Polarized light scattering for rapid observation of bacterial size changes.

The effect of changing growth conditions on the diameter of rod-shaped bacteria was studied in vivo with the use of polarized light scattering. The value of a ratio of scattering matrix elements was measured as a function of scattering angle at various times after nutritional "upshift" for two strains of Escherichia coli cells. The peak locations of the scattering function were calibrated against the diameter for rod-shaped bacteria. The peaks moved toward smaller angles as a function of time after upshift, indicating that the diameter was increasing. Under special conditions, substantial peak shifts occurred within a few minutes of growth condition change, indicating a rapid onset of growth in diameter. The rate of increase of the diameters after upshift was obtained from the angular shift of peak location. This rate was approximately 14 nm/min for E. coli K12 and approximately 9 nm/min for E. coli B/r at 37 degrees C. The rate of diameter increase is smaller at lower temperatures. Experiments with Bacillus megaterium showed that any diameter change after nutritional upshift at 37 degrees C is limited to at most a very small increase, at least for the strain and medium tested.

Measuring diameters of rod-shaped bacteria in vivo with polarized light scattering.

The angular function for elements of the Mueller matrix for polarized light scattering from suspensions of microorganisms is known to be reproducible for different growths of a given bacterial strain in the log (or exponential) phase of growth. The reason for this, the stability of the size and shape distribution for cells, is briefly discussed. Experiments were performed using suspensions of two different strains of Escherichia coli cells in log phase and measuring the angular dependence of the Mueller matrix ratio S34/S11. Calculations were then performed using the coupled dipole approximation to model electromagnetic scattering from particles where the shape of an individual cell was approximated by a cylinder capped with hemispheres of the same radius as the cylinder. Using previously measured values for the length distribution and index of refraction of the cells, the calculated scattering curve was found to fit the measured curve very well. The values obtained for the cell diameters were quite close to diameters previously measured by optical microscopy. Thus this method provides a rapid and convenient method for monitoring bacterial diameters in vivo even when there is an appreciable distribution of bacterial lengths in the population.

Low power laser irradiation alters the rate of regeneration of the rat facial nerve.

Low power laser irradiation has been reported to cause biological effects due to the photochemical and/or photophysical action of the radiation. This study determined quantitatively if transcutaneous low power laser irradiation can affect the regeneration of the rat facial nerve. The facial nerve was crushed unilaterally in anesthetized rats and transcutaneously irradiated daily with a laser beam directed at the area of the crush injury. Laser treatment began on the day of the crush injury and was continued daily for 7, 8, or 9 days. Preliminary experiments determined the most effective wavelength, laser power, length of irradiation, and treatment schedule. The wavelengths examined were 361, 457, 514, 633, 720, and 1064. The laser powers and lengths of irradiation examined ranged from 8.5 to 40 mW and 13 to 120 min. Irradiation treatment was done daily, on alternating days and on the first 4 days postcrush. The most effective laser parameters for the low power treatment included daily irradiation with a helium-neon (HeNe) or argon pumped tunable dye laser a wavelength of 633 nm, with a power of 8.5 mW for 90 minutes (45.9 J, 162.4 J/cm2). The number of horseradish peroxidase (HRP) labeled neurons in the facial motor nucleus was used as an assay of the degree of regeneration. In rats in which the facial nerve was crushed but not irradiated, the average number of HRP labeled neurons in the facial nucleus was 22 on day 7 postcrush, 54 on day 8, 116 on day 9, and 1,149 on day 10. After HeNe or argon pumped tunable dye laser irradiation, the average number of HRP-labeled neurons increased to 34 on day 7 postcrush, 148 on day 8, and 1,725 on day 9. There was a statistically significant difference between the control and irradiated rats on day 9 postcrush (P < 0.01). These data indicate that transcutaneous low power irradiation with the lasers and parameters involved in this study increased the rate of regeneration of rat facial nerve following crush injury.

In vivo measure of average bacterial cell size from a polarized light scattering function.

A particular combination of elements of the Mueller matrix for scattering of polarized light given by (S34 + S14)/(S11 + S13) identical to (S34/S11)++ is measured vs angle at a wavelength of 633 nm for randomly oriented suspensions of several species of bacteria in different stages of growth. (This combination of elements is dominated in the present measurements by the behavior of the normalized S34 matrix element, as is indicated by the notation defined on the right side of the equation.) The resulting graph in each case shows an oscillating function of angle. This function is compressed toward smaller angles when the bacteria are in the exponential phase of growth in comparison with results for a suspension of the same bacteria in the stationary (starving-smaller cells) phase of growth. Microscopic measurements were made to determine, for each case, the average dimensions of the bacterial population. Graphs were then plotted of the peak positions from the Mueller matrix function plots vs either cell length or cell diameter. The function was shown to be strongly correlated with cell diameter under the conditions of this experiment and poorly correlated with cell length. The measurements were shown to have a sensitivity to changes in average diameter of about 20 nm.

Bone ablation with Er:YAG and CO2 laser: study of thermal and acoustic effects.

A pulsed Er:YAG laser at 2.94 microns and a superpulsed CO2 laser at 10.6 microns are used to investigate bone ablation applications in otolaryngology. Quantitative measurements of mass removal and the ablation depth of cat skull bone and rat femur are presented with the Er:YAG laser at fluences of 9-117 J/cm2. Histological results show that the minimal thermal injury zone from the edge of the lesion is 5-10 microns. Comparison of the photoacoustic and thermal effects during the ablation process indicates that the temperature rise from the 10.6-microns light was higher than that from the 2.94-microns light but that the photoacoustic wave amplitude produced with the Er:YAG laser was higher than that with the CO2 laser. The fluence used for the efficient ablation of bone tissues produces a photoacoustic wave ranging from 100 to 120 dB. The ear can tolerate this level for a short time period. Results of this study suggest that the Er:YAG laser can be an important surgical tool in otolaryngology.

Er:YAG laser ablation of enamel and dentin of human teeth: determination of ablation rates at various fluences and pulse repetition rates.

A pulsed Er:YAG laser (2.94 microns) was used to determine ablation depths per pulse of laser energy at 2 Hz and 5 Hz in human teeth cross sections of enamel and dentin. Ablation depths per pulse at 2 Hz in enamel of intact human teeth were measured and compared to ablation depths per pulse determined in enamel cross sections at 2 Hz. Close correlation was observed for ablation depths per pulse of laser energy between teeth cross sections and intact teeth for enamel. Photographs of lased holes at 2 Hz and 5 Hz indicated minimal thermal effects in enamel at fluences below 80 J/cm2. Minimal thermal effects in dentin were noted below 74 J/cm2. Scanning Electron Microscopy (SEM) pictures of lased dentin showed an irregular serrated surface. Results of this study suggest that the Er:YAG laser can effectively ablate enamel and dentin with minimal thermal effects at 2 Hz and 5 Hz.

Dependence of subcellular damage on various laser beam parameters.

A cw argon laser operating at the multiline UV TEM00 mode was used to create lesions in the nucleoli of fibroblast cells. Laser powers emitted through the objective were varied from 5-8 mW and laser pulse durations ranged from 50 to 600 ms. Our study showed lesion diameters ranging from 0.7 microns to 2.9 microns and an average lesion diameter of 1.77 microns. All cells were exposed to at most a laser spotsize of 2.2 microns diameter and for a laser spotsize of this diameter, we calculated that the central 18.3% of the total energy was in the central area of 0.7 microns diameter. We also found that as little as 0.3 mJ of energy could produce a lesion. No absolute threshold correlation was found between the size or type of lesion and the laser parameters used.

Comparison of the erbium-yttrium aluminum garnet and carbon dioxide lasers for in vitro bone and cartilage ablation.

The in vitro bone- and cartilage-ablation characteristics of the solid-state erbium:yttrium aluminum garnet laser were compared to those of the carbon dioxide laser. Ablations of fresh, frozen cadaver septal cartilage and maxillary sinus bone were performed using total energies between 1 and 6 J. Specimens were studied using hematoxylin and eosin stain and digitized, computer-assisted measurements of 35-mm photographs. Erbium-yttrium aluminum garnet-ablated bone averaged 5 microns of adjacent tissue thermal injury, compared with 67 microns with carbon dioxide-ablated bone. Erbium-yttrium aluminum garnet-ablated cartilage averaged 2 microns of adjacent tissue thermal injury, compared with 21 microns with the carbon dioxide-ablated cartilage. The tissue-ablation characteristics of the erbium-yttrium aluminum garnet laser are promising for future otolaryngologic applications.

Reproducibility and sensitivity of polarized light scattering for identifying bacterial suspensions.

Reproducibility and sensitivity of polarized light scattering measurements are explored for a particular rodlike bacterial species in different growth conditions. The angle dependence of certain Mueller scattering matrix elements (primarily S(34)/S(11)) is used to measure these features. The angular S(34)/S(11) scattering pattern was found to be strongly correlated with bacterial cell size. In view of the large changes found for different growth conditions, it is concluded that much improvement in the technique of recognizing scattering patterns would be needed before the angle dependence of a given Mueller matrix element can be unequivocally associated with a given bacterial strain. The sensitivity of S(34)/S(11) patterns (at a wavelength of 633 nm) to changes in internal structure of the bacteria was tested by following the pattern after virus particles were produced inside the individual bacteria by an induction procedure. No appreciable changes occurred up until the time the bacteria were lysed (destroyed) by the process.

Hazardous ranges of laser beams and their reflections from targets.

The hazardous range of laser beams and their reflections from surfaces must be known when one uses lasers outdoors in areas where individuals could be exposed above the applicable exposure limits. In this paper, we describe formulas that can be used to calculate these hazardous ranges, and a few experimental data are presented that indicate the validity of the formulas and the underlying assumptions. The hazardous range depends on the laser parameters, the exposure limit, the atmospheric attenuation, the distance between the laser and reflector, the dimensions of the reflector, the angle of incidence on the reflector, and the surface parameters such as the reflection coefficient and flatness of the surface. The hazardous range of some commonly used lasers by the U.S. Army extends from 5 to 25 km for unaided viewing.

Critique on the analysis of photorepair in chick-embryo fibroblasts.

It is shown that the data by Van de Merwe and Bronk on photoreactivation of chick-embryo fibroblasts do not support the particular application of the Rupert -Harm model used in the paper by Pendrys [6]. Further, it is shown that for blacklight intensities greater than 17 W/m2 at 365 nm, there is approximate reciprocity (i.e. the extent of photoreactivation of available dimers is determined primarily by total blacklight fluence).

The shape of the blacklight dose photoreactivation curve for chick embryo fibroblasts.

The curves of UV (254 nm) induced pyrimidine dimers (endonuclease sensitive sites) vs. photoreactivating blacklight (365 nm) dose for cultured chick embryo fibroblasts reveal several new features. When the cells are incubated in the dark at 37 degrees following UV (254 nm) treatment, the efficiency of subsequent photorepair increases for the first few hours post-UV. The efficiency then remains approximately constant for several hours. Photorepair data obtained during this later period were plotted as the logarithm of dimer-enzyme complexes available for photoreactivation vs. blacklight (365 nm) dose. For a fixed damaging UV (254 nm) dose, the resulting curve has a shoulder of approximately 6-10 kJ/m2 followed by a straight line portion with a slope of magnitude about 1.5 X 10(-4) m2/J for UV doses up to 15 J/m2. For higher UV doses the shoulder remains about the same, but the slope decreases in magnitude. The shoulder is interpreted to indicate that a light-dependent step is necessary to activate the enzyme. The decrease in slope with increased UV dose together with some split photoreactivation dose experiments suggests that some site-to-site motion and multiple site function of the photorepair enzyme molecules may come into play at the higher levels of damage, but the evidence indicates that these complications are relatively unimportant at low UV doses.

Increased efficiency of photoreversal of UV-induced dimers in the DNA of chick embryo fibroblasts with post-UV dark time.

The kinetics of photoreversal of UV-induced dimers in the DNA of early passage chick embryo fibroblasts was studied by monitoring disappearance of UV-endonuclease-sensitive sites. Photorepair was found to increase in efficiency when cells were incubated in the dark for several hours at 37 degrees C following the dimer-inducing short-wavelength (254 nm) UV treatment, but prior to the photoreactivating black light (365 nm). Following a UV dose of 10 J/m2 it took at least 4 h in the dark to saturate this effect. This UV dose inserts roughly 2.4 dimers/10(7) daltons of DNA. Dark repair removes about 0.08 dimers/h/10(7) daltons. After 6 h in the dark, exposure to black light removes an additional 1.4 dimers/10(7) daltons leaving about 0.5 dimers unaffected by this treatment. After saturation of the dark effect, the amount of photoreactivation depends only on total black light fluence and not on fluence rate for the range of rates studied. This indicates that during 30 min, the maximum time of black light exposure, no appreciable reattachment of the photorepair molecule to additional unrepaired dimer sites occurs. We estimate that the number of effective photorepair molecules per chick chick cell is at least of the order of 2 x 10(5).