Materials needed to calibrate a Light microscope or Stereo microscope.
The scale on the microscope slide can be used to determine the field of view in your microscope and to calibrate the ocular scale for each objective. On a zoom stereoscope choose specific zoom settings (magnifications) to calibrate. When you are finished calibrating the scope I recommend you write or print the results and tape the paper to your microscope or nearby it for quick reference.
Most microscope dealers offer both the micrometer slides and the reticles. Before ordering an eyepiece reticle, measure the inner diameter of your microscope eyepiece. Stereo microscopes tend to have larger diameter tubes. Another device that fits into the eyepiece tube that can be used for precise measurements is a filar micrometer. These cost hundreds of dollars. They allow you to move a line over the scale and are also used in astronomy (their use is not described here).
Calibration of a light microscope involves determining the distance (in microns) that each division of the reticle represents for each objective attached to your microscope. For stereoscopes each division of the reticle may represent 1 or more millimeters and if you have a zoom scope you may want to mark the zoom settings on your scope where you calibrated the reticle. Rotating the eyepiece with the reticle allows you to orient the scale in different directions for measuring.
Calibrating a Microscope in order to Measure Specimens
There are two simple ways to measure the size of objects with a microscope. First, I will describe an easy way which can be used to estimate the size of a subject and can be used to measure moving subjects. The second method requires calibrating each objective and using the eyepiece reticle to measure the dimensions of the specimen in reticle divisions and then converting this value to microns. With a stereomicroscope you will follow the same procedure, but use a ruler with a millimeter scale to calibrate the ocular reticle.
The disadvantage of using an eyepiece reticle is that it is not easy to measure subjects that are moving (e.g. ciliates). Large ciliates and other organisms must be pinned down by drawing water from under the coverslip. Alternatively you can fix, anesthetize, or kill the organisms though these procedures can alter the organisms shape, size and color. I prefer to measure the dimensions of live organisms that are gently pinned under the coverslip.
⦁ Easy method for Estimating an Organisms Size
This first method for measuring subjects only requires a microscope slide with a micrometer scale. You will put the micrometer slide on the microscope stage, focus on it with each objective starting from low to high power and measure the field of view (diameter). For low magnification objectives you will need to move the 1 mm scale several times to determine the overall diameter of the field of view in mm, which is then multiplied by 1000 to convert to microns.
A mechanical stage is useful for moving the micrometer scale. Record the total width of the field of view for each objective in millimeters and convert to microns (Table 1). With a 5X objective, the 1 mm scale fits 5 times within the diameter of my microscope, therefore the field of view is 5 mm or 5,000 microns. At 10X the scale fits 2.3 times so the field is 2.3 mm or 2300 microns. With a 20X objective the field of view is 1,150 microns and at 40X it’s 560 microns (on my microscope). For each microscope, the objective and eyepiece used will affect the numbers you get. If you have a 60X and/or 100X objective, calibrate those as well. In my measurements I used 10X wide field eyepieces. If I used 15X or 20X eyepieces I would get different values so would need to calibrate the objectives for different magnification eyepieces. The 10X eyepieces are used most often.
|Objective magnification||Field of view diameter|
|2.5X||10 mm = 10,0000 microns|
|5.0X||5 mm = 5,000 microns|
|10X||2.3 mm = 2300 microns|
|20X||1.15 mm = 1,150 microns|
|40X||0.56 mm = 560 microns|
Once the field of view is determined for each objective, one can estimate an organism’s size by determining the fractional width of the field of view that it occupies. If for instance a ciliate that fits approximately 1/2 the field of view with the 20X objective having width of 1,150 microns then it would be 575 microns, if 1/3 the width is about 383 microns. This method permits you to estimate the size of an organism within ± 10% of its actual size, which is often accurate enough to help you identify some organisms.
Fig. 5. Above image shows a Paramecium caudatum whose size can be estimated by the fraction of the field of view it covers once the field of view for a particular objective has been measured. If the organism fills the field of view equal to 1000 microns A) the paramecium is about 1000 microns long. In B) the Paramecium is about ½ the field diameter therefore it is 500 microns and in C) the Paramecium covers about 1/3 the field and is 330 microns long. This technique can be used to estimate the size of moving organisms.
⦁ Precise way of Calibrating a Microscope and Measuring Organisms Dimensions
For the precise method of measuring subjects, you will need a reticle scale that fits into one of your microscope eyepieces. The reticle requires a retainer ring that screws into the eyepiece and holds it in place. The reticle has a scale on it divided into small divisions. The distance between the divisions is not important only that they are regularly spaced. The reticle scale usually occupies about half the field of view.
To calibrate the reticle scale you need to place the micrometer slide on your microscope stage and focus on it. Start with the lowest power objective and then move on to your higher power objectives. The microscope slide is divided into known divisions the smallest being 0.010 mm or 10 microns. When you view the reticle scale over top of the scale on the microscope slide you need to determine how many divisions of the micrometer slide match one division in the reticle.
Fig. 6. The diagram above shows how you line up the scale in your eyepiece reticle markings with the 1 mm scale on your microscope slide. One division of the reticule scale = 10 divisions on the microscope slide. Thus one reticle division = 100 microns with a 10X objective (i.e. 100X).
You repeat this procedure with each of your objectives until you have a list for all your objectives. Below is a calibration for each of my objectives on one of my microscopes. I print this and attach it to my microscope.
5X objective 1 reticle division = 21 µm
10X objective; 1 reticle division = 10 µm.
20X objective; 1 reticle division = 5 µm.
40X objective; 1 reticle division = 2.5 µm.
100X objective; 1 division = 1 µm.
Once the microscope is calibrated you can measure the size of subjects with the eyepiece reticle in divisions and then convert the reticle divisions to microns.
Fig. 7. The image above is of stained human blood cells and the image includes the reticle scale. The scale has been calibrated for a 100X oil immersion objective (1000X) where one division of the scale is equal to 1 micron. Using the scale red blood cells range from 9-12 microns in diameter and the large white blood cell (neutrophil) is 18 microns in diameter in this picture. Most objects e.g. red blood cells have some variability in their size.
Fig. 8. In the photograph above the eyepiece scale is positioned and photographed over the organism, a rotifer Platyias quadricornis. The length of the rotifer is 44 divisions with a 10X objective, where the magnification it was photographed was 100X. If each division on the eyepiece ocular has been determined to be 10 microns then the length of the rotifer is 44 division x 10 microns = 440 microns.
You only need to calibrate your microscope once, unless you change objectives or eyepieces. Once the microscope is calibrated you can accurately measure the dimensions of a specimen. While taking a series of photographs, I take note of the objective used and the size of the specimen in reticle divisions. I calculate the size in microns later and use the real size of the specimen to make scale bars in Photoshop for the digital photographs as described below.
Putting scale bars on photomicrographs
When someone indicates a picture is magnified 400X this is only an approximation since the size of the picture will change when viewed on different computer screens, tablets, and cell phones or after the image has been cropped. It only suggests that the photomicrograph was taken with a 40X objective. However, if an image has a scale bar any changes in the image size will be reflected by changes in the size of the scale bar. Scale bars are also required on photomicrographs for scientific publications.
Putting a scale bar on an image can be done using software that has been calibrated for each objective in a manner similar to calibrating your microscope. Image J is free software that will do this. Some software however, may not allow the user to reposition the scale bar, change the color or thickness of the bar. For these reasons I prefer to use an image editor (Adobe Photoshop) though some other image editors may also work (see references for alternative methods and software). I provide an overview of how scale bars can be added using Photoshop (due to space limitations it is not a detailed step by step tutorial).
Method to calculate the length of the Scale bar in pixels to add to a photograph
⦁ You must first determine the size of the subject in microns with your microscope.
⦁ Open the image in Photoshop > Select the ruler tool > measure the subjects’ known dimension in pixels with the ruler tool.
⦁ Knowing the size of the subject in microns and in pixels (px) you can calculate the size of a scale bar in pixels and place it on the picture. Choose the length of the scale bar you want to add in microns (µm). E.g.
You measure the size of the object in pixels with a ruler tool in Photoshop. You already know the true size of the subject from measuring it using the calibrated reticle. Decide what the size you want the scale bar to show 10, 50, 100, 200 microns etc. Solve for X which is the size of the scale bar in pixels. Finally, you draw a line “bar” the length you calculated in pixels and add a text label.
Add text to the scale bar e.g. 100 microns or 100 µm or other units. The Greek symbol for micron = µm or just µ and can be added using Photoshops’ glyphs palette found under the Windows menu (Windows > glyphs) or by pressing Alt-230 on your numeric keyboard to get µ. Familiarity with Photoshop is required for the method I use, but this method can be accomplished with other image editing software as well. Below I present a series of screen shots where I describe an overview of the process.
Fig. 9. Above image shows a summary of the steps to make a scale bar 50 microns long in Photoshop. Subject is a Desmid Cosmarium sp. Differential Inference microscopy (DIC).
Fig. 10. Step 1 shown above. Open photomicrograph in Adobe Photoshop (these methods can be done in all recent versions of Photoshop). Select View > rulers to turn the rulers on around the picture. Right click on the ruler and select units > pixels. Select at the top menu Window > info to open the info palette and Window > layers to open layer palette to see the Photoshop palettes shown above.
Fig. 11. Step 2 above. Use the ruler tool in the tool bar and open the info box (Window > info). Dragging the ruler from A to B records the length in pixels. Use this value to solve for the length of a scale bar 50 microns which is 969.5 pixels.
Fig. 11. Step 3 above. Once you determined the width of the desmid in pixels you can calculate the length of the scale bar in pixels as shown above. Use the ruler tool to measure this length. I added two blue guidelines on the photograph to show the width of the scale that will be drawn (the guides just make it easier to draw the correct width of the scale bar). The guides do not show in the final image.
Fig. 12. Above select the pencil tool with a square brush, select color and width of the line, then draw the line between the guides you created. The bar in this instance is 50 microns. If the line is made twice as long, the scale bar would represent 100 microns.
Examples of Photomicrographs with Scale bars
Above unknown aquatic insect larva photographed using dark-field microscopy.
Desmid Micrasterius sp DIC microscopy.
Calibrating a microscope allows the dimensions of objects or organisms to be measured. Knowing the field of view makes it possible to estimate the size of the specimen by estimating the fraction of the field of view it occupies. A more accurate measuring method involves measuring a subject with an eyepiece reticle that has been calibrated as described above. The reticle can then be used to measure the size of the subject by measuring how many divisions it is with a particular objective and then converting the number of divisions to microns. In order to add scale bars to a photomicrograph you first need to measure the specimen size in microns after calibrating your scope. Some software programs allow you to calibrate the objectives and then automatically add scale bars to an image (e.g. Image J). Using Photoshop or other image editor involves measuring the size of the subject first in microns, then determining the length of the organism in in pixels, and finally calculating the length of the scale bar in pixels that you want to add. A scale bar can then be drawn the correct length with the pencil tool. The length of the scale bar involves simple math to calculate. Scale bars are required on photomicrographs destined for scientific publications in journals. Scale bars can also be added to movies so the audience knows the size of the specimens being viewed.
Motic Images 3.0
Motic Reticle https://moticmicroscopes.com/products/bareticle-micrometer
Image J Software: https://imagej.nih.gov/ij/
Adding scale bars to images with Image J https://kaplinskylab.domains.swarthmore.edu/scalebar.htm#
G. Mazo (2021I): A toolkit and Image J Plugin to quickly transform microscope images into scientific figures. PLoS ONE 16(11): e0240280. https://doi.org/10.1371/journal.pone.0240280
Making scale bars using Photoshop CS3 (2016) Digital Microscopy Center, University of Washington - PDF https://depts.washington.edu/digmicro/process_pdf/Making%20Figures%20for%20Publication.pdf
Photoshop for Scale Bar Tutorial - YouTube video: https://www.youtube.com/watch?v=runYCBxxaGI
Photoshop Tutorial : How to add scale bars – PDF https://labs.mcdb.ucsb.edu/weimbs/thomas/sites/labs.mcdb.ucsb.edu.weimbs.thomas/files/docs/scale-bars.pdf
K. Peifley (2015) How to Add Scale bars to Your Images: Zen software and Image J software.