Assignment 1, Part 4: Measuring magnification and bead size

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20.309: Biological Instrumentation and Measurement

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Measure the microscope's magnification

Example images included by past students in their Week 1 report: (top) Air Force target, (center) Silica spheres and dust, (bottom) Ronchi Ruling

Measuring the magnification of your microscope is a good way to verify that your instrument is functioning well. You should measure the magnification of any microscope you plan to use for making quantitative measurements of size. Use the measured value in your calculations, not the number printed on the objective. Consider the uncertainty in your measurement.

  1. Connect the 10x objective to your microscope and image the microruler calibration slide.
    • You will find the objectives in the west drawers of the lab. Be sure to pick up a RMS to SM1 adaptor from the neighboring bin to be able to thread the objective into the Thorlabs cage plate.
    • The microruler calibration slide has tick marks that are 10 um apart. Every 100 um, there is a longer tick mark.
    • Make sure that the side of the microruler with the pattern on it faces the objective. Imaging through the thick glass causes distortion and many other troubles.
  2. Start the live preview using the UsefulImageAcquisition tool:
    • Familiarize yourself with the UsefulImageAcquisition tool and how to save and display images by reading through these instructions.
  3. Ensure that the camera's field of view is approximately centered in the objective's field of view.
    • The objective has a larger FOV than the camera. Use the adjustment knobs on mirror M1 to traverse the objective's FOV horizontally and vertically. The FOV is approximately circular. Find a spot near the middle.
  4. Record an image of the microruler.
    • Once your happy with the image on the live preview, click the Acquire button.
    • Display the image and use imdistline or the data cursor to measure a known distance between rulings in your image and compute the magnification.
    • When choosing a distance to measure, consider the factors that influence the uncertainty of your measurement.
  5. Save your images in a .mat file for later use in MATLAB or as a PNG image for use in your report or other programs (Did you read the instructions??).
  6. Repeat the magnification measurement for the 40x and 100x objectives.
    • With the 100x objective, you may want to substitute the microruler with a Ronchi Ruling, a grating with 600 line pairs per millimeter. Why is it not wise to use the Ronchi Ruling with the 10x objective?
  7. Using your magnification measurements and the known size of the CCD camera, calculate the field of view (FOV) of the microscope for each objective.

  • Display an example image of the ruler at each magnification, and
  • Make a table of displaying the nominal magnification, object height, image height, actual magnification and FOV (see example below). Don't forget to include appropriate units. Report the length and width of the FOV (in distance units), not its area (in distance units squared).

Nominal Magnification Object height Image height Actual magnification FOV

Measure particle size

Example image of 3.2 μm beads using the instructor microscope. Submit picture to replace this!

Now that you know the magnification of your instrument, use it to measure the size of some microscopic objects as imaged with the 40x objective lens only. Slides with 7.2 μm, 3.2 μm and 1 μm silica microspheres are available in the lab.

  1. Image 7.2 μm, 3.2 μm and 1 μm silica microspheres as described in the magnification measurement procedure (40x objective only).
  2. Measure and report the average size and uncertainty of the spheres in each sample. How many spheres should you measure?

  1. Display an example image of each bead size.
  2. Include a table containing the average size and uncertainty of the spheres in each sample, and the number of samples measured.
  3. In one or two sentences, explain how you chose the number of samples to measure.

Microscope storage

During the microscopy lab, approximately seven thousand optical components will be taken from stock, assembled into microscopes, and properly returned to their assigned places. Please observe the following:

  • Store your microscope in one of the cubby holes in 16-336 (not in the lab). If you use one of the high shelves, get somebody to help you lift.
  • Keep all of the boxes for the optics you use with your instrument to simplify putting things away.
  • Take a blue bin to store loose items (such as lens boxes) in.
  • Stages, CCD cameras, neutral density filters and barrier filters stay at the lab station. Do not store these with your microscope.
  • Return objective lenses to the drawer when you are not using them. (Do not store them with your microscope.)
  • The stages are very expensive. Always lift from the bottom.
  • If you break something (or discover something pre-broken for you), do not return it to the component stock. Give all broken items to an instructor. You will not be penalized for breaking something, but not reporting may be looked upon less kindly.

Back to Assignment 1.