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1.
Int J Mol Sci ; 22(12)2021 Jun 17.
Artigo em Inglês | MEDLINE | ID: mdl-34204524

RESUMO

The aim of this work is to develop a biomimetic interface between the natural tooth tissue and the restorative composite and to study it on the basis of synchrotron micro-FTIR mapping and multidimensional processing of the spectral data array. Using hierarchical cluster analysis of 3D FTIR data revealed marked improvements in the formation of the dentine/adhesive/dental hybrid interface using a biomimetic approach. The use of a biomimetic strategy (application of an amino acid-modified primer, alkaline calcium and a nano-c-HAp-modified adhesive) allowed the formation of a matrix that can be structurally integrated with natural dentine and dental composite. The biomimetic hybrid layer was characterised by homogeneous chemical composition and a higher degree of conversion of the adhesive during polymerisation, which should provide optimal integration of the dental composite with the dentine.


Assuntos
Biomimética , Odontologia , Espectroscopia de Infravermelho com Transformada de Fourier , Síncrotrons , Engenharia Tecidual , Dente , Biomimética/métodos , Odontologia/métodos , Humanos , Teste de Materiais , Nanotecnologia , Imagem Óptica/métodos
2.
Molecules ; 26(12)2021 Jun 19.
Artigo em Inglês | MEDLINE | ID: mdl-34205289

RESUMO

The inadvertent severing of a ureter during surgery occurs in as many as 4.5% of colorectal surgeries. To help prevent this issue, several near-infrared (NIR) dyes have been developed to assist surgeons with identifying ureter location. However, the majority of these dyes exhibit at least some issue that precludes their widespread usage such as high levels of uptake in other tissues, overlapping emission wavelengths with other NIR dyes used for other fluorescence-guided surgeries, and/or rapid excretion times through the ureters. To overcome these limitations, we have synthesized and characterized the spectral properties and biodistribution of a new series of PEGylated UreterGlow derivatives. The most promising dye, UreterGlow-11 was shown to almost exclusively excrete through the kidneys/ureters with detectable fluorescence observed for at least 12 h. Additionally, while the excitation wavelength is similar to that of other NIR dyes used for cancer resections, the emission is shifted by ~30 nm allowing for discrimination between the different fluorescence-guided surgery probes. In conclusion, these new UreterGlow dyes show promising optical and biodistribution characteristics and are good candidates for translation into the clinic.


Assuntos
Abdome/cirurgia , Imagem Óptica/métodos , Espectroscopia de Luz Próxima ao Infravermelho/métodos , Ureter/cirurgia , Animais , Fluorescência , Corantes Fluorescentes/metabolismo , Humanos , Rim/cirurgia , Camundongos , Distribuição Tecidual/fisiologia , Ureter/metabolismo
3.
Methods Mol Biol ; 2276: 193-202, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34060042

RESUMO

Brain is one of the most energy-demanding organs. Energy in the form of ATP is produced in brain cells predominantly in oxidative phosphorylation coupled to mitochondrial respiration. Any alteration of the mitochondrial metabolism or prolonged ischemic or anoxic conditions can lead to serious neurological conditions, including neurodegenerative disorders. Assessment of mitochondrial metabolism is important for understanding physiological and pathological processes in the brain. Bioenergetics in central nervous system is dependent on multiple parameters including neuron-glia interactions and considering this, in vivo or ex vivo, the measurements of mitochondrial metabolism should also be complimenting the experiments on isolated mitochondria or cell cultures. To assess the mitochondrial function, there are several key bioenergetic parameters which indicate mitochondrial health. One of the major characteristics of mitochondria is the mitochondrial membrane potential (ΔΨm) which is used as a proton motive force for ATP production and generated by activity of the electron transport chain. Major donor of electrons for the mitochondrial respiratory chain is NADH. Here we demonstrate how to measure mitochondrial NADH/NAD(P)H autofluorescence and ΔΨm in acute brain slices in a time-dependent manner and provide information for the identification of NADH redox index, mitochondrial NADH pool, and the rate of NADH production in the Krebs cycle. Additionally, non-mitochondrial NADH/NADPH autofluorescence can signify the level of activity of the pentose phosphate pathway.


Assuntos
Encéfalo/metabolismo , Potencial da Membrana Mitocondrial/fisiologia , Mitocôndrias/metabolismo , NADP/metabolismo , NAD/metabolismo , Imagem Óptica/métodos , Animais , Química Encefálica , Mitocôndrias/química , NAD/análise , NADP/análise , Oxirredução , Fosforilação Oxidativa
4.
Methods Mol Biol ; 2276: 259-270, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34060048

RESUMO

Mitochondrial dysfunction contributes to various injuries and diseases. A mechanistic understanding of how dysfunctional mitochondria modulates metabolism is of paramount importance. Three-dimensional (3D) optical cryo-imager is a custom-designed device that can quantify the volumetric bioenergetics of organs in small animal models. The instrument captures the autofluorescence of bioenergetics indices (NADH and FAD) from tissues at cryogenic temperature. The quantified redox ratio (NADH/FAD) is used as an optical indicator of mitochondrial redox state.


Assuntos
Flavina-Adenina Dinucleotídeo/análise , Imageamento Tridimensional/métodos , Rim/química , Mitocôndrias/química , NAD/análise , Imagem Óptica/métodos , Animais , Criopreservação , Metabolismo Energético , Flavina-Adenina Dinucleotídeo/metabolismo , Secções Congeladas , Rim/metabolismo , Rim/patologia , Mitocôndrias/metabolismo , Mitocôndrias/patologia , NAD/metabolismo , Oxirredução
5.
Methods Mol Biol ; 2276: 285-303, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34060050

RESUMO

Changes to mitochondrial architecture are associated with various adaptive and pathogenic processes. However, quantification of changes to mitochondrial structures is limited by the yet unmet challenge of defining the borders of each individual mitochondrion within an image. Here, we describe a novel method for segmenting primary brown adipocyte (BA) mitochondria images. We describe a granular approach to quantifying subcellular structures, particularly mitochondria in close proximity to lipid droplets: peridroplet mitochondria. In addition, we lay out a novel machine-learning-based mitochondrial segmentation method that eliminates the bias of manual mitochondrial segmentation and improves object recognition compared to conventional thresholding analyses. By applying these methods, we discovered a significant difference between cytosolic and peridroplet BA mitochondrial H2O2 production and validated the machine-learning algorithm in BA via norepinephrine-induced mitochondrial fragmentation and comparing manual analyses to the automated analysis. This approach provides a high-throughput analysis protocol to quantify ratiometric probes in subpopulations of mitochondria in adipocytes.


Assuntos
Adipócitos Marrons/metabolismo , Ensaios de Triagem em Larga Escala/métodos , Processamento de Imagem Assistida por Computador/métodos , Gotículas Lipídicas/metabolismo , Aprendizado de Máquina , Mitocôndrias/metabolismo , Imagem Óptica/métodos , Adipócitos Marrons/citologia , Algoritmos , Humanos , Gotículas Lipídicas/química , Mitocôndrias/ultraestrutura
6.
J Vis Exp ; (172)2021 06 05.
Artigo em Inglês | MEDLINE | ID: covidwho-1278530

RESUMO

As the COVID-19 pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to evolve, it has become evident that the presence of neutralizing antibodies against the virus may provide protection against future infection. Thus, as the creation and translation of effective COVID-19 vaccines continues at an unprecedented speed, the development of fast and effective methods to measure neutralizing antibodies against SARS-CoV-2 will become increasingly important to determine long-term protection against infection for both previously infected and immunized individuals. This paper describes a high-throughput protocol using vesicular stomatitis virus (VSV) pseudotyped with the SARS-CoV-2 spike protein to measure the presence of neutralizing antibodies in convalescent serum from patients who have recently recovered from COVID-19. The use of a replicating pseudotyped virus eliminates the necessity for a containment level 3 facility required for SARS-CoV-2 handling, making this protocol accessible to virtually any containment level 2 lab. The use of a 96-well format allows for many samples to be run at the same time with a short turnaround time of 24 h.


Assuntos
Anticorpos Neutralizantes/análise , Anticorpos Antivirais/análise , Teste Sorológico para COVID-19/métodos , COVID-19/imunologia , Imagem Óptica/métodos , SARS-CoV-2/imunologia , Glicoproteína da Espícula de Coronavírus/imunologia , Vacinas contra COVID-19/imunologia , Humanos , Testes de Neutralização , Vírus da Estomatite Vesicular Indiana/imunologia
7.
Int J Mol Sci ; 22(10)2021 May 14.
Artigo em Inglês | MEDLINE | ID: mdl-34069002

RESUMO

Precise measurement of particulate matter (PM) on skin is important for managing and preventing PM-related skin diseases. This study aims to directly visualize the deposition and penetration of PM into human skin using a multimodal nonlinear optical (MNLO) imaging system. We successfully obtained PM particle signals by merging two different sources, C-C vibrational frequency and autofluorescence, while simultaneously visualizing the anatomical features of the skin via keratin, collagen, and elastin. As a result, we found morphologically dependent PM deposition, as well as increased deposition following disruption of the skin barrier via tape-stripping. Furthermore, PM penetrated more and deeper into the skin with an increase in the number of tape-strippings, causing a significant increase in the secretion of pro-inflammatory cytokines. Our results suggest that MNLO imaging could be a useful technique for visualizing and quantifying the spatial distribution of PM in ex vivo human skin tissues.


Assuntos
Imagem Multimodal/métodos , Imagem Óptica/métodos , Material Particulado/análise , Dermatopatias/diagnóstico , Pele/metabolismo , Humanos , Dermatopatias/metabolismo
8.
J Vis Exp ; (172)2021 06 05.
Artigo em Inglês | MEDLINE | ID: mdl-34152313

RESUMO

As the COVID-19 pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to evolve, it has become evident that the presence of neutralizing antibodies against the virus may provide protection against future infection. Thus, as the creation and translation of effective COVID-19 vaccines continues at an unprecedented speed, the development of fast and effective methods to measure neutralizing antibodies against SARS-CoV-2 will become increasingly important to determine long-term protection against infection for both previously infected and immunized individuals. This paper describes a high-throughput protocol using vesicular stomatitis virus (VSV) pseudotyped with the SARS-CoV-2 spike protein to measure the presence of neutralizing antibodies in convalescent serum from patients who have recently recovered from COVID-19. The use of a replicating pseudotyped virus eliminates the necessity for a containment level 3 facility required for SARS-CoV-2 handling, making this protocol accessible to virtually any containment level 2 lab. The use of a 96-well format allows for many samples to be run at the same time with a short turnaround time of 24 h.


Assuntos
Anticorpos Neutralizantes/análise , Anticorpos Antivirais/análise , Teste Sorológico para COVID-19/métodos , COVID-19/imunologia , Imagem Óptica/métodos , SARS-CoV-2/imunologia , Glicoproteína da Espícula de Coronavírus/imunologia , Vacinas contra COVID-19/imunologia , Humanos , Testes de Neutralização , Vírus da Estomatite Vesicular Indiana/imunologia
9.
Methods Mol Biol ; 2274: 25-35, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34050459

RESUMO

Visualizing progression through the cell cycle provides valuable information for the study of development, tissue maintenance, and dysregulated growth in proliferative diseases, such as cancer. Developments in fluorescent biosensors have facilitated dynamic tracking of molecular processes, including the cell cycle. The genetically encoded set of fluorescent indicators, Fucci4, enables the visualization of transitions between each cell cycle phase. Here, we describe a method to track progression through each cell cycle phase using Fucci4 in live epifluorescence imaging. In principle, this approach can be adapted to in vitro time-lapse imaging of any four spectrally resolvable fluorescent indicators.


Assuntos
Técnicas Biossensoriais/métodos , Ciclo Celular , Fluorescência , Proteínas Luminescentes/metabolismo , Microscopia de Fluorescência/métodos , Imagem Óptica/métodos , Divisão Celular , Células HeLa , Humanos
10.
Methods Mol Biol ; 2274: 127-138, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34050468

RESUMO

Spectral overlaps in fluorescence (FL) and bioluminescence (BL) commonly cause optical cross talks. The present protocol introduces five different lineages of coelenterazine (CTZ) analogues, which have selectivity to a specific luciferase, and thus cross talk-free. For example, some CTZ analogues with ethynyl or styryl groups display dramatically biased BL to specific luciferases and pH by modifying the functional groups at the C-2 and C-6 positions of the imidazopyridinne backbone of CTZ. The optical cross talk-free feature is exemplified with the multiplex system, which simultaneously illuminated antiestrogenic and rapamycin activities without optical cross talks. This unique protocol contributes to specific and high-throughput BL imaging of multiple optical readouts in mammalian cells without optical contamination.


Assuntos
Alcinos/química , Imidazóis/química , Luciferases/metabolismo , Substâncias Luminescentes/química , Medições Luminescentes/métodos , Imagem Óptica/métodos , Pirazinas/química , Estirenos/química , Animais , Células COS , Chlorocebus aethiops , Moduladores de Receptor Estrogênico/farmacologia , Imunossupressores/farmacologia , Luciferases/efeitos dos fármacos , Sirolimo/farmacologia
11.
Methods Mol Biol ; 2274: 141-154, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34050469

RESUMO

Visualization of virus-infected cells is usually performed by immunostaining with an antiviral antibody. On the other hand, we established an easy method for fluorescence (FL) imaging of cells infected with influenza A and B viruses and some paramyxoviruses without the need for cell fixation and an antiviral antibody. These viruses and the cells they have infected express the viral surface enzyme "neuraminidase" or "hemagglutinin-neuraminidase" that shows sialidase activity. Sialidase activity is fluorescently visualized by using a sialidase fluorogenic probe developed in our previous study. The probe enables histochemical FL imaging of the virus-infected cells and is applicable to virus isolation and detection of an influenza virus resistant to antiinfluenza drugs of sialidase inhibitors.


Assuntos
Antivirais/farmacologia , Farmacorresistência Viral , Fluorescência , Neuraminidase/metabolismo , Imagem Óptica/métodos , Infecções por Orthomyxoviridae/metabolismo , Orthomyxoviridae/enzimologia , Animais , Células COS , Chlorocebus aethiops , Cães , Células Madin Darby de Rim Canino , Neuraminidase/genética , Orthomyxoviridae/efeitos dos fármacos , Orthomyxoviridae/isolamento & purificação , Infecções por Orthomyxoviridae/tratamento farmacológico , Infecções por Orthomyxoviridae/virologia , Células Vero
12.
Methods Mol Biol ; 2274: 169-179, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34050471

RESUMO

Microtubules (MTs) are important targets for imaging in living cells because of their vital roles in cellular processes. The dynamics (polymerization/depolymerization) of MTs has been imaged in living cells by utilizing MT-targeted drugs as scaffolds. We previously developed a unique MT-binding motif derived from a MT-associated protein, Tau. The Tau-derived peptide (TP) binds to the inner surface of MTs without inhibiting the dynamics of MTs. We introduce a new protocol for live-cell imaging of MTs by using fluorescently labeled TP. We exemplify that tetramethylrhodamine (TMR)-labeled TP (TP-TMR) is spontaneously internalized into HepG2 cells and binds to intracellular MTs, enabling visualization of MTs in living cells. TP-TMR shows no apparent effects on polymerization/depolymerization of MTs and no cytotoxicity. Thus, the peptide-based approach is useful for long-term imaging of MTs.


Assuntos
Fluorescência , Microtúbulos/metabolismo , Imagem Óptica/métodos , Fragmentos de Peptídeos/metabolismo , Rodaminas/química , Proteínas tau/metabolismo , Sobrevivência Celular , Células Hep G2 , Humanos , Fragmentos de Peptídeos/química
13.
Methods Mol Biol ; 2274: 247-259, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34050477

RESUMO

The present protocol demonstrates a novel mammalian cell imaging platform exerting a bioluminescence resonance energy transfer (BRET) system. This platform achieves a ~300 nm blue-to-near infrared shift of the emission (NIR-BRET) with the development of a unique coelenterazine (CTZ) derivative named BBlue2.3 and a fusion reporter protein probe named iRFP-RLuc8.6-535SG. The best NIR-BRET shift was achieved by tuning the blue emission peak of BBlue2.3 to a Soret band of the iRFP. In mammalian cells, BBlue2.3 emits light that is ~50-fold brighter than DeepBlueC in cell imaging when combined with RLuc8.6-535SG. This NIR-BRET platform is sufficiently brighter to be used for imaging live mammalian cells at single-cell level, and also for imaging metastases in deep tissues in live mice without generating considerable autoluminescence. This unique optical platform provides the brightest NIR-BLI template that can be used for imaging a diverse group of cellular events in living subjects.


Assuntos
Neoplasias da Mama/patologia , Transferência Ressonante de Energia de Fluorescência/métodos , Imidazóis/química , Luciferases/metabolismo , Medições Luminescentes/métodos , Imagem Óptica/métodos , Pirazinas/química , Espectroscopia de Luz Próxima ao Infravermelho/métodos , Animais , Apoptose , Neoplasias da Mama/metabolismo , Proliferação de Células , Feminino , Humanos , Substâncias Luminescentes/química , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de Xenoenxerto
14.
Methods Mol Biol ; 2274: 261-270, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34050478

RESUMO

Bioluminescence resonance energy transfer (BRET) is a commonly used assay system for studying protein-protein interactions. The present protocol introduces a conceptually unique ligand-activatable BRET system (termed BRET9), where a full-length artificial luciferase variant 23 (ALuc23), acting as the energy donor, is sandwiched in between a protein pair of interest, FRB and FKBP, and further linked to a fluorescent protein as the energy acceptor for studying protein-protein interaction. A specific ligand, rapamycin, which initiates intramolecular interactions of FRB and FKBP inside the probe, which develops molecular strain in the sandwiched ALuc23 to complete its folding, thus, the probe system greatly enhances both the overall bioluminescence (BL) spectrum and the BRET signal in the far-red (FR) region. This new BRET system provides a robust ligand-activatable platform that efficiently reports FR-BL signals in mammalian cells.


Assuntos
Neoplasias da Mama/patologia , Transferência Ressonante de Energia de Fluorescência/métodos , Luciferases/metabolismo , Medições Luminescentes/métodos , Imagem Óptica/métodos , Mapeamento de Interação de Proteínas/métodos , Proteínas de Ligação a Tacrolimo/metabolismo , Apoptose , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/metabolismo , Proliferação de Células , Feminino , Humanos , Imunossupressores/farmacologia , Substâncias Luminescentes/química , Sirolimo/farmacologia , Serina-Treonina Quinases TOR/metabolismo , Células Tumorais Cultivadas
15.
Methods Mol Biol ; 2274: 271-279, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34050479

RESUMO

A method to generate small amount of reactive oxygen species (ROSs) at intracellular targeted region has great potential to manipulate the function of particular proteins. The present protocol introduces a fusion protein that consisted of firefly luciferase (FLuc), photosensitizer protein KillerRed and F-actin-targeting peptide Lifeact (Lifeact-KillerFirefly) to generate ROSs in the vicinity of F-actin and found that morphological change in F-actin structure was induced by the fusion protein after luciferin treatment. This manipulating and imaging method is of use to analyze the role of the locally generated ROSs on the function of intracellular proteins.


Assuntos
Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Luciferases de Vaga-Lume/metabolismo , Medições Luminescentes/métodos , Fármacos Fotossensibilizantes/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Transferência Ressonante de Energia de Fluorescência/métodos , Células HEK293 , Humanos , Substâncias Luminescentes/química , Imagem Óptica/métodos
16.
Methods Mol Biol ; 2274: 281-294, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34050480

RESUMO

Optogenetic calcium sensors enable the imaging in real-time of the activities of single or multiple neurons in brain slices and in vivo. Bioluminescent probes engineered from the natural calcium sensor aequorin do not require illumination, are virtually devoid of background signal, and exhibit wide dynamic range and low cytotoxicity. These probes are thus well suited for long-duration, whole-field recordings of multiple neurons simultaneously. Here, we describe a protocol for monitoring and analyzing the dynamics of neuronal ensembles using whole-field bioluminescence imaging of an aequorin-based sensor in brain slice.


Assuntos
Equorina/química , Técnicas Biossensoriais/métodos , Encéfalo/metabolismo , Cálcio/metabolismo , Substâncias Luminescentes/química , Medições Luminescentes/métodos , Neurônios/metabolismo , Animais , Transferência Ressonante de Energia de Fluorescência/métodos , Camundongos , Vias Neurais , Imagem Óptica/métodos
17.
Methods Mol Biol ; 2274: 305-314, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34050482

RESUMO

Bioluminescence resonance energy transfer (BRET) is an energy transfer phenomenon from a luciferase donor to a fluorescence acceptor and serves as an indicator of protein-protein interaction or protein proximity. BRET imaging is a powerful tool in the investigation of signaling proteins because it enables spatial analysis of such protein interactions. Here, we describe a method exerting high-resolution BRET imaging by combining bright-light output luciferases, such as NanoLuc , photon-counting EM-CCD, and unique algorithms for image correction and denoising.


Assuntos
Transferência Ressonante de Energia de Fluorescência/métodos , Luciferases/metabolismo , Medições Luminescentes/métodos , Microscopia de Fluorescência/métodos , Imagem Óptica/métodos , Fótons , Câmaras gama , Células HEK293 , Humanos , Processamento de Imagem Assistida por Computador , Substâncias Luminescentes/química
18.
Methods Mol Biol ; 2255: 27-42, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34033092

RESUMO

Cellular signals to resist apoptosis have been attributed as one of the mechanisms of tumorigenesis. Hence, apoptosis is a cardinal target for drug development in cancers, and several antitumor drugs have been designed to induce apoptosis in tumor cells. Recently, venetoclax, a Bcl2 inhibitor that induces apoptosis, has been approved by the FDA for the treatment of CLL and SLL patients. Proapoptotic antitumor drugs have been traditionally developed and tested, targeting apoptosis in tumor cells. The mechanism of such drug actions has been functionally connected to the mechanism of apoptosis. The identification of apoptosis in a tumor cell takes into account different characteristics in several steps of apoptosis. Thus, it is understandable that modes of identification of apoptosis observed in tumor cells in a laboratory have also been tuned to different characteristics in several parameters of apoptosis. Here, we present a detailed methodology for a triple-parameter-based co-fluorescence imaging to identify apoptosis in live tumor cells. The procedure involves co-fluorescence staining specific for three cardinal features of apoptosis in live cells. The procedure is simple, time-sensitive, and can be performed successfully in a laboratory-friendly manner.


Assuntos
Apoptose , Neoplasias da Mama/patologia , Fluorescência , Laboratórios/estatística & dados numéricos , Mitocôndrias/metabolismo , Imagem Óptica/métodos , Neoplasias Ovarianas/patologia , Neoplasias da Mama/metabolismo , Caspases/metabolismo , Membrana Celular/metabolismo , Feminino , Humanos , Processamento de Imagem Assistida por Computador , Neoplasias Ovarianas/metabolismo , Fosfatidilserinas/metabolismo , Células Tumorais Cultivadas
19.
Nat Commun ; 12(1): 3150, 2021 05 25.
Artigo em Inglês | MEDLINE | ID: mdl-34035297

RESUMO

Super-resolution imaging has been revolutionizing technical analysis in various fields from biological to physical sciences. However, many objects are hidden by strongly scattering media such as biological tissues that scramble light paths, create speckle patterns and hinder object's visualization, let alone super-resolution imaging. Here, we demonstrate non-invasive super-resolution imaging through scattering media based on a stochastic optical scattering localization imaging (SOSLI) technique. After capturing multiple speckle patterns of photo-switchable point sources, our computational approach utilizes the speckle correlation property of scattering media to retrieve an image with a 100-nm resolution, an eight-fold enhancement compared to the diffraction limit. More importantly, we demonstrate our SOSLI to do non-invasive super-resolution imaging through not only static scattering media, but also dynamic scattering media with strong decorrelation such as biological tissues. Our approach paves the way to non-invasively visualize various samples behind scattering media at nanometer levels of detail.


Assuntos
Difusão Dinâmica da Luz/métodos , Imagem Óptica/métodos , Processos Estocásticos
20.
Nat Commun ; 12(1): 2977, 2021 05 20.
Artigo em Inglês | MEDLINE | ID: mdl-34016996

RESUMO

When exploring new environments animals form spatial memories that are updated with experience and retrieved upon re-exposure to the same environment. The hippocampus is thought to support these memory processes, but how this is achieved by different subnetworks such as CA1 and CA3 remains unclear. To understand how hippocampal spatial representations emerge and evolve during familiarization, we performed 2-photon calcium imaging in mice running in new virtual environments and compared the trial-to-trial dynamics of place cells in CA1 and CA3 over days. We find that place fields in CA1 emerge rapidly but tend to shift backwards from trial-to-trial and remap upon re-exposure to the environment a day later. In contrast, place fields in CA3 emerge gradually but show more stable trial-to-trial and day-to-day dynamics. These results reflect different roles in CA1 and CA3 in spatial memory processing during familiarization to new environments and constrain the potential mechanisms that support them.


Assuntos
Região CA1 Hipocampal/fisiologia , Região CA3 Hipocampal/fisiologia , Células de Lugar/fisiologia , Percepção Espacial/fisiologia , Memória Espacial/fisiologia , Animais , Técnicas de Observação do Comportamento , Comportamento Animal/fisiologia , Região CA1 Hipocampal/citologia , Região CA1 Hipocampal/diagnóstico por imagem , Região CA3 Hipocampal/citologia , Região CA3 Hipocampal/diagnóstico por imagem , Craniotomia , Microscopia Intravital/instrumentação , Microscopia Intravital/métodos , Masculino , Camundongos , Microscopia Confocal/instrumentação , Microscopia Confocal/métodos , Modelos Animais , Imagem Óptica/instrumentação , Imagem Óptica/métodos
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