Name: Vídeo: Video-rate Scanning Confocal Microscopy and Microendoscopy
Uploaded: 2011-10-20T13:00:00
Duration: 14 min 10 s

Os autores gostariam de agradecer pelo seu apoio ThorLabs deste projeto. AJN gostaria de agradecer o apoio de uma Pós-Graduação NSF Fellowship. Este trabalho foi parcialmente financiado pelos Institutos Nacionais de Saúde através do Programa Novo Diretor do NIH Innovator Award, conceder número 1 DP2 OD007096-01. Informações sobre o Programa New Innovator Award está em <a href="http://nihroadmap.nih.gov/newinnovator/">http://nihroadmap.nih.gov/newinnovator/</a> . Os autores gostariam de agradecer a Tom Hayes para a utilização do laboratório de Eletrônica Harvard.

<ol>
	<li>Pawley, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Handbook of biological confocal microscopy. , 985-985 (2006).</li><li>Lippincott-Schwartz, J., Snapp, E., Kenworthy, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Studying+protein+dynamics+in+living+cells.">Studying protein dynamics in living cells.</a> Nature Reviews Molecular Cell Biology. 2, 444-456 (2001).</li><li>Klonis, N., Rug, M., Harper, I., Wickham, M., Cowman, A., Tilley, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescence+photobleaching+analysis+for+the+study+of+cellular+dynamics.">Fluorescence photobleaching analysis for the study of cellular dynamics.</a> European Biophysics Journal. 31, 36-51 (2002).</li><li>Stephens, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Light+Microscopy+Techniques+for+Live+Cell+Imaging.">Light Microscopy Techniques for Live Cell Imaging.</a> Science. 300, 82-86 (2003).</li><li>McMahon, A., Supatto, W., Fraser, S. E., Stathopoulos, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+Analyses+of+Drosophila+Gastrulation+Provide+Insights+into+Collective+Cell+Migration.">Dynamic Analyses of Drosophila Gastrulation Provide Insights into Collective Cell Migration.</a> Science. 322, 1546-1550 (2008).</li><li>Wallingford, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dishevelled+controls+cell+polarity+during+Xenopus+gastrulation.">Dishevelled controls cell polarity during Xenopus gastrulation.</a> Nature. 405, 81-85 (2000).</li><li>Laemmel, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fibered+Confocal+Fluorescence+Microscopy+(Cell-viZio)+Facilitates+Extended+Imaging+in+the+Field+of+Microcirculation.">Fibered Confocal Fluorescence Microscopy (Cell-viZio) Facilitates Extended Imaging in the Field of Microcirculation.</a> Journal of Vascular Research. 41, 400-411 (2004).</li><li>Moussata, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+confocal+laser+endomicroscopy.">The confocal laser endomicroscopy.</a> Acta Endoscopica. 39, 448-451 (2010).</li><li>Dunbar, K., Canto, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Confocal+endomicroscopy.">Confocal endomicroscopy.</a> Current Opinion in Gastroenterology. 24, 631-637 (2008).</li><li>Udovich, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spectral+background+and+transmission+characteristics+of+fiber+optic+imaging+bundles.">Spectral background and transmission characteristics of fiber optic imaging bundles.</a> Applied optics. 47, 4560-4568 (2008).</li></ol>

Produção deste video video-este era patrocinado pela Thorlabs, Inc.

Este sistema de imagem de vídeo-rate faz uso de um espelho ressonante galvanométricos operacional em cerca de 8 kHz. Espelhos de ressonância pode ser bastante alto quando trabalhar a plena potência, e seu tom alto pode ser incômodo ou até mesmo perigoso em tempos de exposição suficiente. Apesar de não demonstrado aqui, é recomendado para proteger o espelho ressonante galvanométricos dentro de uma caixa transparente de reduzir significativamente o volume do sistema e / ou para usar equipamentos adequados de proteção auditiva, como tampões de ouvido. O espelho de ressonância galvanométricos scans em um padrão sinusoidal. No entanto, os cartões framegrabber lido em sinal assumindo uma velocidade de varredura completamente linear em ambas as direções horizontal e vertical. Desde uma varredura senoidal desacelera nas bordas da digitalização, os artefatos de compressão de imagem pode ser observada ao longo do eixo de imagem rápido (horizontal). Uma forma de minimizar este problema é propositadamente conduzir o ressonante Galvo faixa de varredura espelho significativamente maior do que odiâmetro da lente relay. Ao fazer isso, apenas varrer o quase linear central do padrão de leitura sinusoidal irá percorrer a amostra, minimizando distorções de imagem. Outra abordagem seria de pós-processamento de imagens coletadas para linearizar o eixo rápido. Isso pode ser feito por imagem um padrão conhecido fluorescentes (como uma grade) e usando as dimensões padrão conhecido para criar um script de processamento que unwarps as imagens coletadas. Este sistema de digitalização especial foi projetado com a finalidade de imagem in vivo, que muitas vezes exige uma posição vertical orientada microscópio de vídeo-rate. Para experimentos com imagens de celular, microscópios invertidos são mais tipicamente usado. O projeto aqui apresentado pode ser facilmente alterada para construir tal um microscópio invertido, tudo que é necessário é uma rotação da final espelho de diâmetro 2 &quot;. Em vez de orientar o espelho para direcionar o feixe de varredura para baixo, o espelho pode direcionar o feixe para cima. Colocar a lente objetiva umat a mesma distância do espelho, juntamente com um estágio de amostra permitiria imagem em uma geometria invertida. Se o sistema de imagem está sendo construída unicamente para imagens microendoscópica, não há razão para &quot;dobrar&quot; o projeto microscópio verticalmente em tudo. Em vez disso, o sistema de digitalização inteira pode ser construída sobre uma placa de montagem horizontal único com o paralelo lente objetiva orientada para a mesa óptica. Note-se que o microscópio nesta compilação usa uma configuração fixa pinhole, enquanto este prevê a maior simplicidade e facilidade de construir o alinhamento, os usuários que desejam um sistema mais versátil pode considerar a incorporação de uma pinhole variável, como pode ser encontrado na maioria dos comerciais microscópios confocal. Ao permitir que o usuário ajuste o tamanho da pinhole para compensar as amostras de diferentes intensidade de emissão, isto permite que o usuário para melhor otimizar o equilíbrio entre a força do sinal ea resolução de uma dada amostra. O choice de fibra de imagem selecionada para o microscópio é importante. Recomendamos o uso de fibras imagem Sumitomo coerente devido ao seu afastamento do núcleo de fibra perto e autofluorescência relativamente baixo. Fibras de imagem fabricado pela Fujikura foram encontrados para ter grandes quantidades de autofluorescência 10, que pode sobrecarregar fracos sinais de fluorescência de uma amostra e limitar a sensibilidade final do microendoscope. Sumitomo fibras fabricadas, como o 8-30N utilizado nesta configuração particular, têm muito mais baixos níveis de autofluorescência do que seus equivalentes Fujikura. Enquanto feixes de fibras leeched pode ser considerado atraente para microendoscopy, o seu design tipicamente lugares núcleos de fibra individuais muito longe, o que significa que os núcleos de fibra objetos pouco de amostra, deixando de fora as regiões significativas de interesse potencial. Finalmente, deve-se notar que, enquanto o microscópio descrito aqui será útil em uma variedade de in vitro e in vivo applications e pode ser criado por uma fração do custo de um sistema full-featured comercial, ele não tem recursos como detecção de luz transmitida, uma ocular para a visão, ou um caminho do feixe para não-confocal campo amplo de epifluorescência. Embora seja possível construir um sistema com estas características a partir do zero, os leitores que desejam um sistema deste tipo pode desejar modificar um sistema comercial existente para atender suas necessidades ao invés de dar início a uma inteiramente nova construção.

<table border="1"><tbody><tr><td> Nome da parte </td><td> Fabricante </td><td> Número do Item </td><td> Especificações </td><td> Quantidade </td></tr><tr><td> 515 nm filtro passa-banda </td><td> Chroma </td><td> HQ515/50M </td><td> 46 FWHM </td><td> 1 </td></tr><tr><td> Acromática Doublet Lens Dia 25,4 milímetros. FL x 50mm, revestimento MgF2 </td><td> Edmund Optics </td><td> NT49-766 </td><td></td><td> 1 </td></tr><tr><td> Acromática Doublet Lens Dia 25,4 milímetros. FL x 76,2 mm, revestimento MgF2 </td><td> Edmund Optics </td><td> NT49-768 </td><td></td><td> 1 </td></tr><tr><td> Acromática Doublet Lens Dia 25,4 milímetros. x 88,9 milímetros FL, Revestimento MgF2 </td><td> Edmund Optics </td><td> NT49-769 </td><td></td><td> 2 </td></tr><tr><td> Dia Lente acromática Doublet 50mm. FL x 300mm, revestimento MgF2 </td><td> Edmund Optics </td><td> NT45-179 </td&gt; <td></td><td> 1 </td></tr><tr><td> 8 kHz R Scanner óptico de alta freqüência </td><td> Electro-Optical Products Corporation (EOPC) </td><td> SC-30 </td><td> 8 kHz </td><td> 1 </td></tr><tr><td> Driver de AGC </td><td> Electro-Optical Products Corporation (EOPC) </td><td> ACG: 8K </td><td></td><td></td></tr><tr><td> H7422-PA Módulo Fotosensor </td><td> Hamamatsu </td><td> H7422-PA </td><td> Limitação de corrente recomendada </td><td> 1 </td></tr><tr><td> M9012 Alimentação </td><td> Hamamatsu </td><td> M9012 </td><td> Para uso com H7422-PA </td><td> 1 </td></tr><tr><td> HC PL APO CS Objetivo </td><td> Leica </td><td> 11506284 </td><td> 10x/0.40 </td><td> 1 </td></tr><tr><td> Solios Cartão Framegrabber eA / XA </td><td> Matrox </td><td> Solios eA / XA </td><td> MIL software necessário; OEM interliga recomendado </td><td> 1 </td></tr><tr><td> Fonte de alimentação 12V </td><td&gt; Meanwell </td><td> LPV-100-12 </td><td> +12 V, 8.5A </td><td> 1 </td></tr><tr><td> Lente objetiva de 5x Microscópio </td><td> Newport </td><td> M-5X </td><td> 0,10 NA, Comprimento 25,4 milímetros Focal </td><td> 1 </td></tr><tr><td> Fibra imagem coerente </td><td> Sumitomo </td><td> 8-30N </td><td></td><td> 1 </td></tr><tr><td> 1 / 4 &quot;Parafuso -20 e Kit Hardware </td><td> ThorLabs </td><td> HW-KIT2 </td><td></td><td> 1 </td></tr><tr><td> 100 mm Montado Pinhole </td><td> ThorLabs </td><td> P100S </td><td> Ideal para a construção de filtros espaciais </td><td> 1 </td></tr><tr><td> 30 milímetros Grampo Cube Gaiola </td><td> ThorLabs </td><td> B6C </td><td></td><td> 1 </td></tr><tr><td> 30 milímetros Cube Sistema Cage, 4-Way </td><td> ThorLabs </td><td> C4W </td><td></td><td> 1 </td></tr><tr><td> 406 nm, 5 mW, B Pin Code, fibra SM Diode Laser Pigtailed, FC / PC </td><td> ThorLabs </td><td> LPS-406-FC </td><td> Produtos obsoletos; substituído por LP405-SF10 </td><td> 1 </td></tr><tr><td> 5 Minute-Epoxy, uma onça </td><td> ThorLabs </td><td> G14250 </td><td></td><td> 1 </td></tr><tr><td> 6 Axis cinemática Optic Monte </td><td> ThorLabs </td><td> K6X </td><td></td><td> 1 </td></tr><tr><td> 8-32 Parafuso e Kit Hardware </td><td> ThorLabs </td><td> HW-KIT1 </td><td></td><td> 1 </td></tr><tr><td> Parafuso de fixação 8-32 e Kit Hardware </td><td> ThorLabs </td><td> HW-KIT3 </td><td></td><td> 1 </td></tr><tr><td> Adaptador com Rosca externa RMS e roscas internas SM1 </td><td> ThorLabs </td><td> SM1A4 </td><td></td><td> 1 </td></tr><tr><td> Adj. FC / PC e FC / APC Collimator, f = 2,0 mm, ARC: 400-600 nm </td><td> ThorLabs </td><td> CFC-A-2X </td><td> f = 2,0 milímetros </td><td> 1 </td></tr><tr><td> Adaptador Colimador ajustável Fibra, SM1 Threaded </td><td> ThorLabs </td><td> AD9.5F </td><td></td><td> 1 </td></tr><tr><td> Breadboard de alumínio, 12 &quot;x 18&quot; x 1 / 2 &quot; </td><td> ThorLabs </td><td> MB1218 </td><td> 1 / 4 &quot;-20 Threaded </td><td> 2 </td></tr><tr><td> Diode Laser de bancada / TEC Controlador </td><td> ThorLabs </td><td> ITC4001 </td><td> 1 A/96 W </td><td> 1 </td></tr><tr><td> DMLP 425 nm Espelho longa-Pass Dichroic </td><td> ThorLabs </td><td> DMLP425 </td><td></td><td> 1 </td></tr><tr><td> Monte cinemática para Ø1 &quot;Optics </td><td> ThorLabs </td><td> KM100 </td><td></td><td> 3 </td></tr><tr><td> LD / TEC montagem para Diodos ThorLabs Fiber-Pigtailed Laser </td><td> ThorLabs </td><td> LM9LP </td><td></td><td> 1 </td></tr><tr><td> Montagem de Lente de Ø18 MM Optics </td><td> ThorLabs </td><td> LMR18 </td><td> Um anel de retenção incluído </td><td> 1 </td></tr><tr><td> Montagem da lente para 2 &quot;Optics </td><td> ThorLabs </td><td> LMR2S </td><td> Com internos e externos threading; anel de fixação incluída </td><td> 2 </td></tr><tr><td> Mini Series Gaiola Assembléia Rod, 6 &quot;mm, Long Ø4, Quantidade 1. </td><td> ThorLabs </td><td> SR6 </td><td></td><td> 4 </td></tr><tr><td> Ø1.0 &quot;Post Pillar Pedestal, 8-32 Taps, um&quot; Long </td><td> ThorLabs </td><td> RS1P8E </td><td></td><td> 4 </td></tr><tr><td> Ø1 &quot;Pilar Mensagem Extensão Comprimento, = 0,5 </td><td> ThorLabs </td><td> RS05 </td><td></td><td> 4 </td></tr><tr><td> Ø1 &quot;Pilar Mensagem Extensão Comprimento, = 0,75&quot; </td><td> ThorLabs </td><td> RS075 </td><td></td><td> 4 </td></tr><tr><td> Ø1 &quot;Protected Prata Espelho, 3,2 mm de espessura </td><td> ThorLabs </td><td> ME1-P01 </td><td></td><td> 1 </td></tr><tr><td> Ø1 &quot;SM1 rotação do elemento ajustável foco, L = 1&quot; </td><td> ThorLabs </td><td> SM1V10 </td&gt; <td></td><td> 1 </td></tr><tr><td> Ø2 &quot;Protected Prata Espelho, 3,2 mm de espessura </td><td> ThorLabs </td><td> ME2-P01 </td><td></td><td> 2 </td></tr><tr><td> P100S - Ø100 mM Mounted Pinhole </td><td> ThorLabs </td><td> P100S </td><td></td><td> 1 </td></tr><tr><td> Polaris Deriva Low Ø1 &quot;cinemática Espelho Monte </td><td> ThorLabs </td><td> POLARIS-K1 </td><td> Drift baixo </td><td> 1 </td></tr><tr><td> SM1 Lens Tube, L = 1 &quot; </td><td> ThorLabs </td><td> SM1L-10 </td><td> Um anel de retenção incluído </td><td> 4 </td></tr><tr><td> SM1 Threaded 30 Placa Gaiola mm, 0,35 &quot;Thick </td><td> ThorLabs </td><td> CP02 </td><td></td><td> 2 </td></tr><tr><td> SM1 a M25 Adaptador Componente Óptico de Threading </td><td> ThorLabs </td><td> SM1A24 </td><td> Externa e interna Threads SM1 M25.5x0.5 Tópicos </td><td> 1 </td></tr><tr><td> Diam Boca pequenaeter Galvo Sistema </td><td> ThorLabs </td><td> GVSM001 </td><td></td><td> 1 </td></tr><tr><td> Fork aperto pequena </td><td> ThorLabs </td><td> CF125 </td><td> 25/01 &quot;escareados slot, universal </td><td> 15 </td></tr><tr><td> Sistema de filtro espacial </td><td> ThorLabs </td><td> KT310 </td><td> Pinhole vendido separadamente </td><td> 1 </td></tr><tr><td> TE-Cooled montagem para 5,6 e 9 milímetros Lasers </td><td> ThorLabs </td><td> TCLDM9 </td><td></td><td> 1 </td></tr><tr><td> Suporte Vertical para Breadboards </td><td> ThorLabs </td><td> VB01 </td><td> Cada </td><td> 2 </td></tr><tr><td> Plano Apochromat- </td><td> Zeiss </td><td> 1101-957 </td><td> 20x/0.75 NA </td><td> 1 </td></tr></tbody></table>

video-rate scanning confocal microscopy and microendoscopy

Microscopia confocal tem se tornado uma ferramenta valiosa na biologia e nas ciências biomédicas, permitindo rápido, de alta sensibilidade e alta resolução de seccionamento óptico de sistemas complexos. Microscopia confocal é rotineiramente usado, por exemplo, para estudar alvos celulares específicos 1, monitor dinâmica em células vivas 2-4, e visualizar a evolução tridimensional de organismos inteiros 5,6. Extensões de sistemas de imagem confocal, como microendoscopes confocal, permitem alta resolução de imagem in vivo 7 e estão actualmente a ser aplicado a imagem da doença e diagnóstico em clínicas 8,9. Microscopia confocal tridimensional fornece resolução criando os chamados &quot;seções óptica&quot; utilizando óptica geométrica simples. Em um microscópio de campo amplo padrão, de fluorescência gerada a partir de uma amostra é coletada por uma lente objetiva e retransmitidas diretamente para um detector. Enquanto aceitarcapaz de amostras de imagem fina, as amostras de espessura embaçada por fluorescência gerada acima e abaixo do plano objetivo focal. Em contraste, microscopia confocal permite corte, virtual óptico de amostras, rejeitando out-of-foco de luz para construir de alta resolução representações tridimensionais de amostras. Microscópios confocal a conseguir este feito através de uma abertura confocal no caminho do feixe de detecção. A fluorescência de uma amostra coletada pelo objectivo é retransmitida de volta através dos espelhos de varredura e através do espelho primário dicróicas, um espelho para refletir cuidadosamente selecionados comprimentos de onda mais curtos, como o feixe de laser de excitação durante a passagem do tempo, Stokes-shifted emissão de fluorescência. Este sinal de longo comprimento de onda de fluorescência é então passado para um par de lentes de ambos os lados de um diafragma que é posicionado em um avião exatamente conjugado com o plano focal da lente objetiva. Fótons coletados a partir do volume focal do objeto são colimadospela lente objetiva e estão focados pelas lentes confocal através do pinhole. Fluorescência gerado acima ou abaixo do plano focal, portanto, não ser colimada corretamente, e não vai passar pelo pinhole confocal 1, a criação de uma secção óptica na qual apenas a luz do foco do microscópio é visível. (Fig. 1). Assim, o pinhole age efetivamente como uma abertura virtual no plano focal, restringindo a emissão detectada apenas uma localização espacial limitada. Comercial moderno microscópios confocal oferecer aos usuários uma operação totalmente automatizada, fazendo procedimentos de imagem anteriormente complexo relativamente simples e acessível. Apesar da flexibilidade e potência desses sistemas, comercial microscópios confocal não são adequados para todas as tarefas de imagem confocal, como muitos em aplicações de imagem in vivo. Sem a capacidade de criar sistemas de imagem personalizada para atender suas necessidades, experiências importantes podem ficar fora da reaçãoh para muitos cientistas. Neste artigo, nós fornecemos um método passo-a-passo para a construção completa de um costume, sistema de imagem de vídeo de taxa de confocal a partir de componentes básicos. O microscópio vertical será construído utilizando um espelho ressonante galvanométricos para fornecer o eixo de varredura rápida, enquanto um padrão de velocidade de espelho ressonante galvanométricos irá verificar o eixo lento. Para criar um feixe preciso digitalizados no foco da lente objetiva, esses espelhos serão posicionados nos planos chamados telecêntrica usando quatro lentes relay. Detecção confocal será realizada usando um padrão, tubo fotomultiplicador off-the-shelf (PMT), e as imagens serão capturadas e exibidas usando um cartão de framegrabber Matrox eo software incluído.

Watch this Scientific Journal Video about Video-rate Scanning Confocal Microscopy and Microendoscopy at JoVE.com

Video-rate Scanning Confocal Microscopy and Microendoscopy

A construção completa de um costume, sistema de imagens em tempo real confocal de varredura é descrito. Este sistema, que pode ser facilmente utilizado para vídeo-microscopia e taxa de microendoscopy, permite uma variedade de geometrias de imagem e aplicações que não sejam acessíveis usando o padrão comercial sistemas confocal, em uma fração do custo.

Vídeo taxa de microscopia de varredura confocal e Microendoscopy

Confocal microscopy has become an invaluable tool in biology and the biomedical sciences, enabling rapid, high-sensitivity, and high-resolution optical ...

video-rate-scanning-confocal-microscopy-and-microendoscopy

Research

JoVE Journal

Bioengineering

27.7K visualizações.  Harvard University. A construção completa de um costume, sistema de imagens em tempo real confocal de varredura é descrito. Este sistema, que pode ser facilmente utilizado para vídeo-microscopia e taxa de microendoscopy, permite uma variedade de geometrias de imagem e aplicações que não sejam acessíveis usando o padrão comercial sistemas confocal, em uma fração do custo.

Vídeo: Video-rate Scanning Confocal Microscopy and Microendoscopy

High-resolution Fiber-optic Microendoscopy for in situ Cellular Imaging

Many biological and clinical studies require the longitudinal study and analysis of morphology and function with cellular level resolution. Traditionally, multiple experiments are run in parallel, with individual samples removed from the study at sequential time points for evaluation by light microscopy. Several intravital techniques have been developed, with confocal, multiphoton, and second harmonic microscopy all demonstrating their ability to be used for imaging in situ 1. With these systems, however, the required infrastructure is complex and expensive, involving scanning laser systems and complex light sources. Here we present a protocol for the design and assembly of a high-resolution microendoscope which can be built in a day using off-the-shelf components for under US$5,000. The platform offers flexibility in terms of image resolution, field-of-view, and operating wavelength, and we describe how these parameters can be easily modified to meet the specific needs of the end user.
We and others have explored the use of the high-resolution microendoscope (HRME) in in vitro cell culture 2-5, in excised 6 and living animal tissues 2,5, and in human tissues in vivo 2,7. Users have reported the use of several different fluorescent contrast agents, including proflavine 2-4, benzoporphyrin-derivative monoacid ring A (BPD-MA) 5, and fluoroscein 6,7, all of which have received full, or investigational approval from the FDA for use in human subjects. High-resolution microendoscopy, in the form described here, may appeal to a wide range of researchers working in the basic and clinical sciences. The technique offers an effective and economical approach which complements traditional benchtop microscopy, by enabling the user to perform high-resolution, longitudinal imaging in situ.

High-resolution Fiber-optic Microendoscopy for in situ Cellular Imaging

In many biological and clinical situations it is advantageous to study cellular processes as they evolve in their native microenvironment. Here we describe the assembly and use of a low-cost fiber-optic microscope which can provide real time imaging in cell culture, animal studies, and clinical patient studies.

De alta resolução de fibra óptica para Microendoscopy In situ Celular imagem

Many biological and clinical studies require the longitudinal study and analysis of morphology and function with cellular level resolution.  ...

Bioengenharia

Low-Cost Cryo-Light Microscopy Stage Fabrication for Correlated Light/Electron Microscopy

The coupling of cryo-light microscopy (cryo-LM) and cryo-electron microscopy (cryo-EM) poses a number of advantages for understanding cellular 
dynamics and ultrastructure. First, cells can be imaged in a near native environment for both techniques. Second, due to the vitrification process, samples are
 preserved by rapid physical immobilization rather than slow chemical fixation. Third, imaging the same sample with both cryo-LM and cryo-EM provides correlation of
 data from a single cell, rather than a comparison of "representative samples". While these benefits are well known from prior studies, the widespread use of correlative 
 cryo-LM and cryo-EM remains limited due to the expense and complexity of buying or building a suitable cryogenic light microscopy stage. Here we demonstrate the 
 assembly, and use of an inexpensive cryogenic stage that can be fabricated in any lab for less than $40 with parts found at local hardware and grocery stores. This 
 cryo-LM stage is designed for use with reflected light microscopes that are fitted with long working distance air objectives. For correlative cryo-LM and cryo-EM 
 studies, we adapt the use of carbon coated standard 3-mm cryo-EM grids as specimen supports. After adsorbing the sample to the grid, previously established protocols 
 for vitrifying the sample and transferring/handling the grid are followed to permit multi-technique imaging. As a result, this setup allows any laboratory with a 
 reflected light microscope to have access to direct correlative imaging of frozen hydrated samples.

We demonstrate the fabrication of a low-cost cryogenic stage designed to fit most reflected light microscopes. This lab-built cryogenic stage enables efficient and reliable correlative imaging between cryo-light and cryo-electron microscopy.

Low-Cost Cryo-Light Fabricação Stage Microscopia de Luz Correlacionados / Microscopia Eletrônica

The coupling of cryo-light microscopy (cryo-LM) and cryo-electron microscopy (cryo-EM) poses a number of advantages for understanding cellular 
dynamics ...

Specimen Preparation, Imaging, and Analysis Protocols for Knife-edge Scanning Microscopy

Major advances in high-throughput, high-resolution, 3D microscopy techniques have enabled the acquisition of large volumes of neuroanatomical data at submicrometer resolution. One of the first such instruments producing whole-brain-scale data is the Knife-Edge Scanning Microscope (KESM)7, 5, 9, developed and hosted in the authors' lab. KESM has been used to section and image whole mouse brains at submicrometer resolution, revealing the intricate details of the neuronal networks (Golgi)1, 4, 8, vascular networks (India ink)1, 4, and cell body distribution (Nissl)3. The use of KESM is not restricted to the mouse nor the brain. We have successfully imaged the octopus brain6, mouse lung, and rat brain. We are currently working on whole zebra fish embryos. Data like these can greatly contribute to connectomics research10; to microcirculation and hemodynamic research; and to stereology research by providing an exact ground-truth. 
In this article, we will describe the pipeline, including specimen preparation (fixing, staining, and embedding), KESM configuration and setup, sectioning and imaging with the KESM, image processing, data preparation, and data visualization and analysis. The emphasis will be on specimen preparation and visualization/analysis of obtained KESM data. We expect the detailed protocol presented in this article to help broaden the access to KESM and increase its utilization.

The full process from brain specimen preparation to serial sectioning imaging using the Knife-Edge Scanning Microscope, to data visualization and analysis is described. This technique is currently used to acquire mouse brain data, but it is applicable to other organs, other species.

Preparação de amostras, Imaging, and Analysis Protocolos para Faca de ponta microscopia de varredura

Major advances in high-throughput, high-resolution, 3D microscopy techniques have enabled the acquisition of large volumes of neuroanatomical data at ...

Correlative Light- and Electron Microscopy Using Quantum Dot Nanoparticles

A method is described whereby quantum dot (QD) nanoparticles can be used for correlative immunocytochemical studies of human pathology tissue using widefield fluorescence light microscopy and transmission electron microscopy (TEM). To demonstrate the protocol we have immunolabeled ultrathin epoxy sections of human somatostatinoma tumor using a primary antibody to somatostatin, followed by a biotinylated secondary antibody and visualization with streptavidin conjugated 585 nm cadmium-selenium (CdSe) quantum dots (QDs). The sections are mounted on a TEM specimen grid then placed on a glass slide for observation by widefield fluorescence light microscopy. Light microscopy reveals 585 nm QD labeling as bright orange fluorescence forming a granular pattern within the tumor cell cytoplasm. At low to mid-range magnification by light microscopy the labeling pattern can be easily recognized and the level of non-specific or background labeling assessed. This is a critical step for subsequent interpretation of the immunolabeling pattern by TEM and evaluation of the morphological context. The same section is then blotted dry and viewed by TEM. QD probes are seen to be attached to amorphous material contained in individual secretory granules. Images are acquired from the same region of interest (ROI) seen by light microscopy for correlative analysis. Corresponding images from each modality may then be blended to overlay fluorescence data on TEM ultrastructure of the corresponding region.

A method is described whereby quantum dot (QD) nanoparticles can be used for correlative immunocytochemical studies of epoxy embedded human pathology tissue. We employ commercial antibody fragment conjugated QDs that are visualized by widefield fluorescence light microscopy and transmission electron microscopy.

Correlativa luz e de Microscopia Eletrônica Usando Quantum Dot Nanopartículas

A method is described whereby quantum dot (QD) nanoparticles can be used for correlative immunocytochemical studies of human pathology tissue using ...

Multimodal Imaging and Spectroscopy Fiber-bundle Microendoscopy Platform for Non-invasive, In Vivo Tissue Analysis

Recent fiber-bundle microendoscopy techniques enable non-invasive analysis of in vivo tissue using either imaging techniques or a combination of spectroscopy techniques. Combining imaging and spectroscopy techniques into a single optical probe may provide a more complete analysis of tissue health. In this article, two dissimilar modalities are combined, high-resolution fluorescence microendoscopy imaging and diffuse reflectance spectroscopy, into a single optical probe. High-resolution fluorescence microendoscopy imaging is a technique used to visualize apical tissue micro-architecture and, although mostly a qualitative technique, has demonstrated effective real-time differentiation between neoplastic and non-neoplastic tissue. Diffuse reflectance spectroscopy is a technique which can extract tissue physiological parameters including local hemoglobin concentration, melanin concentration, and oxygen saturation. This article describes the specifications required to construct the fiber-optic probe, how to build the instrumentation, and then demonstrates the technique on in vivo human skin. This work revealed that tissue micro-architecture, specifically apical skin keratinocytes, can be co-registered with its associated physiological parameters. The instrumentation and fiber-bundle probe presented here can be optimized as either a handheld or endoscopically-compatible device for use in a variety of organ systems. Additional clinical research is needed to test the viability of this technique for different epithelial disease states.

Multimodal Imaging and Spectroscopy Fiber-bundle Microendoscopy Platform for Non-invasive, In Vivo Tissue Analysis

The assembly and use of a multimodal microendoscope is described which can co-register superficial tissue image data with tissue physiological parameters including hemoglobin concentration, melanin concentration, and oxygen saturation. This technique can be useful for evaluating tissue structure and perfusion, and can be optimized for individual needs of the investigator.

Multimodal Imaging and Platform microendoscopy Espectroscopia de fibra pacote para não-invasiva,<em&gt; In Vivo</em&gt; Análise Tissue

Recent fiber-bundle microendoscopy techniques enable non-invasive analysis of in vivo tissue using either imaging techniques or a combination of ...

Dissection, MicroCT Scanning and Morphometric Analyses of the Baculum

Modern morphometrics provides powerful methods to quantify size and shape variation. A basic requirement is a list of coordinates that define landmarks; however such coordinates must represent homologous structures across specimens. While many biological objects consist of easily identified landmarks to satisfy the assumption of homology, many lack such structures. One potential solution is to mathematically place semi-landmarks on an object that represent the same morphological region across specimens. Here, we illustrate a recently developed pipeline to mathematically define semi-landmarks from the mouse baculum (penis bone). Our methods should be applicable to a wide range of objects.

Many biological structures lack easily definable landmarks, making it difficult to apply modern morphometric methods. Here we illustrate methods to study the mouse baculum (a bone in the penis), including dissection and microCT scanning, followed by computational methods to define semi-landmarks that are used to quantify size and shape variation.

Dissecação, digitalização MicroCT e morfométricas Análises do Baculum

Modern morphometrics provides powerful methods to quantify size and shape variation. A basic requirement is a list of coordinates that define landmarks; ...

Mammalian Cell Division in 3D Matrices via Quantitative Confocal Reflection Microscopy

The study of how mammalian cell division is regulated in a 3D environment remains largely unexplored despite its physiological relevance and therapeutic significance. Possible reasons for the lack of exploration are the experimental limitations and technical challenges that render the study of cell division in 3D culture inefficient. Here, we describe an imaging-based method to efficiently study mammalian cell division and cell-matrix interactions in 3D collagen matrices. Cells labeled with fluorescent H2B are synchronized using the combination of thymidine blocking and nocodazole treatment, followed by a mechanical shake-off technique. Synchronized cells are then embedded into a 3D collagen matrix. Cell division is monitored using live-cell microscopy. The deformation of collagen fibers during and after cell division, which is an indicator of cell-matrix interaction, can be monitored and quantified using quantitative confocal reflection microscopy. The method provides an efficient and general approach to study mammalian cell division and cell-matrix interactions in a physiologically relevant 3D environment. This approach not only provides novel insights into the molecular basis of the development of normal tissue and diseases, but also allows for the design of novel diagnostic and therapeutic approaches.

This protocol efficiently studies mammalian cell division in 3D collagen matrices by integrating synchronization of cell division, monitoring of division events in 3D matrices using live-cell imaging technique, time-resolved confocal reflection microscopy and quantitative imaging analysis.

Divisão celular dos mamíferos em matrizes 3D através de microscopia de reflexão Confocal quantitativa

The study of how mammalian cell division is regulated in a 3D environment remains largely unexplored despite its physiological relevance and therapeutic ...

Multimodal Volumetric Retinal Imaging by Oblique Scanning Laser Ophthalmoscopy (oSLO) and Optical Coherence Tomography (OCT)

While fluorescence imaging is widely used in ophthalmology, a large field of view (FOV) three-dimensional (3D) fluorescence retinal image is still a big challenge with the state-of-the-art retinal imaging modalities because they would require z-stacking to compile a volumetric dataset. Newer optical coherence tomography (OCT) and OCT angiography (OCTA) systems overcome these restrictions to provide three-dimensional (3D) anatomical and vascular images, but the dye-free nature of OCT cannot visualize leakage indicative of vascular dysfunction. This protocol describes a novel oblique scanning laser ophthalmoscopy (oSLO) technique that provides 3D volumetric fluorescence retinal imaging. The setup of the imaging system generates the oblique scanning by a dove tail slider and aligns the final imaging system at an angle to detect fluorescent cross-sectional images. The system uses the laser scanning method, and therefore, allows an easy incorporation of OCT as a complementary volumetric structural imaging modality. In vivo imaging on rat retina is demonstrated here. Fluorescein solution is intravenously injected to produce volumetric fluorescein angiography (vFA).

Multimodal Volumetric Retinal Imaging by Oblique Scanning Laser Ophthalmoscopy oSLO and Optical Coherence Tomography OCT

Here, we present a protocol to get a large field of view (FOV) three-dimensional (3D) fluorescence and OCT retinal image by using a novel imaging multimodal platform. We will introduce the system setup, the method of alignment, and the operational protocols. In vivo imaging will be demonstrated, and representative results will be provided.

Multimodal volumétrico Retinal Imaging pelo oblíquo digitalização Laser Oftalmoscopia (oSLO) e a tomografia de coerência óptica (OCT)

While fluorescence imaging is widely used in ophthalmology, a large field of view (FOV) three-dimensional (3D) fluorescence retinal image is still a big ...

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 ...

Near Simultaneous Laser Scanning Confocal and Atomic Force Microscopy (Conpokal) on Live Cells

Techniques available for micro- and nano-scale mechanical characterization have exploded in the last few decades. From further development of the scanning and transmission electron microscope, to the invention of atomic force microscopy, and advances in fluorescent imaging, there have been substantial gains in technologies that enable the study of small materials. Conpokal is a portmanteau that combines confocal microscopy with atomic force microscopy (AFM), where a probe &#8220;pokes&#8221; the surface. Although each technique is extremely effective for the qualitative and/or quantitative image collection on their own, Conpokal provides the capability to test with blended fluorescence imaging and mechanical characterization. Designed for near simultaneous confocal imaging and atomic force probing, Conpokal facilitates experimentation on live microbiological samples. The added insight from paired instrumentation provides co-localization of measured mechanical properties (e.g., elastic modulus, adhesion, surface roughness) by AFM with subcellular components or activity observable through confocal microscopy. This work provides a step by step protocol for the operation of laser scanning confocal and atomic force microscopy, simultaneously, to achieve same cell, same region, confocal imaging, and mechanical characterization.

Near Simultaneous Laser Scanning Confocal and Atomic Force Microscopy Conpokal on Live Cells

Presented here is the protocol of the Conpokal technique, which combines confocal and atomic force microscopy into a single instrument platform. Conpokal provides same cell, same region, near simultaneous confocal imaging and mechanical characterization of live biological samples.

Microscopia confocal e força atômica (Conpokal) em células vivas

Techniques available for micro- and nano-scale mechanical characterization have exploded in the last few decades. From further development of the scanning ...