Difference between revisions of "Assignment 1 Overview: Transillumination microscopy"

From Course Wiki
Jump to: navigation, search
(Assignment details)
Line 51: Line 51:
 
# Part 1: [[Assignment 1, Part 1: Pre-lab questions|A few questions to answer before you start your lab work.]];
 
# Part 1: [[Assignment 1, Part 1: Pre-lab questions|A few questions to answer before you start your lab work.]];
 
# Part 2: [[Assignment 1, Part 2: Optics bootcamp|Some warm-up lab exercises]];
 
# Part 2: [[Assignment 1, Part 2: Optics bootcamp|Some warm-up lab exercises]];
# Part 3: You will [[Assignment 1, Part 3: Microscope design|design your microscope]]; and finally you will
+
# Part 3: You will [[Assignment 1, Part 3: Microscope design|build a microscope]]; and finally you will
# Part 4: [[Assignment 1, Part 4: Building your transilluminated microscope|Build a transilluminated microscope]] and measure its magnification.
+
# Part 4: [[Assignment 1, Part 4: Building your transilluminated microscope|measure its magnification]].
  
 
You will add fluorescence capability in the next part of the lab.
 
You will add fluorescence capability in the next part of the lab.

Revision as of 15:27, 24 August 2017

20.309: Biological Instrumentation and Measurement

ImageBar 774.jpg


Mens et Manus.jpg

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 report turned in on Stellar.

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.[1] McClintock was awarded the Nobel Prize in Physiology or Medicine in 1983 for her discovery.

An example microscope made by the instructors will be available in the lab for you to examine. Make sure to construct your microscope well. Mechanical stability will be crucial for the particle tracking experiments in the last part of the lab. The required stability specification will be achieved through good design and careful construction — not by indiscriminate over-tightening of screws.

Assignment details

This assignment has 4 parts:

  1. Part 1: 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.

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

Turn in all of your work on Stellar in a single PDF file named <lastname><firstname>Assignment1.pdf.

Background reading

In this part of the lab you will jump right in to building a full-fledged microscope. The following online materials provide useful background.

References

  1. See, for example: McClintock, B. The origin and behavior of mutable loci in maize. PNAS. 1950; 36:344-355. [1], [2], and Endersby, Jim. A Guinea Pig's History of Biology. Cambridge, Massachusetts: Harvard University Press; 2007.