The Size of Things

An important step in understanding new scientific concepts is learning the scales of the problem. How fast do processes occur? Over what spatial scales? How much energy is consumed? In our courses we always begin by looking at various cells and organisms to discern the overall size, sizes of organelles, and rates of whole-cell and intracellular movement, using a variety of light and fluorescence microscopy techniques.

First, students learn how to use bright-field microscopy to calibrate different magnifications on the microscope, enabling them to relate what they see in the microscope to physical distances. A lithographic graticule with 10um markers is imaged at different magnifications and a calibration curve is generated as shown below.

While absolute units of measurement are supremely useful, there is some merit in simply having a 'feel' for the scale of the objects one is considering. In the prokaryotic setting the standard object is E. coli to which all other prokaryotic cells can be compared; likewise the yeast S. cerevisiae is the prototypical eukaryotic cell. As such, after calibration the students always begin by observing the size of these two organisms, with an emphasis on putting scale bars on all of their pictures.

As an exercise in fluorescence microscopy, the students take pictures of bovine pulmonary cells in TRITC, FITC and DAPI fluorescence modes to visualize the actin, mitochondria and nucleus respectively.

The students then use Matlab(c) to combine the images into a fluoro-colored image of the entire cell.

TRITC ---------------------- FITC---------------------- DAPI

C. elegans is a particulary important species in genetic studies. We simply like to use them as dynamic organisms for understanding the size and motility of multi-cellular organisms. Some students took the initiative to measure the correlation between the geometric features of the nematode (length and wavelength).

We became interested in the algorithm employed by the nematode in its hunt for food. Below is a real-time movie of a worm hunting around the petri dish for food (E. coli).

Nowadays it's easy to order a wide array of organisms from a variety of suppliers, but sometimes it's just fun to see what you have in the backyard. We took 1ml of mirky water from a pond on campus and found a veritable zoo of interesting multi and single celled organisms. Here's a small sample:

Heliozoan - notice about 1/3 of the way through the movie, some sort of cavity closes on the right-hand side. The dendritic structures are actually bundles of actin filaments used for motility.

An unidentified flagellate in the process of budding a new cell - flailing in vain. We had considered putting this video to some kind of dance beat.

This amazingly long algae has a well defined cellular frequency. It was great to watch it travese steadily across the field of view.

Stentor is an incredibly large and dynamic single-celled organism, with various organelles visible within the cell using simple brightfield microscopy. There are also fine, fast moving cilia around the "mouth" which make amazing flow patterns (see The Rate of Things).

Dictyostelium have an amazing life cycle, during which they sometimes act as indepedent cellular organisms, but under the right conditions will differentiate into fruiting bodies. Below are two great pictures showing a fruiting body that has recently burst to release new, viable cells. For 'slug' formation see The Rate of Things.

Spirulina is another slow moving algae with an amazingly regular single-helix structure. Using phase constrast microscopy, students characterized this helix.