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1.
Nat Commun ; 11(1): 4854, 2020 09 25.
Artigo em Inglês | MEDLINE | ID: mdl-32978383

RESUMO

Chronic imaging of neuronal networks in vitro has provided fundamental insights into mechanisms underlying neuronal function. Current labeling and optical imaging methods, however, cannot be used for continuous and long-term recordings of the dynamics and evolution of neuronal networks, as fluorescent indicators can cause phototoxicity. Here, we introduce a versatile platform for label-free, comprehensive and detailed electrophysiological live-cell imaging of various neurogenic cells and tissues over extended time scales. We report on a dual-mode high-density microelectrode array, which can simultaneously record in (i) full-frame mode with 19,584 recording sites and (ii) high-signal-to-noise mode with 246 channels. We set out to demonstrate the capabilities of this platform with recordings from primary and iPSC-derived neuronal cultures and tissue preparations over several weeks, providing detailed morpho-electrical phenotypic parameters at subcellular, cellular and network level. Moreover, we develop reliable analysis tools, which drastically increase the throughput to infer axonal morphology and conduction speed.


Assuntos
Rede Nervosa/fisiologia , Neurônios/fisiologia , Imagem Óptica/métodos , Análise de Célula Única/métodos , Animais , Axônios , Encéfalo , Células Cultivadas , Células-Tronco Pluripotentes Induzidas , Camundongos , Microeletrodos , Modelos Animais , Rede Nervosa/diagnóstico por imagem , Imagem Óptica/instrumentação , Ratos , Ratos Wistar , Gravação em Vídeo
2.
Nat Commun ; 11(1): 4192, 2020 08 21.
Artigo em Inglês | MEDLINE | ID: mdl-32826886

RESUMO

Bioluminescence imaging has been widely used in life sciences and biomedical applications. However, conventional bioluminescence imaging usually operates in the visible region, which hampers the high-performance in vivo optical imaging due to the strong tissue absorption and scattering. To address this challenge, here we present bioluminescence probes (BPs) with emission in the second near infrared (NIR-II) region at 1029 nm by employing bioluminescence resonance energy transfer (BRET) and two-step fluorescence resonance energy transfer (FRET) with a specially designed cyanine dye FD-1029. The biocompatible NIR-II-BPs are successfully applied to vessels and lymphatics imaging in mice, which gives ~5 times higher signal-to-noise ratios and ~1.5 times higher spatial resolution than those obtained by NIR-II fluorescence imaging and conventional bioluminescence imaging. Their capability of multiplexed imaging is also well displayed. Taking advantage of the ATP-responding character, the NIR-II-BPs are able to recognize tumor metastasis with a high tumor-to-normal tissue ratio at 83.4.


Assuntos
Trifosfato de Adenosina/metabolismo , Medições Luminescentes/métodos , Metástase Neoplásica/diagnóstico por imagem , Imagem Óptica/métodos , Animais , Técnicas Biossensoriais , Linhagem Celular Tumoral , Feminino , Transferência Ressonante de Energia de Fluorescência/instrumentação , Transferência Ressonante de Energia de Fluorescência/métodos , Xenoenxertos , Humanos , Medições Luminescentes/instrumentação , Camundongos , Imagem Óptica/instrumentação , Neoplasias Ovarianas/diagnóstico por imagem
3.
Nat Commun ; 11(1): 3881, 2020 08 04.
Artigo em Inglês | MEDLINE | ID: mdl-32753572

RESUMO

Cells typically respond to chemical or physical perturbations via complex signaling cascades which can simultaneously affect multiple physiological parameters, such as membrane voltage, calcium, pH, and redox potential. Protein-based fluorescent sensors can report many of these parameters, but spectral overlap prevents more than ~4 modalities from being recorded in parallel. Here we introduce the technique, MOSAIC, Multiplexed Optical Sensors in Arrayed Islands of Cells, where patterning of fluorescent sensor-encoding lentiviral vectors with a microarray printer enables parallel recording of multiple modalities. We demonstrate simultaneous recordings from 20 sensors in parallel in human embryonic kidney (HEK293) cells and in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs), and we describe responses to metabolic and pharmacological perturbations. Together, these results show that MOSAIC can provide rich multi-modal data on complex physiological responses in multiple cell types.


Assuntos
Técnicas Biossensoriais/métodos , Células-Tronco Pluripotentes Induzidas/metabolismo , Microscopia de Fluorescência/métodos , Miócitos Cardíacos/metabolismo , Imagem Óptica/métodos , Potenciais de Ação/efeitos dos fármacos , Antagonistas Adrenérgicos beta/farmacologia , Técnicas Biossensoriais/instrumentação , Cálcio/química , Proteínas de Fluorescência Verde/metabolismo , Células HEK293 , Humanos , Peróxido de Hidrogênio/farmacologia , Concentração de Íons de Hidrogênio , Células-Tronco Pluripotentes Induzidas/citologia , Mitocôndrias/metabolismo , Miócitos Cardíacos/citologia , Miócitos Cardíacos/fisiologia , Imagem Óptica/instrumentação , Oxidantes/farmacologia , Oxirredução/efeitos dos fármacos , Propanolaminas/farmacologia
4.
Nat Commun ; 11(1): 4052, 2020 08 13.
Artigo em Inglês | MEDLINE | ID: mdl-32792510

RESUMO

Turn-on fluorescence imaging is routinely studied; however, turn-on chemiluminescence has been rarely explored for in vivo imaging. Herein, we report the design and validation of chemiluminescence probe ADLumin-1 as a turn-on probe for amyloid beta (Aß) species. Two-photon imaging indicates that ADLumin-1 can efficiently cross the blood-brain barrier and provides excellent contrast for Aß plaques and cerebral amyloid angiopathy. In vivo brain imaging shows that the chemiluminescence signal of ADLumin-1 from 5-month-old transgenic 5xFAD mice is 1.80-fold higher than that from the age-matched wild-type mice. Moreover, we demonstrate that it is feasible to further dually-amplify signal via chemiluminescence resonance energy transfer (DAS-CRET) using two non-conjugated smart probes (ADLumin-1 and CRANAD-3) in solutions, brain homogenates, and in vivo whole brain imaging. Our results show that DAS-CRET can provide a 2.25-fold margin between 5-month-old 5xFAD mice and wild type mice. We believe that our strategy could be extended to other aggregating-prone proteins.


Assuntos
Peptídeos beta-Amiloides/química , Luminescência , Animais , Medições Luminescentes/métodos , Camundongos , Imagem Molecular/métodos , Imagem Óptica/métodos , Agregados Proteicos
5.
PLoS One ; 15(8): e0236397, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32756566

RESUMO

Self-contained imaging systems are versatile instruments that are becoming a staple in cell culture laboratories. Many of these machines possess motorized stages and on-stage incubators that permit programmable imaging of live cells that make them a sensible tool for high-throughput applications. The EVOS imaging system is such a device and is capable of scanning multi-well dishes and stitching together multiple adjacent fields to produce coherent individual images of each well. Automated batch analysis and quantification of these tiled images does however require off-loading files to other software platforms. Our initial attempts to quantify tiled images captured on an EVOS device was plagued by some expected-and other unforeseeable-issues that arose at nearly every stage of analysis. These included: high background, illumination and stitching artifacts, low contrast, noise, focus inconsistencies, and image distortion-all of which negatively impacted processing efficiency. We have since overcome these obstacles and have created a rigorous cell counting pipeline for analyzing images captured by the EVOS scan function. We present development and optimization of this automated pipeline and submit it as an effective and facile tool for accurately counting cells from tiled images.


Assuntos
Técnicas de Cultura de Células/métodos , Rastreamento de Células/métodos , Processamento de Imagem Assistida por Computador/métodos , Software , Humanos , Células MCF-7 , Imagem Óptica/métodos
6.
PLoS One ; 15(7): e0236245, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32706818

RESUMO

We have previously demonstrated that endothelial targeting of gold nanoparticles followed by external beam irradiation can cause specific tumor vascular disruption in mouse models of cancer. The induced vascular damage may lead to changes in tumor physiology, including tumor hypoxia, thereby compromising future therapeutic interventions. In this study, we investigate the dynamic changes in tumor hypoxia mediated by targeted gold nanoparticles and clinical radiation therapy (RT). By using noninvasive whole-body fluorescence imaging, tumor hypoxia was measured at baseline, on day 2 and day 13, post-tumor vascular disruption. A 2.5-fold increase (P<0.05) in tumor hypoxia was measured two days after combined therapy, resolving by day 13. In addition, the combination of vascular-targeted gold nanoparticles and radiation therapy resulted in a significant (P<0.05) suppression of tumor growth. This is the first study to demonstrate the tumor hypoxic physiological response and recovery after delivery of vascular-targeted gold nanoparticles followed by clinical radiation therapy in a human non-small cell lung cancer athymic Foxn1nu mouse model.


Assuntos
Carcinoma Pulmonar de Células não Pequenas , Neoplasias Pulmonares , Nanopartículas Metálicas/uso terapêutico , Hipóxia Tumoral , Células A549 , Animais , Carcinoma Pulmonar de Células não Pequenas/irrigação sanguínea , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/radioterapia , Ouro/uso terapêutico , Humanos , Neoplasias Pulmonares/irrigação sanguínea , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/radioterapia , Camundongos , Camundongos Nus , Imagem Óptica/métodos , Hipóxia Tumoral/efeitos dos fármacos , Hipóxia Tumoral/efeitos da radiação , Ensaios Antitumorais Modelo de Xenoenxerto
7.
Nat Commun ; 11(1): 2933, 2020 06 10.
Artigo em Inglês | MEDLINE | ID: mdl-32523065

RESUMO

Optical probes operating in the second near-infrared window (NIR-II, 1,000-1,700 nm), where tissues are highly transparent, have expanded the applicability of fluorescence in the biomedical field. NIR-II fluorescence enables deep-tissue imaging with micrometric resolution in animal models, but is limited by the low brightness of NIR-II probes, which prevents imaging at low excitation intensities and fluorophore concentrations. Here, we present a new generation of probes (Ag2S superdots) derived from chemically synthesized Ag2S dots, on which a protective shell is grown by femtosecond laser irradiation. This shell reduces the structural defects, causing an 80-fold enhancement of the quantum yield. PEGylated Ag2S superdots enable deep-tissue in vivo imaging at low excitation intensities (<10 mW cm-2) and doses (<0.5 mg kg-1), emerging as unrivaled contrast agents for NIR-II preclinical bioimaging. These results establish an approach for developing superbright NIR-II contrast agents based on the synergy between chemical synthesis and ultrafast laser processing.


Assuntos
Imagem Óptica/métodos , Fotoquímica/métodos , Corantes Fluorescentes , Nanopartículas/química , Pontos Quânticos
8.
Science ; 368(6496): 1265-1269, 2020 06 12.
Artigo em Inglês | MEDLINE | ID: mdl-32527834

RESUMO

CRISPR-Cas systems provide versatile tools for programmable genome editing. Here, we developed a caged RNA strategy that allows Cas9 to bind DNA but not cleave until light-induced activation. This approach, referred to as very fast CRISPR (vfCRISPR), creates double-strand breaks (DSBs) at the submicrometer and second scales. Synchronized cleavage improved kinetic analysis of DNA repair, revealing that cells respond to Cas9-induced DSBs within minutes and can retain MRE11 after DNA ligation. Phosphorylation of H2AX after DNA damage propagated more than 100 kilobases per minute, reaching up to 30 megabases. Using single-cell fluorescence imaging, we characterized multiple cycles of 53BP1 repair foci formation and dissolution, with the first cycle taking longer than subsequent cycles and its duration modulated by inhibition of repair. Imaging-guided subcellular Cas9 activation further facilitated genomic manipulation with single-allele resolution. vfCRISPR enables DNA-repair studies at high resolution in space, time, and genomic coordinates.


Assuntos
Proteína 9 Associada à CRISPR , Sistemas CRISPR-Cas , Clivagem do DNA/efeitos da radiação , Reparo do DNA/genética , Edição de Genes/métodos , Análise de Célula Única/métodos , Quebras de DNA de Cadeia Dupla , Células HEK293 , Histonas/metabolismo , Humanos , Luz , Proteína Homóloga a MRE11/genética , Imagem Óptica/métodos , Fosforilação
9.
Food Chem ; 331: 127221, 2020 Nov 30.
Artigo em Inglês | MEDLINE | ID: mdl-32540697

RESUMO

Herein, a two-photon (TP) ratiometric fluorescent probe (NpFA) was developed for detecting formaldehyde (FA) in real food samples, living onion tissues and zebrafish by fluorescence resonance energy transfer (FRET) strategy. Specifically, a TP fluorophore as the donor and a FA turn-on naphthalimide fluorophore as the acceptor were connected by a non-conjugated linker to construct the TP-FRET-based NpFA, which exhibited a target-modulated ratiometric fluorescence response to FA rapidly with high selectivity and sensitivity during 65 s, and a large ratio ~5-fold enhancement at I550/I410 after addition of FA, displaying ~60-fold enhancement at 550 nm and a quite low DOC of 5.8 ± 0.2 nM. Moreover, NpFA has a good imaging resolution and depth of deep tissue penetration. Therefore, based on the above results, NpFA has the capability to be a useful tool for investigating FA in real samples application, and we also hope NpFA will further study of the physiological and pathological function of FA.


Assuntos
Corantes Fluorescentes/química , Análise de Alimentos/métodos , Formaldeído/análise , Microscopia Confocal/métodos , Imagem Óptica/métodos , Peixe-Zebra/metabolismo , Animais , Cromatografia em Camada Delgada , Transferência Ressonante de Energia de Fluorescência , Formaldeído/metabolismo , Células HeLa , Humanos
10.
J Surg Oncol ; 122(2): 226-233, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32436255

RESUMO

BACKGROUND AND OBJECTIVES: Recently, PINPOINT, a novel laparoscopic fusion indocyanine green fluorescence imaging (IGFI) system has become available for laparoscopic liver resection. This study aims to characterize fluorescence patterns of intrahepatic cholangiocarcinoma (ICC) using the negative counterstaining method in laparoscopic anatomical hepatectomies of ICC. METHODS: Eleven consecutive patients, diagnosed with intrahepatic cholangiocarcinoma and underwent laparoscopic liver resection between April 2017 and December 2018, were retrospectively reviewed. A laparoscopic IGFI navigation system was used to characterize fluorescence patterns of ICC with intraoperative liver segment demarcation by means of negative counterstaining. RESULTS: Fusion IGFI of ICC was successfully obtained from all 11 patients from the surgical specimens. The fluorescence patterns of ICC can be categorized into rim-type fluorescence and segmental fluorescence, depending on tumor growth. In eight patients, indocyanine green fluorescence imaging was used to identify the hepatic lobes or segments by negative counterstaining. In six cases, a valid and persistent demarcation was achieved intraoperatively. CONCLUSION: Laparoscopic IGFI system could identify different types of ICC lesions and may facilitate real-time navigation for laparoscopic anatomic liver resection of ICC.


Assuntos
Neoplasias dos Ductos Biliares/diagnóstico por imagem , Neoplasias dos Ductos Biliares/cirurgia , Colangiocarcinoma/diagnóstico por imagem , Colangiocarcinoma/cirurgia , Verde de Indocianina/administração & dosagem , Imagem Óptica/métodos , Idoso , Idoso de 80 Anos ou mais , Corantes/administração & dosagem , Estudos de Viabilidade , Feminino , Humanos , Período Intraoperatório , Laparoscopia , Masculino , Pessoa de Meia-Idade , Projetos Piloto , Cuidados Pré-Operatórios/métodos , Estudos Retrospectivos , Coloração e Rotulagem/métodos
11.
Lancet Gastroenterol Hepatol ; 5(8): 753-764, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32416764

RESUMO

BACKGROUND: Complete surgical resection remains the primary curative option for pancreatic ductal adenocarcinoma, with positive margins in 30-70% of patients. In this study, we aimed to evaluate the use of intraoperative tumour-specific imaging to enhance a surgeon's ability to detect visually occult cancer in real time. METHODS: In this single-centre, open-label, single-arm study, done in the USA, we enrolled patients who had clinically suspicious or biopsy-confirmed pancreatic ductal adenocarcinomas and were scheduled for curative surgery. Eligible patients were 19 years of age or older with a life expectancy of more than 12 weeks and a Karnofsky performance status of at least 70% or an Eastern Cooperative Oncology Group or Zubrod level of one or lower, who were scheduled to undergo curative surgery. Patients were sequentially enrolled into each dosing group and 2-5 days before surgery, patients were intravenously infused with 100 mg of unlabelled panitumumab followed by 25 mg, 50 mg, or 75 mg of the near-infrared fluorescently labelled antibody (panitumumab-IRDye800CW). The primary endpoint was to determine the optimal dose of panitumumab-IRDye800CW in identifying pancreatic ductal adenocarcinomas as measured by tumour-to-background ratio in all patients. The tumour-to-background ratio was defined as the fluorescence signal of the tumour divided by the fluorescence signal of the surrounding healthy tissue. The dose-finding part of this study has been completed. This study is registered with ClinicalTrials.gov, NCT03384238. FINDINGS: Between April, 2018, and July, 2019, 16 patients were screened for enrolment onto the study. Of the 16 screened patients, two (12%) patients withdrew from the study and three (19%) were not eligible; 11 (69%) patients completed the trial, all of whom were clinically diagnosed with pancreatic ductal adenocarcinoma. The mean tumour-to-background ratio of primary tumours was 3·0 (SD 0·5) in the 25 mg group, 4·0 (SD 0·6) in the 50 mg group, and 3·7 (SD 0·4) in the 75 mg group; the optimal dose was identified as 50 mg. Intraoperatively, near-infrared fluorescence imaging provided enhanced visualisation of the primary tumours, metastatic lymph nodes, and small (<2 mm) peritoneal metastasis. Intravenous administration of panitumumab-IRDye800CW at the doses of 25 mg, 50 mg, and 75 mg did not result in any grade 3 or higher adverse events. There were no serious adverse events attributed to panitumumab-IRDye800CW, although four possibly related adverse events (grade 1 and 2) were reported in four patients. INTERPRETATION: To our knowledge, this study presents the first clinical use of panitumumab-IRDye800CW for detecting pancreatic ductal adenocarcinomas and shows that panitumumab-IRDye800CW is safe and feasible to use during pancreatic cancer surgery. Tumour-specific intraoperative imaging might have added value for treatment of patients with pancreatic ductal adenocarcinomas through improved patient selection and enhanced visualisation of surgical margins, metastatic lymph nodes, and distant metastasis. FUNDING: National Institutes of Health and the Netherlands Organization for Scientific Research.


Assuntos
Antineoplásicos Imunológicos/administração & dosagem , Benzenossulfonatos/administração & dosagem , Carcinoma Ductal Pancreático/cirurgia , Indóis/administração & dosagem , Imagem Óptica/métodos , Neoplasias Pancreáticas/patologia , Panitumumabe/administração & dosagem , Adulto , Idoso , Idoso de 80 Anos ou mais , Carcinoma Ductal Pancreático/diagnóstico por imagem , Carcinoma Ductal Pancreático/tratamento farmacológico , Feminino , Humanos , Infusões Intravenosas/métodos , Período Intraoperatório , Metástase Linfática/diagnóstico por imagem , Masculino , Margens de Excisão , Pessoa de Meia-Idade , Estadiamento de Neoplasias/métodos , Países Baixos/epidemiologia , Neoplasias Peritoneais/diagnóstico por imagem , Neoplasias Peritoneais/secundário
12.
Neuron ; 106(3): 369-387, 2020 05 06.
Artigo em Inglês | MEDLINE | ID: mdl-32380050

RESUMO

Tissue clearing and light-sheet microscopy have a 100-year-plus history, yet these fields have been combined only recently to facilitate novel experiments and measurements in neuroscience. Since tissue-clearing methods were first combined with modernized light-sheet microscopy a decade ago, the performance of both technologies has rapidly improved, broadening their applications. Here, we review the state of the art of tissue-clearing methods and light-sheet microscopy and discuss applications of these techniques in profiling cells and circuits in mice. We examine outstanding challenges and future opportunities for expanding these techniques to achieve brain-wide profiling of cells and circuits in primates and humans. Such integration will help provide a systems-level understanding of the physiology and pathology of our central nervous system.


Assuntos
Encéfalo/citologia , Imageamento Tridimensional/métodos , Imagem Óptica/métodos , Coloração e Rotulagem/métodos , Animais , Encéfalo/fisiologia , Humanos , Microscopia/métodos
13.
Surgery ; 168(1): 178-184, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32223983

RESUMO

BACKGROUND: Fluorescence-based enhanced reality is a software that provides quantitative fluorescence angiography by computing the fluorescence intensity time-to-peak after intravenous indocyanine green. Hyperspectral imaging is a contrast-free, optical imaging modality which measures tissue oxygenation. METHODS: In 8 pigs, an ischemic bowel segment created by dividing the arcade branches was imaged using hyperspectral imaging and fluorescence-based enhanced reality. Tissue oxygenation values were acquired through a hyperspectral imaging system. Subsequently, fluorescence angiography was performed using a near-infrared laparoscopic camera after intravenous injection of 0.2 mg/kg of indocyanine green. The time-to-peak fluorescence signal was analyzed through a proprietary software to realize a perfusion map. This was overlaid onto real-time images to obtain fluorescence-based enhanced reality. Simultaneously, 9 adjacent regions of interest were selected and superimposed onto the real-time video, thereby obtaining hyperspectral-based enhanced reality. Fluorescence-based enhanced reality and hyperspectral-based enhanced reality were superimposed allowing a comparison of both imaging modalities. Local capillary lactate levels were sampled at the regions of interest. Two prediction models using the local capillary lactate levels were extrapolated based on both imaging systems. RESULTS: For all regions of interest, the mean local capillary lactate levels were 4.67 ± 4.34 mmol/L, the mean tissue oxygenation was 45.9 ± 18.9%, and the mean time-to-peak was 10 ± 9.4 seconds. Pearson's test between fluorescence-based enhanced reality-time-to-peak and hyperspectral imaging-tissue oxygenation at the corresponding regions of interest gave an R = -0.66 (P < .0001). The hyperspectral imaging lactate prediction model proved more accurate than the fluorescence-based enhanced reality-based model (P < .0001). CONCLUSION: Bowel perfusion was quantified using hyperspectral imaging and fluorescence angiography. Hyperspectral imaging yielded more accurate results than fluorescence angiography. Hyperspectral-based enhanced reality may prove to be a useful, contrast-free intraoperative tool to quantify bowel ischemia.


Assuntos
Angiofluoresceinografia , Verde de Indocianina , Enteropatias/diagnóstico por imagem , Isquemia/diagnóstico por imagem , Imagem Óptica/métodos , Animais , Ácido Láctico/análise , Masculino , Oxigênio/análise , Suínos
14.
Invest Ophthalmol Vis Sci ; 61(4): 36, 2020 04 09.
Artigo em Inglês | MEDLINE | ID: mdl-32334431

RESUMO

Purpose: To examine the extent of visual function abnormality outside the dark lesion on short-wavelength fundus autofluorescence (SW-AF), and its correlation with background SW-AF features and optical coherence tomography (OCT) in recessive Stargardt disease (STGD1). Methods: Forty-nine eyes of 25 participants in the ProgStar (the Natural History of the Progression of Atrophy Secondary to Stargardt Disease) study at our center were included. Patients underwent microperimetry (both threshold and dense scotoma mapping), OCT, SW-AF, and visual acuity testing. The Fisher's exact test, the χ2 test, and unpaired t-tests were used to analyze the data. Results: Of 40 eyes without central fixation, 33 (82%) placed fixation remote (most ≥5°) from the dense scotoma edge, despite good intervening retinal sensitivity. OCT findings accounted for the remote fixation in 75%. Eighteen (37%) of all 49 eyes had dense scotoma extending past the dark lesion border. OCT was not adequate to define the edge of the scotoma. Of the 49 eyes, 28 (57%) had the mottled background pattern, 10 (20%) had the uniform pattern, and 11 (22%) had the other pattern, with >75% of eyes in each pattern having remote fixation. The dense scotoma exceeded the dark lesion primarily in the mottled pattern. The two eyes of each patient were concordant in all features. Conclusions: Functional abnormalities in STGD1 extend past the SW-AF dark lesion. The disruption of the ellipsoid zone shows that photoreceptor abnormality extends peripheral to the dark lesion, and it explains in part the remote fixation pattern and the dense scotoma exceeding the dark lesion. This has implications for clinical trials for STGD1.


Assuntos
Imagem Óptica/métodos , Doença de Stargardt/diagnóstico por imagem , Doença de Stargardt/patologia , Tomografia de Coerência Óptica/métodos , Transtornos da Visão/diagnóstico , Adulto , Estudos de Coortes , Fundo de Olho , Humanos , Pessoa de Meia-Idade , Prognóstico , Estudos Prospectivos , Medição de Risco , Escotoma/diagnóstico por imagem , Escotoma/patologia , Índice de Gravidade de Doença , Transtornos da Visão/etiologia , Acuidade Visual , Testes de Campo Visual/métodos
15.
Yakugaku Zasshi ; 140(4): 513-519, 2020.
Artigo em Japonês | MEDLINE | ID: mdl-32238634

RESUMO

Repair of injured tissues requires angiogenesis, the growth of new blood vessels from pre-existing ones. Cutaneous wound healing is a complex and dynamic process by which skin tissue repairs itself after injury; however, how endothelial cells and pericytes form new blood vessels during cutaneous wound angiogenesis remains unclear. We recently developed a fluorescence-based live imaging system to analyze cutaneous wound angiogenesis in adult zebrafish. Employing this system, we found that endothelial cells and pericytes remain in a quiescent state in normal skin tissue, whereas cutaneous injury immediately activates both types of cells to induce angiogenesis. At 2 days post-injury (dpi), the injured vessels elongated, and some uninjured vessels became tortuous and began to sprout new branches. Then, vessel sprouting, elongation, bifurcation, and anastomosis progressively occurred to form the tortuous and disorganized vascular networks observed at 6 dpi. Thereafter, blood vessel tortuosity gradually decreased through the regression of excessive vessels, thereby leading to the formation of well-organized vessel networks at 42 dpi. Pericytes are thought to detach from the vessel wall to promote endothelial cell sprouting upon the induction of angiogenesis. However, not only endothelial cells but also pericytes proliferated to form pericyte-covered tortuous blood vessels in response to cutaneous injury, revealing an unexpected role of pericytes in cutaneous wound angiogenesis. Therefore, this live-imaging system for adult zebrafish is anticipated to make a valuable contribution to research advancements in understanding the angiogenesis that occurs during tissue repair.


Assuntos
Neovascularização Fisiológica/fisiologia , Imagem Óptica/métodos , Fenômenos Fisiológicos da Pele , Cicatrização/fisiologia , Animais , Células Endoteliais/fisiologia , Humanos , Modelos Animais , Pericitos/fisiologia , Peixe-Zebra
16.
J Vis Exp ; (157)2020 03 27.
Artigo em Inglês | MEDLINE | ID: mdl-32281971

RESUMO

Amyloid fibrils are associated with a number of neurodegenerative diseases such as Huntington's, Parkinson's, or Alzheimer's disease. These amyloid fibrils can sequester endogenous metastable proteins as well as components of the proteostasis network (PN) and thereby exacerbate protein misfolding in the cell. There are a limited number of tools available to assess the aggregation process of amyloid proteins within an animal. We present a protocol for fluorescence lifetime microscopy (FLIM) that allows monitoring as well as quantification of the amyloid fibrilization in specific cells, such as neurons, in a noninvasive manner and with the progression of aging and upon perturbation of the PN. FLIM is independent of the expression levels of the fluorophore and enables an analysis of the aggregation process without any further staining or bleaching. Fluorophores are quenched when they are in close vicinity of amyloid structures, which results in a decrease of the fluorescence lifetime. The quenching directly correlates with the aggregation of the amyloid protein. FLIM is a versatile technique that can be applied to compare the fibrilization process of different amyloid proteins, environmental stimuli, or genetic backgrounds in vivo in a non-invasive manner.


Assuntos
Caenorhabditis elegans/metabolismo , Fluorescência , Imagem Óptica/métodos , Animais
17.
World Neurosurg ; 139: 343, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32272265

RESUMO

Fluorescence-guided resection of brain tumors using 5-aminolevulinic acid (5-ALA) has been established for high-grade gliomas. Recently, its application for the resection of low grade tumors and benign lesions including meningioma has been suggested in the literature.1 Achieving a Simpson grade I resection in meningioma surgery is associated with a lower rate of recurrence.2,3 Although meningiomas are mostly benign and well-circumscribed lesions, they can be locally aggressive, invading brain parenchyma and other critical structures. In these cases, 5-ALA-guided resection may help maximize the extent of tumor resection and limit disruption of normal structures. In this video, we present 3 cases demonstrating the use of 5-ALA-induced fluorescence to alleviate 3 specific challenges in meningioma resection: 1) to aid visualization with a minimally invasive approach, 2) to distinguish recurrent tumor from scar tissue from prior treatments, and 3) to ensure that no viable tumor cells remain on the surface of a critical artery. The first patient is a 60-year-old woman who was found to have an incidental left sphenoid wing meningioma on magnetic resonance imaging. We elected for an extended lateral orbital craniotomy through a transpalpebral approach. The second patient is a 72-year-old man with recurrent left occipital parietal meningioma who underwent a parietal craniotomy. The third case was a 62-year-old woman with a foramen magnum meningioma encircling the left vertebral artery. These cases demonstrate the utility of 5-ALA in a variety of challenges associated with resection of meningiomas (Video 1).


Assuntos
Ácido Aminolevulínico , Neoplasias Meníngeas/cirurgia , Meningioma/cirurgia , Procedimentos Neurocirúrgicos/métodos , Imagem Óptica/métodos , Idoso , Feminino , Humanos , Masculino , Microcirurgia , Pessoa de Meia-Idade , Neoplasias da Base do Crânio/cirurgia
19.
Nat Commun ; 11(1): 1868, 2020 04 20.
Artigo em Inglês | MEDLINE | ID: mdl-32313067

RESUMO

In today's clinics, a cell-resolution view of the cornea can be achieved only with a confocal microscope (IVCM) in contact with the eye. Here, we present a common-path full-field/spectral-domain OCT microscope (FF/SD OCT), which enables cell-detail imaging of the entire ocular surface in humans (central and peripheral cornea, limbus, sclera, tear film) without contact and in real-time. Real-time performance is achieved through rapid axial eye tracking and simultaneous defocusing correction. Images contain cells and nerves, which can be quantified over a millimetric field-of-view, beyond the capability of IVCM and conventional OCT. In the limbus, palisades of Vogt, vessels, and blood flow can be resolved with high contrast without contrast agent injection. The fast imaging speed of 275 frames/s (0.6 billion pixels/s) allows direct monitoring of blood flow dynamics, enabling creation of high-resolution velocity maps. Tear flow velocity and evaporation time can be measured without fluorescein administration.


Assuntos
Angiografia/instrumentação , Angiografia/métodos , Córnea/diagnóstico por imagem , Tomografia de Coerência Óptica/instrumentação , Tomografia de Coerência Óptica/métodos , Adulto , Engenharia Biomédica/instrumentação , Engenharia Biomédica/métodos , Velocidade do Fluxo Sanguíneo , Córnea/patologia , Desenho de Equipamento , Feminino , Humanos , Limbo da Córnea/diagnóstico por imagem , Limbo da Córnea/patologia , Masculino , Microscopia/métodos , Imagem Óptica/instrumentação , Imagem Óptica/métodos , Software , Adulto Jovem
20.
PLoS One ; 15(4): e0231488, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32315347

RESUMO

PURPOSE: Diagnosis and resection of indeterminate pulmonary nodules (IPNs) is a growing challenge with increased utilization of chest computed tomography. Photoacoustic (PA) -guided surgical resection with local injection of indocyanine green (ICG) may have utility for IPNs that are suspicious for lung cancer. This preclinical study explores the potential of PA imaging (PAI) to detect ICG-labeled tumors. MATERIALS AND METHODS: ICG uptake by H460 lung cancer cells was evaluated in vitro. A phantom study was performed to analyze PA signal intensity according to ICG concentration and tissue thickness/depth using chicken breast. PA signals were measured up to 48 hours after injection of ICG (mixed with 5% agar) into healthy subcutaneous tissue, subcutaneous H460 tumors and right healthy lung in nude mice. RESULTS: Intracellular ICG fluorescence was detected in H460 cells co-incubated with ICG in vitro. The concentration dependence of the PA signal was logarithmic, and PA signal decline was exponential with increasing tissue depth. The PA signal of 2 mg/mL ICG was still detectable at a depth of 22 mm in chicken breast. The PA signal from ICG mixed with agar was detectable 48 hours post injection into subcutaneous tissue and subcutaneous H460 tumors in nude mice. Similar features of PA signals from ICG-agar in mice lung were obtained. CONCLUSION: The results from this preclinical study suggests that PAI of injected ICG-agar may be beneficial for identifying deeply located tumors. These features may be valuable for IPNs.


Assuntos
Nódulos Pulmonares Múltiplos/diagnóstico por imagem , Nódulos Pulmonares Múltiplos/patologia , Técnicas Fotoacústicas/métodos , Animais , Linhagem Celular Tumoral , Feminino , Fluorescência , Humanos , Verde de Indocianina/administração & dosagem , Camundongos , Camundongos Nus , Imagem Óptica/métodos , Imagens de Fantasmas
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