The Scale of Things

By. Nicholas Ballor and Oren Schaedel


January 6, 2006

The error bars in the following images were drawn in Adobe Photoshop (R);
they were drawn to scale based upon a pixel-distance
calibration made using a graticule standard.  Please note that the accuracy
of the 1000X magnification error is questionable and is likely a slight underestimate.

The first sequence of images below were taken to illustrate the relative size of various organisms.
All images were collected using a Mitocam 1000 1.3 M camera in combination with
Olympus CX41 microscopes or, in the case of C. elegans, Olympus SZ61 stereoscopes.

E. Coli at 400X MagnificationE. coli at 40x Magnification
E. Coli at 1000X Magnification E. coli at 100X Magnification
Stentor at 100X Magnification Stentor at 10X Magnification

Stentor is a trumpet shaped, single-celled protozoan.  It is the largest single celled organism discovered to date.
Stentor at 400X MagnificationStentor at 40X Magnification
Stentor at 400X Magnification Stentor at 40X Magnification
C. elegans at 1.5X Magnification C. elegans at 1.5X Magnification

Below is a representative image of a graticule used to make the  distance-pixel calibrations necesary for the construction of the scale bars used in the above images.  A simple correlation between known length and number of pixels using an image analysis program (ImageJ, for example).  

Image of Graticule

After aquiring the above images, an anaysis was completed to determine if any relationship exists between either wavelength or apmlitude of the nematodes' swim pattern and their length.

The data used in these plots was gathered using the image analysis software in Matlab.  The program used was fairly crude, but we were nonetheless able to make two important observations based upon the data it generated.
Image illustrating wavelength and amplitude calculations
Correlation Between Amplitude of Swim Pattern and Worm Length470

The correlation coefficient for this regression is 0.5891, which indicates that there is some correlation between amplitude and worm length.  This makes sense intuitively because one would expect longer worms to be capable of forming larger arcs than smaller worms.
Correlation of Wavelength of Swim Pattern with Worm Length

The correlation coefficient for this regression was 0.8193, which is larger than that calculated for the amplitude with worm length.  This correlation may be an indication of the mechanical properties of the nematode worm and restrictions on motion.  It would seem to indicate that longer worms are either stiffer and less able to bend than their smaller kin or somehow benefit from making wider turns along their length during their motion.

The experiments reported below were conducted to gain a familiarity
with the time scale of cellular events.

The above video shows the initial stages in the development of a Lytechinus variegatusegg following firtilization.  The halo that can be seen surrounding the central opaque region of the egg is the firtilization envelope, which is a barrier impermeable to sperm and helps to protect the egg from the deliterious event of polyspermy (described below).  The above image was constructed using ImageJ.
Image of Sea Urchin Used in Experiment

An image of a sea urchin used in this experiment, as viewed from the mouth.  Source:  These sea urchins were obtained off the coast of Florida.

The fertilization protocol was as follows:

  • Inject a 1M KCl solution into a sea urchin's shell.  Distribute ~1 ml evenly into the urchin's three membrane defined chambers.  This is done using a syringe.  Inject the solution, at a ~90 degree angle to the shell's axis of symmetry, evenly into three locations (corresponding to each of the sea urchin's chambers) spaced by around 120 degrees. 
  • After injecting the KCl, shake the sea urchin briefly to mix the solution evenly.  Wait until the sea urchin begins to eject gametes from the side of the shell opposite the mouth.
  • Collect the gametes on a petri dish and immediately determine whether or not they are eggs or sperm using a microscope.  If sperm, continue to collect it on the petri dish and keep it free from saltwater.  If eggs, place the shell inverted into a beaker full of seawater and allow the eggs to fall to the bottom of the beaker -- the shell should be partially submerged in the water. 
  • Acting quickly, dilute the sperm 1:1000 in seawater.  Also pipette some of the egg suspension (enough solution to cover the bottom of your coverslip) onto a microscope slide.  Be careful not to overcrowd the slide with eggs -- there should be around 1 to 3 eggs on average for every microscope viewing window at 10X.   Cover the solution with a microscope slide that has small wedges of modeling clay at each of its four corners just suspending it above the eggs to avoid crushing them.  
  • Add 10 to 100 ul of the dilute sperm solution to the microscope slide with the eggs.  
  • Observe the firtilization process under the 10X objective using a microscope. 
  • Take images of the sample at regular intervals so that they may later be merged into a time-lapse video.
 Note:  It is important to adjust the concentration of sperm such that polyspermy (multiple sperm firtilizing one egg) and overcrowding of eggs is avoided.  Both situations can alter developmental behavior.

Below are shown some images of the gametes used in this experiment:

Image of Gametes

During the experiment, the time at which each cell division occured was recorded.  The observed time spacing for each division are listed below:  
  • First division:             53 minutes
  • Second division:          26 minutes
  • Third division:            23 minutes
  • Fourth Division:         25 minutes

The process of each division took approximately 6 minutes from initiation to completion.

The crossectional are of the individual cells before and after each cell division were measure using ImageJ.  The crossectional area was calculated directly from the number of pixels found to be contained within each cell.  Cell boundaries were defined arbitrarily according to the best judgement of the observer.   We have assumed that each division is symmetric.  It appears that the total are is approximately cut in half, as would be expected, after each division.  Measurements taken for the first two divisions of the cell are given in the bar graph below.

Individual Cell Crossectional Area

The crossectional area of the entire egg, as defined by the  envelope, was also measured before and after each cell division. The crossectional area was calculated directly from the number of pixels found to be contained within the inner boundary of the firtilization envelope.  This boundary was defined arbitrarily according to the best judgement of the observer.     Measurements taken for the first two divisions of the cell are given in the bar graph below.  It was observed that the size of the egg does not change substantially during the early developmental stages of the sea urchin. 

Egg Cross Sectional Area