LM - 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:

1. External diameter of the reticle disc.
2. 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.
3. 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 12 microns.

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.

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 4000 microns (4mm) to coincide with or read a 100-micron 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 10 microns, then each division = 24.2857 microns. 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

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.

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.