How To Measure Microns With A Microscope?

microscope calibration and measurement accessories

• light microscopes can magnify specimens about 1000x and resolve objects down to 0.2 microns (200 nm, nm = 0.000001 mm).
• light microscopes capable of super resolution can detect objects to about 20 nm.
• most light microscopes are used to measure organisms such as their length and width to aid in identification.
• measuring objects with a light microscope or even a stereo microscope is straight forward, but you will need a few accessories and you will need to calibrate your microscope.
• once your microscope is calibrated you can add scale bars to photographs with an image editing program like photoshop.
• there is software that permits you to calibrate each objective and then it will automatically add scale bars to the picture (e.g. image j).

• one mm scale on a microscope slide. the scale is divided into 0.010 mm.
• the micrometer scale is required to calibrate a light microscope.
• the picture above is magnified 100x and shows the 1 mm scale on the micrometer slide.
• it is divided into 100 divisions the smallest division is equal to 0.010 mm or 10 microns.
• photo above shows an eyepiece reticule with a scale etched on its surface.
• the most common reticles have a horizontal scale; some reticles have both horizontal and vertical markings.
• reticles come in different diameters and different markings e.g. some can measure angles, others have grids and some only have a cross on them.

field of view diameter

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

• 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.

eyepiece reticle calibration

• 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).
• 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.

• 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.

reticle division calculation

• eyepiece reticle is a small piece of glass with a ruler or grid imposed on it that fits into the microscope eyepiece.
• when looking through the microscope, the reticle image is imposed upon your specimen image.
• most often the reticle is used to make measurements or count particles.
• when using a microscope eyepiece reticle with different objective lenses, the distance between the lines on the reticle changes.
• in order to determine the distance between the lines on the reticle you are using, you will want to use this formula for calculation:

formula

reticle division / objective lens value = distance between lines on reticle at this objective value

example

if you are using a 5mm reticle with 100 divisions, your reticle division is 5/100 = 0.05mm

4x objective

0.05mm / 4 = 0.0125mm when using the 4x objective.

10x objective

0.05mm / 10 = 0.005mm when using the 10x objective.

40x objective

0.05mm / 40 = 0.00125mm when using the 40x objective.

100x objective

0.05mm / 100 = 0.0005mm (or 0.5um) when using the 100x objective.

• you need to make this calculation for each objective lens used.

stage micrometers

• linear measurements require the comparison of the object to be measured with a standardized scale, such as a ruler.
• in utilizing eyepiece reticles or micrometer eyepieces for measurements in the microscope, the arbitrary units of the transfer scale (reticle), which is superimposed upon the specimen image, must be converted to absolute units, such as millimeters or micrometers.
• calibration of the reticle scale graduations is commonly performed by imaging a stage micrometer with the same objective to be used for specimen measurements.
• a proper calibration involves determining an absolute distance on the stage micrometer, imaged in place of a specimen, which corresponds to one division of the scale in the eyepiece reticle.
• this value is often referred to as themicrometer value, orcalibration factor, for that particular objective.
• once the value has been determined, the size of any specimen feature may be calculated by multiplying the number of eyepiece reticle divisions spanned by the feature with the calibration factor for the objective in use.

• micrometers commonly have a graduated scale either one or two millimeters in length, subdivided into units that are one-tenth millimeter in length (100 micrometer units).
• each 100 micrometer unit is further subdivided into ten equal sections, resulting in the smallest graduation representing ten micrometers.
• although photographically produced micrometers are adequate for routine work, especially at lower magnifications, their lines are too ragged along the edges and are too wide for accurate measurements, or for use at high magnification.
• these micrometers should be restricted to rough measurements at low magnifications.

calibration procedure

1. place a stage micrometer on the microscope stage and bring the micrometer scale into focus using the microscope coarse and fine focus control knobs.
2. detecting the scale and translating it into the center of the viewfield is facilitated by the use of a low power objective to first locate the circle surrounding the scale, and then the scale itself.
3. the ring encircling the micrometer scale is visible with the naked eye and should be used to position the stage micrometer in the center of the microscope optical path (stage aperture).
4. rotate the desired objective into position and ensure that both scales (the stage micrometer and the eyepiece reticle) are visible in the viewfield in simultaneous focus.
5. for the most accurate measurements, utilize the largest possible range of divisions on both scales.
6. finally, determine the apparent length of the eyepiece scale in reference to the divisions on the stage micrometer.

• the calibration procedure just described must, of course, be repeated for each objective that is to be employed for linear measurements.
• it should be noted that magnification varies by a few percent for similar objectives (even from the same manufacturer) inscribed with the same magnification factor (for example, 10x), so each objective should be independently measured.
• if the microscope is regularly used with a number of different objectives, it may be more convenient to plot calibration curves for each objective in graphical form.
• this provides an easy mechanism to rapidly determine feature sizes while working with the microscope, without having to repeat the arithmetic when applying the micrometer values for all of the objectives used to conduct measurements.

scale bars for photomicrographs

1. open photomicrograph in adobe photoshop (these methods can be done in all recent versions of photoshop).
2. select view > rulers to turn the rulers on around the picture.
3. right click on the ruler and select units > pixels.
4. 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.
5. use the ruler tool in the tool bar and open the info box (window > info).
6. dragging the ruler from a to b records the length in pixels.
7. use this value to solve for the length of a scale bar 50 microns which is 969.5 pixels.
8. once you determined the width of the desmid in pixels you can calculate the length of the scale bar in pixels as shown above.
9. use the ruler tool to measure this length.
10. 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.
• a scale bar adds size information and professionalism to the photograph.

measuring microscope display

• a micrometer, also referred to as a micron, is a microscopic measurement unit equivalent to 0.001 millimeters or 0.000039 inches.
• there are a variety of way to measure microns with a measuring microscope, depending on your microscope and configuration.
• in the toolmakers configuration of the stm7 measuring microscope, operators simply place their sample on the microscope’s stage and then locate the feature on the sample that they want to measure.
• when moving across the sample to an end point, the measuring microscope display shows the distance measured in all three axes.

measurement accuracy and limits

• the minimum resolvable distance in an optical microscope is approximately 0.2 micrometers (under optimal circumstances), a linear measurement below this value cannot be accurately determined.
• it is critical that both scales (reticle and stage micrometer) are imaged as sharply as possible, and as previously suggested, it is preferable to utilize as many divisions of the stage micrometer as can be observed in the field of view for the calibration.
• the alignment of the lines of the eyepiece reticle with those of the micrometer should be made consistently from the same edge of the stage micrometer line rules, and not at the center (which cannot be reproducibly identified).
• the quality of the graduations on a stage micrometer has a significant effect on the accuracy with which a calibration can be conducted, and this is especially true at high magnifications.
• micrometers produced by processes such as thin film deposition usually have much finer lines with better-defined edges than those produced photographically, and can provide improved accuracy and precision.
• if an objective having some field distortion must be utilized, restricting measurements to the central portion of the viewfield will minimize measurement errors.