10 445 303 (40x/6B,adjust.f/eyeglasses) 24.5mm S6 Series?/S8APO 10 447 280 (10x/20B,adjust.f/eyeglasses) 24.5mm EZ Series Leitz Wetzlar Periplan GF10x/18 21mm Brand Eyepiece Reticle Diameter Model ZEISS E‐PL 10x/20 Br. Foc 26mm PL 10x/21 Br foc 26mm PL 10x/23 Br. Foc 26mm WF 10x/18 23mm Primostar PL16x/16 Br. Foc 21mm W 25x/10 foc 21mm Kpl‐W 10x/18 [#46 40 43‐9902] 21mm 872e Brand Eyepiece Reticle Diameter Model MEIJI TECNO EM series 10x 25mm IM7000 series 10x 25mm ML2000 series 10x 21mm ML5000 series 10x 21mm ML7000 series 10x 21mm ML8000 series 10x 21mm ML8500 series 10x 21mm MT4000 series 10x 21mm MT5000 series 10x 25mm MT6000 series 10x 25mm MT9000 series 10x 21mm RZ series 10x 25mm RZ series 15x 19mm RZ series 20X 19m TC5000 seies 10x 25mm TM200 all series 19mm TM400 all series 19mm Brand Eyepiece Reticle Diameter Model MOTIC 100FL 10x 21mm 100FLA 10x 21mm 100FLAQ 10x 21mm B1‐211 10x 21mm B1‐220 10x 21mm B1‐223 10x 21mm M Series 10x 23mm S‐10‐P 10x 23mm S‐20‐2L 10x 23mm SL‐40‐PB 10x 23mm SMZ‐140 10x 23mm SMZ‐143 10x 23mm ST‐30‐2L 10x 23mm ST‐39 10x 23mm 10x 24mm BA 210, BA 310 10x 24mm AE 2000 Brand Eyepiece Reticle Diameter Model LABOMED Prefix 512, 312 or 913 21mm Prefix 912, 414, 914 or 612 26mm Prefix 123, 125 or 127 28mm Brand Bausch Lomb, Cambridge Inst. The following list shows the Bausch & Lomb reticle part number with the Pyser equivalent or nearest match. All reticles are specified as 21mm diameter. Part Number Description ProSciTech Part Number Pyser Description 31‐16‐01 Scale 5mm in 0.05mm PYS01B21203 NE5 Eyepiece Reticle, 5mm in 0.05mm divisions 31‐16‐02 Scale 10mm in 0.1mm. PYS01B21201 NE1 Eyepiece Reticle, 10mmin 0.1mm divisions 31‐16‐04 Scaled to measure 0.001" Would be made to special order with calibration factor 31‐16‐05 Scale 5mm in 0.05mm PYS01B21203 NE5 Eyepiece Reticle, 5mm in 0.05mm divisions 31‐16‐07 Each least division 0.005mm @ 1x objective power PYS01B21219 NE31 Eyepiece Reticle 31‐16‐08 Scaled to measure 0.001" at 3x mag Would be made to special order with calibration factor 31‐16‐09 Micro Disc Would need image information to match pattern 31‐16‐11 0.5mm squares PYS01B21208 OR01B21207 NE10A Eyepiece Reticle, 10 x 10 grid of 0.5mm squares, indexed or NE10 Eyepiece Reticle, net grid of 0.5mm squares 31‐16‐12 1mm squares PYS01B21210 OR01B21209 NE11A Eyepiece Reticle, 10 x 10 grid of 1mm squares, indexed or NE11 Eyepiece Reticle, net grid of 1mmsquares 31‐16‐71 100 x 1mm square grid PYS01B21210 NE11A Eyepiece Reticle, 10 x 10 grid of 1mm squares, indexed 31‐16‐30 Crossline, 0.080mm line width PYS01B21234 NE81 Crossline Eyepiece Reticle, 0.040mm line width 31‐16‐42 Scaled to measure 0.001" at 3x mag Would need image information to match pattern 31‐16‐43 100 divisions of 0.1mm PYS01B21021 NE1 Eyepiece Reticle, 10mmin 0.1mm divisions 31‐16‐44 Scaled to measure 0.01mm at 7x mag Would be made to special order with calibration factor 31‐16‐45 Grid of 64 squares PYS01A21218 NE29 Eyepiece Reticle, Whipple grid 31‐16‐46 Protractor reticle, 180 deg PYS01B21216 NE25 Eyepiece Reticle, half protractor 31‐16‐47 General purpose reticle Would need image information to match pattern 31‐16‐87 Stage micrometer ruled to 0.005" PYS02A00402 S4 Stage Micrometer, 0.1" in 0.001" divisions 31‐16‐89 Stage micrometer ruled to 0.001" PYS02A00402 S4 Stage Micrometer, 0.1" in 0.001" divisions 31‐16‐90 Stage micrometer ruled to 0.01mm PYS02A00404 S8 Stage Micrometer, 1mm in 0.01mm divisions 31‐16‐99 Stage micrometer ruled to 0.01mm PYS02A00404 S8 Stage Micrometer, 1mm in 0.01mm divisions 31‐16‐35 Micrometer scale 0.001" PYS01B21214 NE20 Eyepiece Reticle, 0.1" in 0.001" divisions
Fused Quartz
Properties of Fused Quartz Silica is found almost everywhere in nature, it represents almost 1/3 the mass of the earth's crust. Vitreous Silica is the generic term used to describe all types of silica glass, with manufacturers referring to the material as either Fused Quartz or Fused Silica. Manufactured by fusing naturally occurring crystalline silica, either sand or rock crystal, a wide range of products are available that may be opaque, translucent or transparent. If the silicon dioxide is synthetically derived, the material produced is commonly called Synthetic Fused Silica. Vitreous Silica, in all its forms, offers a variety of properties such as: Permeability Extreme Hardness Very Low Coefficient of Thermal Expansion Resistance to High Temperature High Chemical Purity High Corrosion Resistance Extensive Optical Transmission from Ultra-Violet to Infra-Red Excellent Electrical Insulation Qualities Remarkable Stability Under Atomic Bombardment Properties Density 2.2 x 103 kg/m3 Hardness 5.5-6.5 Mohs' Scale 570KHN100 Design Tensile Strength 4.8 x 107 Pa (N/m²) Design Compressive Strength Greater than 1.1 x 109 Pa Bulk Modulus 3.7 x 1010 Pa Rigidity Modulus 7.2 x 1010 Pa Young's Modulus 7.2 x 1010 Pa Poisson's Ratio 0.17 Coefficient of Thermal Expansion 5.5 x 10-7 m/m °K (293°K - 593°K) Thermal Conductivity (20°C) 1.4 W/m °K Specific Heat (20°) 670 J/kg °K Softening Point 1956° Annealing Point 1488° Strain Point 1393° Electrical Resistivity 7(107)ohm-m Dielectric Properties (293°K and 1 MHz) Constant 3.75 Strength 5 x 107 V/m Loss Factor Less than 4 x 10-4 Dissipation Factor Less than 1 x 10-4 Index of Refraction 1.4585 Constrigence (Nu value) Fused Quartz 67.56 Velocity of Sound-Shear Wave 3.75 x 103 m/s Velocity of Sound-Compression Wave 5.90 x 103 m/s Sonic Attenuation Less than 11 db/m MHz Permeability Constants (cm mm/cm sec cm of Hg - 700°C/973°K) Helium 210 x 10-10 Hydrogen 21 x 10-10 Deuterium 17 x 10-10 Neon 905 x 10-10 TRACE IMPURITIES TYPE (PPM) AI AS B Ca Cd Cr Cu Fe K Li GE 124® 14 <.002 <0.2 0.4 <0.01 <0.05 <0.05 0.2 0.6 0.6 GE 214® 14 <.002 <0.2 0.4 <0.01 0.05 <0.05 0.2 0.6 0.6 NSG OZ® 40 - - 2.5 - - .50 0.9 1.7 .06 TYPE (PPB) Ag Al As Au B Ba Be Bi Ca Cd Corning 7980 ® <150 -40 <5 n.d. <100 <14 <5 <10 <20 n.d. K Li Mg Mn Mo Na Ni P Sb Sr <21 <1 <25 <10 <5 <150 <7 <100 <5 <3 TRACE IMPURITIES TYPE (PPM) Mg Mn Na Ni P Sb Ti Zr *OH- GE 124® 0.1 <0.05 0.7 <0.1 <0.2 <0.003 1.1 0.8 <5 GE 214® 0.1 <0.05 0.7 <0.1 <0.2 <0.003 1.1 0.8 <5 NSG OZ® 0.3 .03 2.5 - - - 0.8 - 200 TYPE (PPB) Co Cr Cu Fe Ga Corning 7980 ® <10 <1 <13 <15 n.d. Ti U V Zn Zr <40 <1 <10 <30 <30 PRESSURE CALCULATIONS INTERNAL PRESSURE CALCULATIONS RUPTURE FORMULA FOR TUBING Because fused quartz is used in applications involving internal pressures, it is helpful to know the maximum pressure that can be applied to a selected fused quartz tube. The formula at right can approximate this information at room temperature. S = pr/t Where:S = Hoop Stress in Pa p = Working Pressure (Pa) r0 = Inside Radius (mm) t = Wall Thickness (mm) This formula can not be used when internal pressure exceeds 100 psi. RUPTURE PRESSURE CALCULATIONS FOR DISCS AND PLATES Determining pressure differential is required for many applications of stressed fused quartz discs, plates and sight glasses. The formulas below can be used for room temperature applications of parts having either clamped or unclamped edges. A = Unsupported Area in sq/inches T = Thickness (inches) F = Safety Factor (7) M = Modulus of Rupture (7,000 psi) P = Pressure (psi) THE ABOVE PRESSURE CALCULATIONS ARE RECOMMENDATIONS ONLY. ACTUAL PRESSURE POINTS MAY VARY DEPENDING ON USER APPLICATIONS FUSED QUARTZ PROPER USAGE GUIDELINES Cleaning The cleaning of fused quartz is critical before it is used in any application. The fused quartz should be cleaned by placing it in a 7% maximum solution of ammonium bifluoride for no more than ten (10) minutes, or a 10% volume maximum solution of hydrofloric acid for no more than five (5) minutes. After cleaning, using the above method, the fused quartz should be rinsed in deionised or distilled water and then dried. Running in Procedure In order to increase resistance to devitrification and sag of your quartzware, an even layer of cristobalite must be formed on the outer surface of quartz tubes. Expose a new tube to a temperature of up to 1200°C and rotate it 90° every two (2) hours for the first 12 to 24 hours. Storage Space permitting, fused quartz should be stored in its original shipping container. If that is not practical, at least the wrapping should be retained. In the case of tubing, the end coverings should be kept in place until the product is used. This protects the ends from chipping and keeps out dirt and moisture which could compromise the purity and performance of the tubing. THESE PRODUCTS ARE ANNEALED Both quartz and silica glass are annealed at approximately 1150°C. However, they reach a strain point at about 1120°C. These glass products, if rapidly cooled after use at temperatures above this strain point, will develop strain again. Special care should be taken when using large sized products. JOINING FUSED QUARTZ AND OTHER MATERIALS Quartz and silica glass only slightly expand with increases in temperature, in contrast with other materials. Care must be taken when these glass products are connected to other materials and the temperature rises, in order to avoid the development of cracks. CARE MUST BE TAKEN DURING FURNACE INSERTION Quartz and silica glass feature low thermal conductivity. If the glass product comes too close to a heating element, or is put in direct contact with a flame, it may become locally heated and develop cracks. Long glass tubes may also deform at temperatures of 1100°C or higher. Care should be taken to support both glass types, expecially large-sized products. DEVITRIFICATION Devitrification of quartz and silica glass means transition from a metastable (vitrified) state to a stable crystallised state of cristobalite. Devitrification occurs when the product is used at high temperatures over a long period of time, or it is heated while impurities adhere to its surface. Even very small impurities on the surface can have a major influence. Under such conditions, devitrification may even occur at temperatures of 1000°C or less. This hardly ever occurs at temperatures of 1150°C or less, if the glass surface is perfectly clean. Devitrification usually starts when the temperature rises to 1200°C or higher, then further develops as the temperature increases.
Light Microscopy Definitions
Abbreviations APO = apochromatic CCD = charge-coupled device CMOS = Complementary metal-oxide-semiconductor LWD = long working distance NA - numerical aperture P = plan Ph = Phase contrast S = spring loaded (sometimes called R for retractable) UW = ultra wide field (also UWF, or SWF for super wide field) WD = working distance WF = wide field Binocular vs. trinocular vs. stereo microscopes A binocular compound microscope has a single optical channel split into two paths brought to the eyes. A trinocular microscope has one optical channel split into 3 paths, two for the eyes and one for a camera. Usually the image for the camera eyepiece is diverted from one of the binocular eyepieces. A stereo or dissecting microscope has a separate optical channel for each eye which allows three-dimensional viewing. Photos taken with a stereo microscope use only one eyepiece or one binocular eyepiece image is diverted to the phototube; photos are not in stereo. To produce stereo-pair photos requires two images taken at about 10° to each other with the same spot in the centre of the image or two images taken from the both the normal binoc eyepieces. If a video camera is fitted and the image is observed on a monitor, a single eyepiece microscope may be sufficient, however, most professional microscopes are at least binocular and, for more versatile photo capability, trinocular. Objectives A microscope has two sets of lenses, the eyepiece (or ocular) and the objective. Eyepiece lenses are simple; they merely focus the light coming up the barrel of the microscope, whereas the objectives do the serious magnification. Descriptions of objective lenses for compound microscopes can seem complex at first. Objectives have different levels of correction for chromatic (colour) and spherical aberration, these are explained below. It should be noted that the outer 35%, 20%, or 5% of the field of view will not be out of focus or blurred - but if aberrations occur, they will be found there. Achromatic Achromatic objectives have been corrected for colour aberration and have a flat field of focus in the middle 65% of the field of view. Semi-plan Semi-plan objectives give a flatter field of view, with 80% of the field of view in focus; edges would require slight refocusing. These lenses also correct for chromatic aberration. Plan Plan objectives have about 95% of the field of view in flat focus. These lenses also correct for chromatic aberration. Plan lenses are superior, but more expensive. Apochromatic Achromatic lenses are corrected for chromatic (colour) aberrations by focusing two wavelengths of light onto the same plane. Apochromatic (APO) lenses focus three wavelengths on the same plane. These lenses are much more expensive and are only necessary for the most demanding applications. Immersion Oil Only used at very high magnifications, an objective lens described with the suffix (Oil) can be used at a very short working distance with a layer of immersion oil between the specimen and the lens. The oil is optically similar to glass, reducing refraction. For more immersion oil details see our online page i1. S (S) in an objective description denotes a spring loaded retraction that saves slides and objectives from collision damage. Sometimes called R for retractable. Explanation of colour codes and numbers on objectives Example objective appearance: Coloured ring 10/0.25 160/0.17 Coloured ring: red = 4x yellow = 10x blue = 40x white = 100x 10/0.25: 10 = magnification (10x) 0.25 = numerical aperture 160/0.17: 160 is a DIN (German Standard) measurement in mm of the tube length required for this lens 0.17 is the thickness in mm of the required coverglass (0.17 is No. 1 coverglass) Understanding an infinity microscope A basic microscope is a long empty tube with a lens system at each end. Typically, the objective lens produces parallel beams of light within the tube and the eyepieces focus these for our eyes. With an infinity-corrected system, an additional lens system is contained within the tube itself. This allows the microscope objective lens to be positioned further away from the specimen, which results in greater working distance and a safer position for the lens. Infinity-corrected objectives must be used with an infinity-corrected tube which means that such objectives are not interchangeable with those from ordinary microscopes. Phase contrast microscopy A phase contrast microscope is one that does not require stained specimens, but instead enhances the contrast of near-transparent specimens. This makes it possible to view living cells and tissues and a variety of low-contrast specimens such as protozoans, bacteria and sperm tails. As light travels through a transparent medium, its amplitude and phase are altered. Amplitude gives rise to colour, but the human eye cannot discern changes in phase. In bright-field microscopes the information carried by phase is lost. Frits Zernike realised that it was necessary to induce a phase shift in relation to a reference beam. He etched concentric circles on a glass plate and inserted this into the optical path of the microscope. This allows the phase of the light passing through the specimen to be inferred from the intensity of the image produced. The phase contrast technique proved to be such an advancement in microscopy that Zernike was awarded the Nobel prize (physics) in 1953. Some compound microscopes are equipped as, and some may be converted to, phase contrast instruments. Changes required are special strain-free objectives and matching condenser phase plates for each objective power. Phase objectives are identified with 'PH'. Fluorescence illuminator - Wave Length Reflected Light Source Excitation Dichroic Mirror Barrier Filter G EX510-560 DM575 BA590 B EX450-490 DM505 BA520 BV EX400-440 DM455 BA470 V EX380-420 DM430 BA450 U EX330-380 DM400 BA420 Y EX540-580 DM595 BA600-660
Making Slides Hydrophobic and Dust Repellent
Coating with HMDS to make slides hydrophobic and dust repellent Silanisation of glass microscope slides using hexamethyldisil Azane (HMDS) can make slides hydrophobic dust repellent. Simply place the separated slides in a container like a wide mouth glass jar with an aluminium or Teflon lined cap. Add a few drops of HMDS and it will vaporise overnight in the sealed jar and react with the silanols on the surface. If the container is left slightly ajar in an oven at 70°C overnight, then any residual ammonia will also dissipate. Open the jar in a fume-hood and remove the slides. Due to a molecular (invisible) coating they will now be very hydrophobic, repel dust particles and bead water. Products like Rainex® and Aquapels® also work but cannot be applied in the vapour state and a thicker, perhaps streaky film results. Please note that HexamethylsilOXane is NOT the same material. HMDS is simple, easy to apply, and safely handled. Use gloves, a hood, and respirator when handling these materials. Let the dropper and the opened jar with HMDS evaporate overnight. Do not inhale any HMDS vapours! Read the MSDS information on handling. Despite these warnings I consider HMDS is a safer product than chlorosilane - which I have experienced to spontaneously explode. From a Microscopy Listserver posting, with permission from Paul Beauregard Beauregard beaurega@westol.com
Microscope Eyepiece Magnification
Standard 10x eyepieces (oculars) are supplied with all ProSciTech microscopes. Other magnifications are available. 15x and 20x eyepieces are commonly used in stereo microscopes, these increase magnification. The objective is the more important lens group; eyepieces can only enlarge existing information projected by the objective. All our eyepieces are high quality, plan achromatic lens and can be placed in other microscopes (brands and types), be they stereo or compound microscopes ‐ if they have the right diameter. Our stereo microscopes use 30mm diameter eyepieces. Confusingly, some brands use 30.5mm eyepieces. The common standard for compound microscopes is 23.2mm, but some manufacturers use either of the larger size eyepieces on compound microscopes. It can be hard to tell the difference between a $50 and $400 eyepiece; brand name tags are very expensive. Final magnification in microscopy depends on several factors. In stereo or macro systems the magnification at the eye is the magnification of the objective multiplied by that of the eyepiece. If an additional lens is added to the front of the objective, both, photo and viewing magnifications, whereas the use of different eyepieces changes viewed magnifications only. Photo magnification is subject to other, additional factors. Final magnification: Compound microscopes, objective x eyepieces = Final magnification Zoom stereo microscopes, objective x eyepieces x zoom range (min and max) = Final magnification Example: The standard OXTL series microscope is equipped with a 1x objective, 10x eyepiece and has a zoom range of 0.7x ‐ 4.5. Minimum magnification = 1 x 10 x 0.7 = 7x Maximum magnification = 1 x 10 x 4.5 = 45x This OXTL microscope has standard magnification range of 7x ‐ 45x
Polishing Fibre Optic Connectors
ProSciTech offers complete solutions for fibre optic connector polishing ‐ with a full line of high performance precision abrasives, backed by the technical support of our experienced fibre optics specialists. We're ready to help you generate the finish you need on a wide variety of connectors. We can optimise your polishing operation to produce cost-effective results. 3M™ Lapping Films are available in sheets, discs and rolls for use on any type of polishing equipment. These films are especially designed for use in factory or field applications. 3M™ Lapping Film Precisely graded minerals are coated on a high strength, 3 mil polyester backing to provide a uniform, consistent finish. Available in silicon carbide film for glass and epoxy removal, and in aluminium oxide for levelling and polishing steps. Available in 0.05 ‐ 30 micron grades, with or without PSA (Pressure Sensitive Adhesive) backing. 3M™ Diamond Lapping Film 661X This standard Diamond Lapping Film is comprised of tightly graded diamond minerals uniformly coated on a polyester film backing. It is able to cut and polish hard ceramic ferrules and glass fibres at the same rate and to the same level. Used to radius ferrule connectors or to refine the finish in preparation for the final polish. Available in 0.1 ‐ 30 micron grades, with or without PSA backing. 3M™ Diamond Lapping Film 661XU Tightly graded diamond mineral is precision coated on a polyester film backing. This durable construction provides consistent results throughout the life of the product. Available in 0.5, 1, 3 and 6 micron grades, with or without PSA backing. 3M™ Diamond Lapping Film ‐ Type H ‐ 662XW Designed for radiusing and levelling operations that require added durability. Diamond Lapping Film ‐ Type H has a thicker diamond coating with a higher diamond concentration and a tougher resin for an increased cut rate and longer life. Type H lasts 2 to 3 times longer than standard DLF. Available in 0.5, 1, 1.5, 3, 6 and 9 micron grades, with or without PSA backing. 3M™ Diamond Lapping Film 660XV This long life, precision coated 3M™ Diamond Lapping Film is the newest and most durable film in 3M's Diamond Lapping Film product family. It combines a high cut rate with a great finish, and is designed for use on slower rpm polishing machines. Available in 1, 3, 6, and 9 micron grades. Selection Guide 3M's technical service representatives suggest using these polishing guidelines for polishing ceramic singlemode or multimode fibre optic connectors. In the top chart, locate your connector type, then refer to tables A, B and C below to select one of the options for each step. These recommended sequences provide typical starting points. Your actual sequences may vary depending on your polishing equipment and finish requirements. 3M™ Polishing Film helps you consistently meet geometry and fibre height requirements in your MT connector polishing operation. Precisely graded minerals coated on a fibrous backing enable you to generate fibre protrusion and attain the proper ferrule geometry. 3M Polishing Film can provide: Control of fibre protrusion Less cleaning than a slurry process High throughput Low rejects De‐Nub Mineral Available Micron Sizes 3M™ Diamond Lapping Films, 661X, 662XW, 661XU, 660XV Diamond M3203‐30 ‐ 30µm ‐ Green Epoxy Removal Mineral Available Micron Sizes 3M™ Diamond Lapping Films, 661X, 662XW, 661XU, 660XV Diamond M3203‐09 ‐ 9µm ‐ Blue M3203‐15 ‐ 15µm ‐ Orange (common for angle grinding) Refine (Level Connector) Mineral Available Micron Sizes 3M™ Diamond Lapping Films, 661X, 662XW, 661XU, 660XV Diamond M3203‐005 ‐ 0.5µm ‐ Manila M3203‐01 ‐ 1µm ‐ Lavender M3203‐03 ‐ 3µm ‐ Pink M3203‐06 ‐ 6µm ‐ Brown Polish (Finish) Mineral Available Micron Sizes 3M™ Diamond Lapping Films, 661X, 662XW, 661XU, 660XV Diamond M3203‐001 ‐ 0.1µm ‐ Green
Quartz Slides and Coverslips
Optical properties of fused Quartz Optical transmission properties provide a means for distinguishing among various types of vitreous silica as the degree of transparency reflects material purity and the method of manufacture. Specific indicators are the UV cutoff and the presence or absence of bands at 245nm and 2.73µm. The UV cutoff ranges from about 155 to 175nm for a 10mm thick specimen and for pure fused quartz is a reflection of material purity. The presence of transition metallic impurities will shift the cutoff toward longer wavelengths. When desired, intentional doping, e.g., with Ti in the case of Type 219, may be employed to increase absorption in the UV. The absorption band at 245nm characterises a reduced glass and typifies material made by electric fusion. If a vitreous silica is formed by a "wet" process, either flame fusion or synthetic material, for example, the fundamental vibrational band of incorporated structural hydroxyl ions will absorb strongly at 2.73µm. UV Cutoff As the transmission curve in Figure 24 illustrates, GE Type 214 fused quartz has a UV cutoff (1 mm thickness) at <160nm, a small absorption at 245nm and no appreciable absorption due to hydroxyl ions. Type 219, which contains approximately 100ppm Ti, has a UV cutoff at about 230 nm for a 1 mm thick sample. The IR edge falls between 4.5 and 5.0µm for a 1mm thick sample. Tables XIV and XV detail the percent transmittance for Types 214 and 124 fused quartz, including the losses caused by reflections at both surfaces. Values represent a 1mm thick Type 214 sample and a 10mm thick Type 124 sample. Type 124 fused quartz is a very efficient material for the transmission of infrared radiation. Its infrared transmission extends out to about 4µm with little absorption in the "water band" at 2.73µm. Conversion to other thicknesses can be accomplished with the following formula: T = (1-R) 2e - at Where: T = percent transmission expressed as a decimal. R = surface reflection loss for one surface. e = base of natural logarithms a = absorption coefficient, cm-1-1 t = thickness, cm (Data for GE Fused Quartz) SOME PHYSICAL CONSTANTS OF CLEAR FUSED QUARTZ Density: 2.2g./c.c. Hardness: 4.9(Mohs') Thermal Conductivity: .0033 g. cal./cm.²/sec./°C/cm. Specific Heat: .18 g. cal./gm. Softening Point: (approx.) 1665°C Annealing Point: (approx.) 1140°C
Resolution and Physical Image Size in C-mount Cameras
Magnification versus Resolution The below table is about a photograph's resolution capacity, which relates both to pixels and output image size. If a camera's resolution is insufficient, an image cannot be sharp. The other factors affecting image quality are the microscope's performance, preparation or suitability of specimen and the resolution of the unaided human eye (able to distinguish two points 0.1mm apart). Code Image Size Computer Screen (72 DPI) Print (300 DPI) 300 DPI/4 ODCMA35 350KB 8.8" 2.1" 0.5" ODCM0130C 1.3MP 17" 4.2" 1" ODCM0310C 3MP 28.4" 6.8" 1.7" ODCM0510C 5MP 36" 8.6" 2.2" ODCM0900C 9MP 48.4" 11.6" 2.9" ODCM1400C 14MP 55.5" 13.3" 3.3" DPI - Dots Per Inch Modern quality lenses should never be a limiting factor here. However, using a compound microscope under oil immersion, or with the 'high dry' lens, the image could be enlarged past 1000 x, and past the limit of resolution imposed by the wave-length of 'white' light. This would exceed the useful magnification which is possible, and the image cannot be sharp. Other reasons for an unsharp image are bad focus, thick specimens (using a compound microscope 2µm sections give best resolution) or specimen movement. It must be clearly understood that microscope or specimen related problems are totally divorced from the performance of a camera. How many Pixels do I require? The above table is a guide for selecting a camera size (pixels) which suits your requirements. These cameras mostly differ in resolution - number of pixels. Note that magnifications always compare lengths; with cameras we consider the number of pixels along the longer axis divided by dpi. Since computer screens (they measure the diagonal) and pixels are given in inches we have retained that measure here; to convert inches to mm multiply by 25.4. The column '72' relates to common computer screen and Internet resolution. So if the requirement is to display images on the screen or post these on the Internet, they may be enlarged to the indicated inches. Requirements for printing are more stringent and many publications require 300 dpi, which curtails 'enlargeability'. Additionally, in many photos the subject, does not fill the frame and when printing, part of the original is cropped. Sometimes this is deliberate as under a dissecting microscope, depth-of-field (or apparent focus) is most effectively increased by taking the photograph at a lower power and then enlarging by printing the centre part of the image only. The last column '300 dpi/4' assumes that only a quarter of the photograph is printed, which would greatly increase depth-of-field, but lower resolution markedly - unless the print is small. Further Information en.wikipedia.org/wiki/Dots_per_inch en.wikipedia.org/wiki/Pixels_per_inch en.wikipedia.org/wiki/Image_resolution en.wikipedia.org/wiki/Display_resolution Image Resolution Calculator
Schott Glass Technical Data
Description Schott D263M is a colorless borosilicate glass with a very low iron content. It meets requirements laid down in ISO 8255-1. It has high spectral transmission, excellent flatness and a refractive index finely adapted to microscopes. Very good resistance to chemical attack. Fire polished. Applications The characteristics of this special composition substrate glass makes it suitable for a variety of applications, including: Microscope cover glass Thin film substrate Touch control panels Solar cells Sensors Mechanical Properties Density p in g/cm3 2.51 Stress optical coefficient C in 1.02 · 10-12 m2/N 3.4 Young's Modulus E in kN/mm2 72.9 Poisson number µ 0.208 Modulus of torsional shear G in kN/mm2 30.1 Knoop hardness HK100 590 Electrical Properties Dielectric constant Ԑr at 1 MHz 6.7 Dielectrical loss factor tan δ at 1 MHz 61·10-4 Electrical volume resistance pD in Ω · cm at defined temperatures pD for alternating current 50 Hz θ = 250°C 1.6 · 108 θ = 350°C 3.5 · 106 Thermal Characteristics Viscosity and Temperature Description Viscosity log Ƞ [dPas] Temperature θ [°C] Strain point 14.5 529 Annealing point 13.0 557 Softening point 7.6 736 Transformation temperature in °C 557 Expansion factor α Coefficient of thermal expansion α (20-300°C) in 10-6 K-1 (static measurement) 7.2 Spectral Transmittance (d=0.15mm) Optical Properties of Schott D263M Refractive indicies ne (λ = 546.1nm): 1.5255 ±0.0015 nD (λ = 589.3nm): 1.5230 Above value ve: 55 The light transmittance for a thickness of 0.15 mm is TVD65 in % (d=0.15mm) = 91.7 ± 0.3%. In the visible range of the spectrum D26 M is without absorption. The excellent UV absorption properties make D263M an ideal material for use in fluorescence microscopy. Thickness* Dimension of Schott D263M No. 0 = (0.08 - 0.12mm) No. 1 = (0.13 -0.16mm) No. 1.5 = (0.16 - 0.19mm) No. 2 = (0.19 - 0.23mm) *Not all thicknesses available in all sizes Chemical Properties of Schott D263M Hydrolytic resistance: (DIN ISO 719) Hydrolytic class: HGB 1 Equivalient of alkali (Na2O) per gram of glass grains in µg/g: 20 Acid resistance (DIN 12 116) Acid class: S 2 Half surface weight loss after 6 hours mg/dm2: 1.4 Alkali resistance: (DIN ISO 695) Class: A 2 Surface weight loss after 3 hours mg/dm2: 88 Chemical Composition of Schott D263M SiO2 = 64.1% B2O3 = 8.4% Al2O3= 4.2% Na2O = 6.4% K2O = 6.9% MgO - CaO - BaO - ZnO = 5.9% TiO2 = 4.0% As2O3 - Sb2O3 = 0.1%
Stereo Dissection Microscopes
Avoiding Reflections for Stereo Dissection Microscopes The following article is in reference to coins, however it can be adapted to suit any sort of reflective sample. Change the liquid to suit your sample type. Because the surface of a coin has many angles and polarised light does not work well on metals, it may seem an intractable problem. Actually it's easy: just place the coin in a suitable shallow dish and cover the coin with water ‐ or alcohol if you are loath to use water on pristine coins. The light now no longer is reflected by the angled metal but only by the liquid's flat surface. Apply a light at roughly 30° degrees and the reflection exits at the same angle. Therefore your photo will be totally reflection free. If you normally use ring light illumination consider adding a fibre light source with gooseneck to have greater control over the angle of light. See our full collection of light sources. Tip: Place a coloured cloth under the dish for a uniform background.
Utermöhl Chambers
Instructions for combined plate chamber after Utermöhl The small ledge in the centre hole of the base plate has to be greased with a very thin layer of silicone grease or vaseline. The 0.2mm thick bottom glass is now inserted into the threaded ring(AISI 316 stainless steel) and the ring is screwed in the base plate with the ring tool. If necessary, use some silicone grease or vaseline on the inside of the threaded ring. The base plate is mounted into the holder. The square base of the tubular chamber (settling chamber) is also greased with a thin layer of silicone grease. The tubular chamber is placed right over the hole in the base plate. The sample is fed into the tubular chamber and a round top glass is pushed right over the sample. There must be NO air bubbles under the top glass. When the sedimentation of the sample is finished, push the tubular chamber towards one side of the base plate and the content can be emptied into a sink or similar. A square cover glass is pushed over the hole in the base plate to cover the sample. The sample is now ready for microscopic examination. Important: Do not use alcohol for cleaning acrylic parts.
UV Light Exposure Limits
Depending on specific application there may be operator exposure to UV light. The manual does not have any warnings on UV exposure but the TLV for the spectral region of these lamps is that the total irradiance incident upon the unprotected skin or eyes should not exceed and intensity of 1mW/cm² for periods greater than 17 minutes. Blak‐Ray® Lamps deliver high intensity long‐wave (365nm) ultraviolet light. Blak‐Ray® UV Spot Light with 100W lamp has an intensity of 21.7 mW/cm² at 5cm and 8.9 mW/cm² at 25cm distance. Blak‐Ray® UV Tube Light with two 15W lamps has an intensity of 1.6 mW/cm² at 15cm. Depending on how the lamp is used including distance, reflective surfaces, exposure time, etc. this TLV could be exceeded. If there is any potential for the eyes and face exposure to exceed the above stated exposure to UV radiation, a polycarbonate face shield that complies with the ANSI Z87.1‐2003 UV certification must be worn to protect the eyes and face. Ordinary prescription eyeglasses may not block UV radiation. UV certified goggles and safety glasses will protect the eyes but not the operator's skin.