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Imaging turbid media is range limited. In contrast, sensing the medium's optical properties is possible in larger depths using the iterative multi-plane optical properties extraction technique. It analyzes the reconstructed reemitted light phase image. The root mean square of the phase image yields two graphs with opposite behaviors that intersect at µ's,cp. These graphs enable the extraction of a certain range of the reduced scattering coefficient, µ's. Here, we aim to extend the range of µ's detection by optical magnification. We use a modified diffusion theory and show how µ's,cp shifts with the varying magnification. The theoretical results were tested experimentally, showing that the technique can be adapted to different ranges of µ's by changing the magnification.
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We report on SwissSPAD2, an image sensor with 512×512 photon-counting pixels, each comprising a single-photon avalanche diode (SPAD), a 1-bit memory, and a gating mechanism capable of turning the SPAD on and off, with a skew of 250ps and 344ps, respectively, for a minimum duration of 5.75ns. The sensor is designed to achieve a frame rate of up to 97,700 binary frames per second and sub-40ps gate shifts. By synchronizing it with a pulsed laser and using multiple successive overlapping gates, one can reconstruct a molecule's fluorescent response with picosecond temporal resolution. Thanks to the sensor's number of pixels (the largest to date) and the fully integrated gated operation, SwissSPAD2 enables widefield FLIM with an all-solid-state solution and at relatively high frame rates. This was demonstrated with preliminary results on organic dyes and semiconductor quantum dots using both decay fitting and phasor analysis. Furthermore, pixels with an exceptionally low dark count rate and high photon detection probability enable uniform and high quality imaging of biologically relevant fluorescent samples stained with multiple dyes. While future versions will feature the addition of microlenses and optimize firmware speed, our results open the way to low-cost alternatives to commercially available scientific time-resolved imagers.
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Nanoparticle-based contrast agents have been used as an imaging tool for selectively detecting cancerous processes. We aimed to evaluate the detection sensitivity of reflection measurements of gold nanorods (GNRs) bio-conjugated to anti-epidermal growth factor receptor (GNRs-EGFR) monoclonal antibodies in discriminating benign from premalignant and malignant human oral lesions. Tissue sections incubated with GNRs-EGFR and the reflectance spectrum was measured using hyperspectral microscopy. Reflectance intensity increased with the progression of the disease, lowest in the control group and increasing as the dysplastic changes increase (P<0.001 for linear trend of grade). Intensity was significantly higher in the moderate and severe dysplasias and cancer patients than in the controls and mild dysplasia (t test P=0.0003, Mann-Whitney P<0.0001). The GNRs reflection measurements can discriminate benign and mild dysplastic lesions from the more severe dysplasia and invasive cancer, suggesting an objective, not dependent on the qualification of a technician and with less interpretation errors.
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Anticorpos Monoclonais/química , Carcinoma de Células Escamosas/diagnóstico , Ouro/química , Neoplasias Bucais/diagnóstico , Nanotubos/química , Adulto , Idoso , Idoso de 80 Anos ou mais , Receptores ErbB/antagonistas & inibidores , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Adulto JovemRESUMO
In this study we report the use of gold nanorods (GNRs) as absorption contrast agents in the diffusion reflection (DR) method for the in vivo detection of atherosclerotic injury. The early detection and characterization of atherosclerotic vascular disease is considered to be one of the greatest medical challenges today. We show that macrophage cells, which are major components of unstable active atherosclerotic plaques, uptake gold nanoparticles, resulting in a change in the optical properties of tissue-like phantoms and a unique DR profile. In vivo DR measurements of rats that underwent injury of the carotid artery showed a clear difference between the DR profiles of the injured compared with healthy arteries. The results suggest that DR measurements following GNRs administration represent a potential novel method for the early detection of atherosclerotic vascular disease.
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Aterosclerose/diagnóstico por imagem , Meios de Contraste/química , Imagem Molecular/métodos , Nanotubos/química , Animais , Aterosclerose/fisiopatologia , Meios de Contraste/administração & dosagem , Ouro/administração & dosagem , Ouro/química , Humanos , Radiografia , RatosRESUMO
Significance: Fluorescence lifetime imaging (FLI) plays a pivotal role in enhancing our understanding of biological systems, providing a valuable tool for non-invasive exploration of biomolecular and cellular dynamics, both in vitro and in vivo. Its ability to selectively target and multiplex various entities, alongside heightened sensitivity and specificity, offers rapid and cost-effective insights. Aim: Our aim is to investigate the multiplexing capabilities of near-infrared (NIR) FLI within a scattering medium that mimics biological tissues. We strive to develop a comprehensive understanding of FLI's potential for multiplexing diverse targets within a complex, tissue-like environment. Approach: We introduce an innovative Monte Carlo (MC) simulation approach that accurately describes the scattering behavior of fluorescent photons within turbid media. Applying phasor analyses, we enable the multiplexing of distinct targets within a single FLI image. Leveraging the state-of-the-art single-photon avalanche diode (SPAD) time-gated camera, SPAD512S, we conduct experimental wide-field FLI in the NIR regime. Results: Our study demonstrates the successful multiplexing of dual targets within a single FLI image, reaching a depth of 1 cm within tissue-like phantoms. Through our novel MC simulation approach and phasor analyses, we showcase the effectiveness of our methodology in overcoming the challenges posed by scattering media. Conclusions: This research underscores the potential of NIR FLI for multiplexing applications in complex biological environments. By combining advanced simulation techniques with cutting-edge experimental tools, we introduce significant results in the non-invasive exploration of biomolecular dynamics, to advance the field of FLI research.
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Imagem Óptica , Fótons , Simulação por Computador , Imagens de Fantasmas , CorantesRESUMO
The near-infrared (NIR) range of the electromagnetic (EM) spectrum offers a nearly transparent window for imaging tissue. Despite the significant potential of NIR fluorescence-based imaging, its establishment in basic research and clinical applications remains limited due to the scarcity of fluorescent molecules with absorption and emission properties in the NIR region, especially those suitable for biological applications. In this study, we present a novel approach by combining the widely used IRdye 800NHS fluorophore with gold nanospheres (GNSs) and gold nanorods (GNRs) to create Au nanodyes, with improved quantum yield (QY) and distinct lifetimes. These nanodyes exhibit varying photophysical properties due to the differences in the separation distance between the dye and the gold nanoparticles (GNP). Leveraging a rapid and highly sensitive wide-field fluorescence lifetime imaging (FLI) macroscopic set up, along with phasor based analysis, we introduce multiplexing capabilities for the Au nanodyes. Our approach showcases the ability to differentiate between NIR dyes with very similar, short lifetimes within a single image, using the combination of Au nanodyes and wide-field FLI. Furthermore, we demonstrate the uptake of Au nanodyes by mineral-oil induced plasmacytomas (MOPC315.bm) cells, indicating their potential for in vitro and in vivo applications.
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Non-invasive and high spatiotemporal resolution mythologies for the diagnosis and treatment of disease in clinical medicine promote the development of modern medicine. Ultrasound (US) technology provides a non-invasive, real-time, and cost-effective clinical imaging modality, which plays a significant role in chemical synthesis and clinical translation, especially in in vivo imaging and cancer therapy. On the one hand, the US treatment is usually accompanied by cavitation, leading to high temperature and pressure, so-called "hot spot", playing a significant role in sonochemical-based colloidal synthesis. Compared with the classical nucleation synthetic method, the sonochemical synthesis strategy presents high efficiency for the fabrication of colloidal nanocrystals due to its fast nucleation and growth procedure. On the other hand, the US is attractive for in vivo and medical treatment, with applications increasing with the development of novel contrast agents, such as the micro and nano bubbles, which are widely used in neuromodulation, with which the US can breach the blood-brain barrier temporarily and safely, opening a new door to neuromodulation and therapy. In terms of cancer treatment, sonodynamic therapy and US-assisted synergetic therapy show great effects against cancer and sonodynamic immunotherapy present unparalleled potentiality compared with other synergetic therapies. Further development of ultrasound technology can revolutionize both chemical synthesis and clinical translation by improving efficiency, precision, and accessibility while reducing environmental impact and enhancing patient care. In this paper, we review the US-assisted sonochemical synthesis and biological applications, to promote the next generation US technology-assisted applications.
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Nanopartículas , Terapia por Ultrassom , Humanos , Ultrassonografia , Fenômenos Químicos , TecnologiaRESUMO
Near-infrared (NIR) J-aggregates attract increasing attention in many areas, especially in biomedical applications, as they combine the advantages of NIR spectroscopy with the unique J-aggregation properties of organic dyes. They enhance light absorption and have been used as effective biological imaging and therapeutic agents to achieve high-resolution imaging or effective phototherapy in vivo. In this work, we present novel J-aggregates composed of the well-known cyanine molecules. Cyanines are one of the few types of molecules whose absorption and emission can be shifted over a broad spectral range, from the ultraviolet (UV) to the NIR regime. They can easily transform into J-aggregates with narrow absorption and emission peaks, which is accompanied by a red shift in their spectra. In this work, we show, for the first time, that the tricarbocyanine dye (IR 820) has two sharp fluorescence emission bands in the NIR-II region with high photostability. These emission bands can be tuned to a desired wavelength in the range of 1150-1560 and 1675 nm, with a linear dependence on the excitation wavelength. Cryogenic transmission electron microscopy (cryo-TEM) images are presented, and combined with molecular modeling analysis, they confirm IR 820 π-stacked self-assembled fibrous structures.
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Fluorescence-based imaging has an enormous impact on our understanding of biological systems. However, in vivo fluorescence imaging is greatly influenced by tissue scattering. A better understanding of this dependence can improve the potential of noninvasive in vivo fluorescence imaging. In this article, we present a diffusion model, based on an existing master-slave model, of isotropic point sources imbedded in a scattering slab, representing fluorophores within a tissue. The model was compared with Monte Carlo simulations and measurements of a fluorescent slide measured through tissue-like phantoms with different reduced scattering coefficients (0.5-2.5 mm-1 ) and thicknesses (0.5-5 mm). Results show a good correlation between our suggested theory, simulations and experiments; while the fluorescence intensity decays as the slab's scattering and thickness increase, the decay rate decreases as the reduced scattering coefficient increases in a counterintuitive manner, suggesting fewer fluorescence artifacts from deep within the tissue in highly scattering media.
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Corantes Fluorescentes , Simulação por Computador , Espalhamento de Radiação , Imagens de Fantasmas , Método de Monte CarloRESUMO
Cardiovascular disease (CVD) is a major cause of death and disability worldwide. A real need exists in the development of new, improved therapeutic methods for treating CVD, while major advances in nanotechnology have opened new avenues in this field. In this paper, we report the use of gold nanoparticles (GNPs) coated with high-density lipoprotein (HDL) (GNP-HDL) for the simultaneous detection and therapy of unstable plaques. Based on the well-known HDL cardiovascular protection, by promoting the reverse cholesterol transport (RCT), injured rat carotids, as a model for unstable plaques, were injected with the GNP-HDL. Noninvasive detection of the plaques 24 h post the GNP injection was enabled using the diffusion reflection (DR) method, indicating that the GNP-HDL particles had accumulated in the injured site. Pathology and noninvasive CT measurements proved the recovery of the injured artery treated with the GNP-HDL. The DR of the GNP-HDL presented a simple and highly sensitive method at a low cost, resulting in simultaneous specific unstable plaque diagnosis and recovery.
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BACKGROUND AND OBJECTIVE: Lately in phototherapy the use of diodes instead of lasers was suggested for economical and practical reasons. It has been argued that lasers have no preference over diodes since they lose their coherence once penetrating biological tissues. However, this point has never been experimentally proven. In this work we, for the first time, have experimentally validated the conditions affecting the spatial coherence of a laser illumination going through a biological tissue. STUDY DESIGN/MATERIALS AND METHODS: In our experiments we measured the spatial coherence of the light passing through phantoms containing intralipid and ink component as well as through uncooked turkey meat. We do this measuring the changes of the contrast of the speckle patterns generated due to laser illumination. Flow tunnels inside the phantoms were generated by needles in two different diameters. The measurements were performed for varied integration time, varied thickness of phantoms, and for varied flow rates. The measurement system included two excitation sources: a green doubled Nd:YAG laser at wavelength of 532 nm and an ultra high power green LED at a wavelength of 520 nm. RESULTS: It was experimentally validated that the thickness of the tissue does not change the coherence while there is no flow. Furthermore, the flow velocity and the flow volumetric rate highly affect the coherence length. Previously developed mathematical expression, in which the contrast depends on the correlation and the exposure time, was found to be compatible with the obtained experimental results. CONCLUSIONS: We found that the coherence of the laser is not lost when the light goes through a static tissue but it is partially lost when there is a flow of fluid through the tissue. The volumetric flow rate is directly correlated to the loss of spatial coherence. Higher flow rate produces shorter coherence length.
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Modelos Biológicos , Fototerapia/instrumentação , Tomografia de Coerência Óptica/instrumentação , Animais , Lasers , Imagens de Fantasmas , Fototerapia/métodos , Doses de Radiação , Sensibilidade e Especificidade , TurquiaRESUMO
BACKGROUND AND OBJECTIVES: According to earlier in vitro low level laser therapy (LLLT) studies, wavelengths in the red and near infrared range, that are absorbed by cytochrome oxidase, stimulate cell growth and hence wound healing. Wavelengths in the blue region that are absorbed by flavins were found to exert a bactericidal effect that is very important for treating infected wounds. However, as far as therapeutic application of light is concerned, penetration into the tissue must be considered. For this purpose we estimated the penetration depth as a function of the relevant wavelengths, using the formulae of the photon migration model for skin tissue. METHODS: We use the photon diffusion model, which is an analytical model for describing light transfer in biological tissues. We refer to the most common chromophores in human tissue and evaluate their volume fraction and concentration in skin cells. These empirically estimated mean wavelength-dependent absorption coefficients are then substituted in the theoretical expressions for the optical penetration depth in the tissue. The wavelengths, for which the penetration depth is the highest, are the optimal wavelengths to be used in wound healing treatments. RESULTS: Our model suggests that the optimal wavelengths for therapeutic treatments are in the red region with a local maximum at 730 nm. As to the blue region, a local maximum at 480 nm was found. CONCLUSION: Light at 480 nm should be used for treating infected wounds followed by 730 nm light for enhancing wound closure.
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Lasers , Modelos Biológicos , Doses de Radiação , Cicatrização/efeitos da radiação , Difusão , Fibroblastos/metabolismo , Hemoglobinas/metabolismo , Humanos , Fótons , Pele/citologia , Pele/metabolismoRESUMO
BACKGROUND: Visible light-based stimulation using low-intensity lasers, LEDs, and broadband visible light devices has been recently introduced for therapy of human tissues in the absence of exogenous photosensitizers. Nitric oxide (NO) formation might be a potential mechanism for photobiomodulation because it is synthesized in cells by nitric oxide synthase (NOS), which contains both flavin and heme groups that absorb visible light. NO synthesis may also result from increased reactive oxygen species (ROS), which are found in various cell cultures following visible light illumination. NO is mainly known for inducing blood vessel dilation by endothelial cells, and in sperm cells NO is considered as an important agent in acrosome reaction and capacitation process, which are essential for successful fertilization. PURPOSE: To study NO formation in endothelial and sperm cells following visible light irradiation. MATERIALS AND METHODS: Sperm and endothelial cells were illuminated with broadband visible light, 400-800 nm, 130 mW/cm(2), for 5 minutes. During illumination, the endothelial cells were incubated in PBS free of Ca(+2) and Mg(+2), and the sperm cells were incubated in NKM buffer, to induce "stress conditions." NO production was quantified by using the Griess reagent which reacts with nitrite in the medium to yield an Azo compound which has an absorption band at 540 nm. RESULTS: Visible light illumination increased NO concentration both in sperm and endothelial cells. Blue light was more effective than red. Light-induced NO occurred only when endothelial cells were incubated in PBS free of Ca(+2) and Mg(+2), and in sperm cells, only when incubated in NKM. CONCLUSION: Light induces NO formation in endothelial and sperm cells. In endothelial cells, NO formation may explain previous results demonstrating enhanced wound healing and pain relief following illumination. In illuminated sperm cells, NO formation may account for the enhanced fertilization rate.
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Células Endoteliais/metabolismo , Luz , Óxido Nítrico/metabolismo , Espermatozoides/metabolismo , Animais , Bovinos , Endotélio Vascular/metabolismo , MasculinoRESUMO
Diffusion reflectance spectroscopy measurements targeted with gold nanoparticles (GNPs) can identify residual cutaneous squamous cell carcinoma (SCC) in excision borders. Human SCC specimens were stained with hematoxylin and eosin to identify tumor borders, and reflected onto an unstained deparaffinized section. Diffusion reflection of three sites (normal and SCC) were measured before and after GNPs targeting. Hyperspectral imaging showed a mean of 2.5 sites with tumor per specimen and 1.2 tumor-free (p < 0.05, t-test). GNPs were detected in 25/30 tumor sites (sensitivity 83.3%, false-negative rate 16.6%) and 12/30 non-tumor sites (specificity 60%, false-positive rate 40%). This study verifies the use of nanotechnology in identifying SCC tumor margins. Diffusion reflection scanning has high sensitivity for detecting the residual tumor.
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Fluorescence lifetime imaging (FLI) is increasingly recognized as a powerful tool for biochemical and cellular investigations, including in vivo applications. Fluorescence lifetime is an intrinsic characteristic of any fluorescent dye which, to a large extent, does not depend on excitation intensity and signal level. In particular, it allows distinguishing dyes with similar emission spectra, offering additional multiplexing capabilities. However, in vivo FLI in the visible range is complicated by the contamination by (i) tissue autofluorescence, which decreases contrast, and by (ii) light scattering and absorption in tissues, which significantly reduce fluorescence intensity and modify the temporal profile of the signal. Here, we demonstrate how these issues can be accounted for and overcome, using a new time-gated single-photon avalanche diode array camera, SwissSPAD2, combined with phasor analysis to provide a simple and fast visual method for lifetime imaging. In particular, we show how phasor dispersion increases with increasing scattering and/or decreasing fluorescence intensity. Next, we show that as long as the fluorescence signal of interest is larger than the phantom autofluorescence, the presence of a distinct lifetime can be clearly identified with appropriate background correction. We use these results to demonstrate the detection of A459 cells expressing the fluorescent protein mCyRFP1 through highly scattering and autofluorescent phantom layers. These results showcase the possibility to perform FLI in challenging conditions, using standard, bright, visible fluorophore or fluorescence proteins.
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Atherosclerosis (AS), the leading cause of morbidity and mortality in cardiovascular disease, needs an early detection for treatment and prevention of fatal events. Here, for the first time, we applied gold nanorods (GNRs)-assisted diffusion reflection (DR), a noninvasive technique for in vivo detection of AS in a high-fat-diet-induced c57bl mouse model, which resembles the manifestation of AS in humans. DR simply detects the change in light reflection profile of tissue due to the accumulation of GNRs in the AS plaques and enables clear detection of AS lesions in carotid and femoral arteries of these hyperlipidemic mice. After 24 hours post-GNRs injection, DR showed the highest efficiency of AS detection. Moreover, the sensitivity of the DR method is much higher than computed tomography (CT) and is comparable to ex vivo high-resolution CT. Our results strongly suggest that the DR method can detect early atherosclerotic lesions in a sensitive and specific manner.
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Aterosclerose/diagnóstico , Ouro/química , Hiperlipidemias/diagnóstico , Nanomedicina/métodos , Nanotubos/química , Animais , Difusão , Modelos Animais de Doenças , Hiperlipidemias/diagnóstico por imagem , Hiperlipidemias/patologia , Camundongos , Camundongos Endogâmicos C57BL , Sensibilidade e Especificidade , Tomografia Computadorizada por Raios XRESUMO
In this work, we present a novel, simple, and highly accurate three-dimensional (3D) diffusion reflection (DR) imaging system and method for the detection of accumulation sites of gold nanorods (GNRs) within the tissue. GNRs are intensively used for diagnosis purposes of varied diseases, mainly because of their ability to well absorb visible light, which introduces them as terrific contrast agents in various imaging and theranostics methods. Lately, these GNRs unique absorption properties have served in DR intensity-based measurements, suggesting a novel diagnostic tool, DR-GNRs. In this paper, we show a new measurement system and method for DR, based on its radial collection from the tissue. These radial measurements enabled a unique 3D presentation of the DR-GNR, introducing the dimensions ρ for the radius, θ for the angle, and Γ for the reflected intensity. On the basis of the diffusion model, which enables to correlate between the sample's optical properties and its reflectance, a unique, radial map is presented. This map introduces the slopes of the DR curves in each measured angle, which are linearly correlated with the tissue's optical properties and with the GNRs concentrations within the tissue, thus enables the exact radial localization of the GNRs in the sample. We show the detection of macrophage accumulation in tissue-like phantoms, as well as the localization of unstable plaques in hyperlipidemic mice, in vivo. This highly accurate, powerful technology paves the way toward a real-time detection method that can be successfully integrated in the rapid increasing field of personalized medicine.
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A critical challenge arising during a surgical procedure for tumor removal is the determination of tumor margins. Gold nanorods (GNRs) conjugated to epidermal growth factor receptors (EGFR) (GNRs-EGFR) have long been used in the detection of cancerous cells as the expression of EGFR dramatically increases once the tissue becomes cancerous. Optical techniques for the identification of these GNRs-EGFR in tumor are intensively developed based on the unique scattering and absorption properties of the GNRs. In this study, we investigate the distribution of the GNRs in tissue sections presenting squamous cell carcinoma (SCC) to evaluate the SCC margins. Air scanning electron microscopy (airSEM), a novel, high resolution microscopy is used, enabling to localize and actually visualize nanoparticles on the tissue. The airSEM pictures presented a gradient of GNRs from the tumor to normal epithelium, spread in an area of 1 mm, suggesting tumor margins of 1 mm. Diffusion reflection (DR) measurements, performed in a resolution of 1 mm, of human oral SCC have shown a clear difference between the DR profiles of the healthy epithelium and the tumor itself.
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Carcinoma de Células Escamosas/patologia , Cetuximab/química , Nanopartículas Metálicas/química , Neoplasias Bucais/patologia , Nanotubos/química , Idoso , Carcinoma de Células Escamosas/metabolismo , Cetuximab/farmacologia , Receptores ErbB/metabolismo , Feminino , Ouro/química , Humanos , Microscopia Eletrônica de Varredura/métodos , Neoplasias Bucais/metabolismo , Ligação ProteicaRESUMO
In this study, we report a potential noninvasive technique for the detection of vulnerable plaques using scatter analyses with flow cytometry (FCM) method combined with the diffusion reflection (DR) method. The atherosclerotic plaques are commonly divided into two major categories: stable and vulnerable. The vulnerable plaques are rich with inflammatory cells, mostly macrophages (MΦ), which release enzymes that break down collagen in the cap. The detection method is based on uptake of gold nanorods (GNR) by MΦ. The GNR have unique optical properties that enable their detection using the FCM method, based on their scattering properties, and using the DR method, based on their unique absorption properties. This work demonstrates that after GNR labeling of MΦ, 1) the FCM scatter values increased up to 3.7-fold with arbitrary intensity values increasing from 1,110 to 4,100 and 2) the DR slope changed from an average slope of 0.196 (MΦ only) to an average slope of 0.827 (MΦ labeled with GNR) (P<0.001 for both cases). The combination of FCM and DR measurements provides a potential novel, highly sensitive, and noninvasive method for the identification of atherosclerotic vulnerable plaques, aimed to develop a potential tool for in vivo tracking.
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Ouro/química , Macrófagos/metabolismo , Nanotubos/química , Imagem Óptica/métodos , Ouro/farmacocinética , Humanos , Imagens de Fantasmas , Placa AteroscleróticaRESUMO
The composition of atherosclerotic (AS) plaques is crucial concerning rupture, thrombosis and clinical events. Two plaque types are distinguished: stable and vulnerable plaques. Vulnerable plaques are rich in inflammatory cells, mostly only M1 macrophages, and are highly susceptible to rupture. These plaques represent a high risk particularly with the standard invasive diagnosis by coronary angiography. So far there are no non-invasive low-risk clinical approaches available to detect and distinguish AS plaque types in vivo. The perspective review introduces a whole work-flow for a novel approach for non-invasive detection and classification of AS plaques using the diffusion reflection method with gold nanoparticle loaded macrophages in combination with flow and image cytometric analysis for quality assurance. Classical biophotonic methods for AS diagnosis are summarized. Phenotyping of monocytes and macrophages are discussed for specific subset labelling by nanomaterials, as well as existing studies and first experimental proofs of concept for the novel approach are shown. In vitro and in vivo detection of NP loaded macrophages (MΦ). Different ways of MΦ labelling include (1) in vitro labelling in suspension (whole blood or buffy coat) or (2) labelling of short-term MΦ cultures with re-injection of MΦ-NP into the animal to detect migration of the cells in the plaques and (3) in vivo injection of NP into the organism.