MULTIPHOTON MICROSCOPY: A NEW APPROACH, IN PHYSIOLOGICAL STUDIES AND PATHOLOGICAL DIAGNOSIS FOR OPHTHALMOLOGY

2009 ◽  
Vol 02 (01) ◽  
pp. 45-60 ◽  
Author(s):  
CHIU-MEI HSUEH ◽  
WEN LO ◽  
SUNG-JAN LIN ◽  
TSUNG-JEN WANG ◽  
FUNG-RUNG HU ◽  
...  
Keyword(s):  
Second Harmonic ◽  
Healing Process ◽  
Multiphoton Microscopy ◽  
In Vivo Studies ◽  
Wound Healing Process ◽  
Label Free ◽  
Multiphoton Imaging ◽  
Morphological Alterations ◽  
Corneal Scar

Multiphoton microscopy (MPM), with the advantages of improved penetration depth, decreased photo-damage, and optical sectioning capability, has become an indispensable tool for biomedical imaging. The combination of multiphoton fluorescence (MF) and second-harmonic generation (SHG) microscopy is particularly effective in imaging tissue structures of the ocular surface. This work is intended to be a review of advances that MPM has made in ophthalmic imaging. The MPM not only can be used for the label-free imaging of ocular structures, it can also be applied for investigating the morphological alterations in corneal pathologies, such as keratoconus, infected keratitis, and corneal scar. Furthermore, the corneal wound healing process after refractive surgical procedures such as conductive keratoplasty (CK) can also be studied with MPM. Finally, qualitative and quantitative SHG microscopy is effective for characterizing corneal thermal denaturation. With additional development, multiphoton imaging has the potential to be developed into an effective imaging technique for in vivo studies and clinical diagnosis in ophthalmology.

scholarly journals Stimulation of wound healing process through ROS/RNS signals indirectly generated by N2/Ar micro-plasma - in vitro and in vivo studies

2015 ◽  
Vol 18 (2) ◽  
pp. 29-37
Author(s):  
Hien Thi Minh Ngo ◽  
Linh Quang Huynh ◽  
Liao Jiunn Der ◽  
Thuy Ngu Son Nguyen
Keyword(s):  
Healing Process ◽  
In Vivo Studies ◽  
Wound Healing Process ◽  
Fibroblast Cells ◽  
Plasma Exposure ◽  
Burn Wound ◽  
Degree Burn ◽  
Plasma Exposure Time

In this work, non-thermal N2/Ar micro-plasma was applied to fibroblast cells and second degree burn in mice to investigate the bio-safety and bioefficiency of micro-plasma device for studying wound healing process. The chosen parameters of the device were the addition of 0.5% N2 in argon plasma and RF supplied power of 17 W and 13 W in vitro and in vivo studies, respectively. Firstly, micro-plasma was applied to fibroblast cells and the induced biological effect was studied in vitro. The result showed that cells number increased three folds for plasma exposure time of 5 or 10 sec, followed by cell culture for 48 hrs. The cell coverage rate rose 20% for the same plasma exposure time, followed by cell culture for 6 or 12 hrs. Secondly, micro-plasma was applied to the second degree burn wound mice, followed by related ex vivo and in vivo assessments. For the former, 0.5% N2/Ar micro-plasma was competent to generate ROS/RNS signals for advancing healing process by the increase of ROS/RNS concentration around the plasma-exposed wound bed. The induced effect is most probably correlated with the angiogenesis and epithelialization processes of the burn wound on mice.

scholarly journals Label-free multiphoton imaging allows brain tumor recognition based on texture analysis—a study of 382 tumor patients

2020 ◽  
Vol 2 (1) ◽  
Author(s):  
Ortrud Uckermann ◽  
Roberta Galli ◽  
Georg Mark ◽  
Matthias Meinhardt ◽  
Edmund Koch ◽  
...  
Keyword(s):  
Brain Tumors ◽  
Second Harmonic ◽  
Multiphoton Microscopy ◽  
Recurrent Glioblastoma ◽  
Image Resolution ◽  
Label Free ◽  
Multiphoton Imaging ◽  
Linear Discriminant ◽  
High Grade Astrocytoma ◽  
Texture Parameters

Abstract Background Label-free multiphoton microscopy has been suggested for intraoperative recognition and delineation of brain tumors. For any future clinical application, appropriate approaches for image acquisition and analysis have to be developed. Moreover, an evaluation of the reliability of the approach, taking into account inter- and intrapatient variability, is needed. Methods Coherent anti-Stokes Raman scattering (CARS), two-photon excited fluorescence (TPEF), and second-harmonic generation were acquired on cryosections of brain tumors of 382 patients and 28 human nontumor brain samples. Texture parameters of those images were calculated and used as input for linear discriminant analysis. Results The combined analysis of texture parameters of the CARS and TPEF signal proved to be most suited for the discrimination of nontumor brain versus brain tumors (low- and high-grade astrocytoma, oligodendroglioma, glioblastoma, recurrent glioblastoma, brain metastases of lung, colon, renal, and breast cancer and of malignant melanoma) leading to a correct rate of 96% (sensitivity: 96%, specificity: 100%). To approximate the clinical setting, the results were validated on 42 fresh, unfixed tumor biopsies. 82% of the tumors and, most important, all of the nontumor samples were correctly recognized. An image resolution of 1 µm was sufficient to distinguish brain tumors and nontumor brain. Moreover, the vast majority of single fields of view of each patient’s sample were correctly classified with high probabilities, which is important for clinical translation. Conclusion Label-free multiphoton imaging might allow fast and accurate intraoperative delineation of primary and secondary brain tumors in combination with endoscopic systems.

scholarly journals Label-Free Multiphoton Microscopy: Much More Than Fancy Images

2021 ◽  
Vol 22 (5) ◽  
pp. 2657
Author(s):  
Giulia Borile ◽  
Deborah Sandrin ◽  
Andrea Filippi ◽  
Kurt I. Anderson ◽  
Filippo Romanato
Keyword(s):  
Harmonic Generation ◽  
Biomedical Research ◽  
Quantitative Methods ◽  
Second Harmonic ◽  
Multiphoton Microscopy ◽  
Basic Research ◽  
Label Free ◽  
Multiphoton Imaging ◽  
Third Harmonic ◽  
Research Fields

Multiphoton microscopy has recently passed the milestone of its first 30 years of activity in biomedical research. The growing interest around this approach has led to a variety of applications from basic research to clinical practice. Moreover, this technique offers the advantage of label-free multiphoton imaging to analyze samples without staining processes and the need for a dedicated system. Here, we review the state of the art of label-free techniques; then, we focus on two-photon autofluorescence as well as second and third harmonic generation, describing physical and technical characteristics. We summarize some successful applications to a plethora of biomedical research fields and samples, underlying the versatility of this technique. A paragraph is dedicated to an overview of sample preparation, which is a crucial step in every microscopy experiment. Afterwards, we provide a detailed review analysis of the main quantitative methods to extract important information and parameters from acquired images using second harmonic generation. Lastly, we discuss advantages, limitations, and future perspectives in label-free multiphoton microscopy.

scholarly journals Real-Time Multidepth Multiphoton Microscopy Using Pixel-to-Pixel Focus-Switching

2020 ◽  
Vol 10 (20) ◽  
pp. 7173
Author(s):  
Yifan Qin ◽  
Deying Chen ◽  
Yuanqin Xia
Keyword(s):  
Real Time ◽  
Imaging System ◽  
Second Harmonic ◽  
Multiphoton Microscopy ◽  
Cost Effective ◽  
Axial Direction ◽  
Three Dimensions ◽  
Mouse Lung ◽  
Label Free

Multiphoton microscopy is a well-established technique for biomedical applications, but real-time multidepth multimodal multiphoton microscopy using non-imaging detection has barely been discussed. We demonstrate a novel label-free imaging system capable of generating multimodal multiphoton signals at different focal planes simultaneously. Two spatially overlapped and temporally interlaced beams are obtained by applying cost-effective electro-optic modulator (EOM)-based fast-switching light paths. The switching beams have different divergence properties, enabling imaging at different depths into samples. The EOM is synchronized to the pixel clock from the microscope, achieving pixel-to-pixel focus-switching. The capability of the imaging system is demonstrated by performing real-time multidepth two-photon fluorescence (TPF) and second-harmonic generation (SHG) imaging of freshly excised mouse lung lobes. TPF and SHG images are acquired at two wavelength ranges. One is between 415 and 455 nm, and the other is between 495 and 635 nm. The microenvironment of pulmonary alveoli is depicted by the distributions of both elastin fibers visualized by TPF and collagen fibers illustrated by SHG. Macrophages residing inside apparent alveolar lumens are also identified by TPF, which shows that the imaging system is capable of localizing biological objects in three dimensions and has the potential of monitoring in vivo cellular dynamics in the axial direction.

scholarly journals Rifampicin-Loaded Alginate-Gelatin Fibers Incorporated within Transdermal Films as a Fiber-in-Film System for Wound Healing Applications

Membranes ◽  
2020 ◽  
Vol 11 (1) ◽  
pp. 7
Author(s):  
Ameya Sharma ◽  
Vivek Puri ◽  
Pradeep Kumar ◽  
Inderbir Singh
Keyword(s):  
Wound Healing ◽  
Transmission Rate ◽  
Wound Care ◽  
Healing Process ◽  
In Vivo Studies ◽  
Wound Healing Process ◽  
Uptake Study ◽  
Tissue Proliferation ◽  
Water Transmission

The various biological and molecular cascades including different stages or phases such as inflammation, tissue proliferation, and remodeling phases, which significantly define the wound healing process. The natural matrix system is suggested to increase and sustain these cascades. Biocompatible biopolymers, sodium alginate and gelatin, and a drug (Rifampicin) were used for the preparation of fibers into a physical crosslinking solution using extrusion-gelation. The formed fibers were then loaded in transdermal films for wound healing applications. Rifampicin, an antibiotic, antibacterial agent was incorporated into fibers and afterwards the fibers were loaded into transdermal films. Initially, rifampicin fibers were developed using biopolymers including alginate and gelatin, and were further loaded into polymeric matrix which led to the formation of transdermal films. The transdermal films were coded as TF1, TF2, TF3 and TF4.The characterization technique, FTIR, was used to describe molecular transitions within fibers, transdermal films, and was further corroborated using SEM and XRD. In mechanical properties, the parameters, such as tensile strength and elongation-at-break (extensibility), were found to be ranged between 2.32 ± 0.45 N/mm2 to 14.32 ± 0.98 N/mm2 and 15.2% ± 0.98% to 30.54% ± 1.08%. The morphological analysis firmed the development of fibers and fiber-loaded transdermal films. Additionally, physical evaluation such as water uptake study, water transmission rate, swelling index, moisture content, and moisture uptake study were executed to describe comparative interpretation of the formulations developed. In vivo studies were executed using a full thickness cutaneous wound healing model, the transdermal films developed showed higher degree of contraction, i.e., 98.85% ± 4.04% as compared to marketed formulation (Povidone). The fiber-in-film is a promising delivery system for loading therapeutic agents for effective wound care management.

scholarly journals Healing Efficacy of an EGF Impregnated Triple Gel Based Wound Dressing: In Vitro and In Vivo Studies

2014 ◽  
Vol 2014 ◽  
pp. 1-10 ◽  
Author(s):  
Najmeh Khanbanha ◽  
Fatemeh Atyabi ◽  
Azade Taheri ◽  
Fatemeh Talaie ◽  
Mirgholamreza Mahbod ◽  
...  
Keyword(s):  
Wound Healing ◽  
Topical Application ◽  
Healing Process ◽  
Tissue Growth ◽  
In Vivo Studies ◽  
Wound Healing Process ◽  
Beneficial Effects ◽  
Linear Viscoelastic

To accomplish an ideal wound healing process which promotes healthy tissue growth with less scaring, a novel gel based topical drug delivery system composed of 3 different polymers chitosan, dextran sulfate, and polyvinylpyrrolidone K30 (CDP) was prepared. The physicochemical properties of the prepared gels were investigated in vitro. Gels showed a maximum swelling ratio of 50 ± 1.95 times of dried gel in PBS at pH 7.4. The swelling ratios increase in acidic and alkaline pH to 55.3 ± 1.75 and 65.5 ± 2.42, respectively. In the rheological test, prepared gels revealed viscoelastic properties and a small linear viscoelastic region of 0.166%. In vivo wound healing promoting activities of CDP gels containing 20 μg/mL EGF were evaluated on surgically induced dermal wounds in rats using pathologic examination. The application of CDP gel with incorporated EGF significantly reduced the defect on the rat’s skin and enhanced epithelial healing compared with the topical application of the EGF-free CDP gel. The results clearly substantiate the beneficial effects of the topical application of CDP containing EGF in the acceleration of healthy wound healing process with less scarring.

scholarly journals Development of a Topical Insulin Polymeric Nanoformulation for Skin Burn Regeneration: An Experimental Approach

2021 ◽  
Vol 22 (8) ◽  
pp. 4087
Author(s):  
Maria Quitério ◽  
Sandra Simões ◽  
Andreia Ascenso ◽  
Manuela Carvalheiro ◽  
Ana Paula Leandro ◽  
...  
Keyword(s):  
Wound Healing ◽  
Healing Process ◽  
In Vitro Release ◽  
Peptide Hormone ◽  
Plga Nanoparticles ◽  
Wound Healing Process ◽  
Target Area ◽  
Encapsulation Process

Insulin is a peptide hormone with many physiological functions, besides its use in diabetes treatment. An important role of insulin is related to the wound healing process—however, insulin itself is too sensitive to the external environment requiring the protective of a nanocarrier. Polymer-based nanoparticles can protect, deliver, and retain the protein in the target area. This study aims to produce and characterize a topical treatment for wound healing consisting of insulin-loaded poly-DL-lactide/glycolide (PLGA) nanoparticles. Insulin-loaded nanoparticles present a mean size of approximately 500 nm and neutral surface charge. Spherical shaped nanoparticles are observed by scanning electron microscopy and confirmed by atomic force microscopy. SDS-PAGE and circular dichroism analysis demonstrated that insulin preserved its integrity and secondary structure after the encapsulation process. In vitro release studies suggested a controlled release profile. Safety of the formulation was confirmed using cell lines, and cell viability was concentration and time-dependent. Preliminary safety in vivo assays also revealed promising results.

In Vivo Testing of Sericin-Polyurethane Nanofiber Mats for Wound Healing in Rats

2017 ◽  
Vol 751 ◽  
pp. 581-585 ◽  
Author(s):  
Piyaporn Kampeerapappun ◽  
Pornpen Siridamrong
Keyword(s):  
Wound Healing ◽  
Healing Process ◽  
Active Agent ◽  
L929 Cell ◽  
Wound Healing Process ◽  
Original Size ◽  
Nanofiber Mats ◽  
Dressing Materials

The objective of this study was to investigate sericin-polyurethane nanofiber cover (SUC) for wound dressing materials in a rat skin. Sericin-polyurethane blended nanofibers were fabricated by using electrospinning. The composition of 3%w/v polyurethane in ethanol and 19% w/v sericin were blended and electrospun at 15 kV, 20 cm from tip to collector with a feed rate of 6.2 ml/hr. The mats, approximately 1.5 mm thick, were sterile by gamma irradiation with a radiation dose of 15 kGy. The samples of in vitro and in vivo testing were separated into three groups; gauze, polyurethane nanofiber cover (UC), and SUC. In vitro cultured L929 cell lines were investigated with inverted microscope. It was found that cells migrated to SCU. For in vivo tests, the remaining wound in rats was measured on day 2-14 after excision. Compared to original size of wound samples, the size of the wound remained 24% for SUC, 33% for gauze, and 34% for UC at day 8. The sericin, an active agent, contained in SUC mats was about 5 µl at 1.5 ×1.5 cm. It can be concluded that sericin is non-toxic to cells and can promote wound healing process in rats.

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