This study investigated the use of clinoptilolite zeolites (CZs) as the drug adsorbent and their ability to release 5- Aminolevulinic acid (5-ALA) and porphyrin (Zn-TBut3 PyP) in the presence of biomolecules, by absorption and fluorescence spectrometry and multiphoton spectroscopy. About one-third of the porphyrin adsorbed on to CZs was released in the presence of biomolecule such as insulin. In-vitro measurements show that the skin absorbed the dye more than 1.5 times for ALA-CZ and porphyrin-CZ complex compared to non-CZ form. The measurements on chicken skin were also performed using multiphoton microscopy which compared the penetration of the dye into the skin in the presence and absence of CZ. These results demonstrate the potential of CZ in the efficient adsorption and biomolecule favoured release of 5-ALA and porphyrin in biological systems.
A developed temporal focusing-based multiphoton microscope (TFMPM) has a digital micromirror device (DMD) which is adopted not only as a blazed grating for light spatial dispersion but also for patterned illumination simultaneously. Herein, the TFMPM has been extended to implement spatially modulated illumination at structured frequency and orientation to increase the beam coverage at the back-focal aperture of the objective lens. The axial excitation confinement (AEC) of TFMPM can be condensed from 3.0 μm to 1.5 μm for a 50% improvement. Furthermore, a multiline scanning mechanism based on the DMD can push its AEC nearly equivalent to line scanningbased TFMPM to 1.5 μm from optimal 3.0 μm of the conventional TFMPM. By using the TFMPM with structured illumination and multiline scanning, reconstructed biotissue images according to the condensed AEC structured illumination are shown obviously superior in contrast and better scattering suppression.
Adsorption of photodynamic active dyes (hypericin, chlorin e6, methylene blue, ALA and fluorescein) and they release from clinoptilolite type of zeolite (CZ) in the presence of biomolecules, such as collagen, albumin and hemoglobin, were quantitatively investigated by absorption and fluorescence spectrometry. Using multiphoton microscopy, we demonstrated that effective inducing of two-photon excited luminescence and second harmonic generation signals in nano/micro-particles of CZ by femtosecond near-infrared laser excitation can be successfully utilized in multiphoton imaging of CZ particles and the dye adsorption processes. In addition, CZ magnetic (MCZ) particles were fabricated and proposed as a promising material for drug delivery and controlled release in biological systems. It is shown that the temperature of the aqueous suspension of MCZ of 2 cm3 can be increased by 7 ºC for 3 minutes by an induction heater. Furthermore, it was demonstrated that MCZ particles have strong inhibition and destruction effects on insulin and lysozyme fibrillization, and the protein amyloid formation is inhibited in a dose-dependent manner. Optical spectrometry and multiphoton microscopy are effective approaches that may reveal potential of magnetic zeolites in drug delivery and biomedical imaging of cancer photodynamic therapy and hyperthermia.
Intravital multiphoton microscopy was used to study hepatobiliary metabolism in chronic pathologies of the liver. Through the use of the probe molecule 6-carboxyfluorescein diacetate (6-CFDA), the effects of liver fibrosis, fatty liver, and hepatocellular carcinoma on the metabolic capabilities of mouse liver were investigated. After the acquisition of time lapse images, a first order kinetic model was used to calculate rate constant resolved images of various pathologies. It was found the ability of the liver to metabolically process the probe molecules varies among different pathologies, with liver fibrosis and fatty liver disease negatively impacted the uptake, processing, and excretion of molecules.
Hypericin (Hyp) has received attention due to its high phototoxicity against viruses and anti-tumor photoactivity. Using
two-photon imaging, we demonstrated that Hyp induced photosensitized modification of collagen fibers in native tissues.
Dynamics of photo-processes was monitored by time-lapse multiphoton imaging. We showed that Hyp–mediated
processes in collagen tissues may be used for the selective modification of collagen fibers.
Hepatycotes in the liver may appear similar in morphology, however, heterogeneities may exist in cellular metabolism.
In this study, in vivo imaging of 6-carboxfluorescein diacetate (6-CFDA) metabolism in the liver was studied. We used
two-photon fluorescence microscopy and hepatic window to provide quantification in studying hepatocellular
metabolism. This model not only provides a potential platform for future study in hepatic responses and regulations, but
also contributes to the fine-tuning of organ-specific functions so as to open up a new era of exciting discoveries.
Time-lapsed, three-dimensional multiphoton microscopy showed that application of air-drying and glycerol to animal
tissue induced a well-expressed optical clearing. The effect was dynamic, reversible process, and can be used to enhance
capabilities of nonlinear imaging.
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.
Collagen, the main structural protein in vertebrates, possesses different structural organization that is responsible for
specific functions of the tissues. Polarization dependence of the second harmonic generation (SHG) signal on spatial
orientation of optically nonlinear materials, such as collagen, provides information on characteristic organization and
architecture not available from intensity measurements alone. Here we describe a simple approach for determining
both the azimuth and elevation angles of collagen fiber orientation in biological tissues. Azimuth angle of the fiber
orientation is determined as an orthogonal angle to the laser polarization direction, when laser-induced total SHG
signal is minimal, whereas the elevation angle is estimated from the ratio of the minimal SHG intensity to the
intensity when laser polarization and fiber directions are parallel to each other. Using this approach pixel-resolved
mapping of the spatial orientation of collagen fibers in tendon and cornea is demonstrated. The new approach may be
used for analyzing of biological tissues in vivo. Spatial orientation mapping method provides additional information
concerning fiber organization, and may be incorporated into nonlinear optical imaging systems.
We discuss the recent advances in the development and applications of second-order susceptibility as a contrast mechanism in optical microscopy for biological tissues. We review nonlinear optical methods and approaches for differentiation of tissue structures and discrimination of normal and pathological skin tissues, which have been demonstrated for the potential use in clinical diagnosis. In addition, the potential of second-order susceptibility imaging, encompassing applications in differentiating various types of collagen molecules for clinical diagnosis, is demonstrated. Finally, we discuss future development and application of this technique.
Collagen is the major component of skin, tendon, cartilage, cornea, and, as a main structural protein it is the key
determinant of thermo-mechanical properties of collagen-rich tissues in mammals. Thermal damage of chicken dermis
and tendon, bovine leg tendon, and other collagen contained tissues were investigated with the use of second harmonic
generation (SHG) and two-photon excited auto-fluorescence microscopy and spectroscopy. Samples were heating in a
temperature-controlled water bath in the temperature range 18-90° C. SHG time-lapse imaging and analysis of intensity
decay showed that the collagen thermal destruction depended on both temperature and heating time, and can be modeled
by the Arrhenius equation. Temporal decay of SHG signal from the chicken dermis was single exponential during
isothermal treatment at temperatures above 60º C that allowed to determine activation energy and frequency factor of
skin collagen denaturation. Furthermore, two-exponential decay and partially reversible change in SHG intensity were
registered during the tendon thermal treatment. A simple laser system and procedure is proposed for a real-time
monitoring of collagen fiber thermal modification within a microscopic volume of 1 nl.
Optical-based microscopy plays an important role in various scientific fields such as physics, chemistry and biology. Second harmonic generation (SHG) microscopy has become one of the indispensable tools for biomedical imaging for the last decade because the signal generated from SHG is sensitive to the objective structure and this amazing non-invasive method can also directly observe the objective without using extra fluorescent labels, especially for collagen molecules. As the most abundant protein in animals, collagen is responsible for a number of important structural and functional roles in vertebrates. For certain diseases, it has been shown that collagen fiber diameter has a significant variation and thus as a vital symptom for diagnosis. Moreover, collagen diameter is also a key parameter for fibrogenesis studying. Therefore, the determination of collagen fiber diameter is important for studying biophysical processes and identifying bioengineering applications. In this study, we investigated various collagen fibril thicknesses and the corresponding forward (FSHG) and backward (BSHG) second harmonic signal intensity variation. Our result exhibits that SHG intensity can quantify describe the relative collagen fibril thickness alteration, which also indicates the coherent effect difference between FSHG and BSHG. This approach demonstrates the capability of SHG imaging in providing collagen mechanical information and that may be applied in the evaluation of advancing collagen issues in vivo.
In this work, we applied multi-photon microscopy to image and characterize the extent of tissue
glycation. Specifically, features of multi-photon autofluorescence (MPAF) and second harmonic
generation (SHG) images were presented. Our study shows that multiphoton imaging is capable of
providing qualitative and quantitative information of the extent of tissue glycation in ribose treated
samples including bovine sclera and vessel and that this approach may be useful for clinical
evaluation of advanced glycation endproducts (AGEs) formation.
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.
We used second harmonic generation (SHG) microscopy to image and quantify the structural changes of bovine corneal
edema. Forward SHG (FWSHG) and backward SHG (BWSHG) signals were simultaneously collected from normal and
edematous bovine corneas to reveal their morphological differences. In SHG imaging, edematous corneas can be
characterized by uneven expansion in the lamellar interspacing and increased lamellar thickness in posterior stroma
(depth > 200 μm), while the anterior stroma composed of interwoven collagen architecture remained unaffected. Our
work demonstrate the capability of SHG imaging in providing morphological information for the investigation of corneal
edema biophysics and its potential application in the in vivo evaluation of advancing corneal edema.
Conventionally, the diagnosis of hepatocellular carcinoma (HCC) is performed by qualitative
examination of histopathological specimens, which takes times for sample preparation in fixation,
section and stain. Our objective is to demonstrate an effective and efficient approach to apply
multiphoton microscopy imaging the HCC specimens, with the advantages of being optical section,
label-free, subcellular resolution, minimal invasiveness, and the acquisition of quantitative information
at the same time. The imaging modality of multiphoton autofluorescence (MAF) was used for the
qualitative imaging and quantitative analysis of HCC of different grades under ex-vivo, label-free
conditions. We found that while MAF is effective in identifying cellular architecture in the liver
specimens, and obtained quantitative parameters in characterizing the disease. Our results demonstrates
the capability of using tissue quantitative parameters of multiphoton autofluorescence (MAF), the
nuclear number density (NND), and nuclear-cytoplasmic ratio (NCR) for tumor discrimination and that
this technology has the potential in clinical diagnosis of HCC and the in-vivo investigation of liver tumor development in animal models.
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.
We used the combination of multiphoton autofluorescence (MAF), forward second-harmonic generation (FWSHG), and backward second-harmonic generation (BWSHG) imaging for the qualitative and quantitative characterization of thermal damage of ex vivo bovine cornea. We attempt to characterize the structural alterations by qualitative MAF, FWSHG, and BWSHG imaging in the temperature range of 37 to 90°C. In addition to measuring the absolute changes in the three types of signals at the stromal surface, we also performed image correlation analysis between FWSHG and BWSHG and demonstrate that with increasing thermal damage, image correlation between FWSHG and BWSHG significantly increases. Our results show that while MAF and BWSHG intensities may be used as preliminary indicators of the extent of corneal thermal damage, the most sensitive measures are provided by the decay in FWSHG intensity and the convergence of FWSHG and BWSHG images.
Controllable, non-ablative photo-processing of collagen, elastic, muscle and cotton fibers was achieved by femtosecond
Ti:Sa laser. Fibers were cut, bended and welded by the infrared laser and simultaneously imaged using second harmonic
generation and two-photon auotoflourescence microscopy.
We utilize multiphoton microscopy for the label-free diagnosis of noncancerous, lung adenocarcinoma (LAC), and lung squamous cell carcinoma (SCC) tissues from humans. 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 noncancerous lung tissues. Specifically, noncancerous 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) 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. Our study shows that nonlinear optical microscopy can assist in differentiating and diagnosing pulmonary cancer from noncancerous tissues.
The fruit fly Drosophila melanogaster is one of the most valuable organisms in studying genetics and developmental biology. To gain insight into Drosophila development, we successfully acquired label-free, in vivo images of both developing muscles and internal organs in a stage 2 larva using the minimally invasive imaging modality of multiphoton autofuorescence (MAF) and second harmonic generation (SHG) microscopy. We found that although MAF is useful in identifying structures such as the digestive system, trachea, and intestinal track, it is the SHG signal that allowed the investigation of the muscular architecture within the developing larva. Our results suggest that multiphoton microscopy is a powerful in vivo, label-free imaging technique to examine Drosophila physiology and may be used for developmental studies.
The effects of off-axis optical aberration in multiphoton microscopy and the resulting lateral and axial image inhomogeneity are investigated. The lateral inhomogeneity of the scanning field is demonstrated by second harmonic generation (SHG) imaging of fasciae and two-photon fluorescence (TPF) microscopy of thin fluorescent samples. Furthermore, refractive index mismatch-caused intensity attenuation of the TPF signal at central and peripheral regions of the scanning frame is measured using homogeneous 10-µM sulforhodamine B samples with refractive indexes of 1.33 and around 1.465. In addition to characterizing image field convexity, we also found that image resolution degrades away from the optical axis. These effects need to be accounted for in both qualitative and quantitative multphoton imaging applications.
By using two-photon autofluorescence (TPAF) and second harmonics generation (SHG), we imaged hepatocellular
carcinoma (HCC) biopsies from the human patients and compared them with the conventional histological biopsies. We
found that multiphoton microscopy may be used to obtain label-free images of liver tissues and may be developed into
an effective diagnostic tool for liver diseases.
The fruit fly Drosophila melanogaster is one of the most valuable organisms in genetic and
developmental biology studies. Drosophila is a small organism with a short life cycle, and is
inexpensive and easy to maintain. The entire genome of Drosophila has recently been sequenced (cite
the reference). These advantages make fruit fly an attractive model organism for biomedical researches.
Unlike humans, Drosophila can be subjected to genetic manipulation with relative ease. Originally,
Drosophila was mostly used in classical genetics studies. In the model era of molecular biology, the
fruit fly has become a model organ for developmental biology researches. In the past, numerous
molecularly modified mutants with well defined genetic defects affecting different aspects of the
developmental processes have been identified and studied. However, traditionally, the developmental
defects of the mutant flies are mostly examined in isolated fixed tissues which preclude the observation
of the dynamic interaction of the different cell types and the extracellular matrix. Therefore, the ability
to image different organelles of the fruit fly without extrinsic labeling is invaluable for Drosophila
biology. In this work, we successfully acquire in vivo images of both developing muscles and axons of
motor neurons in the three larval stages by using the minimially invasive imaging modality of
multiphoton (SHG) microscopy. We found that while SHG imaging is useful in revealing the muscular
architecture of the developing larva, it is the autofluorescence signal that allows label-free imaging of
various organelles to be achieved. Our results demonstrate that multiphoton imaging is a powerful
technique for investigation the development of Drosophila.
Multi-wavelength (670, 805, 848 and 905 nm), multi-detector device for non-invasive measurement of biochemical components concentration in human or animal tissues, combining the methods of conventional pulse-oximetry and near infrared spectroscopy, is developed. The portable and clinically applicable system allows to measure heart pulse rate, oxygen saturation of arterial hemoglobin (pulse-oximetry method) and local absolute concentration of oxyhemoglobin, deoxyhemoglobin and oxidized cytochrome aa3 or other IR absorbed compounds (NIRS method). The system can be applied in monitoring of oxygen availability and utilization by the brain in neonatal and adults, neuro- traumatology, intensive care medicine, transplantation and plastic surgery, in sport, high-altitude and aviation medicine.
Photodynamic activity of several dyes on Drosophila melanogaster at different temperatures (15-35°C) inside of test-tubes was investigated. Both phototoxic sensitizers (chlorin e6, methylene blue, etc. -group A) and non active compounds (hemoglobin, brilliant green, pyronine, etc.-group B) were used. Dyes of 10-5-10-3 M concentration were added to the food for drosophila 24 hours before irradiation. Solar radiation, narrow-band halogen lamps, LEDs and laser were used as a photo-stimulator. Irradiation parameters: I ≤ 45mW/cm2 and 0.2<tirr<1.5 h. Without using dyes in the control test-tubes at all doses of irradiation and temperatures applied percentage of survived insects was approximately 100%. In the darkness with the use of all dyes observations also indicated no damage to the insects. At the temperatures up to 25°C when using dyes of group B insects were not affected at all, while with the dyes of group A findings showed dose-dependent insect mortality. At high temperatures (30-35°C) when using group B dyes flies were losing their mobility and in the case of group A dyes the drosophila’s survival value sharply dropped. Combination of dyes from A group with some dyes from B group leads to the partial disappearance of photodynamic effect. This, probably, is concerned with the toxic photoproduct suppression by the inactive dye. Experimental model of drosophila allows to investigate photosensitization impact within wide temperature range, to find out the processes, when using combination of dyes, as well as to study photodynamic effect on reproductive functions of insects.
A novel detection method for malignant tumour detection through specific accumulation of far-red emitting photosensitisers, has been developed. The set-up, based on He-Ne (633 nm) excitation was tested with phantom malignancies models using photosensitisers on chicken tissue. Intensity vs. depth of tissue measurements as well as photobleaching measurements were performed. Point by point fluorescence imaging indicated that the formation size is not significantly distorted with respect to depth of location.
Under pulsed nanosecond laser irradiation of type I pure collagen over the 760 - 1070 nm spectral range, optical up- conversion of the incident radiation is observed. More specifically irradiation of collagen at 1064, 901, 892, 828, 785 and 766 nm produces monochromatic second harmonic signals at half the original wavelengths i.e. 532, 451, 446, 414, 393 and 383 nm respectively. The dependence of the second harmonic signal on the excitation intensity was found to be quadratic [log(I532) equals 1.92*log(I1064]. A weaker third harmonic signal was also observed from collagen at 355 nm when irradiated by 1064 nm nanosecond pulses. This signal was found to bear a near cubic dependence upon the irradiation intensity ]log(I356) equals 2.53*log(I1064)]. The polar distribution of the second harmonic radiation was recorded for both pure fibrous collagen as well a for collagen diluted in acetic acid and forming a semitransparent dry film. In the latter case, significant optical behavior was demonstrated, potentially important for further studies of these nonlinear phenomena and for novel applications.
A computerized fiber-optic spectrofluorometer based on a multifrequency Nd:YAG laser ((lambda) equals 355, 440, 532 and 660 nm, f equals 25 Hz, E equals 1 - 10 mJ, (tau) equals 12 ns) for tissue fluorescence registration in vivo and ex vivo has been developed. The less intensive fluorescence from a tumor of Sarcoma-45 bearing animal model in comparison with the surrounding normal tissue was observed at the spectral region around 450 nm. The influence of reabsorption, energy transfer and other physical factors on tumor fluorescence, sensitized by Photohem (hematoporphyrin derivative), disodium salt of fluorescein (FL) and chlorin e6 (Chl) was investigated. The pharmacokinetic behavior of Chl in different organs and tumors of the animal models has been estimated. The most intensive Chl fluorescence of tumor tissue was observed at 18 hours after photosensitizer injection. The maximum of the tumor-to-healthy tissue ratio of fluorescence was reached 10 at 27 hours after pigment injection. The fluorescence spectra from different types of human tumors after i/v injection with FL or topical application of ALA were studied. A simple model of Nd:YAG laser system for tumor fluorescence diagnosis has been elaborated. Advantages of the laser fluorescence diagnosis of malignant tumors by solid state multifrequency Nd:YAG laser and the increase in accuracy and specificity of this method is discussed.
The photoinduced effects of hypericin, a polycyclic quinone, on collagen has been investigated. It was found that after laser irradiation at both 532 nm and 337 nm, the spectral form of triple helix structure collagen fluorescence, changed to a spectral profile bearing resemblance to that of its polypeptide single chain counterpart, gelatin, or heated collagen. The effect of Chlorin e6 on collagen was also investigated and proved to be dissimilar to that of hypericin and not indicative of profound structural alterations. Second Harmonic Generation (SHG) of 1064 nm- nanosecond laser radiation in collagen was studied. While it was very efficient for pure collagen, the signal intensity was found to diminish by at least an order of magnitude after hypericin photosensitization or heating. The above noted fluorescence spectra form alteration was also observed in a smaller scale in collagen rich chicken tissue (tendon). Non sensitized chicken tendon tissue exhibited very efficient SHG, unlike skin and artery samples.
The interaction of aromatic amino acids, superoxide dismutase (SOD) and bovine serum albumin (BSA) with chlorin e6 (Chl) and formation of complexes were studied. The decrease of the efficiency of photochemical destruction in the absence of oxygen was observed. The changes of SOD absorption spectra in UV region agree with our former results on irreversible decrease of protein's tryptophan (Trp) luminescence under irradiation. At the same time no changes in enzymatic activity of irradiated SOD in the presence of Chl were detected. The irradiation effect on K+ and H+ ions movement across the membrane of erythrocytes loaded with Chl was studied.
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