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Enter keyword or # Calibrating a Microscope

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Digital Camera Adapters To properly calibrate your reticle with a stage micrometer, align the
Digital Microscopes (beginning) of the stage micrometer with the zero line (beginning) of the
carefully scan over until you see the lines line up again. You can then u
Forensic ratio to determine the value that each line represents in your re
Gout Microscopes
In the example above, the eyepiece micrometer (reticle image) is on the
Industrial stage micrometer image is on the bottom. The stage micrometer is 1 mm
100 divisions so each division of the stage micrometer is one one-hundre
Inverted (0.01mm or 10 um). Hint, you move the decimal point over three places t
Metallurgical change mm to micrometers.

Polarizing The eyepiece micrometer is divided into 100 units. We don't need to kno
distance between marks on it.
Professional Biological
Stereo / Low Power When the zero marks are lined up, scan across and look for a convenien
the lines converge again. If you look at the 30 mark on the reticle, you w
Students - Cordless close alignment with the stage micrometer. How many divisions? Did y
You are right! And, if each line is 10um wide, what will 20 lines equal
Students - Elementary
200um.
Students - High School
Now it is just a simple math ratio. 30 divisions of the reticle (eyepiece m
Students - University equal 200 micrometers. So what does one division on the reticle equal? L
Teaching Handbooks to 200 as one is to X. Remember how to do a ratio? Two fractions, 30 ov
1 over X. Cross multiply, you get 30X=200um, solve for X by dividing b
Teaching Videos
Video Flex Imaging 30 and X equals 6.7 um. Notice that they line up again at 60 but alignm
one at 90. If we use 90 and 61 (610um) we get 6.8um. The wider the inter
accurate your results should be.

Remember, this distance between reticle lines is only good for that p
objective lens and it may not come out to be a nice round number. Whe
to a different objective, you must recalibrate.

Quiz time: Our stage micrometer has a line 1mm long with 100 divisions
that each division is one one-hundredth of a mm (.01mm or 10um). Whe
it with the reticle, you notice that the lines converge at 8 and again at 1
choose 16. At the 16 mark on the reticle, we notice 60 lines on the stage
What does each mark on the reticle represent?

If you got 37.5 um, give yourself an A!

For further information you may want to view this link about eyepie
calibration.

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Microscopy and related methods: [overview; types; bright field microscopy] [dark
field optics] [phase contrast] [oil immersion] [differential interference contrast]
[measuring] [using a counting chamber] [wet (Vaseline) mount]
Recommended studies: See "Lab guides" microscopy/invertebrates

Measurement with the Light

 Microscope
Your microscope may be equipped with a scale (called a reticule) that is built into
one eyepiece. The reticule can be used to measure any planar dimension in a
microscope field since the ocular can be turned in any direction and the object of
interest can be repositioned with the stage manipulators. To measure the length of an
object note the number of ocular divisions spanned by the object. Then multiply by
the conversion factor for the magnification used. The conversion factor is different at
each magnification. Therefore, when using a reticule for the first time, it is necessary
to calibrate the scale by focusing on a second micrometer scale (a stage micrometer)
placed directly on the stage.

Conversion factor
Identify the ocular micrometer. A typical scale consists of 50 - 100 divisions. You
may have to adjust the focus of your eyepiece in order to make the scale as sharp as
possible. If you do that, also adjust the other eyepiece to match the focus. Any ocular
scale must be calibrated, using a device called a stage micrometer. A stage
micrometer is simply a microscope slide with a scale etched on the surface. A typical
micrometer scale is 2 mm long and at least part of it should be etched with divisions
of 0.01 mm (10 µm).

Suppose that a stage micrometer scale has divisions that are equal to 0.1 mm, which
is 100 micrometers (µm). Suppose that the scale is lined up with the ocular scale, and
at 100x it is observed that each micrometer division covers the same distance as 10
ocular divisions. Then one ocular division (smallest increment on the scale) = 10 µm
at 100 power. The conversion to other magnifications is accomplished by factoring
in the difference in magnification. In the example, the calibration would be 25 µm at
40x, 2.5 µm at 400x, and 1 µm at 1000x.

Some stage micrometers are finely divided only at one end. These are particularly
useful for determining the diameter of a microscope field. One of the larger divisions
is positioned at one edge of the field of view, so that the fine part of the scale ovelaps
the opposite side. The field diameter can then be determined to the maximum
available precision.

Estimating and reporting dimensions

Be aware that even under the best of circumstances the limit of resolution of your
microscope is 1 or 2 µm (or worse) at any dry magnification, and 0.5 µm or so using
oil immersion. No directly measured linear dimension or value that is calculated
from a linear dimension should be reported with implied accuracy that is better than
that. That includes means, surface areas, volumes, and any other derived values. For
example, suppose you measure the length of a flagellum on a Chlamydomonas cell at
400x, and determine that it covered 3 1/2 ocular divisions. The length is directly
calculated as 3.5 divisions times 2.5 µm per division, which comes out to 8.75 µm.
You know, however, that at 400x the absolute best you can do is to estimate to the
nearest µm, so before reporting this measurement round it to 9 micrometers (not 9.0,
which would imply an accuracy to the nearest 0.1 µm). For more information on
reporting uncertain quantities see our Resources section (analytical resources).

The calculation of a volume is subject to error propagation, namely the magnification

of an error when deriving a figure from one or more measured variables. For
example, suppose you measure the length and diameter of an object to be 65 and 30
micrometers, respectively, assuming a cylindrical shape. The volume is given by the
formula v = ¼r2l, where r = radius and l = length. The formula gives a volume of 45,
946 µm3. The volume isn't accurate to the nearest cubic micrometer, however.

Let's make the very optimistic assumption that the measurement of 65 micrometers is
indeed accurate to the nearest 1 µm. Then the number 65 means "greater than 64.5
and less than 65.5." The number 30 really means "greater than or equal to 29.5 and
less than or equal to 30.5." The smaller set of measurements yields a volume of
44,085 µm3, while the larger yields a volume of 47,855 µm3. False precision would
be implied even if one reported a volume of 46,000 µm3, obtained by rounding the
middle measurement. It would probably be better to report a range in this case, of
44,000 to 48,000 µm3. By the way, 46,000µm3 is 0.046 mm3, which probably
represents a better choice of units in this case.
Making assumptions
In many areas of experimental science, including biosciences, the ability to estimate
and make reasonable assumptions is a valuable skill. In order to make some
quantitative estimates, particularly of volumes, you will have to make assumptions
regarding the shape of some organisms. For example, if a specimen appears round,
you would likely make your volume calculation based on the assumption that the
specimen is a perfect sphere. For something like a Paramecium you might assume a
cylindrical shape in order to simplify your estimate, while remaining aware that you
could be way off the mark.

A specimen such as Chaos (Pelomyxa) carolinensis represents a real challenge.

Ameoboid organisms are irregularly shaped most of the time. Is it flat on the slide, or
does it extend up toward the coverslip? Perhaps it is attached to both. What model do
you use as a basis for volume estimation? Is it best to assume a particular shape and
take measurements at different times? Is it best to estimate a maximum and
minimum for each possible dimension and obtain a range of possible volumes?
Remember, you are only asked to estimate. Sometimes the best estimates have a
potential error of more than an order of magnitude.

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Created by David R. Caprette (caprette@rice.edu), Rice University 11 May 00
Updated 13 May 05