Characterization of photodamage to escherichia coli in optical traps

Optical tweezers (infrared laser-based optical traps) have emerged as a powerful tool in molecular and cell biology. However, their usefulness has been limited, particularly in vivo, by the potential for damage to specimens resulting from the trapping laser. Relatively little is known about the origin of this phenomenon. Here we employed a wavelength-tunable optical trap in which the microscope objective transmission was fully characterized throughout the near infrared, in conjunction with a sensitive, rotating bacterial cell assay. Single cells of Escherichia coli were tethered to a glass coverslip by means of a single flagellum: such cells rotate at rates proportional to their transmembrane proton potential (. J. Mol. Biol. 138:541-561). Monitoring the rotation rates of cells subjected to laser illumination permits a rapid and quantitative measure of their metabolic state. Employing this assay, we characterized photodamage throughout the near-infrared region favored for optical trapping (790-1064 nm). The action spectrum for photodamage exhibits minima at 830 and 970 nm, and maxima at 870 and 930 nm. Damage was reduced to background levels under anaerobic conditions, implicating oxygen in the photodamage pathway. The intensity dependence for photodamage was linear, supporting a single-photon process. These findings may help guide the selection of lasers and experimental protocols best suited for optical trapping work.     

Identified taste bud cell proliferation in the perihatching chick [published erratum appears in Chem Senses 1998 Aug;23(4):459]

Developing taste buds in the anterior mandibular floor of perihatching chicks were studied by high voltage electron microscopic autoradiography in order to identify proliferating gemmal cell types. Montaged profiles of 29 taste buds in five cases euthanized between embryonic day 21 and posthatching day 2 were analyzed after a single [3H]thymidine injection administered on embryonic day 16, 17 or 18. Results showed that dark cells comprised 55% of identified (n = 900 cells) and 62% of labeled (n = 568 cells) gemmal cells as compared with light, intermediate, basal or perigemmal bud cells. Dark cells had both a greater (P < 0.05) number of labeled cells and a greater amount of label (grains/nucleus) than the other four bud cell types, irrespective of injection day. The nuclear area (micron 2) of dark cells was not significantly larger (P > 0.05) than that of the other gemmal cell types and therefore cannot account for the greater amount for label in the dark cells. Interestingly, only dark cells showed a positive correlation (P < 0.003) between amount of label and nuclear area. Results suggest that, during the perihatching period of robust cell proliferation, dividing dark cells may give rise primarily, but not exclusively, to dark cell progeny.