Use the eyepiece reticule and measure a distance on the stage micrometer. Lets say we measure from the .2 to the .3 on the stage micrometer. The measurement we get is 1mm, we counted 10 division when we measured, so each division of the eyepiece reticule measures .01 mm.
This is the best way to calibrate a reticule since we are comparing a unknown to a known. This method eliminates any discrepancies caused by magnifications not being exact. Fact is that nothing is perfect so using a know standard is a good idea. To calibrate a reticule with out a standard you need to know the total magnification of the system up to the reticule. Since the eyepiece is above the reticule we ignore the eyepiece. Look at it this way if you use a magnifying glass to look at a ruler you don't take the magnifying glass into account. To get the total magnification multiply the magnification of the objective times the magnification of the tube, if any. If there is no tube magnification then the tube magnification is one. Divide the total magnification (TM) into the distance between lines on the reticule. The distance between lines (DL) can be found by dividing he number of lines into the length of the reticule. So the apearant distance between line = DL/TM. To calibrate a 10X objective on a microscope with a tube factor of 1, a microscope with no tube factor, first find the distance between marks. If the eyepiece reticule is a 10mm divided 100 times then the distance between marks is .1mm. Divide .1mm by the total magnification which is 10 and you get .01 mm. This is the measurement distance between each mark.
This method works but not as well as using a standard. Both methods need to be done for each objective you are going to use. Always use a high quality standared and use a metric standard for metric reticules and an English standard for English. Trying to convert only induces error. If you need really exact measurements from a reticule you can use a stage micrometer that is traceable to the National Institute for Standards and Technology (NIST). They insure that the standard is accurate by comparing it to known standards. Usually this is not necessary since micrometers measurements are not all that accurate any way. This brings up the issue of relative or absolute measurement. Relative measurement is used to report percentage differences against some other condition. An example of relative measurement would be measuring the average size of normal cells and abnormal cells in a specimen and reporting the percentage difference not the measured sizes. An absolute measurement is reporting the actual size of the object based on some standard measurement such as inches or meters. Absolute measurement is used for things such as parts. Industry is interested in the true diameter of drill bits not the variance. Biology based applications use relative size a lot since this can show changes. Once a microscope is calibrated it is calibrated for life unless you change the configuration. Frequent recalibration of a reticule is a waste of time and money. Some medical inspection persons require this and it is a total crock. If accuracy is a real concern you should use a video based measuring system or a filar micrometer. Filar micrometers have a moving line visible in an eyepiece that is driven by a micrometer drum. The distance is read of the micrometer drum or of a digital display. These work very well if the specimen is rectilinear. They are used a lot in industry.
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The problem with reticules is that specimens don't always begin and end at a reticule line. You have to move things around to get the measurement. This leads to interpalation errors. If you do a lot of measuring reticules are hard to use. They also don't do area of irregular objects well at all. Were your eye is centered in the field will also influence the measurement. The end result is that two different persons will read a reticule differently. This difference may be significant or it may not. If it is you will need to use another system. Video systems are more accurate and easier to use for very exact measurements. There are three general types of video measuring systems, video filars, interactive video and fully automatic. Each has its uses and problems and manufacturers make a wide range of each system. All video systems use a video camera, mostly black and white. The camera is connected to a computer or digital system, think of it as a black box. The black box does something and out pops the magic number. You see all of this on the monitor. This means that you have to set the microscope up for video. If the microscope isn't set up properly the system will be very difficult to use. A video filar micrometer shows you the image and either two or four lines. These lines are used to box the specimen or indicate the start and stop point for a length measurement. If the specimen is rectilinear or supposed to be this is an excellent tool. If you are measuring holes, boxes or lines you need to look at one of these. Interactive video systems usually use a computer with a board installed to show you the specimen and allow you to draw a line around the specimen. As you draw you see the line. Once you get close to the end the system closes the line and ends the measurement. Now the computer takes over. Using the array of points stored in it the computer calculates the information you ask it to. Normally these systems can do lengths, areas, perimeters and form factors. Form factor tell you something about the shape of the object. These are as accurate as your drawing. This can be quite accurate. Sometimes they are the only way to do an application. These days they are an excellent trade of since there cost is low and the accuracy is high. However if you need to measure a lot of things they can be very slow. If you want to count large numbers of things they may be no help at all. Automatic systems are very fast. They can count thousands of items in seconds. These counts can be linked to sizes and statistical analysis done at modern computer speeds. This sound like this is what every one needs but there are problems. What they do they do well and what they don't they don't do at all. Fully automatic systems use the density or color of the specimen to automatically measure multiple points in the specimen. A image capture board in a computer changes the image to an array of numbers based on the density or the color of the specimen. You then tell the system what constitutes the objects you want to measure based on the density or color. This is called "thresholding" and the success or failure of the application is based on it. The real world problems are many. The major problem is things in the specimen having the same density as things you want to measure. Most analyzers have "filters" that try to eliminate unwanted objects by shape, size and other criteria. Filters usually are employed after the actual measurement is taken. If the object doesn't meet the criteria for the object you want to measure it is thrown out of the set of objects that will be reported. On modern computers this takes very little time, you probably wont notice it. Another problem is touching objects. People are very good at distinguishing touching objects but automatic systems aren't. Each system has ways to reduce the problem but none of them eliminates it. What automatic systems do very will is simple images with lots of contrast. If your specimen is like this or if you can make it like this with different preparation then an automatic system will work very well. If you have complex requirement for a system it may not work at all. Before you buy an image analyser see it in operation. These are complex programs and need to be thoroughly examined. If it doesn't do the application while you are looking at it then don't buy it. It is common practice to send the manufacturer of an analyser a specimen with a detailed description of what you want measured. Let them try the application before they show up in your lab. If they can do it then take a long look at it, if not no hard feelings. Most modern automatic image analyzers use an IBM or Macintosh computer. However some system still use a proprietary computer. I wouldn't consider a proprietary system. In fact a lot of manufacturers now just make the software and support a range of image boards. The problem with proprietary computers is that computer technology is progressing rapidly. How would you like to be stuck with an old computer in a system when you could get a new, faster computer at a local store? Service is another problem. Computers service and parts are readily available for major computer brands. A proprietary make puts you at the mercy of the manufacturer. If an automatic image analyser sound like an image processor you are right and have read the last section! Most image analyzers can do image processing. Frequently image analyzers have macro languages or self teaching modes so that you can pre-program a whole processing-analyzing function. Each analyser whether it is interactive, automatic or a filar will have a calibration routine. In industry it is common to use a N.I.S.T. traceable standard. Using a traceable standard makes all plants and suppliers systems measure identically.