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1.
Chinese Journal of Biotechnology ; (12): 620-631, 2022.
Article in Chinese | WPRIM | ID: wpr-927732

ABSTRACT

Genetic code expansion (GCE) allows the incorporation of unnatural amino acids into proteins via using stop codons. GCE may achieve site-specific labeling of proteins in combination with the click reaction. Compared with other labeling tools such as fluorescent proteins and tagged antibodies, the compound molecules used in protein labeling by GCE technology are smaller, and therefore, may less interfere the conformational structure of proteins. In addition, through click reaction, GCE allows a 1:1 stoichiometric ratio of the target protein molecule and the fluorescent dye, and the protein can be quantified based on the fluorescence intensity. Thus, GCE technology has great advantages in the researches that require the exposition of living cells under high laser power for longer time, for example, in the context of single molecule tracing and super-resolution microscopic imaging. Meanwhile, this technology lays the foundation for improving the accuracy of positioning and molecule counting in the imaging process of living cells. This review summarized the GCE technology and its recent applications in functionally characterizing, labeling and imaging of proteins.


Subject(s)
Amino Acids/chemistry , Fluorescent Dyes/chemistry , Genetic Code , Proteins/chemistry
2.
Chinese Journal of Biotechnology ; (12): 2678-2687, 2021.
Article in Chinese | WPRIM | ID: wpr-887833

ABSTRACT

Fluorescence imaging has been widely used in the fields of biomedicine and clinical diagnosis. Compared with traditional fluorescence imaging in the visible spectral region (400-760 nm), near-infrared (NIR, 700-1 700 nm) fluorescence imaging is more helpful to improve the signal-to-noise ratio and the sensitivity of imaging. Highly-sensitive fluorescent probes are required for high-quality fluorescence imaging, and the rapid development of nanotechnology has led to the emergence of organic dyes with excellent fluorescent properties. Among them, organic fluorescent probes with the advantages of high safety, good biocompatibility, and high optical stability, are more favorable than inorganic fluorescent probes. Therefore, NIR fluorescence imaging assisted with organic fluorescent probes can provide more structural and dynamic information of biological samples to the researchers, which becomes a hot spot in the interdisciplinary research field of optics, chemistry and biomedicine. This review summarizes the application of NIR organic fluorescent probes in cervical cancer imaging. Several typical organic fluorescent probes (such as indocyanine green, heptamethine cyanine dye, rhodamine and polymer fluorescent nanoparticles) assisted NIR fluorescence imaging and their applications in cervical cancer diagnosis were introduced, and the future development and application of these techniques were discussed.


Subject(s)
Female , Fluorescent Dyes , Humans , Nanoparticles , Optical Imaging , Polymers , Uterine Cervical Neoplasms/diagnostic imaging
4.
Article in Chinese | WPRIM | ID: wpr-774190

ABSTRACT

The convective polymerase chain reaction (CCPCR) uses the principle of thermal convection to allow the reagent to flow in the test tube and achieve the purpose of amplification by the temperature difference between the upper and lower portions of the test tube. In order to detect the amplification effect in real time, we added a fluorophore to the reagent system to reflect the amplification in real time through the intensity of fluorescence. The experimental results show that the fluorescence curve conforms to the S-type trend of the amplification curve, but there is a certain jitter condition due to the instability of the thermal convection, which is not conducive to the calculation of the cycle threshold (CT value). In order to solve this problem, this paper uses the dynamic method, using the double S-type function model to fit the curve, so that the fluorescence curve is smooth and the initial concentration of the nucleic acid can be deduced better to achieve the quantitative purpose based on the curve. At the same time, the PSO+ algorithm is used to solve the double s-type function parameters, that is, particle swarm optimization (PSO) algorithm combined with Levenberg-Marquardt, Newton-CG and other algorithms for curve fitting. The proposed method effectively overcoms PSO randomness and the shortcoming of traditional algorithms such as Levenberg-Marquardt and Newton-CG which are easy to fall into the local optimal solution. The of the data fitting result can reach 0.999 8. This study is of guiding significance for the future quantitative detection of real-time fluorescent heat convection amplification.


Subject(s)
Algorithms , Fluorescence , Fluorescent Dyes , Polymerase Chain Reaction
5.
Article in English | WPRIM | ID: wpr-764067

ABSTRACT

Glutathione (GSH) is a major antioxidant in cells, and plays vital roles in the cellular defense against oxidants and in the regulation of redox signals. In a previous report, we demonstrated that stem cell function is critically affected by heterogeneity and dynamic changes in cellular GSH concentration. Here, we present a detailed protocol for the monitoring of GSH concentration in living stem cells using FreSHtracer, a real-time GSH probe. We describe the steps involved in monitoring GSH concentration in single living stem cells using confocal microscopy and flow cytometry. These methods are simple, rapid, and quantitative, and able to demonstrate intracellular GSH concentration changes in real time. We also describe the application of FreSHtracer to the sorting of stem cells according to their GSH content using flow cytometry. Typically, microscopic or flow cytometric analyses of FreSHtracer and MitoFreSHtracer signals in living stem cells take ~2~3 h, and the fractionation of stem cells into subpopulations on the basis of cellular GSH levels takes 3~4.5 h. This method could be applied to almost every kind of mammalian cell with minor modifications to the protocol described here.


Subject(s)
Flow Cytometry , Fluorescent Dyes , Glutathione , Methods , Microscopy, Confocal , Oxidants , Oxidation-Reduction , Population Characteristics , Stem Cells
6.
Rev. Ciênc. Méd. Biol. (Impr.) ; 17(3): 359-368, nov 19, 2018. ilus
Article in Portuguese | LILACS | ID: biblio-1247808

ABSTRACT

Introdução: a regeneração e o reparo de tecidos ósseos perdidos é objeto de estudo da Bioengenharia Tecidual. O uso de biomateriais substitutos ósseos biomiméticos visa estimular os sistemas celulares e bioquímicos para restabelecer de modo mais eficiente o tecido ósseo nos casos de sua reconstrução. Ao investigar o processo de remodelação, é vital identificar áreas de novo crescimento para avaliar a eficácia dos biomateriais implantados e respectivos regimes de tratamento. A avaliação qualitativa e quantitativa da regeneração óssea pode ser realizada através da aplicação de marcadores como o Xilenol, a Tetraciclina, a Calceína e a Alizarina. A administração desses marcadores de forma associada possibilita ainda marcar sequencialmente camadas de nova deposição e remodelação durante o reparo. Objetivo: estabelecer um protocolo para utilização dos marcadores fluorescentes de reparo ósseo xilenol, tetraciclina, calceína e alizarina, em ratos. Metodologia: foram utilizados 35 ratos da linhagem Wistar, machos adultos, com massa corpórea entre 350 e 400g, e idade aproximada de 4 a 5 meses, distribuídos randomicamente em 5 grupos experimentais, submetidos à confecção de defeito ósseo circular de 8 mm em região de calvária, e administração dos diferentes marcadores segundo os grupos; XO ­ Xilenol; Ca ­ Calceína; Al ­ Alizarina; Te ­ Tetraciclina; C ­ Controle. Após 15 dias de experimento, os animais foram eutanasiados e as calvárias processadas e analisadas por histomorfometria, microscopia de epifluorescência e microscopia de fluorescência. Resultados: todos protocolos empregados para utilização dos marcadores fluorescentes xilenol, calceína, alizarina e tetracicilina foram úteis para identificar área de deposição mineral durante o período analisado de regeneração óssea em ratos. As imagens obtidas pela microscopia de fluorescência revela a presença dos marcadores incorporados à matriz óssea neoformada, no entanto a utilização da Alizarina e Calceína dentro dos protocolos testados mostraram-se mais eficientes. Conclusão: os protocolos testados nesse estudo apresentaram-se viáveis para utilização em pesquisas envolvendo marcadores de regeneração óssea, com resultados superiores para Alizarina e Calceína


Introduction: The regeneration and repair of lost bone tissues is the subject of a study of Tissue Bioengineering. The use of biomimetic biomaterial bone substitutes aims to stimulate the cellular and biochemical systems to restore more efficiently the bone tissue in the cases of its reconstruction. When investigating the remodeling process, it is vital to identify areas of new growth to evaluate the efficacy of implanted biomaterials and their treatment regimens. The qualitative and quantitative evaluation of bone regeneration can be performed through the use of markers such as Xylenol, Tetracycline, Calcein and Alizarin. The administration of such markers in an associated manner also makes it possible to sequentially mark layers of new deposition and remodeling during the repair. Objective: to establish a protocol for the use of fluorescent xylenol, tetracycline, calcein and alizarin bone repair markers in rats. Metodology: thirtyfive male adult Wistar rats with a body mass ranging from 350 to 400 g and approximately 4 to 5 months old were randomly assigned to 5 experimental groups submitted to a circular bone defect of 8 mm in the region of calvaria, and administration of the different markers according to the groups; XO ­ Xylenol; Ca ­ Calcein; Al-Alizarin; Te ­ Tetracycline; C ­ Control. After 15 days of experiment, the animals were euthanized and the calvaria processed and analyzed by histomorphometry, epifluorescence microscopy and fluorescence microscopy. Results: all protocols used for fluorescence markers xylenol, calcein, alizarin and tetracycline were useful to identify area of mineral deposition during the analyzed period of bone regeneration in rats. The images obtained by fluorescence microscopy revealed the presence of the markers incorporated into the neoformed bone matrix, however the use of Alizarin and Calcein within the protocols tested were more efficient. Conclusion: the protocols tested in this study were feasible for use in research involving markers of bone regeneration, with superior results for Alizarin and Calcein.


Subject(s)
Animals , Male , Rats , Bone Regeneration/drug effects , Tissue Engineering/methods , Fluorescent Dyes/pharmacology , Tetracycline/pharmacology , Xylenes/pharmacology , Random Allocation , Pilot Projects , Rats, Wistar , Disease Models, Animal , Microscopy, Fluorescence
7.
Braz. dent. j ; 29(4): 325-334, July-Aug. 2018. tab, graf
Article in English | LILACS | ID: biblio-974170

ABSTRACT

Abstract Several techniques have been proposed for vertical bone regeneration, and many of them use bone autogenous and allogeneic grafts. The purpose of this study was to compare demineralised freeze-dried bone allografts (DFDBA), fresh-frozen (FF) allografts, autogenous bone grafts to find differences between volumetric and histological quantity of bone formation and vertical bone growth dynamic. A vertical tissue regeneration bone model was performed in rabbit calvarias under general anaesthesia. Four hollow cylinders of pure titanium were screwed onto external cortical bone calvarias in eight rabbits. Each one of the cylinders was randomly filled with one intervention: DFDBA, FF, autogenous bone, or left to be filled with blood clot (BC) as control. Allogeneic grafts were obtained from a ninth animal following international standardised protocols for the harvesting, processing, and cryopreservation of allografts. Autogenous graft was obtained from the host femur scraping before adapting hollow cylinders. Animals were euthanized at 13 weeks. Vertical volume was calculated after probe device measurements of the new formed tissue inside the cylinders and after titanium cylinders were removed. Histomorphometry and fluorochrome staining were used to analyse quantity and dynamic of bone formation, respectively. Results showed that DFDBA and fresh-frozen bone improved the velocity and the quantity of bone deposition in distant portions of the basal plane of grafting. Remaining material in allograft groups was more intense than in autogenous group. Both allografts can be indicated as reliable alternatives for volume gain and vertical bone augmentation.


Resumo Várias técnicas foram propostas para regeneração óssea vertical, e muitas delas usam enxertos ósseos e alogênicos ósseos. O objetivo deste estudo foi comparar os aloenxertos ósseos congelados desmineralizados (DFDBA), os aloenxertos congelados frescos (FF) com os enxertos ósseos autógenos para encontrar diferenças entre o volume, a histologia da formação óssea e a dinâmica do crescimento ósseo vertical. Um modelo ósseo de regeneração tecidual vertical foi realizado em calvarias de coelho sob anestesia geral. Quatro cilindros ocos de titânio puro foram parafusados nas calvarias de osso cortical externo em oito coelhos. Cada um dos cilindros foi preenchido aleatoriamente com uma intervenção: DFDBA, FF, osso autógeno ou com coágulo sanguíneo (BC) como controle. Os enxertos alogênicos foram obtidos a partir de um nono animal seguindo protocolos internacionais padronizados para a coleta, processamento e criopreservação de aloenxertos. O enxerto autógeno foi obtido da raspagem do fêmur do hospedeiro antes de adaptar os cilindros ocos. Os animais foram eutanasiados após 13 semanas. O volume vertical foi calculado após a medição, por meio de sonda milimetrada, do novo tecido formado dentro dos cilindros e após a remoção dos cilindros de titânio. Histomorfometria e coloração com fluorocromios foram utilizados para analisar a quantidade e a dinâmica da formação óssea. Os resultados mostraram que DFDBA e osso fresco congelado melhoraram a velocidade e a quantidade de deposição óssea em porções distantes do plano basal de enxerto. O material remanescente nos grupos de aloenxerto foi mais intenso do que em grupo autógeno. Ambos os aloenxertos podem ser indicados como alternativas confiáveis para ganho de volume e aumento ósseo vertical.


Subject(s)
Animals , Male , Rabbits , Bone Regeneration , Bone Transplantation/methods , Models, Biological , Transplantation, Autologous , Transplantation, Homologous , Bone Screws , Fluorescent Dyes/chemistry , Freeze Drying , Microscopy, Fluorescence
8.
J. appl. oral sci ; 26: e20170470, 2018. graf
Article in English | LILACS, BBO | ID: biblio-954503

ABSTRACT

Abstract The hypothesis of this study was that the peri-implant bone healing of the group of pinealectomized rats would differ from the control group. The samples were subjected to immunohistochemical, microtomographic (total porosity and connectivity density), and fluorochrome (mineralized surface) analyses. Objectives The goal of this study was to investigate the cellular changes and bone remodeling dynamics along the bone/implant interface in pinealectomized rats. Material and Methods The total of 18 adult male rats (Rattus norvegicus albinus, Wistar) was divided into three groups (n=6): control (CO), pinealectomized without melatonin (PNX) and pinealectomized with melatonin (PNXm). All animals were submitted to the first surgery (pinealectomy), except the CO group. Thirty days after the pinealectomy without melatonin, the second surgery was conducted, in which all animals received an implant in each tibia (36 titanium implants with surface treatment were installed - Implalife® São Paulo, SP, Brazil). By gavage, the rats of the PNX group received the vehicle solution, and the procedure. Results Immunohistochemical analysis for runt-related transcription factor 2 (RUNX2), alkaline phosphatase (ALP), osteopontin (OP) and osteocalcin (OC) showed that the bone repair process in the PNXm group was similar to that of the CO group, whereas the PNX group showed a delay. The microtomographic parameters of total porosity [Po(tot)] and bone surface (BS) showed no statistically significant differences, whereas for the connective density (Conn.Dn) a statistical difference was found between the CO and PNXm groups. Fluorochrome analysis of the active mineralized surface showed statistically significant difference between the CO and PNX and between the CO and PNXm groups. Conclusion The absence of the pineal gland impaired the bone repair process during osseointegration, however the daily melatonin replacement was able to restore this response.


Subject(s)
Animals , Male , Pineal Gland/surgery , Osseointegration/drug effects , Bone Density Conservation Agents/pharmacology , Bone-Implant Interface , Melatonin/pharmacology , Tibia/drug effects , Tibia/injuries , Tibia/pathology , Titanium , Immunohistochemistry , Osteocalcin/analysis , Reproducibility of Results , Treatment Outcome , Rats, Wistar , Implants, Experimental , Dental Implantation, Endosseous , Alkaline Phosphatase/analysis , Core Binding Factor Alpha 1 Subunit/analysis , Osteopontin/analysis , X-Ray Microtomography , Fluorescent Dyes
9.
Article in English | WPRIM | ID: wpr-742282

ABSTRACT

This study aimed to develop a new multiplex real-time PCR detection method for 3 species of waterborne protozoan parasites (Cryptosporidium parvum, Giardia lamblia, and Cyclospora cayetanensis) identified as major causes of traveler's diarrhea. Three target genes were specifically and simultaneously detected by the TaqMan probe method for multiple parasitic infection cases, including Cryptosporidium oocyst wall protein for C. parvum, glutamate dehydrogenase for G. lamblia, and internal transcribed spacer 1 for C. cayetanensis. Gene product 21 for bacteriophage T4 was used as an internal control DNA target for monitoring human stool DNA amplification. TaqMan probes were prepared using 4 fluorescent dyes, FAM™, HEX™, Cy5™, and CAL Fluor Red® 610 on C. parvum, G. lamblia, C. cayetanensis, and bacteriophage T4, respectively. We developed a novel primer-probe set for each parasite, a primer-probe cocktail (a mixture of primers and probes for the parasites and the internal control) for multiplex real-time PCR analysis, and a protocol for this detection method. Multiplex real-time PCR with the primer-probe cocktail successfully and specifically detected the target genes of C. parvum, G. lamblia, and C. cayetanensis in the mixed spiked human stool sample. The limit of detection for our assay was 2×10 copies for C. parvum and for C. cayetanensis, while it was 2×10³ copies for G. lamblia. We propose that the multiplex real-time PCR detection method developed here is a useful method for simultaneously diagnosing the most common causative protozoa in traveler's diarrhea.


Subject(s)
Bacteriophage T4 , Cryptosporidium parvum , Cryptosporidium , Cyclospora , Diagnosis , Diarrhea , DNA , Fluorescent Dyes , Giardia lamblia , Giardia , Glutamate Dehydrogenase , Humans , Limit of Detection , Methods , Multiplex Polymerase Chain Reaction , Oocysts , Parasites , Real-Time Polymerase Chain Reaction
10.
Article in English | WPRIM | ID: wpr-713082

ABSTRACT

ICG is widely applied in real-time imaging during abdominal surgery, plastic surgery, as well as oncologic staging and treatment. A twenty-eight year-old female patient was found to have a 4.5 cm solid pseudopapillary neoplasm in the tail of the pancreas. Under ICG-fluorescent pancreatic perfusion-guidance, we easily defined the margin of the pancreatic tumor and secured the resection margin when performing laparoscopic distal pancreatosplenectomy in the patient. No clinically relevant complications, including postoperative pancreatic fistula, were noted. Intravenous ICG can be very easily and quickly detected in the pancreas under near infrared light. This enhanced vision gives strong contrast to the organ compared to a necrotic tumor with poor blood perfusion, such as solid pseudopapillary neoplasm. Based on our current experience, ICG pancreatic perfusion-guided determination of appropriate resection margin is useful and feasible during pancreaticoduodenectomy.


Subject(s)
Female , Fluorescent Dyes , Humans , Indocyanine Green , Pancreas , Pancreatectomy , Pancreatic Fistula , Pancreaticoduodenectomy , Perfusion , Surgery, Plastic , Tail
11.
Acta cir. bras ; 32(6): 440-448, June 2017. tab, graf
Article in English | LILACS | ID: biblio-886209

ABSTRACT

Abstract Purpose: To investigate if fluorescein fluorescent test can predict dehiscence in a model of ischemic colonic anastomosis in rats. Methods: This experimental controlled trial randomly assigned 55 rats to four groups. Anastomoses were performed in non-ischemic colon segments (control group) and in ischemic colon segments measuring 1, 2 or 3 cm long (groups 1, 2 and 3, respectively). Fluorescein was injected and the tissues were examined under ultraviolet light. Seven days later, a second-look surgery was performed to check for the presence or absence of anastomosis dehiscence. Results: Twenty-four rats presented anastomotic dehiscence during the second-look surgery. Reticular and nonfluorescent patterns were significantly associated with the occurrence of anastomotic dehiscence. Fluorescein fluorescence had a sensitivity of 95.8%, specificity of 89.2%, positive predictive value of 88.4%, negative predictive value of 96.2%, and accuracy of 92.3% to predict anastomotic dehiscence. Conclusion: Fluorescein fluorescent test can accurately predict leak in a model of ischemic colonic anastomosis in rats.


Subject(s)
Animals , Male , Rats , Surgical Wound Dehiscence/diagnosis , Anastomosis, Surgical , Colon/surgery , Fluorescein , Fluorescent Dyes , Ischemia/surgery , Wound Healing , Colon/blood supply , Colon/pathology
12.
Mem. Inst. Oswaldo Cruz ; 112(2): 140-145, Feb. 2017. graf
Article in English | LILACS | ID: biblio-841762

ABSTRACT

BACKGROUND Fluorescence in situ hybridisation (FISH) associated with Tyramide Signal Amplification (TSA) using oligonucleotides labeled with non-radioactive fluorophores is a promising technique for detection and differentiation of fungal species in environmental or clinical samples, being suitable for microorganisms which are difficult or even impossible to culture. OBJECTIVE In this study, we aimed to standardise an in situ hybridisation technique for the differentiation between the pathogenic species Paracoccidioides brasiliensis and Paracoccidioides lutzii, by using species-specific DNA probes targeting the internal transcribed spacer-1 (ITS-1) of the rRNA gene. METHODS Yeast and mycelial phase of each Paracoccidioides species, were tested by two different detection/differentiation techniques: TSA-FISH for P. brasiliensis with HRP (Horseradish Peroxidase) linked to the probe 5’ end; and FISH for P. lutzii with the fluorophore TEXAS RED-X® also linked to the probe 5’ end. After testing different protocols, the optimised procedure for both techniques was accomplished without cross-positivity with other pathogenic fungi. FINDINGS The in silico and in vitro tests show no reaction with controls, like Candida and Cryptococcus (in silico) and Histoplasma capsulatum and Aspergillus spp. (in vitro). For both phases (mycelial and yeast) the in situ hybridisation showed dots of hybridisation, with no cross-reaction between them, with a lower signal for Texas Red probe than HRP-TSA probe. The dots of hybridisation was confirmed with genetic material marked with 4’,6-diamidino-2-phenylindole (DAPI), visualised in a different filter (WU) on fluorescent microscopic. MAIN CONCLUSION Our results indicated that TSA-FISH and/or FISH are suitable for in situ detection and differentiation of Paracoccidioides species. This approach has the potential for future application in clinical samples for the improvement of paracoccidioidomycosis patients prognosis.


Subject(s)
Paracoccidioides/classification , Paracoccidioides/genetics , DNA, Fungal , DNA, Ribosomal Spacer , Species Specificity , Oligonucleotide Probes , In Situ Hybridization, Fluorescence , Fluorescence , Fluorescent Dyes
13.
Bauru; s.n; 2017. 143 p. tab, ilus, graf.
Thesis in Portuguese | LILACS, BBO | ID: biblio-880165

ABSTRACT

A adição de corantes fluorescentes a adesivos odontológicos possibilita a investigação da distribuição espacial desses materiais na interface dente-restauração, utilizando-se a microscopia confocal de varredura a laser (MCVL). A literatura indica falta de padronização na aplicação de agentes fluorescentes com tal finalidade. Esse estudo sistematizou estratégias para a adição de rodamina B (RB) e fluoresceína sódica (FS) a um sistema adesivo convencional de três passos, Adper Scotchbond Multi-Purpose (MP), e um autocondicionante de dois passos, Clearfil SE Bond (SE), considerados "padrão-ouro" na Odontologia. Os objetivos principais foram (a) determinar a menor faixa de concentrações de RB e FS necessária para produzir imagens satisfatórias da interface dentina-adesivo e (b) avaliar o efeito da adição desses corantes sobre algumas propriedades das resinas. Os adesivos foram marcados com RB ou FS em concentrações decrescentes (0,5, 0,1, 0,02 e 0,004 mg/mL) por meio de um método de dispersão semidireto. O comportamento fotofísico/ fluorescente dos adesivos marcados foi investigado por espectroscopia de fotoluminescência e MCVL. Paralelamente, avaliaram-se os adesivos quanto ao grau de conversão (GC) e ao ângulo de contato (AC). Tanto os resultados de GC como os de AC foram submetidos à análise de variância com dois fatores (adesivo e tratamento) com α = 0,05, seguida de teste post-hoc de Tukey. Os máximos comprimentos de onda de emissão e de excitação da RB e da FS foram influenciados pelo meio polimérico e pela concentração de corante de modo geral. A MCVL preliminar de amostras de adesivo polimerizado, realizada sob condições experimentais padronizadas, mostrou que o comportamento fluorescente da RB em MP e SE foi muito semelhante na mesma concentração de corante, mas o mesmo não pôde ser dito do comportamento da FS, que foi notavelmente inferior no adesivo autocondicionante, SE, na concentração mais alta. Em dentina, os adesivos preparados com RB nas concentrações-alvo de 0,1 e 0,02 mg/mL apresentaram fluorescência ótima; já aqueles preparados com 0,004 mg/mL produziram fraco sinal. Adesivos preparados com FS a 0,5 mg/mL apresentaram ótima fluorescência na interface de adesão, enquanto que concentração menor desse corante não produziu sinal suficiente. Padrões morfológicos aparentemente atípicos foram observados na interface de adesão, quando da associação do adesivo SE com o corante FS. A adição de RB e FS nas quatro concentrações indicadas aos adesivos MP e SE não afetou o GC nem o AC em comparação com os grupos de controle correspondentes. Em suma, a RB mostra-se um corante mais versátil que a FS na avaliação morfológica das interfaces dentina-MP e dentina-SE via MCVL. A menor faixa de concentrações de RB nos adesivos MP e SE, na qual é possível produzir imagens satisfatórias das interfaces, situa-se entre 0,1­0,02 mg/mL. Já o corante FS deve ser adicionado a esses adesivos a pelo menos 0,5 mg/mL para produzir níveis de fluorescência satisfatórios na interface de adesão. A não ocorrência de efeitos deletérios sobre a polimerização e a molhabilidade das resinas estabelece uma margem de segurança para a incorporação desses agentes fluorescentes (em concentração ≤ 0,5 mg/mL) nesses sistemas monoméricos.(AU)


The addition of fluorescent dyes to dental adhesives makes it possible to investigate the spatial distribution of such resin-based materials in the tooth-restoration interface, using confocal laser scanning microscopy (CLSM). Literature indicates a lack of standardization on the application of fluorescent agents for this purpose. This work presents strategies for adding rhodamine B (RB) and fluorescein sodium salt (FS) to a three-step etch-and-rinse adhesive system, Adper Scotchbond Multi-Purpose (MP), and a two-step self-etching one, Clearfil SE Bond (SE), both regarded as "gold standard" in restorative dentistry. The main objectives were (a) to determine the lowest range of RB and FS concentrations required to produce suitable images of the dentin-adhesive interface via CLSM and (b) to investigate potential effects of addition of these dyes on some resin properties. The adhesives were labeled with RB or FS at decreasing concentrations (0.5, 0.1, 0.02 and 0.004 mg/mL) by means of a semi-direct dispersion method. The photophysical/fluorescent behavior of the labeled resins was investigated by photoluminescence spectroscopy and by CLSM. The adhesives were also investigated with regards to the degree of conversion (DC) and contact angle (CA). A two-way ANOVA of "adhesive" and "treatment" was conducted on DC and CA separately, followed by Tukey's test. The maximum emission and excitation wavelengths of RB and FS were influenced by the host polymer and the dye concentration in general. The preliminary CLSM of cured adhesive samples, performed with standardized settings, showed that the fluorescent behavior of RB in MP and SE was very similar in the same dye concentration, unlike the behavior of FS, which was lower in the self-etching adhesive for the highest dye concentration. In dentin, the adhesives prepared with RB at the target concentrations of 0.1 and 0.02 mg/mL presented optimal fluorescence; those with 0.004 mg/mL produced poor signal. Adhesives prepared with FS at 0.5 mg/mL presented optimal fluorescence at the bonding interface, whereas lower concentrations of FS did not produce sufficient signal. Atypical morphological features were observed at the bonding interface, when adhesive SE was used with FS. The addition of RB and FS at the four decreasing concentrations to adhesives MP and SE did not affect DC or CA compared to the corresponding controls. In short, RB is more versatile than FS for the morphological characterization of dentin-MP and dentin-SE interfaces via MCVL. The lowest range of RB concentrations in adhesives MP and SE that can produce suitable images of the bonding interface lies between 0.1­0.02 mg/mL. The dye FS should be added to these adhesives at 0.5 mg/mL at least to produce satisfactory fluorescence levels at the bonding interface. Since negative effects on polymerization and wettability of the resins were not observed, the use of RB and FS (in concentration ≤ 0.5 mg/mL) together with MP and SE should be reliable in terms of resin properties.(AU)


Subject(s)
Fluorescein/chemistry , Fluorescent Dyes/chemistry , Resin Cements/chemistry , Rhodamines/chemistry , Analysis of Variance , Ethanol/chemistry , Microscopy, Confocal , Reference Values , Reproducibility of Results , Self-Curing of Dental Resins/methods , Spectrometry, Fluorescence , Spectrophotometry, Infrared
14.
Chonnam Medical Journal ; : 83-94, 2017.
Article in English | WPRIM | ID: wpr-788379

ABSTRACT

Heptamethine cyanine dyes are categorized as a class of near infrared fluorescent (NIRF) dyes which have been discovered to have tumor targeting and accumulation capability. This unique feature of NIRF dye makes it a promising candidate for imaging, targeted therapy and also as a drug delivery vehicle for various types of cancers. The favored uptake of dyes only in cancer cells is facilitated by several factors which include organic anion-transporting polypeptides, high mitochondrial membrane potential and tumor hypoxia in cancer cells. Currently nanotechnology has opened possibilities for multimodal or multifunctional strategies for cancer treatment. Including heptamethine cyanine dyes in nanoparticle based delivery systems have generally improved its theranostic ability by several fold owing to the multiple functionalities and structural features of heptamethine dyes. For this reason, nanocomplexes with NIRF heptamethine cyanine dye probe are preferred over non-targeting dyes such as indo cyanine green (ICG). This review sums up current trends and progress in NIRF heptamethine cyanine dye, including dye properties, multifunctional imaging and therapeutic applications in cancer.


Subject(s)
Hypoxia , Coloring Agents , Drug Delivery Systems , Fluorescent Dyes , Membrane Potential, Mitochondrial , Nanoparticles , Nanotechnology , Peptides , Theranostic Nanomedicine
15.
Chonnam Medical Journal ; : 95-102, 2017.
Article in English | WPRIM | ID: wpr-788378

ABSTRACT

Although various clinical imaging modalities have been developed to visualize internal body structures and detect abnormal tissues prior to surgical procedures, most medical imaging modalities do not provide disease-specific images in real-time. Optical imaging can provide the surgeon with real-time visualization of the surgical field for intraoperative image-guided surgery. Imaging in the near-infrared (NIR) window (650-900 nm), also known as the “therapeutic window” has high potential by offering low absorbance and scattering in tissues resulting in minimized background autofluorescence. Clinically, optical fluorescence imaging with the targeted contrast agents provides opportunities for significant advances in intraoperative image-guided surgery. There are only two clinically available NIR fluorophores, indocyanine green (ICG) and methylene blue (MB), that support the image-guided surgery. However, neither of them perform in vivo by providing optimum specificity and stability for targeted image guidance. Therefore, it is of paramount importance to develop targeted NIR fluorophores for unmet clinical needs. Using the right combination of an NIR fluorescence imaging system and a targeted fluorophore, the desired target tissues can be imaged to provide real-time fluorescence guidance without changing the field-of-view during surgery. Thus, in a clinical discipline, the development of NIR fluorophores for ‘structure-inherent targeting’ is an unmet need for early phase diagnostics with accurate targeting.


Subject(s)
Contrast Media , Diagnostic Imaging , Fluorescence , Fluorescent Dyes , Indocyanine Green , Methylene Blue , Optical Imaging , Sensitivity and Specificity , Surgery, Computer-Assisted
16.
Chonnam Medical Journal ; : 83-94, 2017.
Article in English | WPRIM | ID: wpr-151399

ABSTRACT

Heptamethine cyanine dyes are categorized as a class of near infrared fluorescent (NIRF) dyes which have been discovered to have tumor targeting and accumulation capability. This unique feature of NIRF dye makes it a promising candidate for imaging, targeted therapy and also as a drug delivery vehicle for various types of cancers. The favored uptake of dyes only in cancer cells is facilitated by several factors which include organic anion-transporting polypeptides, high mitochondrial membrane potential and tumor hypoxia in cancer cells. Currently nanotechnology has opened possibilities for multimodal or multifunctional strategies for cancer treatment. Including heptamethine cyanine dyes in nanoparticle based delivery systems have generally improved its theranostic ability by several fold owing to the multiple functionalities and structural features of heptamethine dyes. For this reason, nanocomplexes with NIRF heptamethine cyanine dye probe are preferred over non-targeting dyes such as indo cyanine green (ICG). This review sums up current trends and progress in NIRF heptamethine cyanine dye, including dye properties, multifunctional imaging and therapeutic applications in cancer.


Subject(s)
Hypoxia , Coloring Agents , Drug Delivery Systems , Fluorescent Dyes , Membrane Potential, Mitochondrial , Nanoparticles , Nanotechnology , Peptides , Theranostic Nanomedicine
17.
Chonnam Medical Journal ; : 95-102, 2017.
Article in English | WPRIM | ID: wpr-151398

ABSTRACT

Although various clinical imaging modalities have been developed to visualize internal body structures and detect abnormal tissues prior to surgical procedures, most medical imaging modalities do not provide disease-specific images in real-time. Optical imaging can provide the surgeon with real-time visualization of the surgical field for intraoperative image-guided surgery. Imaging in the near-infrared (NIR) window (650-900 nm), also known as the “therapeutic window” has high potential by offering low absorbance and scattering in tissues resulting in minimized background autofluorescence. Clinically, optical fluorescence imaging with the targeted contrast agents provides opportunities for significant advances in intraoperative image-guided surgery. There are only two clinically available NIR fluorophores, indocyanine green (ICG) and methylene blue (MB), that support the image-guided surgery. However, neither of them perform in vivo by providing optimum specificity and stability for targeted image guidance. Therefore, it is of paramount importance to develop targeted NIR fluorophores for unmet clinical needs. Using the right combination of an NIR fluorescence imaging system and a targeted fluorophore, the desired target tissues can be imaged to provide real-time fluorescence guidance without changing the field-of-view during surgery. Thus, in a clinical discipline, the development of NIR fluorophores for ‘structure-inherent targeting’ is an unmet need for early phase diagnostics with accurate targeting.


Subject(s)
Contrast Media , Diagnostic Imaging , Fluorescence , Fluorescent Dyes , Indocyanine Green , Methylene Blue , Optical Imaging , Sensitivity and Specificity , Surgery, Computer-Assisted
18.
J. appl. oral sci ; 24(4): 317-324, July-Aug. 2016. tab, graf
Article in English | LILACS, BBO | ID: lil-792589

ABSTRACT

ABSTRACT Objective This study investigated the effect of the fluorescent dye rhodamine B (RB) for interfacial micromorphology analysis of dental composite restorations on water sorption/solubility (WS/WSL) and microtensile bond strength to dentin (µTBS) of a 3-step total etch and a 2-step self-etch adhesive system. Material and Methods The adhesives Adper Scotchbond Multi-Purpose (MP) and Clearfil SE Bond (SE) were mixed with 0.1 mg/mL of RB. For the WS/WSL tests, cured resin disks (5.0 mm in diameter x 0.8 mm thick) were prepared and assigned into four groups (n=10): MP, MP-RB, SE, and SE-RB. For µTBS assessment, extracted human third molars (n=40) had the flat occlusal dentin prepared and assigned into the same experimental groups (n=10). After the bonding and restoration procedures, specimens were sectioned in rectangular beams, stored in water and tested after seven days or after 12 months. The failure mode of fractured specimens was qualitatively evaluated under optical microscope (x40). Data from WS/WSL and µTBS were assessed by one-way and three-way ANOVA, respectively, and Tukey’s test (α=5%). Results RB increased the WSL of MP and SE. On the other hand, WS of both MP and SE was not affected by the addition of RB. No significance in µTBS between MP and MP-RB for seven days or one year was observed, whereas for SE a decrease in the µTBS means occurred in both storage times. Conclusions RB should be incorporated into non-simplified DBSs with caution, as it can interfere with their physical-mechanical properties, leading to a possible misinterpretation of bonded interface.


Subject(s)
Humans , Rhodamines/chemistry , Water/chemistry , Dentin-Bonding Agents/chemistry , Dentin/drug effects , Fluorescent Dyes/chemistry , Solubility , Surface Properties , Tensile Strength , Time Factors , Materials Testing , Reproducibility of Results , Dental Bonding/methods , Microscopy, Confocal , Composite Resins/chemistry , Resin Cements/chemistry , Dental Restoration Failure
19.
Rev. Soc. Bras. Med. Trop ; 49(3): 279-285, tab, graf
Article in English | LILACS | ID: lil-785796

ABSTRACT

Abstract: INTRODUCTION: The genus Flavivirus includes several pathogenic species that cause severe illness in humans. Therefore, a rapid and accurate molecular method for diagnosis and surveillance of these viruses would be of great importance. Here, we evaluate and optimize a quantitative real-time reverse transcription polymerase chain reaction (RT-PCR) method for the diagnosis of the Flavivirus genus. METHODS: We evaluated different commercial kits that use the SYBR Green system for real-time RT-PCR with a primer set that amplifies a fragment of the NS5 flavivirus gene. The specificity and sensitivity of the assay were tested using twelve flaviviruses and ribonucleic acid (RNA) transcribed from the yellow fever virus. Additionally, this assay was evaluated using the sera of 410 patients from different regions of Brazil with acute febrile illness and a negative diagnosis for the dengue virus. RESULTS: The real-time RT-PCR amplified all flaviviruses tested at a melting temperature of 79.92 to 83.49°C. A detection limit of 100 copies per ml was determined for this assay. Surprisingly, we detected dengue virus in 4.1% (17/410) of samples from patients with febrile illness and a supposedly negative dengue infection diagnosis. The viral load in patients ranged from 2.1×107to 3.4×103copies per ml. CONCLUSIONS: The real-time RT-PCR method may be very useful for preliminary diagnoses in screenings, outbreaks, and other surveillance studies. Moreover, this assay can be easily applied to monitor viral activity and to measure viral load in pathogenesis studies.


Subject(s)
Humans , Flavivirus Infections/diagnosis , Flavivirus/genetics , Organic Chemicals , Reagent Kits, Diagnostic , Brazil , RNA, Viral/genetics , Sensitivity and Specificity , Flavivirus Infections/virology , DNA Primers , Reverse Transcriptase Polymerase Chain Reaction/methods , Flavivirus/isolation & purification , Flavivirus/classification , Fluorescent Dyes
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