Vladimir A. Hovhannisyan

Thermally Induced Irreversible Conformational Changes in Collagen Probed by Optical Second Harmonic Generation and Laser-induced Fluorescence

Irreversible thermal conformational changes induced to collagen have been studied by optical methods. More specifically, second harmonic generation (SHG) from incident nanosecond Ng:YAG 1064 nm radiation and laser-induced fluorescence by 337 nm, pulsed nanosecond nitrogen laser excitation, at 405, 410 and 415 nm emission wavelengths were registered at eight temperatures (40°, 50°, 55°, 60°, 65°, 70°, 75° and 80°C) and normalised with respect to the corresponding values at the ambient temperature of 30°C. The heating protocol used in this work, was selected to monitor only permanent changes reflecting in the optical properties of the samples under investigation. In this context, the SHG, directly related to the collagen fibril population in triple helix conformation, indicated on irreversible phase transition around 64°C. On the other hand fluorescence related to the destruction of cross-linked chromophores in collagen, some of which are related to the triple helix tertiary structure, also indicated a permanent phase transition around 63°C. These results are in agreement with previous results from studies with differential scanning calorimetry. However SHG and fluorescence, being non-invasive optical methods are expected to have a significant impact in the fields of laser ablative surgery and laser tissue welding.

Multiple Release Kinetics of Targeted Drug from Gold Nanorod Embedded Polyelectrolyte Conjugates Induced by Near-Infrared Laser Irradiation

The conjugates of gold nanorods and the model drug, fluorescein isothiocyanate (FITC), embedded inside polyelectrolytes (GNRs/FITC@PLE) were synthesized to study the release kinetics of FITC under femtosecond near-infrared (NIR) laser irradiation. The optical and structural properties of GNRs/FITC@PLE conjugates before and after laser treatments were examined using UV-vis spectroscopy, confocal microscopy, and transmission electron microscopy (TEM). The release of FITC from the conjugates was induced by the heat generated from gold nanorods under laser irradiation. The concentration of released FITC was measured as the time of continuous and periodic laser irradiation was varied. Within 5 min of the laser exposure, the release rates of FITC exhibited zero-order and first-order kinetics under continuous and periodic irradiation, respectively. Furthermore, a drug release system was designed based on the conjugates of gold nanorods and the anticancer drug, paclitaxel (PTX), embedded inside polyelectrolytes (GNRs/PTX@PLE). The conjugates were applied for in vitro studies with breast cancer cells. The release of PTX from the conjugates was triggered by NIR laser irradiation, and the inhibition rates of the cells showed strong dependencies on the irradiation modes and time. The results suggested that the multiple releases of PTX from the conjugates can be controlled by laser irradiation within a long period of time. Our system holds great potential for future therapeutic applications on breast cancers.

Second and third optical harmonic generation in type I collagen, by nanosecond laser irradiation, over a broad spectral region

Abstract We report optical up-conversion of pulsed (ns) laser radiation in type I pure collagen, tuneable over a broad excitation spectrum covering the 760–1070 nm wavelength range. We investigated second harmonic generation (SHG) in collagen using λ p =1064, 901, 892, 828, 785 and 766 nm nanosecond pulsed laser excitation and recorded monochromatic signals at λ p /2, i.e. 532, 451, 446, 414, 393 and 383 nm, respectively, corresponding to the SHG optical process. The SHG signal intensity exhibited a quadratic dependence on the excitation radiation (log[ I 532 ]=1.92*log[ I 1064 ]). Furthermore, a weaker third harmonic generation (THG) signal from collagen was also observed at λ p /3 (355 nm) using 1064 nm nanosecond pulsed laser excitation. The THG signal was found to have near-cubic dependence upon the irradiation laser intensity (log[ I 356 ]=2.53*log[ I 1064 ]). The significance of collagen ability to exhibit broadly tuneable second harmonic generation is discussed.

Quantifying thermodynamics of collagen thermal denaturation by second harmonic generation imaging

Time-lapse second harmonic generation (SHG) microscopy was applied for the extraction of thermodynamic parameters of collagen thermal denaturation. We found that at sufficiently high temperatures, temporal dependence of SHG intensity from the isothermal treatment of chicken dermal collagen was single exponential and can be modeled by the Arrhenius equation. Activation energy and the frequency factor of chicken dermal collagen thermal denaturation were determined using temporal decays of SHG intensity at different temperatures. Our results show that time-lapse, high temperature SHG imaging can be used to quantify kinetic properties of collagen thermal denaturation within a microscopic volume of 1 nl.

Differentiation of normal and cancerous lung tissues by multiphoton imaging

In this work, we utilized multiphoton microscopy for the label-free diagnosis of non-cancerous, lung adenocarcinoma (LAC), and lung squamous cell carcinoma (SCC) tissues from human. Our results show that the combination of second harmonic generation (SHG) and multiphoton excited autofluorescence (MAF) signals may be used to acquire morphological and quantitative information in discriminating cancerous from non-cancerous lung tissues. Specifically, non-cancerous lung tissues are largely fibrotic in structure while cancerous specimens are composed primarily of tumor masses. Quantitative ratiometric analysis using MAF to SHG index (MAFSI or SAAID) shows that the average MAFSI for noncancerous and LAC lung tissue pairs are 0.55 ±0.23 and 0.87±0.15 respectively. In comparison, the MAFSIs for the noncancerous and SCC tissue pairs are 0.50±0.12 and 0.72±0.13 respectively. Intrinsic fluorescence ratio (FAD/NADH) of SCC and non-cancerous tissues are 0.40±0.05 and 0.53±0.05 respectively, the redox ratio of SCC diminishes significantly, indicating that increased cellular metabolic activity. Our study shows that nonlinear optical microscopy can assist in differentiating and diagnosing pulmonary cancer from non-cancerous tissues. With additional development, multiphoton microscopy may be used for the clinical diagnosis of lung cancers.

Ex vivo imaging and quantification of liver fibrosis using second-harmonic generation microscopy

Conventionally, liver fibrosis is diagnosed using histopathological techniques. The traditional method is time-consuming in that the specimen preparation procedure requires sample fixation, slicing, and labeling. Our goal is to apply multiphoton microscopy to efficiently image and quantitatively analyze liver fibrosis specimens bypassing steps required in histological preparation. In this work, the combined imaging modality of multiphoton autofluorescence (MAF) and second-harmonic generation (SHG) was used for the qualitative imaging of liver fibrosis of different METAVIR grades under label-free, ex vivo conditions. We found that while MAF is effective in identifying cellular architecture in the liver specimens, it is the spectrally distinct SHG signal that allows the characterization of the extent of fibrosis. We found that qualitative SHG imaging can be used for the effective identification of the associated features of liver fibrosis specimens graded METAVIR 0 to 4. In addition, we attempted to associate quantitative SHG signal to the different METAVIR grades and found that an objective determination of the extent of disease progression can be made. Our approach demonstrates the potential of using multiphoton imaging in rapid classification of ex vivo liver fibrosis in the clinical setting and investigation of liver fibrosis-associated physiopathology in animal models in vivo.

Structural Characterization of Edematous Corneas by Forward and Backward Second Harmonic Generation Imaging

The purpose of this study was to image and quantify the structural changes of corneal edema by second harmonic generation (SHG) microscopy. Bovine cornea was used as an experimental model to characterize structural alterations in edematous corneas. Forward SHG and backward SHG signals were simultaneously collected from normal and edematous bovine corneas to reveal the morphological differences between them. In edematous cornea, both an uneven expansion in the lamellar interspacing and an increased lamellar thickness in the posterior stroma (depth > 200 microm) were identified, whereas the anterior stroma, composed of interwoven collagen architecture, remained unaffected. Our findings of heterogeneous structural alteration at the microscopic scale in edematous corneas suggest that the strength of collagen cross-linking is heterogeneous in the corneal stroma. In addition, we found that qualitative backward SHG collagen fiber imaging and depth-dependent signal decay can be used to detect and diagnose corneal edema. Our work demonstrates that SHG imaging can provide morphological information for the investigation of corneal edema biophysics, and may be applied in the evaluation of advancing corneal edema in vivo.

Photochemical effects and hypericin photosensitized processes in collagen

Emission and excitation spectra of collagen were recorded in the ultraviolet and visible regions. The existence of several types of chromophores absorbing and emitting throughout these spectral regions was observed. It was shown that laser irradiation at 355 and 532 nm caused collagen fluorescence photobleaching by 30%, when the delivered light doses were 9 and 18 J/cm2, respectively. This process of collagen fluorophores photodestruction was found to be a one-photon effect. The effect of hypericin (HYP), a polycyclic quinone, photosensitization on collagen was also studied. Addition of HYP aqueous solution to collagen produced quenching, redshift of the maximum, and broadening of the spectral form of its fluorescence. These effects became more prominent with increasing HYP concentration. The fluorescence of HYP sensitized collagen decreased in a spectrally nonproportional manner during laser irradiation at both 355 and 532 nm.

Multiphoton spectral microscopy for imaging and quantification of tissue glycation

Tissue glycation from diabetes and aging can result in complications such as renal failure, blindness, nerve damage and vascular diseases. In this work, we applied multiphoton microscopy for imaging and characterizing the extent of tissue glycation. The characteristic features of multiphoton autofluorescence (MPAF) and second harmonic generation (SHG) images as well as MPAF spectra of glycated bovine skin, cornea and aorta were acquired. The analysis of MPAF intensity change accompanying the glycation process shows that collagen is more responsive to the formation of autofluorescent advanced glycation endproducts (AGE) than elastic fibers. Changes in spectral features were also used to estimate the rate of glycation in tissues with intrinsic AF. Our study shows that multiphton imaging may be used for the in vitro investigation of the effects of tissue glycation and that this approach may be used for monitoring AGE formation in the clinical setting.

Elucidation of the mechanisms of optical clearing in collagen tissue with multiphoton imaging

Abstract. Optical clearing (OC) is a promising method to overcome limitations in biomedical depth-resolved optical studies. Mechanisms of OC in purified bovine Achilles tendon, chicken skin, and chicken tendon were studied using time-lapsed, three-dimensional second harmonic generation (SHG) and two-photon fluorescence microscopic imaging. Quantified nonlinear optical measurements allowed temporal separation of two processes in collagen OC with glycerol. The first one is a fast process of tissue dehydration accompanied with collagen shrinkage and the second relatively slow process is glycerol penetration into the interfibrillar space of collagen alongside with CF swelling. The use of 50% glycerol induced less-expressed OC via partial substitution of water molecules with glycerol molecules. We also found that phosphate-buffered saline- and glycerol-treatments were reversible, and fiber morphology and SHG signal intensity were recovered after the removal of immersion agents. It was shown that tissue OC was a dynamic process and elucidation of its physical mechanisms may help choose optimal diagnostic, treatment, and modification regimes for collagen-based as well as other types of biomaterials.