Resonance Raman and fluorescence spectroscopy were used to assess increased kynurenine pathway activity in brain samples from Alzheimer’s patients and age-matched controls. Increased activity was seen in areas of the brain involved in Alzheimer’s disease.
Under stress conditions, pro-inflammatory cytokines, such as tumor necrosis factor-alpha, interleukin-1 beta, interleukin 6 and interferon gamma are released. It is known that these cytokines stimulate indoleamine 2,3-dioxygenase (IDO) and tryptophan 2,3-dioxygenase (TDO), which increase tryptophan metabolism through the kynurenine pathway, and that this can cause increased production of neurotoxic compounds. Brain tissues from Alzheimer’s disease patients and agematched controls were investigated using label-free fluorescence spectroscopy. Tryptophan (exc. 280/ em. 340 nm) and its metabolites (N-formyl-L-kynurenine (exc. 325/em. 434 nm), kynurenine (exc. 365/em. 480 nm) and kynurenic acid (exc. 330/em. 390 nm)) have distinct spectral profiles. Preliminary results show a difference in the optical signatures in three important areas of the brain (hippocampus, BA 9, BA 17) between patients with Alzheimer’s disease and agedmatched controls (normal), and a marked relative increase in tryptophan in the Alzheimer’s patients. Thus determinations of tryptophan to tryptophan metabolite ratios could potentially be used to measure IDO and TDO activity and the degree of inflammation in the brain. This label-free optical technique may be useful in the study of Alzheimer’s and other neurodegenerative diseases.
Analyzing spectral or imaging data collected with various optical biopsy methods is often times difficult due to the complexity of the biological basis. Robust methods that can utilize the spectral or imaging data and detect the characteristic spectral or spatial signatures for different types of tissue is challenging but highly desired. In this study, we used various machine learning algorithms to analyze a spectral dataset acquired from human skin normal and cancerous tissue samples using resonance Raman spectroscopy with 532nm excitation. The algorithms including principal component analysis, nonnegative matrix factorization, and autoencoder artificial neural network are used to reduce dimension of the dataset and detect features. A support vector machine with a linear kernel is used to classify the normal tissue and cancerous tissue samples. The efficacies of the methods are compared.
The Resonance Raman (RR) spectra of basal cell carcinoma (BCC) and normal human skin tissues were analyzed using 532nm laser excitation. RR spectral differences in vibrational fingerprints revealed skin normal and cancerous states tissues. The standard diagnosis criterion for BCC tissues are created by native RR biomarkers and its changes at peak intensity. The diagnostic algorithms for the classification of BCC and normal were generated based on SVM classifier and PCA statistical method. These statistical methods were used to analyze the RR spectral data collected from skin tissues, yielding a diagnostic sensitivity of 98.7% and specificity of 79% compared with pathological reports.
Femtosecond (fs) pulse lasers in the near-infrared (NIR) range exhibit very distinct properties upon their interaction with biomolecules compared to the corresponding continuous wave (CW) lasers. Ultrafast NIR laser tissue bonding (LTB) was used to fuse edges of two opposing animal tissue segments in vivo using fs laser photoexcitation of the native vibrations of chomophores. The fusion of the incised tissues was achieved in vivo at the molecular level as the result of the energy–matter interactions of NIR laser radiation with water and the structural proteins like collagen in the target tissues. Nonthermal vibrational excitation from the fs laser absorption by water and collagen induced the formation of cross-links between tissue proteins on either sides of the weld line resulting in tissue bonding. No extrinsic agents were used to facilitate tissue bonding in the fs LTB. These studies were pursued for the understanding and evaluation of the role of ultrafast NIR fs laser radiation in the LTB and consequent wound healing. The fs LTB can be used for difficult to suture structures such as blood vessels, nerves, gallbladder, liver, intestines, and other viscera. Ultrafast NIR LTB yields promising outcomes and benefits in terms of wound closure and wound healing under optimal conditions.
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.
KEYWORDS: Laser tissue interaction, Laser welding, Tissues, In vivo imaging, Skin, Animal model studies, Near infrared, Laser energy, Collagen, Absorption
Near infrared laser tissue welding (LTW) is achieved by subjecting the closely approximated surgically incised tissues to
a laser beam at a wavelength that is absorbed by water in the tissue. Full thickness welds are accomplished with
optimum laser power and penetration depths appropriate for the thickness of welded tissues. No extrinsic cross-linking
or bonding materials are used. The absorbed laser energy increases the entropy of collagen in the tissue. In LTW, tissue
water temperatures transiently rises to approximately 60° C, causing partial denaturing of collagen and other structural
proteins due to breaking of hydrogen bonds, electrostatic interactions and some interchain covalent bonds for a short
duration of time. This is followed by cross linking of proteins on either side of weld line, with reformation of the above
mentioned bonds as the tissue cools, resulting in the formation of water tight full thickness welds. In this study, a cw
fiber laser emitting at 1455 nm, corresponding to absorption by a water vibrational overtone, is used for in vivo LTW of
surgical incisions made in the skin of guinea pigs under general anesthesia. The tensile strength and healing rates of the
welded incisions are compared to suturing of similar incisions. Laser parameters, including power, scanning rates,
exposure area, and exposure duration, are optimized to reduce thermal damage while maintaining tensile strength.
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.
We report an ex-vivo study on Raman spectra of adipose tissue covered by layers of aortic intimal wall tissue with
different thicknesses. The Raman vibration modes of
1435cm-1, 2850cm-1 and 2892cm-1 were investigated for the first
time on fresh porcine aortic adipose tissue with 633 nm laser excitation.
The adipose tissue was taken from adventitial fat grown on aorta walls. The frozen sections of porcine aorta wall tissue
with a thickness of 25μ - 50μ were cut from intimal surface. The samples were prepared by placing the variable numbers
of the aorta intimal wall tissue layers on the top of adipose tissue. The Raman spectra of adipose tissue were
investigated. The changes of intensities of the Raman modes versus thickness of the aorta intimal wall tissue layers were
measured. The total thickness of the aorta intimal wall tissue layers was varied in the range of 50μ - 1800μ.
The main characteristic Raman vibration modes of adipose tissue were found at 1435cm-1, 2850cm-1 and 2892cm-1.
Among them, the intensities of the modes of 2850 cm-1 and 2892 cm-1 are about four-times stronger than that of the
1435cm-1 mode.
The study on Raman vibration modes of 1435cm-1, 2800 cm-1 and 2950 cm-1 may be useful for developing a simple,
inexpensive and accurate optical technique for monitoring the degree of vulnerability of the aorta intimal surface due to
atherogenesis. These three Raman modes can be used as new molecular spectroscopic indicators to monitor in situ the
development of fatty-streaks and lipid core in aorta walls, and determine the thickness change of aorta intimal wall at
different stages of atherogenesis.
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.
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.
In this study, a NIR erbium fiber laser tuned to a water vibrational overtone absorption band at 1455 nm was used to weld directly, in vitro, seventy-six porcine aorta tissues without the need for extrinsic solder materials. The tissues were divided into eleven groups based on the multiple and variable parameters that were used to weld the tissues. The effectiveness of the parameters used in each of the weld groups was evaluated directly at the time of the weld and also by tensile strength measurements done at the termination of the weld. Management of heat produced in tissues is of critical importance for good laser tissue welding (LTW). To address heat
management issues, we report LTW using a transparent cover over the tissue specimen as a heat sink. Multiple scanning helps distribute the laser-generated heat and allows the tissue to cool between scans, reducing thermal damage. Better heat management using a transparent cover slide enhances the welding success. It reduces collateral damage and limits water evaporation and control the buckling of tissue around the line of apposition so that the two pieces that are welded do not move apart along the line of apposition due to buckling pressure and ensure a full-length weld.
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