Este trabalho é apoiado pelo Instituto Nacional do Câncer do Instituto Nacional de Saúde concessão R01 CA140257-01.

<html> NOTA: O consentimento informado foi obtido dos pacientes. Além disso, esta pesquisa foi realizada em conformidade com todas as diretrizes institucionais, nacionais e internacionais para o bem-estar humano. 1. Prepare Computador <ol><li> Vire laptop e conecte USB da Placa de Captura DVI2USB. </li></ol> 2. Prepare o Monitor <ol><li> Conecte o cabo DVI para monitorar e PinP para permitir que o endoscopista para ver vídeos a partir dessas telas ao invés do computador. </li><li> Certifique-se que o monitor está em pé e está previsto para DVI. </li><li> Conecte PinP a parte de trás do sistema Olympus, em seguida, pressione o botão de entrada no sistema Olympus para ver a imagem no monitor grande montada na parede. </li></ol> 3. Laser Diode Motorista Setting <ol><li> Verifique se o driver de diodo laser é desligado. Ele só deve ser ligado a alguns minutos antes de imagem é realizado. </li></ol>e_title &quot;&gt; 4. Power Strip <ol><li> Ligue portátil, processador, controlador de diodo laser, DVI splitter, e placa de captura de Epiphan no filtro de linha. </li></ol> 5. Execute MDE Widefield no Desktop Laptop <ol><li> Hit &#39;Current Folder&#39;. </li><li> Digite o número e as iniciais do paciente e pressione &#39;Criar pasta Paciente&#39;. </li></ol> 6. Prepare Cap e Filtro <ol><li> Ao manusear filtro, sempre utilizar luvas e Kimwipes para minimizar a transferência de óleo e detritos para o filtro. Gaze ou álcool cotonetes pode deixar fibras que podem interferir com a imagem. </li><li> Coloque algumas Kimwipes sobre uma mesa para criar uma plataforma para estabelecer o filtro e tampa. Esta área desinfectada deve ser facilmente acessível para o gastroenterologista durante o procedimento. </li><li> Deite-se o filtro e tampa e manter Kimwipes por perto para uso durante o procedimento. </li></ol> 7. Preparação Paciente <ol><li> Consentimento paciente em usode VFI e corante proflavina antes de chegar à suíte endo. </li><li> Posição do paciente para o procedimento de endoscopia digestiva alta. </li><li> Prossiga com a imagem de luz branca padrão, utilizando um microscópio digital multiespectral (MDM) 7. </li></ol> 8. Insira e Spray proflavina <ol><li> Depois de terminar com a imagem de luz branca, inserir e spray corante proflavina sobre o tecido de interesse. 1-5 mm deve ser suficiente, dependendo da área de tecido Barrett. Ao pulverizar o corante proflavina neste passo, tempo suficiente (pelo menos 1 minuto) é determinada por absorção de tecido suficiente antes VFI. Proflavina, que é coberto por uma aplicação nova droga investigacional da FDA (IND 102217), é um agente de contraste fluorescente que se concentra no núcleo das células, logo após a aplicação. Embora VFI não pode resolver morfologia celular individual, quando as panelas de diodo laser sobre o tecido, a luz refletida permite que o endoscopista a apreciar morpholo glandular geralgy. </li></ol> 9. Ligue Laser Diode <ol><li> Ligue o diodo laser. Faça isso pelo menos 2 min antes do início da imagem. </li></ol> 10. Prepare endoscópio para VFI <ol><li> Retire o endoscópio completamente. </li><li> Antes de lidar com o endoscópio, garantir que o assistente tem dois pares de luvas. </li><li> Já o endoscopista segurar o endoscópio na frente do assistente que fica ao lado da plataforma Kimwipe detalhado acima. </li><li> Com Kimwipes, ter o assistente limpa a ponta do endoscópio. Certifique-se de limpar a superfície frontal levemente e também limpar alguns centímetros ao longo do lado do endoscópio para facilitar o manuseio. </li><li> Após a limpeza, tem o descarte assistente do par exterior de luvas. </li><li> Já o assistente de colocar no filtro. Insira a protuberância cilíndrica curta no filtro em seu buraco complementar no endoscópio. Manter o endoscópio vertical para auxiliar o processo. </li><li> Segurando o filter no lugar, deslize a tampa sobre o filtro e empurrá-lo para baixo sobre a ponta do endoscópio. Certifique-se que a tampa é seguro e empurrou completamente com as bordas lave com o endoscópio. Certifique-se o lábio da tampa prolonga-se ligeiramente sobre a ponta do endoscópio. Por fim, verifique se o filtro ainda está em vigor e lave com a ponta do endoscópio. </li></ol> 11. Insira o endoscópio volta para o esôfago e Imagem 12. Remover endoscópio de Esôfago <ol><li> Usando Kimwipes puxe cuidadosamente a tampa e filtrar fora do endoscópio. Jogue fora a tampa e limpe o filtro para o próximo caso. </li></ol> 13. Filtro limpo <ol><li> Delicadamente, limpe o filtro com Kimwipes. </li><li> Encha um copo pequeno com CyDex e mergulhe o filtro para 12 min. A cada poucos minutos virar o filtro sobre. </li><li> Delicadamente, limpe o filtro com Kimwipes. </li><li> Submergir filtro, desta vez em água estéril. Depois de 5 minutos, use um esguicho bottle e pulverizar água estéril sobre o filtro. </li><li> Coloque filtro em algumas Kimwipes e deixe secar. </li><li> Depois de seco, coloque em recipiente de armazenamento entre Kimwipes. </li></ol>

<ol>
	<li>Modiano, N., Gerson, L. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Barrett's+Esophagus:+Incidence,+etiology,+pathophysiology,+prevention+and+treatment.">Barrett's Esophagus: Incidence, etiology, pathophysiology, prevention and treatment.</a> Therapeutics and Clinical Risk Management. 3, 1035-1145 (2007).</li><li>Brown, L. M., Devesa, S. S., Chow, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Incidence+of+Adenocarcinoma+of+the+esophagus+among+white+Americans+by+sex,+stage,+and+age.">Incidence of Adenocarcinoma of the esophagus among white Americans by sex, stage, and age.</a> Journal of the National Cancer Institute. 100, 1184-1187 (2008).</li><li>Siegel, R., Naishadham, D., Ahmedin, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cancer+Statistics,+2012.">Cancer Statistics, 2012.</a> A Cancer Journal for Clinicians. 62, 10-29 (2012).</li><li>Vieth, M., Ell, C., Gossner, L., May, A., Stolte, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Histological+analysis+of+endoscopic+resection+specimens+from+326+patients+with+Barrett's+esophagus+and+early+neoplasia.">Histological analysis of endoscopic resection specimens from 326 patients with Barrett's esophagus and early neoplasia.</a> Endoscopy. 36, 776-781 (2004).</li><li>Pohl, H., Rosch, T., Vieth, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Miniprobe+confocal+laser+microscopy+for+the+detection+of+invisible+neoplasia+in+patients+with+Barrett's+oesophagus.">Miniprobe confocal laser microscopy for the detection of invisible neoplasia in patients with Barrett's oesophagus.</a> Gut. 57, 1648-1653 (2008).</li><li>Thekkek, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modular+video+endoscopy+for+in+vivo+cross-polarized+and+vital-dye+fluorescence+imaging+of+Barrett's-associated+neoplasia.">Modular video endoscopy for in vivo cross-polarized and vital-dye fluorescence imaging of Barrett's-associated neoplasia.</a> Journal of Biomedical Optics. 18, 026007 (2013).</li><li>Roblyer, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multispectral+optical+imaging+device+for+in+vivo+detection+of+oral+neoplasia.">Multispectral optical imaging device for in vivo detection of oral neoplasia.</a> Journal of Biomedical Optics. 13, 024019 (2008).</li><li>Egger, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biopsy+surveillance+is+still+necessary+in+patients+with+Barrett's+oesophagus+despite+new+endoscopic+imaging+techniques.">Biopsy surveillance is still necessary in patients with Barrett's oesophagus despite new endoscopic imaging techniques.</a> Gut. 52, 18-23 (2003).</li><li>Thekkek, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vital-dye+enhanced+fluorescence+imaging+of+GI+mucosa:+metaplasia,+neoplasia,+inflammation.">Vital-dye enhanced fluorescence imaging of GI mucosa: metaplasia, neoplasia, inflammation.</a> Gastrointestinal Endoscopy. 75, 877-887 (2012).</li><li>Muldoon, T. J., Anandasabapathy, S., Maru, D., Richards-Kortum, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-resolution+imaging+in+Barrett's+esophagus:+a+novel,+low-cost+endoscopic+microscope.">High-resolution imaging in Barrett's esophagus: a novel, low-cost endoscopic microscope.</a> Gastrointestinal Endoscopy. 68, 737-744 (2008).</li></ol>

Não há nada a divulgar.

Com vigilância endoscópica normal, neoplasia em BE é muitas vezes perdida 8 porque metaplasia benigna pode ser indistinguível de displasia de alto grau e adenocarcinoma. Como uma ferramenta que seria melhor permitir gastroenterologistas para remediar esse erro atualmente inevitável, o corante vital reforçada imagens de fluorescência destaca morfologia glandular de um tecido proporcionando assim uma característica distinta para diferenciar os tipos de tecidos. Além disso, através de um campo de visã...

Ao longo dos últimos quarenta anos, a incidência de adenocarcinoma esofágico (EAC) aumentou significativamente 1,2; ainda devido ao diagnóstico tardio, a taxa de sobrevivência de cinco anos é inferior a 20% 3. O padrão atual de vigilância endoscópica em BE, o precursor da EAC, é endoscopia luz branca com biópsias do segmento aleatórios &#39;fórceps quatro quandrant. Infelizmente, esta técnica muitas vezes perde neoplasia, que pode ser plana, sutil e difícil de se diferenciar em padrão de luz branca de imagem 4. Embora tenha havido sucesso no uso de microscopia confocal a laser para destacar características celulares in vivo, lesões ainda pode ser perdida devido à diminuição do campo de visão 5. Ter uma tecnologia de &quot;ponte&quot; que pode destacar as áreas para posterior imagem microendoscópica confocal seria marcadamente valioso. Por conseguinte, uma modalidade de imagem de bandeira vermelha avançado que melhora a capacidade de TARGEt e biópsia neoplasia no início BE seria um instrumento para detectar a EAC, numa fase curável cedo e poderia levar a um tratamento mais eficaz e, posteriormente, melhorou as taxas de sobrevivência. VFI é uma nova técnica que combina imagens de alta resolução epitelial com exógena tópica fluorescente contraste, proflavina, para destacar a morfologia glandular e delinear neoplasia (displasia de alto grau e câncer) no esôfago distal na esperança de melhorar o diagnóstico in vivo 6. Após a excitação do proflavina, que se concentra no núcleo das células, logo após a aplicação, as imagens fluorescentes fornecer resolução espacial de 50 a 100 ^ m e um campo de visão de 2,5 cm, o que permite visualizar endoscopists morfologia glandular. Como resultado, esta abordagem permite gastroenterologistas distinguir metaplasia clássico de Barrett, que tem contínuas, glândulas uniformemente espaçados e uma morfologia homogénea em geral, a partir de BE com neoplasia, o qual tem obliteração da arquitetura glandular. Aqui nós descrevemos o protocolo desta nova técnica com um endoscópio multiespectral, e fornecer resultados representativos para demonstrar a utilidade deste dispositivo em descrever a transformação morfológica de metaplasia benigna para displasia de alto grau e câncer.

<table border="0" cellpadding="0" cellspacing="0" width="650">
	<tbody>
		<tr>
			<td>Filter*</td>
			<td>Schott North America, Inc., Duryea, Pennsylvania</td>
			<td>Not Applicable</td>
			<td>495-nm long-pass filter</td>
		</tr>
		<tr>
			<td>Halo Cap &#8211; Medium*</td>
			<td>Barrx Medical</td>
			<td>CP-002A</td>
			<td></td>
		</tr>
		<tr>
			<td>Processor*</td>
			<td>Pentax</td>
			<td>EPK-i</td>
			<td></td>
		</tr>
		<tr>
			<td>Multispectral Digital Microscope**</td>
			<td>Not Applicable</td>
			<td>Not Applicable</td>
			<td></td>
		</tr>
		<tr>
			<td>Kimwipes</td>
			<td>Kimberly-Clark</td>
			<td>KimTech Science</td>
			<td>S-8115</td>
		</tr>
		<tr>
			<td>Cidex</td>
			<td>Advanced Sterilization Products&#160;</td>
			<td>CIDEX OPA Solution</td>
			<td></td>
		</tr>
		<tr>
			<td>Proflavine hemisulfate (0.01% w/v)</td>
			<td></td>
			<td></td>
			<td>FDA (IND 102,217)</td>
		</tr>
		<tr>
			<td>Laser Diode*</td>
			<td>Nichia Corporation</td>
			<td>Not Applicable</td>
			<td>455-nm</td>
		</tr>
		<tr>
			<td>Image Capture*</td>
			<td>Labview</td>
			<td>Not Applicable</td>
			<td></td>
		</tr>
		<tr>
			<td>Spray Catheter</td>
			<td>Olympus</td>
			<td>Not Applicable</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>*Equipment specifics within Reference 6. **Equipment specifics within Reference 7</td>
		</tr>
	</tbody>
</table>

diagnosis of neoplasia in barrett&#8217;s esophagus using vital-dye enhanced fluorescence imaging

A capacidade de diferenciar metaplasia benigna no esôfago de Barrett (BE) de neoplasia in vivo permanece difícil como os dois tipos de tecido pode ser plana e indistinguível com luz branca só imagem. Como resultado, uma modalidade que destaca arquitectura glandular seria útil para discriminar neoplasia benigna do epitélio na parte distal. VFI é uma nova técnica que utiliza um agente de contraste fluorescente tópica exógena para delinear displasia de alto grau e câncer do epitélio benigno. Especificamente, as imagens fluorescentes fornecer resolução espacial de 50 a 100 ^ m e um campo de visão de 2,5 cm, o que permite visualizar endoscopists morfologia glandular. Após a excitação, metaplasia clássico de Barrett surge como contínuos, glândulas uniformemente espaçados e uma morfologia homogênea geral; em contraste, tecido neoplásico aparece cheia de obliteração completa da estrutura glandular. Aqui nós fornecemos uma visão geral doa instrumentação e enumerar o protocolo desta nova técnica. Enquanto VFI oferece um gastroenterologista com a arquitetura glandular de tecido suspeito, displasia celular não pode ser resolvida com esta modalidade. Como tal, não se pode distinguir morfologicamente metaplasia de Barrett do BE com baixo grau Displasia através desta modalidade de imagem. Por trocando uma diminuição na resolução com um maior campo de visão, este sistema de imagem pode ser usado pelo menos como um dispositivo de imagem de bandeira vermelha para alvejar e biópsia de lesões suspeitas; Ainda, se as medidas de precisão são promissores, VFI pode tornar-se a técnica de imagem padrão para o diagnóstico de neoplasia (definidas como displasia de alto grau ou cancro) na parte distal.

A Figura 1B mostra clássico Esôfago de Barrett sem displasia cercado nas fronteiras por epitélio escamoso normal. Começando com o tecido escamoso mais plana, que está localizado perifericamente e indicado pelas setas azuis, uma zona homogénea de fluorescência monótona está presente sem arquitectura glandular. As setas verdes indicam uma linha verde circular em torno do tecido escamoso. Este esboço é artefacto resultante a partir da tampa do endoscópio. Movendo-se para o tecido localizado cen...

Watch this Scientific Journal Video about Diagnosis of Neoplasia in BarrettÃ¢â‚¬â„¢s Esophagus using Vital-dye Enhanced Fluorescence Imaging at JoVE.com

Diagnosis of Neoplasia in Barrett&#8217;s Esophagus using Vital-dye Enhanced Fluorescence Imaging

-Dye Vital reforçada imagens de fluorescência (VFI) é um romance em técnica vivo, que combina alta resolução epitelial de imagem com contraste fluorescente tópica exógena para destacar morfologia glandular e delinear neoplasia (displasia de alto grau e câncer) no esôfago distal.

Diagnóstico de neoplasia de esôfago de Barrett usando-dye Vital Aprimorado imagens de fluorescência

The ability to differentiate benign metaplasia in Barrett&#8217;s Esophagus (BE) from neoplasia in vivo remains difficult as both tissue types can be flat ...

diagnosis-neoplasia-barretts-esophagus-using-vital-dye-enhanced

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Unit 1

Disorders of the Esophagus

SCIENTISTS IN ACTION

12.0K visualizações.  Icahn School of Medicine at Mount Sinai.  -Dye Vital reforçada imagens de fluorescência (VFI) é um romance em técnica vivo, que combina alta resolução epitelial de imagem com contraste fluorescente tópica exógena para destacar morfologia glandular e delinear neoplasia (displasia de alto grau e câncer) no esôfago distal. 

Vídeo: Diagnosis of Neoplasia in Barrett’s Esophagus using Vital-dye Enhanced Fluorescence Imaging

Three-dimensional Optical-resolution Photoacoustic Microscopy

Optical microscopy, providing valuable insights at the cellular and organelle levels, has been widely recognized as an enabling biomedical technology. As the mainstays of in vivo three-dimensional (3-D) optical microscopy, single-/multi-photon fluorescence microscopy and optical coherence tomography (OCT) have demonstrated their extraordinary sensitivities to fluorescence and optical scattering contrasts, respectively. However, the optical absorption contrast of biological tissues, which encodes essential physiological/pathological information, has not yet been assessable. 
The emergence of biomedical photoacoustics has led to a new branch of optical microscopy optical-resolution photoacoustic microscopy (OR-PAM)1, where the optical irradiation is focused to the diffraction limit to achieve cellular1 or even subcellular2 level lateral resolution. As a valuable complement to existing optical microscopy technologies, OR-PAM brings in at least two novelties. First and most importantly, OR-PAM detects optical absorption contrasts with extraordinary sensitivity (i.e., 100%). Combining OR-PAM with fluorescence microscopy3 or with optical-scattering-based OCT4 (or with both) provides comprehensive optical properties of biological tissues. Second, OR-PAM encodes optical absorption into acoustic waves, in contrast to the pure optical processes in fluorescence microscopy and OCT, and provides background-free detection. The acoustic detection in OR-PAM mitigates the impacts of optical scattering on signal degradation and naturally eliminates possible interferences (i.e., crosstalks) between excitation and detection, which is a common problem in fluorescence microscopy due to the overlap between the excitation and fluorescence spectra. 
Unique for optical absorption imaging, OR-PAM has demonstrated broad biomedical applications since its invention, including, but not limited to, neurology5, 6, ophthalmology7, 8, vascular biology9, and dermatology10. In this video, we teach the system configuration and alignment of OR-PAM as well as the experimental procedures for in vivo functional microvascular imaging.

Optical-resolution photoacoustic microscopy (OR-PAM) is an emerging technology capable of imaging optical absorption contrasts in vivo with cellular resolution and sensitivity. Here, we provide a visualized instruction on the experimental protocols of OR-PAM, including system configuration, system alignment, typical in vivo experimental procedures, and functional imaging schemes.

Tridimensional Optical resolução Microscopia Fotoacústica

Optical microscopy, providing valuable insights at the cellular and organelle levels, has been widely recognized as an enabling biomedical technology. As ...

Bioengenharia

Fluorescence Lifetime Imaging of Molecular Rotors in Living Cells

Diffusion is often an important rate-determining step in chemical reactions or biological processes and plays a role in a wide range of intracellular events. Viscosity is one of the key parameters affecting the diffusion of molecules and proteins, and changes in viscosity have been linked to disease and malfunction at the cellular level.1-3 While methods to measure the bulk viscosity are well developed, imaging microviscosity remains a challenge. Viscosity maps of microscopic objects, such as single cells, have until recently been hard to obtain. Mapping viscosity with fluorescence techniques is advantageous because, similar to other optical techniques, it is minimally invasive, non-destructive and can be applied to living cells and tissues.
Fluorescent molecular rotors exhibit fluorescence lifetimes and quantum yields which are a function of the viscosity of their microenvironment.4,5 Intramolecular twisting or rotation leads to non-radiative decay from the excited state back to the ground state. A viscous environment slows this rotation or twisting, restricting access to this non-radiative decay pathway. This leads to an increase in the fluorescence quantum yield and the fluorescence lifetime. Fluorescence Lifetime Imaging (FLIM) of modified hydrophobic BODIPY dyes that act as fluorescent molecular rotors show that the fluorescence lifetime of these probes is a function of the microviscosity of their environment.6-8 A logarithmic plot of the fluorescence lifetime versus the solvent viscosity yields a straight line that obeys the F&ouml;rster Hoffman equation.9 This plot also serves as a calibration graph to convert fluorescence lifetime into viscosity.
Following incubation of living cells with the modified BODIPY fluorescent molecular rotor, a punctate dye distribution is observed in the fluorescence images. The viscosity value obtained in the puncta in live cells is around 100 times higher than that of water and of cellular cytoplasm.6,7 Time-resolved fluorescence anisotropy measurements yield rotational correlation times in agreement with these large microviscosity values. Mapping the fluorescence lifetime is independent of the fluorescence intensity, and thus allows the separation of probe concentration and viscosity effects. 
In summary, we have developed a practical and versatile approach to map the microviscosity in cells based on FLIM of fluorescent molecular rotors.

Fluorescence Lifetime Imaging (FLIM) has emerged as a key technique to image the environment and interaction of specific proteins and dyes in living cells. FLIM of fluorescent molecular rotors allows mapping of viscosity in living cells.

Imagem Lifetime fluorescência de rotores moleculares em células vivas

Diffusion is often an important rate-determining step in chemical reactions or biological processes and plays a role in a wide range of intracellular ...

High-resolution Spatiotemporal Analysis of Receptor Dynamics by Single-molecule Fluorescence Microscopy

Single-molecule microscopy is emerging as a powerful approach to analyze the behavior of signaling molecules, in particular concerning those aspect (e.g., kinetics, coexistence of different states and populations, transient interactions), which are typically hidden in ensemble measurements, such as those obtained with standard biochemical or microscopy methods. Thus, dynamic events, such as receptor-receptor interactions, can be followed in real time in a living cell with high spatiotemporal resolution. This protocol describes a method based on labeling with small and bright organic fluorophores and total internal reflection fluorescence (TIRF) microscopy to directly visualize single receptors on the surface of living cells. This approach allows one to precisely localize receptors, measure the size of receptor complexes, and capture dynamic events such as transient receptor-receptor interactions. The protocol provides a detailed description of how to perform a single-molecule experiment, including sample preparation, image acquisition and image analysis. As an example, the application of this method to analyze two G-protein-coupled receptors, i.e., &#946;2-adrenergic and &#947;-aminobutyric acid type B (GABAB) receptor, is reported. The protocol can be adapted to other membrane proteins and different cell models, transfection methods and labeling strategies.

This protocol describes how to use total internal reflection fluorescence microscopy to visualize and track single receptors on the surface of living cells and thereby analyze receptor lateral mobility, size of receptor complexes as well as to visualize transient receptor-receptor interactions. This protocol can be extended to other membrane proteins.

De alta resolução espacial e temporal Análise de Receptor Dynamics por uma única molécula de microscopia de fluorescência

Single-molecule microscopy is emerging as a powerful approach to analyze the behavior of signaling molecules, in particular concerning those aspect (e.g., ...

3D Orbital Tracking in a Modified Two-photon Microscope: An Application to the Tracking of Intracellular Vesicles

The objective of this video protocol is to discuss how to perform and analyze a three-dimensional fluorescent orbital particle tracking experiment using a modified two-photon microscope1. As opposed to conventional approaches (raster scan or wide field based on a stack of frames), the 3D orbital tracking allows to localize and follow with a high spatial (10 nm accuracy) and temporal resolution (50 Hz frequency response) the 3D displacement of a moving fluorescent particle on length-scales of hundreds of microns2. The method is based on a feedback algorithm that controls the hardware of a two-photon laser scanning microscope in order to perform a circular orbit around the object to be tracked: the feedback mechanism will maintain the fluorescent object in the center by controlling the displacement of the scanning beam3-5. To demonstrate the advantages of this technique, we followed a fast moving organelle, the lysosome, within a living cell6,7. Cells were plated according to standard protocols, and stained using a commercially lysosome dye. We discuss briefly the hardware configuration and in more detail the control software, to perform a 3D orbital tracking experiment inside living cells. We discuss in detail the parameters required in order to control the scanning microscope and enable the motion of the beam in a closed orbit around the particle. We conclude by demonstrating how this method can be effectively used to track the fast motion of a labeled lysosome along microtubules in 3D within a live cell. Lysosomes can move with speeds in the range of 0.4-0.5 &#181;m/sec, typically displaying a directed motion along the microtubule network8.

In this video protocol we track - at high speed and in three dimensions - fluorescently labeled lysosomes within living cells, using the orbital tracking method in a modified two-photon microscope.

3D Orbital Rastreamento em um microscópio de dois fotões Modificação: Uma Aplicação para o rastreamento de vesículas intracelulares

The objective of this video protocol is to discuss how to perform and analyze a three-dimensional fluorescent orbital particle tracking experiment using a ...

From Fast Fluorescence Imaging to Molecular Diffusion Law on Live Cell Membranes in a Commercial Microscope

It has become increasingly evident that the spatial distribution and the motion of membrane components like lipids and proteins are key factors in the regulation of many cellular functions. However, due to the fast dynamics and the tiny structures involved, a very high spatio-temporal resolution is required to catch the real behavior of molecules. Here we present the experimental protocol for studying the dynamics of fluorescently-labeled plasma-membrane proteins and lipids in live cells with high spatiotemporal resolution. Notably, this approach doesn&#8217;t need to track each molecule, but it calculates population behavior using all molecules in a given region of the membrane. The starting point is a fast imaging of a given region on the membrane. Afterwards, a complete spatio-temporal autocorrelation function is calculated correlating acquired images at increasing time delays, for example each 2, 3, n repetitions. It is possible to demonstrate that the width of the peak of the spatial autocorrelation function increases at increasing time delay as a function of particle movement due to diffusion. Therefore, fitting of the series of autocorrelation functions enables to extract the actual protein mean square displacement from imaging (iMSD), here presented in the form of apparent diffusivity vs average displacement. This yields a quantitative view of the average dynamics of single molecules with nanometer accuracy. By using a GFP-tagged variant of the Transferrin Receptor (TfR) and an ATTO488 labeled 1-palmitoyl-2-hydroxy-sn-glycero-3-phosphoethanolamine (PPE) it is possible to observe the spatiotemporal regulation of protein and lipid diffusion on &#181;m-sized membrane regions in the micro-to-milli-second time range.

Spatial distribution and temporal dynamics of plasma membrane proteins and lipids is a hot topic in biology. Here this issue is addressed by a spatio-temporal image fluctuation analysis that provides conceptually the same physical quantities of single particle tracking, but it uses small molecular labels and standard microscopy setups.

De fast imagens de fluorescência com a Lei difusão molecular em membranas de células vivas em um microscópio comercial

It has become increasingly evident that the spatial distribution and the motion of membrane components like lipids and proteins are key factors in the ...

Electrotaxis Studies of Lung Cancer Cells using a Multichannel Dual-electric-field Microfluidic Chip

The behavior of directional cell migration under a direct current electric-field (dcEF) is referred to as electrotaxis. The significant role of physiological dcEF in guiding cell movement during embryo development, cell differentiation, and wound healing has been demonstrated in many studies. By applying microfluidic chips to an electrotaxis assay, the investigation process is shortened and experimental errors are minimized. In recent years, microfluidic devices made of polymeric substances (e.g., polymethylmethacrylate, PMMA, or acrylic) or polydimethylsiloxane (PDMS) have been widely used in studying the responses of cells to electrical stimulation. However, unlike the numerous steps required to fabricate a PDMS device, the simple and rapid construction of the acrylic micro&#64258;uidic chip makes it suitable for both device prototyping and production. Yet none of the reported devices facilitate the efficient study of the simultaneous chemical and dcEF effects on cells. In this report, we describe our design and fabrication of an acrylic-based multichannel dual-electric-field (MDF) chip to investigate the concurrent effect of chemical and electrical stimulation on lung cancer cells. The MDF chip provides eight combinations of electrical/chemical stimulations in a single test. The chip not only greatly shortens the required experimental time but also increases accuracy in electrotaxis studies.

Many microfluidic devices have been developed for use in the study of electrotaxis. Yet, none of these chips allows the efficient study of the simultaneous chemical and electric-field (EF) effects on cells. We developed a polymethylmethacrylate-based device that offers better-controlled coexisting EF and chemical stimulation for use in electrotaxis research.

Electrotaxis Estudos de pulmão células de câncer usando um Multichannel Dual-elétrico-campo Microfluidic Chip

The behavior of directional cell migration under a direct current electric-field (dcEF) is referred to as electrotaxis. The significant role of ...

Conducting Multiple Imaging Modes with One Fluorescence Microscope

Fluorescence microscopy is a powerful tool to detect biological molecules in situ and monitor their dynamics and interactions in real-time. In addition to conventional epi-fluorescence microscopy, various imaging techniques have been developed to achieve specific experimental goals. Some of the widely used techniques include single-molecule fluorescence resonance energy transfer (smFRET), which can report conformational changes and molecular interactions with angstrom resolution, and single-molecule detection-based super-resolution (SR) imaging, which can enhance the spatial resolution approximately ten&#160;to twentyfold compared to diffraction-limited microscopy. Here we present a customer-designed integrated system, which merges multiple imaging methods in one microscope, including conventional epi-fluorescent imaging, single-molecule detection-based SR imaging, and multi-color single-molecule detection, including smFRET imaging. Different imaging methods can be achieved easily and reproducibly by switching optical elements. This set-up is easy to adopt by any research laboratory in biological sciences with a need for routine and diverse imaging experiments at a reduced cost and space relative to building separate microscopes for individual purposes.

Here we present a practical guide of building an integrated microscopy system, which merges conventional epi-fluorescent imaging, single-molecule detection-based super-resolution imaging, and multi-color single-molecule detection, including single-molecule fluorescence resonance energy transfer imaging, into one set-up in a cost-efficient way.

Realização de vários modos de imagem com um microscópio de fluorescência

Fluorescence microscopy is a powerful tool to detect biological molecules in situ and monitor their dynamics and interactions in real-time. In addition to ...

Visualizing Adhesion Formation in Cells by Means of Advanced Spinning Disk-Total Internal Reflection Fluorescence Microscopy

In living cells, processes such as adhesion formation involve extensive structural changes in the plasma membrane and the cell interior. In order to visualize these highly dynamic events, two complementary light microscopy techniques that allow fast imaging of live samples were combined: spinning disk microscopy (SD) for fast and high-resolution volume recording and total internal reflection fluorescence (TIRF) microscopy for precise localization and visualization of the plasma membrane. A comprehensive and complete imaging protocol will be shown for guiding through sample preparation, microscope calibration, image formation and acquisition, resulting in multi-color SD-TIRF live imaging series with high spatio-temporal resolution. All necessary image post-processing steps to generate multi-dimensional live imaging datasets, i.e. registration and combination of the individual channels, are provided in a self-written macro for the open source software ImageJ. The imaging of fluorescent proteins during initiation and maturation of adhesion complexes, as well as the formation of the actin cytoskeletal network, was used as a proof of principle for this novel approach. The combination of high resolution 3D microscopy and TIRF provided a detailed description of these complex processes within the cellular environment and, at the same time, precise localization of the membrane-associated molecules detected with a high signal-to-background ratio.

An advanced microscope that permit fast and high-resolution imaging of both, the isolated plasma membrane and the surrounding intracellular volume, will be presented. The integration of spinning disk and total internal reflection fluorescence microscopy in one setup allows live imaging experiments at high acquisition rates up to 3.5 s per image stack.

Visualizando a formação de adesão em células por meio de avançados girando a reflexão interna Total disco microscopia de fluorescência

In living cells, processes such as adhesion formation involve extensive structural changes in the plasma membrane and the cell interior. In order to ...

Using Nanoplasmon-Enhanced Scattering and Low-Magnification Microscope Imaging to Quantify Tumor-Derived Exosomes

Infected or malignant cells frequently secrete more exosomes, leading to elevated levels of disease-associated exosomes in the circulation. These exosomes have the potential to serve as biomarkers for disease diagnosis and to monitor disease progression and treatment response. However, most exosome analysis procedures require exosome isolation and purification steps, which are usually time-consuming and labor-intensive, and thus of limited utility in clinical settings. This report describes a rapid procedure to analyze specific biomarkers on the outer membrane of exosomes without requiring separate isolation and purification steps. In this method, exosomes are captured on the surface of a slide by exosome-specific antibodies and then hybridized with nanoparticle-conjugated antibody probes specific to a disease. After hybridization, the abundance of the target exosome population is determined by analyzing low-magnification dark-field microscope (LMDFM) images of the bound nanoparticles. This approach can be easily adopted for research and clinical use to analyze membrane-associated exosome biomarkers linked to disease.

Clinical translation of exosome-derived biomarkers for diseased and malignant cells is hindered by the lack of rapid and accurate quantification methods. This report describes the use of low-magnification dark-field microscope images to quantify specific exosome subtypes in small volume serum or plasma samples.

Usando Nanoplasmon-Enhanced espalhamento e microscópio de baixa ampliação de imagem para quantificar Exosomas derivados do tumor

Infected or malignant cells frequently secrete more exosomes, leading to elevated levels of disease-associated exosomes in the circulation. These exosomes ...

Studying Cavitation Enhanced Therapy

Interest in the therapeutic applications of ultrasound is significant and growing, with potential clinical targets ranging from cancer to Alzheimer&#8217;s disease. Cavitation - the formation and subsequent motion of bubbles within an ultrasound field - represents a key phenomenon underpinning many of these treatments. There remains, however, considerable uncertainty regarding the detailed mechanisms of action by which cavitation promotes therapeutic effects and there is a need to develop reliable monitoring techniques that can be implemented clinically. In particular, there is significant variation between studies in the exposure parameters reported as successfully delivering therapeutic effects and the corresponding acoustic emissions. The aim of this paper is to provide design guidelines and an experimental protocol using widely available components for performing studies of cavitation-mediated bioeffects, and&#160;include real-time acoustic monitoring. It is hoped that the protocol will enable more widespread incorporation of acoustic monitoring into therapeutic ultrasound experiments and facilitate easier comparison across studies of exposure conditions and their correlation to relevant bio-effects.

The presented experimental protocol can be used to perform real time measurements of cavitation activity in a cell culture device with the aim of enabling investigation of the conditions required for successful drug delivery and/or other bioeffects.