Difference between revisions of "Lab Manual: Optical Microscopy"

1665	2009
Robert Hooke's microscope	20.309 student's microscope
Hooke micrograph of cork cells	CCD image of fluorescent labeled intracellular membranes[1]

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^[2]

Introduction

In this lab, 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 4 parts. Each part requires some lab work, some analysis, lots of clear thinking, and a written report. You will submit a short, group report after parts 1-3. The final report should include results from all 4 parts of the lab. You may revise and improve your part 1-3 reports before the final submission. At the end of the lab, you will give an individual, 5 minute, oral presentation to an instructor. The presentation will be followed by ten minutes of questions and answers.

In part 1 of the lab, you will build a compound microscope, determine its magnification, and attempt to measure the size of microscopic objects. The instrument you create in part 1 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 will call to mind Hooke's early quantification of the size of plant cells (see quote above). 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.)

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.

Specimens in part 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 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 bird feathers).

Likely inspired by Micrographia, 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 and Volvox, and also parthenogenesis in aphids. He was truly the first microbiologist.

Barbara McClintock made perhaps the most remarkable discovery that has ever been made with a simple light microscope. She was a talented microscopist who developed a technique that let her visualize and differentiate individual chromosomes in Zea Mays (corn) plant cells. One important element of her method was that she squashed the cells instead of cutting them as Hooke did 300 years earlier. She observed genetic transposition through an optical microscope in 1944, even before the structure of DNA was known. Several decades elapsed before molecular techniques sufficiently sophisticated to confirm her discovery were developed.^[3] McClintock was awarded the Nobel Prize in Physiology or Medicine in 1983 for her discovery.

Part 2 will augment your microscope with fluorescence.

Challenges are the same: resolution, contrast And Hooke could draw. Really ... really ... well.

How to do this lab

This lab is divided into 4 one-week sections. Each section requires some lab work and some data analysis. A group report is required for each section. One group member should submit a PDF format document to Stellar before the deadline. The file name should include the last names of all group members.

An example microscope made by the instructors will be available in the lab for you to examine. Feel free to make improvements on this design. Mechanical stability will be crucial for the particle tracking experiments in parts 3 and 4 of the lab. The required stability specification will be achieved through good design and careful construction — not by indiscriminate over-tightening of screws.

The final report should consist of all 4 sections in a single file. In the final document, you may revise any part of the first three sections without penalty. Only the final report will be graded. You may not skip any of the weekly reports.

Follow the format suggested in the Microscopy report outline.

After the lab work is complete, each student will make a 10 minute, individual presentation to one of the instructors. The presentation will be followed by 5 minutes of open questions and (if you want a good grade) answers.

Microscope design

20.309 microscope block diagram

Brightfield transmitted microscopy is the simplest and most common optical microscopy method. In this technique, photons from an illuminator pass through the sample, where they may be absorbed, diffracted, or refracted. (The sample is usually mounted on a glass slide.) An objective lens on the opposite side of the sample collects the light.

Illumination for epi-fluorescence microscopy reaches the sample through the objective lens — from the same side of the sample that is observed. Epi-fluorescence microscopy is normally used on samples that have been labeled with a fluorescent molecule called a fluorophore. The (narrowband) illumination wavelength must match the absorption characteristic of the fluorophore. After becoming excited by a photon from the illuminator, fluorophores emit photons with a longer wavelength. A dichroic mirror in the microscope reflects the illumination wavelength but allows the emitted photons to pass through.

The imaging path of the microscope includes an objective lens (L1) and a tube lens (L2). The sample is placed at the front focus of L1, producing collimated light that reflects of mirror M1. L2 forms an image of the sample on a CCD camera. In brightfield mode, collimated light from an LED passes through the sample. A green laser illuminates the sample in epi-fluorescence mode. Laser light passes through a Gallilean beam expander (L3 and L4). L5 focuses the laser beam at the back focus of L1. This arrangement provides collimated sample illumination. Dichroic mirror DM reflects the green light toward the sample and allows emitted red light to pass through.

Part 2: fluorescence microscopy

• Read background references
  • Optical detectors, noise, and the limit of detection
• Refer to Optical Microscopy Part 2: Fluorescence Microscopy
  • Add a laser illumination beam path
  • Image fluorescent samples
  • Characterize the fluorescent imaging performance of the microscope
  • Process images
• Turn in Part 2 report

Part 3: particle tracking

• Read background references
  • R. Newburgh, Einstein, Perrin, and the reality of atoms: 1905 revisited, Am. J. Phys. (2006). A modern replication of Perrin's experiment. Has a good, concise appendix with both the Einstein and Langevin derivations.
  • A. Einstein, On the Motion of Small Particles Suspended in Liquids at Rest Required by the Molecular-Kinetic Theory of Heat, Annalen der Physik (1905).
  • M. Haw, Colloidal suspensions, Brownian motion, molecular reality: a short history, J. Phys. Condens. Matter (2002).
  • E. Frey and K. Kroy, Brownian motion: a paradigm of soft matter and biological physics, Ann. Phys. (2005).
  • Random Force & Brownian Motion — 60 Symbols

• Refer to Optical Microscopy Part 3: Particle Tracking
  • Track fixed beads and measure microscope stability
  • Track suspended microspheres
  • Estimate diffusion coefficients in a Newtonian fluid; calculate viscosities
• Turn in Part 3 report

Part 4: cellular microrheology

• Read background references
  • Cellular microrheology
• Refer to: Optical Microscopy Part 4: Microrheology Measurements in Fibroblast Cells
  • Image actin in 3T3 cells before and after exposure to cytochalasin D, an inhibitor of actin polymerization
  • Measure the frequency-dependent storage and loss modulus of 3T3 cells
• Begin Final report (see due date on Stellar)

Optical microscopy lab

Code examples and simulations

• Converting Gaussian fit to Rayleigh resolution
• MATLAB: Estimating resolution from a PSF slide image
• Matlab: Scalebars
• Calculating MSD and Diffusion Coefficients

Background reading

• Geometrical optics and ray tracing
• Physical optics and resolution
• Optical aberrations
• Aperture and field stops
• Optical detectors, noise, and the limit of detection
• Manta G032 camera measurements
• Understanding log plots

References

1. ↑ Onion endothelial cell incubated with FM 4-64 dye (Invitrogen). See class stellar site for protocol. Oh & Yamaguchi, unpublished lab report
2. ↑ 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
3. ↑ 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.