Histology, microscopy, anatomy and disease
Histology, microscopy, anatomy and disease

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Histology, microscopy, anatomy and disease

3 Measurements on the microscope

Sometimes it is important to measure the size of a cell, the distance between cells or the number of cells in a given area. Most light microscopes have the option to add a graticule for determining distances or a grid into the light path, to allow such measurements to take place.

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Transcript

DAVID MALE
In this video, I'll be showing you some more functions of the virtual microscope, and also how to calibrate a microscope using a standard etched slide. This information allows you to measure the size of cells and structures on the sections.
On a real microscope, the magnification is normally described as the magnification of the objective multiplied by the magnification of the eyepiece. For example, using a times 10 objective and a times 10 eyepiece gives a times 100 magnification. But this assumes that you're looking at the section from 30 centimetres away, which is about as close as most people can normally accommodate.
On a virtual microscope, the size of the image will depend on the objective used to produce the image, the camera attached to the microscope, and the size of the screen used to view the image, which could be anything from a mobile phone to a cinema screen. Moreover, the perceived magnification also depends on how far a person is from the screen.
So stating a magnification for a virtual microscope is fairly meaningless. It depends entirely on the device being used to view it. What you really want to know is how big the original item is. And that is one reason for calibrating the microscope.
As well as showing the additional microscope functions, there are some shortcuts which can be done on the virtual microscope, but not on a real microscope. So let's take a look at some of those now.
I have the normal blood smear loaded on the stage again. First, look at this tool icon. If you check the box, sections will automatically be loaded in focus. Not so realistic, but it will save you time. Another shortcut is on the stage view. If you click and drag the red circle, you'll be able to move around the section much more quickly than if you just used the stage movement arrows.
Another bit of information is given by the x- and y-coordinate positions corresponding to the centre of the eyepiece view. In this case, the centre of the view is position x equals 4,445, y equals 3,958. This is useful if you want to describe exactly where something is on the section.
Just above our two boxes, suppose that someone asks you to describe the leukocyte located at position 4,190, 2,400. Then you'd just type those coordinates into the boxes and click the Go button. The microscope takes you immediately to the correct point.
If you now change the objectives, you can see that the cell at that point is a neutrophil. You might want to take a picture of it. In this case, you need to click on the Camera icon, which will grab the eyepiece image. Depending on the device you are using, there are various options to copy, print, or save the image.
Returning to the microscope again, if you want to count cells, the Grid control will put an overlay onto the section, which makes it easier to count the number of cells or other features in a defined area. When doing this, it is conventional to count in anything that overlaps the top or left hand side of a square, and discount anything overlapping the right or bottom sides.
I'll take the grid off now, as I want to show you how to calibrate the microscope. To do this, I first need to load a standard etched slide onto the stage. This slide has been photographed on the real microscope using exactly the same objective and camera as was used on that microscope to take images of the other section shown here. The spaces on this slide are 1 millimetre apart. The spacings between the numbers 5 and 6 are 0.1 millimetre apart.
Let's suppose that I want to calibrate the times 20 objective on the virtual microscope. First, I add a fixed graticule to the eyepiece image. On a real microscope, the graticule would be part of the eyepiece. Now I'm switching to the times 20 objective. I'm going to align the marks on the graticule with the marks on the slide. I can now see that 1, 2, 3, 4, 5, 6, 7.5 marks on the graticule correspond with 0.1 millimetre. This means that with this objective, one whole division on the graticule equals 0.1 millimetre divided by 7.5, which equals 0.013 millimetres or 13 micrometres.
Armed with this information, I can now work out the size of a cell. Let's have another look at that neutrophil, which is at position 4,190, 2,400. I must look at it on the same objective as I used for calibration-- times 20. And you can see that the cell just about occupies one division of the graticule, which implies that it is approximately 13 micrometres in diameter. For convenience, both the grid and the graticule can be rotated.
Remember that calibration has to be done for each objective. When micrographs are produced, it is conventional to add a scale bar. In this case, I'm adding a scale bar corresponding to 50 micrometres to the image. If the scale bar is part of the image, then any change in the size of the image will change the length of the scale bar, so the size of the cells is unambiguous. Finally, I'm going to take the graticule off the image. And now you should try to calibrate the other objectives yourself.
End transcript
 
Interactive feature not available in single page view (see it in standard view).

These elements are usually added by exchanging one of the eyepieces for another with a graticule built into it. Therefore, the appearance of the graticule is fixed according to the eyepiece that is used; changing the objective does not change the appearance of the graticule.

A consequence is that the graticule has to be calibrated for each objective. This is most readily done by viewing a slide that has rulings etched into its surface, and observing how many units on the graticule correspond to, for example, 1 mm on the slide. Such a slide is shown below.

Described image
Figure 11 The appearance of an etched slide on a microscope stage when viewed with an eyepiece that includes a graticule.

The video above shows you how to calibrate an objective using a graticule and etched slide on the virtual microscope, as well as introducing a grid feature that you can apply. The grid can be used to measure the number of items within a particular area, for example, the density of blood vessels or the numbers of cells within a defined area of tissue. These are skills you will use in later parts of the course.

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