Initial Attempt At Trapping Bacteria (Vibrio Cholerae)
To begin using the optical tweezers to perform biological measurements, Vibrio Cholerae bacteria was obtained from the Biophysics department. Particulars about the bacteria are described in under the Vibrio Cholerae section.
Using the objective just to image the bacteria, there were three basic types of bacterial behavior observed. First there were single vibrio cells traversing the slide at very fast velocity, close to 50 microns per second. Next observed were slightly larger organisms, possibly multiple cells linked together, which moved around with considerable energy (wiggling and writhing) but with lower overall velocity across the slide. Finally there were large chains of bacteria that moved very sluggishly throughout the slide.
Next using the IR laser to attempt optical trapping, the sample was scanned to see if any trapping was possible. The fastest level of bacteria never seemed effected by the optical trap. None of these samples were trapped. The medium speed bacteria also was generally too strong and also did not appear to be affected by the optical trap. This could mean that they can easily overpower the piconewton forces, or that they are swimming in a different plane then the focal point of the laser. One of the larger, more vigorous, bacteria was trapped but escaped before any force measurements could be recorded. Finally the large sluggish bacteria were trapped with ease. It was noticed that as time wore on more of these slow chains begin to occur, possibly from the diminishing supply of nutrient broth in the slide which led to more sluggish behavior. A success apart from the trapping was that the IR laser appeared to do no harm to the bacteria and they wriggled away after the trap had been sufficiently weakened.
Motility by definition is an organisms ability to spontaneously and actively, consuming energy in the process. Therefore the organism will exert a certain amount of force when met with resistance such as an optical trap. Using our previous force calibrations, a rough estimate of the escape force could be determined by observing what laser power the bacteria was able to escape the trap. To initially trap bacteria, the IR laser was operating at a relatively high power, about 50 mW. Once the bacteria was trapped, the laser power was slowing turned down until the bacteria was observed to escape. (Keep in mind that only the slowest bacteria succumbed to the trap). It was observed that the bacteria escaped the trap at a laser driver current of 33-39 mA which is after the threshold current of our IR laser. The corresponding laser power was 2-7 mW. Using our force calibrations to relate laser power to trapping force we determined the force generated by the bacteria to escape the optical trap was between 1-5 pico-Newtons. Clearly there is a large margin for error with these calculations, primarily due to the fact that only slow bacteria were trapped and that we assumed our force calibrations for the microspheres to be valid for the bacteria. However, it was a success to relate an escape force to the motion of a biological sample.