scholarly journals Optical Characterization of Sodium Fluorescein In Vitro and Ex Vivo

2021 ◽  
Vol 11 ◽  
Author(s):  
Ran Xu ◽  
Wanda Teich ◽  
Florian Frenzel ◽  
Katrin Hoffmann ◽  
Josefine Radke ◽  
...  
Keyword(s):  
Optical Properties ◽  
Laser Scanning ◽  
Tumor Tissue ◽  
Ex Vivo ◽  
Bathochromic Shift ◽  
Emission Spectra ◽  
Optical Measurements ◽  
Confocal Laser ◽  
Sodium Fluorescein

ObjectiveThe utilization of fluorescein-guided biopsies and resection has been recently discussed as a suitable strategy to improve and expedite operative techniques for the resection of central nervous system (CNS) tumors. However, little is known about the optical properties of sodium fluorescein (NaFl) in human tumor tissue and their potential impact on ex vivo analyses involving fluorescence-based methods.MethodsTumor tissue was obtained from a study cohort of an observational study on the utilization of fluorescein-guided biopsy and resection (n=5). The optical properties of fluorescein-stained tissue were compared to the optical features of the dye in vitro and in control samples consisting of tumor tissue of high-grade glioma patients (n=3) without intravenous (i.v.) application of NaFl. The dye-exposed tumor tissues were used for optical measurements to confirm the detectability of NaFl emission ex vivo. The tissue samples were fixed in 4%PFA, immersed in 30% sucrose, embedded in Tissue-Tek OCT compound, and cut to 10 μm cryosections. Spatially resolved emission spectra from tumor samples were recorded on representative slides with a Confocal Laser Scanning Microscope FV1000 (Olympus GmbH, Hamburg, Germany) upon excitation with λexc = 488 nm.ResultsOptical measurements of fluorescein in 0.9% sodium chloride (NaCl) under in vitro conditions showed an absorption maximum of λmax abs = 479 nm as detected with spectrophotometer Specord 200 and an emission peak at λmax em = 538 nm recorded with the emCCD detection system of a custom-made microscope-based single particle setup using a 500 nm long-pass filter. Further measurements revealed pH- and concentration-dependent emission spectra of NaFl. Under ex vivo conditions, confocal laser scanning microscopy of fluorescein tumor samples revealed a slight bathochromic shift and a broadening of the emission band.ConclusionTumor uptake of NaFl leads to changes in the optical properties – a bathochromic shift and broadening of the emission band – possibly caused by the dye’s high pH sensitivity and concentration-dependent reabsorption acting as an inner filter of the dye’s emission, particularly in the short wavelength region of the emission spectrum where absorption and fluorescence overlap. Understanding the ex vivo optical properties of fluorescein is crucial for testing and validating its further applicability as an optical probe for intravital microscopy, immunofluorescence localization studies, and flow cytometry analysis.

scholarly journals Protein-Protein Interactions between Hepatitis C Virus Nonstructural Proteins

2003 ◽  
Vol 77 (9) ◽  
pp. 5401-5414 ◽  
Author(s):  
Maria Dimitrova ◽  
Isabelle Imbert ◽  
Marie Paule Kieny ◽  
Catherine Schuster
Keyword(s):  
Endoplasmic Reticulum ◽  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Protein Interactions ◽  
Laser Scanning ◽  
Ex Vivo ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Nonstructural Proteins

ABSTRACT Replication of the hepatitis C virus (HCV) genome has been proposed to take place close to the membrane of the endoplasmic reticulum in membrane-associated replicase complexes, as is the case with several other plus-strand RNA viruses, such as poliovirus and flaviviruses. The most obvious benefits of this property are the possibility of coupling functions residing in different polypeptidic chains and the sequestration of viral proteins and nucleic acids in a distinct cytoplasmic compartment with high local concentrations of viral components. Indeed, HCV nonstructural (NS) proteins were clearly colocalized in association with membranes derived from the endoplasmic reticulum. This observation, together with the demonstration of the existence of several physical interactions between HCV NS proteins, supports the idea of assembly of a highly ordered multisubunit protein complex(es) probably involved in the replication of the viral genome. The objective of this study, therefore, was to examine all potential interactions between HCV NS proteins which could result in the formation of a replication complex(es). We identified several interacting viral partners by using a glutathione S-transferase pull-down assay, by in vitro and ex vivo coimmunoprecipitation experiments in adenovirus-infected Huh-7 cells allowing the expression of HCV NS proteins, and, finally, by using the yeast two-hybrid system. In addition, by confocal laser scanning microscopy, NS proteins were clearly shown to colocalize when expressed together in Huh-7 cells. We have been able to demonstrate the existence of a complex network of interactions implicating all six NS proteins. Our observations confirm previously described associations and identify several novel homo- and heterodimerizations.

Dynamics of Cytokine Capture Within Hemoadsorption Beads Used to Treat Sepsis

2009 ◽  
Author(s):  
Jeremy D. Kimmel ◽  
Morgan V. DiLeo ◽  
Isabella E. Valenti ◽  
Gregory A. Gibson ◽  
Simon C. Watkins ◽  
...  
Keyword(s):  
Inflammatory Cytokines ◽  
Laser Scanning ◽  
Medical Condition ◽  
Ex Vivo ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Adsorption Dynamics ◽  
The Us ◽  
Rapid Clearance

Sepsis is a serious medical condition characterized by systemic inflammation caused by infection, and affects more than 750,000 individuals per year in the US, with a mortality rate of approximately 30% [1]. The pathophysiology of sepsis is complex and not entirely understood, but is believed to be related to the dysfunction of multiple interdependent humoral mediator pathways, including redundant release of inflammatory cytokines [2]. Removal of both pro- and anti-inflammatory cytokines from the circulating blood is believed to be a promising therapy for severe sepsis [3]. We are developing an extracorporeal hemoadsorption device to remove cytokines from the blood using a novel, biocompatible, sorbent bead technology. A simple model was developed to characterize cytokine adsorption within hemoadsorption beads [4]. Despite rapid clearance of cytokines with hemoadsorption in an ex vivo murine sepsis model [5], our model analysis predicted that only the outer 20μm of each sorbent bead (avg diam = 450μm) adsorbed cytokine. In this work, we used in vitro column capture experiments and confocal laser scanning microscopy (CLSM) to examine cytokine adsorption dynamics within hemoadsorption beads.

scholarly journals Machine Learning Based Prediction of Squamous Cell Carcinoma in Ex Vivo Confocal Laser Scanning Microscopy

Cancers ◽  
2021 ◽  
Vol 13 (21) ◽  
pp. 5522
Author(s):  
Cristel Ruini ◽  
Sophia Schlingmann ◽  
Žan Jonke ◽  
Pinar Avci ◽  
Víctor Padrón-Laso ◽  
...  
Keyword(s):  
Machine Learning ◽  
Squamous Cell Carcinoma ◽  
Deep Learning ◽  
Cell Carcinoma ◽  
Laser Scanning ◽  
Tumor Tissue ◽  
Ex Vivo ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Tissue Samples

Image classification with convolutional neural networks (CNN) offers an unprecedented opportunity to medical imaging. Regulatory agencies in the USA and Europe have already cleared numerous deep learning/machine learning based medical devices and algorithms. While the field of radiology is on the forefront of artificial intelligence (AI) revolution, conventional pathology, which commonly relies on examination of tissue samples on a glass slide, is falling behind in leveraging this technology. On the other hand, ex vivo confocal laser scanning microscopy (ex vivo CLSM), owing to its digital workflow features, has a high potential to benefit from integrating AI tools into the assessment and decision-making process. Aim of this work was to explore a preliminary application of CNN in digitally stained ex vivo CLSM images of cutaneous squamous cell carcinoma (cSCC) for automated detection of tumor tissue. Thirty-four freshly excised tissue samples were prospectively collected and examined immediately after resection. After the histologically confirmed ex vivo CLSM diagnosis, the tumor tissue was annotated for segmentation by experts, in order to train the MobileNet CNN. The model was then trained and evaluated using cross validation. The overall sensitivity and specificity of the deep neural network for detecting cSCC and tumor free areas on ex vivo CLSM slides compared to expert evaluation were 0.76 and 0.91, respectively. The area under the ROC curve was equal to 0.90 and the area under the precision-recall curve was 0.85. The results demonstrate a high potential of deep learning models to detect cSCC regions on digitally stained ex vivo CLSM slides and to distinguish them from tumor-free skin.

scholarly journals Design and Engineering of “Green” Nanoemulsions for Enhanced Topical Delivery of Bakuchiol Achieved in a Sustainable Manner: A Novel Eco-Friendly Approach to Bioretinol

2021 ◽  
Vol 22 (18) ◽  
pp. 10091
Author(s):  
Agnieszka Lewińska ◽  
Marta Domżał-Kędzia ◽  
Ewa Maciejczyk ◽  
Marcin Łukaszewicz ◽  
Urszula Bazylińska
Keyword(s):  
Human Skin ◽  
Laser Scanning ◽  
Ex Vivo ◽  
Rapeseed Meal ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Biological Investigation ◽  
Vis Spectroscopy

In the present work, we establish novel “environmentally-friendly” oil-in-water nanoemulsions to enhance the transdermal delivery of bakuchiol, the so-called “bioretinol” obtained from powdered Psoralea corylifolia seeds via a sustainable process, i.e., using a supercritical fluid extraction approach with pure carbon dioxide (SC-CO2). According to Green Chemistry principles, five novel formulations were stabilized by “green” hybrid ionic surfactants such as coco-betaine—surfactin molecules obtained from coconut and fermented rapeseed meal. Preliminary optimization studies involving three dispersion stability tests, i.e., centrifugation, heating, and cooling cycles, indicated the most promising candidates for further physicochemical analysis. Finally, nanoemulsion colloidal characterization provided by scattering (dynamic and electrophoretic light scattering as well as backscattering), microscopic (transmission electron and confocal laser scanning microscopy), and spectroscopic (UV–Vis spectroscopy) methods revealed the most stable nanocarrier for transdermal biological investigation. In vitro, topical experiments provided on human skin cell line HaCaT keratinocytes and normal dermal NHDF fibroblasts indicated high cell viability upon treatment of the tested formulation with a final 0.02–0.2 mg/mL bakuchiol concentration. This excellent biocompatibility was confirmed by ex vivo and in vivo tests on animal and human skin tissue. The improved permeability and antiaging potential of the bakuchiol-encapsulated rich extract were observed, indicating that the obtained ecological nanoemulsions are competitive with commercial retinol formulations.

scholarly journals Effect of mRNA Delivery Modality and Formulation on Cutaneous mRNA Distribution and Downstream eGFP Expression

Pharmaceutics ◽  
2022 ◽  
Vol 14 (1) ◽  
pp. 151
Author(s):  
Aditya R. Darade ◽  
Maria Lapteva ◽  
Thomas Hoffmann ◽  
Markus Mandler ◽  
Achim Schneeberger ◽  
...  
Keyword(s):  
Laser Scanning ◽  
Fluorescent Protein ◽  
Ex Vivo ◽  
Intradermal Injection ◽  
Laser Scanning Microscopy ◽  
Egfp Expression ◽  
Confocal Laser ◽  
Skin Sample ◽  
Messenger Ribonucleic Acid

In vitro transcribed messenger ribonucleic acid (mRNA) constitutes an emerging therapeutic class with several clinical applications. This study presents a systematic comparison of different technologies—intradermal injection, microneedle injection, jet injection, and fractional laser ablation—for the topical cutaneous delivery of mRNA. Delivery of Cy5 labeled mRNA and non-labeled enhanced green fluorescent protein (eGFP) expressing mRNA was investigated in a viable ex vivo porcine skin model and monitored for 48 h. Forty 10 µm-thick horizontal sections were prepared from each skin sample and Cy5 labeled mRNA or eGFP expression visualized as a function of depth by confocal laser scanning microscopy and immunohistochemistry. A pixel-based method was used to create a semi-quantitative biodistribution profile. Different spatial distributions of Cy5 labeled mRNA and eGFP expression were observed, depending on the delivery modality; localization of eGFP expression pointed to the cells responsible. Delivery efficiencies and knowledge of delivery sites can facilitate development of efficient, targeted mRNA-based therapeutics.

scholarly journals Skin Targeting of An Optimized Caffeine Nanostructured Lipid Carrier With Improved Efficiency Against Chemotherapy Induced Alopecia

2021 ◽  
Author(s):  
Amna Makky ◽  
Eman Sadddar ◽  
Doaa galaa ◽  
Abeer Khattab
Keyword(s):  
Particle Size ◽  
Laser Scanning ◽  
Ex Vivo ◽  
Entrapment Efficiency ◽  
Tween 80 ◽  
Stability Study ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Nanostructured Lipid Carrier

Abstract The current investigation was designed to develop and optimize caffeine-loaded nanostructured lipid carriers (NLCs) for topical alopecia treatment. Screening of drug solubility in various excipients was executed. The 23 full factorial design was employed for NLCs optimization. Lipid type, surfactant type, and drug concentration were the independent variables. Entrapment efficiency (EE), particle size, polydispersity index (PDI) and % drug release were the chosen responses. Physiochemical evaluation, in vitro release, ex-vivo permeation, and stability study were achieved. The solubility of caffeine in stearic acid and glyceryl monostearate (GMS) was 47.11 ± 3.048 and 32.67 ± 2.955 mg/g, respectively. Oleic acid: garlic oil mixture at ratio 1:1 v/v was the oily phase. Tween 80 and Cremophor EL, Transcutol HP, carbonate buffer (pH 10.8 and ionic strength 200Mm) were chosen as a surfactant, co-surfactant, and aqueous phase, respectively. The optimized formula showed particle size, %EE, PDI, zeta potential of 358nm, 72.55 %, 0.912, -24.8, respectively. The % release was 92.9 ± 4.9 % after 4hours. Confocal laser scanning microscopy showed an improved permeation of caffeine-loaded NLCs to the whole skin layers. The histological examination proved the efficiency of caffeine NLCs optimized formula on promoting hair growth compared to the market formula.

Vacuoles Induced in Mammalian Cells by Helicobacter Cytotoxin are Acidic Organelles

1990 ◽  
Vol 48 (3) ◽  
pp. 168-169
Author(s):  
M. H. Chestnut ◽  
C. E. Catrenich
Keyword(s):  
Acridine Orange ◽  
Mammalian Cells ◽  
Laser Scanning ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Ulcer Disease ◽  
Gastroduodenal Disease ◽  
H Pylori

Helicobacter pylori is a non-invasive, Gram-negative spiral bacterium first identified in 1983, and subsequently implicated in the pathogenesis of gastroduodenal disease including gastritis and peptic ulcer disease. Cytotoxic activity, manifested by intracytoplasmic vacuolation of mammalian cells in vitro, was identified in 55% of H. pylori strains examined. The vacuoles increase in number and size during extended incubation, resulting in vacuolar and cellular degeneration after 24 h to 48 h. Vacuolation of gastric epithelial cells is also observed in vivo during infection by H. pylori. A high molecular weight, heat labile protein is believed to be responsible for vacuolation and to significantly contribute to the development of gastroduodenal disease in humans. The mechanism by which the cytotoxin exerts its effect is unknown, as is the intracellular origin of the vacuolar membrane and contents. Acridine orange is a membrane-permeant weak base that initially accumulates in low-pH compartments. We have used acridine orange accumulation in conjunction with confocal laser scanning microscopy of toxin-treated cells to begin probing the nature and origin of these vacuoles.

The Development of Pemetrexed Diacid-Loaded Gelatin-Cloisite 30B (MMT) Nanocomposite for Improved Oral Efficacy Against Cancer: Characterization, In-Vitro and Ex-Vivo Assessment

2020 ◽  
Vol 17 (3) ◽  
pp. 246-256
Author(s):  
Kriti Soni ◽  
Ali Mujtaba ◽  
Md. Habban Akhter ◽  
Kanchan Kohli
Keyword(s):  
Drug Release ◽  
Oral Delivery ◽  
Ex Vivo ◽  
Confocal Laser ◽  
Release Mechanism ◽  
Scanning Calorimetry ◽  
Sem Images ◽  
Cloisite 30B ◽  
Ft Ir

Aim: The intention of this investigation was to develop Pemetrexed Diacid (PTX)-loaded gelatine-cloisite 30B (MMT) nanocomposite for the potential oral delivery of PTX and the in vitro, and ex vivo assessment. Background: Gelatin/Cloisite 30 B (MMT) nanocomposites were prepared by blending gelatin with MMT in aqueous solution. Methods: PTX was incorporated into the nanocomposite preparation. The nanocomposites were investigated by Fourier Transmission Infra Red Spectroscopy (FT-IR), Differential Scanning Calorimetry (DSC), Scanning Electron Microscope (SEM) X-Ray Diffraction (XRD) and Confocal Laser Microscopy (CLSM). FT-IR of nanocomposite showed the disappearance of all major peaks which corroborated the formation of nanocomposites. The nanocomposites were found to have a particle size of 121.9 ± 1.85 nm and zeta potential -12.1 ± 0.63 mV. DSC thermogram of drug loaded nanocomposites indicated peak at 117.165 oC and 205.816 oC, which clearly revealed that the drug has been incorporated into the nanocomposite because of cross-linking of cloisite 30 B and gelatin in the presence of glutaraldehyde. Results: SEM images of gelatin show a network like structure which disappears in the nanocomposite. The kinetics of the drug release was studied in order to ascertain the type of release mechanism. The drug release from nanocomposites was in a controlled manner, followed by first-order kinetics and the drug release mechanism was found to be of Fickian type. Conclusion: Ex vivo gut permeation studies revealed 4 times enhancement in the permeation of drug present in the nanocomposite as compared to plain drug solution and were further affirmed by CLSM. Thus, gelatin/(MMT) nanocomposite could be promising for the oral delivery of PTX in cancer therapy and future prospects for the industrial pharmacy.

scholarly journals Effect of dissolution rate and subsequent ion release on cytocompatibility properties of borophosphate glasses

2019 ◽  
Vol 5 (1) ◽  
pp. 85-97
Author(s):  
Nusrat Sharmin ◽  
Mohammad S. Hasan ◽  
Md. Towhidul Islam ◽  
Chengheng Pang ◽  
Fu Gu ◽  
...  
Keyword(s):  
Dissolution Rate ◽  
Laser Scanning ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Ion Release ◽  
Scanning Calorimetry ◽  
Cell Culture Study ◽  
Borophosphate Glasses ◽  
Mg63 Cells

AbstractPresent work explores the relationship between the composition, dissolution rate, ion release and cytocompatibility of a series of borophosphate glasses. While, the base glass was selected to be 40mol%P2O5-16mol%CaO-24mol%MgO-20mol%Na2O, three B2O3 modified glass compositions were formulated by replacing Na2O with 1, 5 and 10 mol% B2O3. Ion release study was conducted using inductively coupled plasma atomic emission spectroscopy (ICP-AES). The thermal scans of the glasses as determined by differential scanning calorimetry (DSC) revealed an increment in the thermal properties with increasing B2O3 content in the glasses. On the other hand, the dissolution rate of the glasses decreased with increasing B2O3 content. To identify the effect of boron ion release on the cytocompatibility properties of the glasses, MG63 cells were cultured on the surface of the glass discs. The in vitro cell culture study suggested that glasses with 5 mol% B2O3 (P40B5) showed better cell proliferation and metabolic activity as compares to the glasses with 10 mol% (P40B10) or with no B2O3 (P40B0). The confocal laser scanning microscopy (CLSM) images of live/dead stained MG63 cells attached to the surface of the glasses also revealed that the number of dead cells attached to P40B5 glasses were significantly lower than both P40B0 and P40B10 glasses.

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