Assignment 1 Overview: Transillumination microscopy

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20.309: Biological Instrumentation and Measurement

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I took a good clear piece of Cork, and with a Pen-knife sharpen'd as keen as a Razor, I cut a piece of it off, and thereby left the surface of it exceeding smooth, then examining it very diligently with a Microscope, me thought I could perceive it to appear a little porous; but I could not so plainly distinguish them, as to be sure that they were pores, much less what Figure they were of: But judging from the lightness and yielding quality of the Cork, that certainly the texture could not be so curious, but that possibly, if I could use some further diligence, I might find it to be discernable with a Microscope, I with the same sharp Penknife, cut off from the former smooth surface an exceeding thin piece of it, and placing it on a black object Plate, because it was it self a white body, and casting the light on it with a deep plano-convex Glass, I could exceeding plainly perceive it to be all perforated and porous, much like a Honey-comb, but that the pores of it were not regular; yet it was not unlike a Honey-comb in these particulars.

I told several lines of these pores, and found that there were usually about threescore of these small Cells placed end-ways in the eighteenth part of an Inch in length, whence I concluded there must be neer eleven hundred of them, or somewhat more then a thousand in the length of an Inch, and therefore in a square Inch above a Million, or 1166400. and in a Cubick Inch, above twelve hundred Millions, or 1259712000. a thing almost incredible, did not our Microscope assure us of it by ocular demonstration.

Robert Hooke from Micrographia: or Some Physiological Descriptions of Minute Bodies made by Magnifying Glasses with Observations and Inquiries Thereupon (1665)[1]


Introduction

Example 20.309 microscope.

Over the next few weeks, you will build an optical microscope using lenses, mirrors, filters, optical mounts, CCD cameras, lasers, and other components in the lab. The work is divided into 5 assignments. Each assignment requires some lab work, some analysis, lots of clear thinking, and an individually written answer sheet turned in on Stellar. All of the items you are expected to turn in are indicated by a pencil symbol in the lab manual.


Pencil.png This symbol means that you have to turn something in.


Assignment 1

In this first assignment, you will build a compound microscope, determine its magnification, and attempt to measure the size of microscopic objects. The instrument you create will have a great deal in common with the microscope Robert Hooke built in the mid-1660s. Hooke meticulously documented his microscopic observations and published them in a popular volume called Micrographia in 1665. The measurements you make in part 1 will call to mind Hooke's early quantification of the size of plant cells (see quote at top of page). You will grapple with many of the same challenges Hooke faced: resolution, contrast, field of view, optical aberrations, and obscurity of thick samples. (To overcome the thick sample problem, Hooke used a very sharp knife to cut an "exceeding thin" slice of cork — a technique still in everyday use.)

Robert Hooke's drawing of his microscope apparatus.
Robert Hooke's drawing of a flea.

Hooke spent countless hours hand drawing the breathtaking illustrations for Micrographia. A CCD camera in the image plane of your microscope will provide a huge advantage. You will be able to record micrographs nearly as spectacular as Hooke's in a fraction of a second and with far less skill. (As a young man, Hooke apprenticed as a painter. The guy could draw.)

Specimens in Assignment 1 will be illuminated by an LED that shines light through the sample plane. The illumination will show up as a bright background in your images. The unsurprising name of this method is: transilluminated, bright field microscopy. Transillumination works well for samples that absorb or scatter a lot of light. Most biological samples have low contrast when imaged this way. Despite the limitations of bright field microscopy, many important discoveries were made with this simple method. Hooke was an early discoverer of plant cells, but he was mostly interested in how the cell structure of his cork sample explained the material's unique mechanical properties. He soon trained his microscope on other things (like glass canes, a bloodsucking louse, and feathers).

Likely inspired by Micrographia, a Dutch draper named Anton van Leeuwenhoek honed his lens-making skills and developed his own microscope. Van Leeuwenhoek was intensely interested in the tiny creatures he dubbed "animalcules" that he observed in water, blood, semen, and other specimens. Looking at samples of plaque from his own mouth, van Leeuwenhoek recorded: "I then most always saw, with great wonder, that in the said matter there were many very little living animalcules, very prettily a-moving. The biggest sort. . . had a very strong and swift motion, and shot through the water (or spittle) like a pike does through the water. Looking at the second sort. . . oft-times spun round like a top. . . and these were far more in number." (Sadly, the colorful term "animalcule" did not have as much staying power as "cell.") Van Leeuwenhoek discovered bacteria, protozoa, spermatozoa, rotifers, Hydra, Volvox, and parthenogenesis in aphids. He was truly the first microbiologist.

Barbara McClintock with her microscope

Perhaps the most remarkable discovery ever made with nothing but a simple light microscope was genetic transposition. Barbara McClintock was a talented microscopist who developed a technique that enabled her to distinguish individual chromosomes in Zea mays (corn) plant cells. One important element of her method was that she prepared her samples by squashing them instead of cutting thin slices as Hooke did 300 years earlier. Squashing tended to preserve the chromosomal structure better than slicing. She observed genetic transposition through an optical microscope in 1944, nearly 10 years before the chemical structure of DNA was deciphered. Several decades elapsed before molecular techniques sufficiently sophisticated to confirm her discovery were developed.[2] McClintock was awarded the Nobel Prize in Physiology or Medicine in 1983 for her discovery.

Background reading and resources

You will work with log-log plots in this assignment and future ones. These seem to confuse everybody. Read this page to remind yourself how log-log plots work.

Several microscope manufacturers maintain educational websites, including Nikon's MicroscopyU, Olympus' Microscopy Primer, and the Zeiss online microscopy campus. The content on these sites ranges from basic concepts like Snell's law and Resolution to advanced techniques like supper resolution imaging.

Assignment details

This assignment has 4 parts:

  1. Part 1: Learn about optics and answer a few questions to answer before you start your lab work;
  2. Part 2: Some warm-up lab exercises;
  3. Part 3: You will build a microscope; and finally you will
  4. Part 4: Measure its magnification and the size of some small beads.

You will add fluorescence capability in the next part of the lab.

Submit your work in on Stellar in a single PDF file with the naming convention <Lastname><Firstname>Assignment1.pdf. Here is a checklist of all things you have to turn in:

Pencil.png Make sure to include answers to all the following questions:

Part 1:

  1. Answers to the pre-lab questions listed at the bottom of the Part 1 page

Part 2:

  1. Turn in your measured focal lengths for each lens A through D.
  2. In a table, report the values you measured for $ S_o, S_i, h_o, h_i, $ and $ M $.
  3. Plot $ {1 \over S_i} $ as a function of $ {1 \over f} - {1 \over S_o} $
  4. Plot $ {h_i \over h_o} $ as a function of $ {S_i \over S_o} $
  5. Do the relationships between $ M $, $ S_o $, and $ S_i $ match the theory?
  6. What sources of error affect your measurements?
  7. Plot pixel variance vs mean.
  8. How does noise vary as a function of light intensity?
  9. Did the plot look the way you expected?

Parts 3 and 4:

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


Navigation

Back to 20.309 Main Page

References

  1. Hooke, R. Micrographia: or Some Physiological Descriptions of Minute Bodies made by Magnifying Glasses with Observations and Inquiries Thereupon London:Jo. Martyn, and Ja. Allestry, Printers to the Royal Society; 1665
  2. See, for example: McClintock, B. The origin and behavior of mutable loci in maize. PNAS. 1950; 36:344-355. and Endersby, Jim. A Guinea Pig's History of Biology. Cambridge, Massachusetts: Harvard University Press; 2007.