Changes in collagen in the wound during the healing process of guinea pig skin following surgical incisions and LTW
was evaluated using in vivo, using Raman spectroscopy. Raman spectroscopy provided information regarding the
internal structure of the proteins. After the incisions were closed either by suturing or by LTW the ratio of the Raman
peaks of the amide III (1247 cm-1) band to a peak at 1326 cm-1 used to evaluate the progression of collagen deposition.
Histopathology was used as the gold standard. LTW skin demonstrated better healing than sutured skin, exhibiting
minimal hyperkeratosis, minimal collagen deposition, near-normal surface contour, and minimal loss of dermal
appendages. This work is important to plastic surgery.
The changes of Raman spectra from ex-vivo porcine aorta tissues were studied before and after laser tissue welding
(LTW). Raman spectra were measured and compared for normal and welded tissues in both tunica adventitial and
intimal sides. The vibrational modes at the peak of 1301
cm-1 and the weak shoulder peak of 1264 cm-1 of amide III for
the normal tissue changed to a peak at 1322cm-1 and a relative intense peak at 1264cm-1, respectively, for the welded
tissue. The Raman spectra were analyzed using a linear regression fitting method and compared with characteristic
Raman spectra from proteins and lipids compounds. The relative biochemical molecular composition changes of proteins
(Collagen types I, III, V and Elastin) and lipids for the laser welded tissue were modeled by basis biochemical
component analyses (BBCA) and compared with the normal tissue.
The oxidative stress due to free radicals is implicated in the pathogenesis of tissue damage in diseases such as muscular
dystrophy, Alzheimer dementia, diabetes mellitus, and mitochrondrial myopathies. In this study, the acute oxidative
stress induced changes in nicotinamide adenine dinucleotides in mouse skeletal muscles are studied in vitro using Raman
spectroscopy. Mammalian skeletal muscles are rich in nicotinamide adenine dinucleotides in both reduced (NADH) and
oxidized (NAD) states, as they are sites of aerobic and anaerobic respiration. The relative levels of NAD and NADH are
altered in certain physiological and pathological conditions of skeletal muscles. In this study, near infrared Raman
spectroscopy is used to identify the molecular fingerprints of NAD and NADH in five-week-old mice biceps femoris
muscles. A Raman vibrational mode of NADH is identified in fresh skeletal muscle samples suspended in buffered
normal saline. In the same samples, when treated with 1% H2O2 for 5 minutes and 15 minutes, the Raman spectrum
shows molecular fingerprints specific to NAD and the disappearance of NADH vibrational bands. The NAD bands after
15 minutes were more intense than after 5 minutes. Since NADH fluoresces and NAD does not, fluorescence
spectroscopy is used to confirm the results of the Raman measurements. Fluorescence spectra exhibit an emission peak
at 460 nm, corresponding to NADH emission wavelength in fresh muscle samples; while the H2O2 treated muscle
samples do not exhibit NADH fluorescence. Raman spectroscopy may be used to develop a minimally invasive, in vivo
optical biopsy method to measure the relative NAD and NADH levels in muscle tissues. This may help to detect diseases
of muscle, including mitochondrial myopathies and muscular dystrophies.
Fluorescence and phosphorescence measurements are performed on normal and malignant ex vivo human breast tissues using UV LED and xenon lamp excitation. Tryptophan (trp) phosphorescence intensity is higher in both normal glandular and adipose tissue when compared to malignant tissue. An algorithm based on the ratio of trp fluorescence intensity at 345 nm to phosphorescence intensity at 500 nm is successfully used to separate normal from malignant tissue types. Normal specimens consistently exhibited a low I345/I500 ratio (<10), while for malignant specimens, the I345/I500 ratio is consistently high (>15). The ratio analysis correlates well with histopathology. Intensity ratio maps with a spatial resolution of 0.5 mm are generated in which local regions of malignancy could be identified.
Light scattering experiments and phase contrast microscopy are used to evaluate the aggregate-forming characteristics of simple clay-bacteria mixtures. Colloidal suspensions of negatively charged Pseudomonas syringae (Ps) and Mg2+-, Li+ - or Ca2+ -exchanged smectite (and non-exchanged smectite) are flocculated in neutral (pH 7) aqueous media. Aggregate
formation is monitored using changes in optical transmission. Clustering is observed in all the clay-bacteria preparations. The Li+-substituted clay aggregates average 50-300 microns in diameter, in contrast to the Ca2+- substituted clay that
produces aggregates of 10-50 microns in diameter. Light scattering measurements indicate that aggregates begin forming 3 hours after mixing and that the (larger sized) aggregates exhibit less scattering than a mixture with an equivalent concentration of unattached Ps and clay particles.
Native fluorescence of tissues in the UV and visible spectral regions has been investigated for over two decades. Native fluorescence has been demonstrated to be an accurate tools for distinguish normal tissue from malignant and pre-malignant. Prior investigations have demonstrated that there are several ratio-based algorithms, which can distinguish malignant tissue from normal with high sensitivity and specificity.1 The wavelength combinations used in these ratios isolate the contributions from pairs of tissue fluorophors, one of which is frequently tryptophan (trp), the predominant tissue fluorophore with excitation in the UV (250-300 nm).
In this work, algorithms using a combination of native fluorescence and trp phosphorescence were developed which show promise for providing enhanced detection accuracy. Using optical fibers to collect the emission from the specimen allowed interrogation of small regions of tissue, providing precise spatial information. Using a specially designed setup, specimens were excited in the UV and spectra were collected in the range of 300 to 700 nm. Three main emission bands were selected for analysis: 340 nm (trp fluorescence); 420 - 460 nm band (fluorescence from the extra cellular matrix); and 500 - 520 nm (trp phosphorescence). Normal specimens consistently exhibited a low ratio (<10) of 345 to 500 nm emission intensity while this same ratio was consistently high (>15) for cancer specimens. Creating intensities ratio maps from the tissue allows one to localize the malignant regions with high spatial precision. The study was performed on ex vivo human breast tissues. The ratio analysis correlated well with histopathology.
Light transmission measurements in the wavelength range of 400 to 1000 nm were performed on Bacillus subtilis spores at periodic time intervals after heat-shock induced activation. The Gaussian ray approximation, using a concentric sphere model for the spore coat and spore core, was used to calculate the scattering cross-section of the spores. Analysis of transmission spectra determined that the refractive index of the spore core was 1.515. In the three hours following heat shock, the core refractive index decreased to 1.39, and subsequently remained constant. During the measurements, the spore radius increased from 0.38 microns to 0.6 microns. The results were confirmed by phase contrast microscopy.
Fluorescence and absorption spectroscopy were performed on Bacillus subtilis spores which were heat treated at temperatures ranging from 20°C to 90°C. The tryptophan (trp) emission from the spore suspensions treated at temperatures greater than 60°C was shifted towards longer wavelength as compared to the spores which were heat treated at lower temperatures. These spectral changes were the result of proteins released by the spores into the suspension. Trp residues in the emitted proteins are in a more polar environment and therefore exhibit a larger Stokes shift. Fluorescence and absorption measurements show that the concentration of proteins in the supernatant was greater for spores treated at higher temperatures. Electrophoresis gel analysis showed the presence of a 47 KDa protein in the supernatant.
The fluorescence spectra from Φ6 and Φ12 cystoviruses and their pseudomonad host were investigated. The predominant fluorophore in both the Φ6 and Φ12 viruses and pseudomonas syringae is tryptophan. The emission maxima of the virus emission was found to be at 330 nm, compared to an emission maxima from the bacteria, which typically varies between 337 and 349 nm, depending on growth conditions. This difference Stokes shifts between the viruses and syringae hosts are most likely due to the virus proteins being in a much less polar environment than the bacteria proteins. This difference in Stokes shift was used to monitor the infection process.
The time-resolved and steady-state changes in fluorescence were investigated from one spore-forming (Bacillus subtilis) and four non-spore forming (Escherichia coli, Staphylococcus aureus, Enterococcus faecalis, and Pseudomonas aeruginosa) bacteria subjected to different bactericidal agents. The bactericidal agents were sodium hypochlorite (bleach) hydrogen peroxide, formaldehyde, and UV light exposure. Application of sodium hypochlorite resulted in an almost total lose of fluorescence signal and large decrease in the optical density of the bacterial suspension. Addition of hydrogen peroxide resulted in a 35% decrease in emission intensity fom the Sa and an 85-95% decrease for the other bacteria. Ultraviolet light exposure resulted in a 5-35% decrease in the emission intensity of the tryptophan band. The addition of formaldehyde to the bacteria did not result in significant changes in the steady-state emission intensity, but did shift the tryptophan emission peak position to shorter wavelengths by 3 to 5 nm. Time-resolved fluorescence measurements showed that the fluorescence lifetime of tryptophan in the bacteria could not be described by a single exponential decay, and was similar to that of tryptophan in neutral aqueous solution. Upon addition of formaldehyde to the Gram positive bacteria (Bs and Sa) the strength of the short lifetime component increased dramatically, while for the Gram negative bacteria, a smaller increase was observed. These fluorescence changes reflect the different mechanisms of the bactericidal agents and may provide a useful tool to monitor the effectiveness of disinfectants.
Laser tissue welding involves the partial denaturing and renaturing of the collagen triple helical structure. Although the mechanisms of laser tissue welding are not well understood, water in tissues plays an important role in the process. High quality welding of human and porcine aorta tissue have been achieved using NIR lasers tuned to the water absorption band around 1450 nm. Fluorescence and Raman spectra from welded and non-welded regions are compared for ex vivo human and porcine aorta tissues. The fluorescence from the outer surface of welded aorta was substantially weaker than the fluorescence from the non-welded region. The Raman spectra from the welded and non-welded tissue regions appeared similar in the energies of the observed vibrational levels but the intensity of the fluorescence wing was considerably greater from the outer surface of the welded region as compared to the non-welded region. For the outer surface of the aorta, the emission intensity from the welded region was larger than for the non-welded region.
Optical transmission and light scattering measurements were performed on three species of bacteria with different sizes and shapes (Pseudomonas aeruginosa. Staphylococcus aureus and Bacillus subtilis). The average bacteria size was determined from transmission measurements by using the Gaussian Ray Approximation of Anomalous Diffraction Theory. A rescaled spectra combining multiple angular data was analyzed in the framework of the Rayleigh-Gans theory of light scattering in order to determine particle shape and size distribution. Particle size and shape as determined by both
methods are in good agreement with size and shape measured by scanning
electron microscopy. These results demonstrate that light scattering may be able to detect and identify microbial contamination in the environment.
Native fluorescence emission and excitation spectra were used to monitor changes in Bacillus subtilis (Bs) and Staphylococcus aureus (Sa) subjected to starvation conditions. Initially, the fluorescence spectra from the Bs and Sa was dominated by tryptophan emission. After the second day, a fluorescence band with an emission peak at 410 nm and an excitation peak at 345 nm appeared in the Bs. This emission is from dipicolinic acid, a major constituent of bacterial endospores. The dipicolinic acid intensity increased steadily during the next 2 to 4 days as the number of Bs forming spores increased. No dipicolinic acid signal was observed in the (non-spore forming) Sa. The addition of β-hydroxybutyric acid to either the Bs or Sa
resulted in the emergence of a third band with very strong fluorescence emission maximum at 460 nm and with excitation maxima at 250, 270 and 400 nm. This 460 nm emission was quenched with the addition of Fe2+, indicating that the source of this emission is a siderophore produced by the bacteria.
Laser tissue welding involves the denaturing and partial renaturing of collagen and elastin. Tissues welded with NIR lasers tuned to the 1455 nm water absorption band have demonstrated high tensile strength with minimal collateral damage. To better understand the welding process, welded tissue samples were investigated using fluorescence imaging and Raman spectroscopy. As part of this study, human aorta, and porcine aorta and skin, specimens were investigated. Emission and excitation/emission wavelengths corresponding to tryptophan and collagen emission and slightly weaker emission for wavelengths corresponding to elastin emission. The inner surface an cross-section images of the aortic specimens exhibited a very high degree of uniformity with no indication of the presence of a weld. The Raman spectra from the aortic specimens at the weld site and a few mm away form the weld were very similar. This work indicates the emission and Raman properties of the collagen helix after welding are very similar to native collagen tissue.
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