Este trabalho foi financiado por um subsídio da Swiss National Science Foundation (SNSF; #320030_156019). Os autores receberam apoio financeiros Heidelberg Engineering GmBH, Alemanha.

em todos os procedimentos, BALB/c adulto masculinos e femininos ratos que expressam gfp sob o promotor da Cx3cr1 foram usados 24. Os ratos foram tratados de acordo com a instrução de ARVO para o uso de animais em oftalmologia e visão pesquisa e todos os procedimentos foram aprovados do governo suíço, de acordo com os regulamentos federais suíços em matéria de bem-estar Animal. Os ratos foram anestesiados por uma injeção subcutânea de cloridrato de medetomidina (0,75 mg/kg) e a cetamina (45 mg/kg). Anestesia adequada foi confirmada pelo monitoramento da frequência respiratória e o animal &#39; reflex s contra uma pitada de cauda. No final dos experimentos, os ratos foram sacrificados com inalação de CO 2.
 Nota: realizar cada sessão de imagens tão rapidamente quanto possível (no máximo 20 min), desde a formação da catarata anestesia seguinte poderá prejudicar a visualização da retina 30.
 1. configuração do sistema <ol><li>ligar o computador.</li>
	<li>Ligar a alimentação. A mensagem &#34; módulo de aquisição de início &#34; aparecerá na tela de painel de controle do dispositivo.</li>
	<li>Duplo clique no atalho do software no desktop para operar o software.</li>
	<li>No modo de exibição de banco de dados, clique no &#34; adicionar o paciente &#34; ícone.</li>
	<li>Na janela pop-up, insira todas as informações necessárias (sobrenome, nome, título, data de nascimento, sexo e paciente-ID) e clique em &#34; okey &#34;. Na janela pop definir a curvatura da córnea de 2mm e clique &#34; okey &#34;.</li>
	<li>Colocar uma lente de lâmpada 78D Oftalmologia padrão sem contato fenda na frente a óptica padrão 30 ° e fixá-la de forma segura com fita.</li>
	<li>Certifique-se de que a alavanca à esquerda do dispositivo é posicionada sobre a indicação de &#34; A &#34; para permitir que os IR + OCT e AF de imagem ( Figura 1).</li>
</ol> 2. Preparação de mouse <ol><li>uso uma dose de cloridrato de medetomidina de 0,75 mg/kg e uma ketamina dose de 45 mg/kg em tampão fosfato salino (PBS, pH 7,4) para preparar a solução de anestesia. Assim, para um rato de 20 g, misture 15 µ l de cloridrato de medetomidina com 18 µ l de cetamina para um volume final de 50 µ l de PBS. Armazenar a solução a 4 ° C por até uma semana.</li>
	<li>Segure o mouse pela nuca e aplique uma gota de tropicamida 0.5% + cloridrato de fenilefrina 2,5% em cada olho para conseguir a dilatação da pupila.</li>
	<li>Manter o mouse contido e com uma seringa de insulina anexada a uma agulha 30G, 50 µ l da solução de anestesia de injectar por via subcutânea. Coloque o mouse de volta à sua jaula que é colocada sobre uma almofada de aquecimento. Espere 3-5 min até que o mouse está totalmente sedado. Para avaliar a profundidade anestésica, belisca o rabo do rato. Verificar o reflexo corneal tocando suavemente a córnea de rato com um cotonete. Se nenhum reflexo positivo é observado, continue com as imagens. 
	Nota: É importante monitorar a frequência respiratória durante a anestesia. Se o mouse não está totalmente sedado, a taxa respiratória irá aumentar após um estímulo doloroso.</li>
	<li>Hidrato a córnea de rato com um aplicativo de hidroxipropilmetilcelulose 2% gotas em cada olho.</li>
</ol> 3. SD-OCT <ol><li>Coloque um patch de aquecimento de cerca de 32 ° C na plataforma sob medida anexada no descanso de queixo do dispositivo ( Figura 1).</li>
	<li>a imagem do olho direito, posicione o mouse na parte esquerda da plataforma ( Figura 1). Certifique-se de que a órbita direita do mouse enfrenta a lente e seu corpo encontra-se sujeito na parte esquerda da plataforma.</li>
	<li>Aplicar uma gota de hidroxipropilmetilcelulose no olho direito e cuidadosamente coloque uma lente de contato dioptria + 4 rígida gás permeável nele (poder esférico:-25.00 para +25,00 dioptrias). Armazene a lente de contato em solução salina balanceada (BSS). Para agarrar a lente, usar fórceps de plástico ou silicone para evitar danos.</li>
	<li>Pressione a caixa amarela no canto direito do visor do painel de controle para iniciar o módulo de aquisição (caixa verde na Figura 2A). A caixa amarela vai ficar verde e no menu do painel de controle aparecerá na tela ( Figura 2A).</li>
	<li>Para aquisição de B-scans, selecione a opção IR + OCT no painel de controle ( Figura 2A). Configurações selecionadas são destacadas em azul no painel de controle.</li>
	<li>Select &#34; Retina &#34; sob &#34; aplicação e estrutura &#34; no software e mover a lente para o olho do mouse usando o micromanipulador no dispositivo ( Figura 1). Antes de se concentrar sobre a retina, verificar a indicação &#34; OD &#34; estiver selecionada na parte inferior esquerda da tela. O software identifica automaticamente a esquerda (OS) e olho direito (OD) com base na posição do objectivo. 
	Nota: Se o objetivo é posicionado no meio da plataforma, uma mensagem de erro aparecerá na parte inferior da tela. Nesse caso, volte a posicionar o mouse ligeiramente até que o software reconhece o olho correto.</li>
	<li>Com o botão de foco, foco na retina, até os grandes vasos são claramente vistos na imagem do fundo à esquerda da tela do computador. Mover a câmera rodando o micromanipulador esquerda ou direita mover a câmera nessa direção, ou no sentido horário ou no sentido anti-horário, para mover a câmera, cima ou baixo, respectivamente. 
	Nota: Reposicionar o mouse na plataforma ou mover a lente para cima ou para baixo para alcançar a localização preferencial da cabeça do nervo óptico na imagem infravermelha.</li>
	<li>Rode o botão de sensibilidade no sentido anti-horário para reduzir ou no sentido horário para aumentar o brilho da imagem do fundo. Quando o foco ideal é alcançado, um SD-OCT B-scan aparecerá no lado direito da tela. 
	Nota: Se o B-scan não pode ser visualizado, ajustando o foco, pressione a combinação Ctrl + Alt + Shift + O no teclado. Na janela pop-up, ajustar o valor do braço referência até o B-scan aparece na tela.</li>
	<li>No canto inferior direito da tela, escolha o único scan no menu padrão (única linha na Figura 2B).</li>
	<li>Transformar o micromanipulador do dispositivo ( Figura 1) esquerda, direita, frente ou para trás (para ligar a câmera nessa direção) para garantir que o B-scan localiza-se entre a parte superior e inferior cantos da SD-OCT varrer janela.</li>
	<li>Definir o valor de automática em tempo real (arte) para pelo menos 9 para obter imagens de alta qualidade. 
	Nota: Arte melhora a qualidade de imagem por uma média de vários exames consecutivos. Quanto maior o &#34; arte &#34; valor, quanto maior a relação sinal-ruído e, portanto, a qualidade da imagem. No entanto, aumentando o &#34; arte &#34; de valor, a aquisição de tempo também é aumentado.</li>
	<li>Imprensa o &#34; adquirir &#34; botão ( Figura 2) sobre a tela do painel de controle e adquirir as imagens.</li>
	<li>Re-posicione o mouse para o olho esquerdo da imagem e repita etapas 3.2-3.12.</li>
	<li>Remover a lente de contato com a pinça de plástico/silicone e coloque-o no BSS.</li>
	<li>Hidratar a córnea de rato com uma gota fresca de hidroxipropilmetilcelulose e remova o excesso com um papel absorvente.</li>
	<li>Remover a óptica padrão 30 °, girando-o no sentido anti-horário.</li>
	<li>Montar a lente 55 ° e repita etapas 3.4-3.12 para cada olho.</li>
</ol> 4. Auto fluorescência Imaging <ol><li>sem mover o mouse sobre a plataforma, selecionar IR em painel de controle.</li>
	<li>Com o botão de foco, foco sobre os grandes vasos da retina.</li>
	<li>No painel de controle selecione AF.</li>
	<li>Rode o botão de sensibilidade no sentido anti-horário para reduzir ou no sentido horário para aumentar o brilho da imagem.</li>
	<li>Pressione o botão de sensibilidade e defina o &#34; arte &#34; valor de 67 ou mais para obter alta qualidade imagens.</li>
	<li>Quando o conjunto &#34; arte &#34; valor foi alcançado, pressione &#34; adquirir &#34; no painel de controle para adquirir a imagem. Pressione o botão de sensibilidade novamente para parar uma média. Ajustar o foco, para visualizar as diferentes camadas da retina.</li>
	<li>Remover a lente de 55° e montar a lente de 102° de campo largo. Siga os passos 4.1 &#8211; 4.6 para ambos os olhos de cada rato e para cada mouse.</li>
	<li>Clique &#8220; salvar imagens &#8221; do lado esquerdo superior da janela e clique em &#8220; sair do &#8221;. Para salvar as imagens no computador, selecione o mouse na lista na parte direita da tela clicando duas vezes no nome do rato. Clique duas vezes sobre cada imagem separadamente e, em seguida, clique no ícone do disquete. Salve a imagem como TIF,. bmp,. jpg ou. png na pasta desejada. Para sair, pressione o software &#8220; arquivo &#8221; e &#8220; sair do &#8221;.</li>
</ol> 5. Reversão de anestesia <ol><li>hidratar os olhos de rato com uma gota de hidroxipropilmetilcelulose e retornar o mouse sobre uma almofada de aquecimento.</li>
	<li>Solução de atipamezole preparar uma dose de 2,25 mg/kg para reverter os efeitos sedativos de cloridrato de medetomidina. Para um mouse de 20 g, adicione 9 µ l de atipamezol em um volume final de 150 µ l de PBS. Armazenar a solução a 4 ° C por até uma semana.</li>
	<li>min após injeção de anestesia (passo 2.2), 150 µ l de solução de atipamezole de injectar por via subcutânea.</li>
	<li>Monitorar o mouse até a recuperação da anestesia. Quando o mouse está totalmente recuperado (5-10 min após a injeção de atipamezole) colocá-lo de volta à sua jaula. Recuperação é indicada pela capacidade do animal para transformar seu corpo quando colocada de um lado, recuperar a atividade de caminhar, e reagindo em resposta à estimulação ambiental.</li>
</ol> 6. Medição de espessura da retina manual de imagens SD-OCT <ol><li>abrir a varredura OCT clicando duas vezes sobre o nome do animal.</li>
	<li>Abra o B-scan, obtido com a lente de 30 ° ou ° 55.</li>
	<li>Escolher &#34; perfil de espessura &#34;.</li>
	<li>Imprensa o &#34; editar segmentações de camada &#34; ícone ( Figura 2B). O software automaticamente identifica a limitação interna e membrana de base.</li>
	<li>Se necessário, corrigir manualmente a posição da limitação interna e membrana de base. Para fazer isso, escolha a camada a ser modificada do lado esquerdo da tela e, em seguida, a opção círculo vermelho ( Figura 2B). Mover o círculo com o botão do mouse pressionado para modificar a linha.</li>
	<li>Modificar a linha para posicionar a camada correspondente corretamente. Clique em &#34; salvar e fechar &#34; para sair da janela. 
	Nota: Devido a refletividade de coroide, o software pode identificar a membrana base incorretamente. Assim, é preferível para defini-lo manualmente antes de prosseguir para medições de espessura da retina.</li>
	<li>Escolher &#34; Retina &#34; sob a &#34; camada &#34; opção. Um diagrama da espessura da retina aparecerá na parte inferior da tela.</li>
	<li>Clique em uma posição diferente sobre o diagrama ou sobre o B-scan para ver a espessura da retina (indicada em µm) para a posição selecionada.</li>
	<li>Medir a espessura da retina na distância desejada da cabeça do nervo óptico e copiar os valores em uma planilha.</li>
	<li>Repita os passos 5.1-5.9 para cada rato para a lente de 55 ° a 30 ° e.</li>
	<li>Realizar análise estatística com o software desejado.</li>
</ol>

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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Using+theoretical+models+from+adult+stroke+recovery+to+improve+use+of+non-invasive+brain+stimulation+for+children+with+congenital+hemiparesis.">Using theoretical models from adult stroke recovery to improve use of non-invasive brain stimulation for children with congenital hemiparesis.</a> J Neurophysiol. , (2017).</li><li>Combadiere, C., 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=CX3CR1-dependent+subretinal+microglia+cell+accumulation+is+associated+with+cardinal+features+of+age-related+macular+degeneration.">CX3CR1-dependent subretinal microglia cell accumulation is associated with cardinal features of age-related macular degeneration.</a> J Clin Invest. 117 (10), 2920-2928 (2007).</li><li>Bermudez, M. A., 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=Time+course+of+cold+cataract+development+in+anesthetized+mice.">Time course of cold cataract development in anesthetized mice.</a> Curr Eye Res. 36 (3), 278-284 (2011).</li><li>Toth, C. A., 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=A+comparison+of+retinal+morphology+viewed+by+optical+coherence+tomography+and+by+light+microscopy.">A comparison of retinal morphology viewed by optical coherence tomography and by light microscopy.</a> Arch Ophthalmol. 115 (11), 1425-1428 (1997).</li><li>Ebneter, A., Kokona, D., Jovanovic, J., Zinkernagel, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dramatic+Effect+of+Oral+CSF-1R+Kinase+Inhibitor+on+Retinal+Microglia+Revealed+by+In+Vivo+Scanning+Laser+Ophthalmoscopy.">Dramatic Effect of Oral CSF-1R Kinase Inhibitor on Retinal Microglia Revealed by In Vivo Scanning Laser Ophthalmoscopy.</a> Transl Vis Sci Technol. 6 (2), 10 (2017).</li><li>Gabriele, M. L., 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=Reproducibility+of+spectral-domain+optical+coherence+tomography+total+retinal+thickness+measurements+in+mice.">Reproducibility of spectral-domain optical coherence tomography total retinal thickness measurements in mice.</a> Invest Ophthalmol Vis Sci. 51 (12), 6519-6523 (2010).</li><li>Nakao, S., 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=Wide-field+laser+ophthalmoscopy+for+mice:+a+novel+evaluation+system+for+retinal/choroidal+angiogenesis+in+mice.">Wide-field laser ophthalmoscopy for mice: a novel evaluation system for retinal/choroidal angiogenesis in mice.</a> Invest Ophthalmol Vis Sci. 54 (8), 5288-5293 (2013).</li><li>Wang, N. 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=Origin+of+fundus+hyperautofluorescent+spots+and+their+role+in+retinal+degeneration+in+a+mouse+model+of+Goldmann-Favre+syndrome.">Origin of fundus hyperautofluorescent spots and their role in retinal degeneration in a mouse model of Goldmann-Favre syndrome.</a> Dis Model Mech. 6 (5), 1113-1122 (2013).</li><li>Wang, N. 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=Cellular+origin+of+fundus+autofluorescence+in+patients+and+mice+with+a+defective+NR2E3+gene.">Cellular origin of fundus autofluorescence in patients and mice with a defective NR2E3 gene.</a> Br J Ophthalmol. 93 (9), 1234-1240 (2009).</li><li>Thanos, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sick+photoreceptors+attract+activated+microglia+from+the+ganglion+cell+layer:+a+model+to+study+the+inflammatory+cascades+in+rats+with+inherited+retinal+dystrophy.">Sick photoreceptors attract activated microglia from the ganglion cell layer: a model to study the inflammatory cascades in rats with inherited retinal dystrophy.</a> Brain Res. 588 (1), 21-28 (1992).</li><li>Hughes, E. H., 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=Generation+of+activated+sialoadhesin-positive+microglia+during+retinal+degeneration.">Generation of activated sialoadhesin-positive microglia during retinal degeneration.</a> Invest Ophthalmol Vis Sci. 44 (5), 2229-2234 (2003).</li></ol>

Os autores não têm nada para divulgar.

O presente artigo demonstra um protocolo para a aquisição de B-retina e a imagem latente de gfp microglia positiva distribuição na retina do rato na mesma sessão de imagens. SD-OCT e SLO são cada vez mais usados em modelos animais de doença da retina para fornecer informações de alterações da retina ao longo do tempo,10,14,17,18,21. Com este protoco...

Na oftalmologia experimental, exame de patologia retiniana geralmente é avaliada usando técnicas histológicas. No entanto, histologia requer a eutanásia de animais e pode causar alteração para as propriedades reais do tecido. SD-OCT e SLO são rotineiramente usados em oftalmologia clínica para fins de diagnóstico e de acompanhamento de diversas doenças da retina como edema macular diabético9, neuropatia óptica isquêmica anterior10ou retinite pigmentosa11 . SD-OCT e SLO são técnicas não-invasivas que geram imagens de alta resolução da retina, que são visualizadas através da pupila dilatada sem mais intervenção. SD-OCT fornece informações da estrutura da retina e da espessura da retina através da recolha de dados Retrodispersão para criar imagens transversais da retina, enquanto SLO coleta dados de fluorescência para produzir imagens estereoscópicas de alto contraste da retina. Hoje em dia, ambas as técnicas são cada vez mais utilizadas em oftalmologia experimental usando pequenos roedores12,13,14,15 (ou mesmo zebrafish16,17) e pode fornece tanto informações qualitativas e quantitativas12,17,18,19,20,21.
Acumulação de fluorophores endógena como lipofuscins ou a formação de drusen na retina pode ser visualizada por SLO como sinal fluorescente auto. Esta característica torna SLO uma técnica valiosa para diagnóstico e monitoramento de doenças da retina como a degeneração macular relacionada à idade ou retinite pigmentosa22,23. Na oftalmologia experimental, fluorescência de auto imagem (AF) pode ser usada para a detecção de tipos de células específicas em linhas de rato de repórter. Por exemplo, ratos heterozigotos para a expressão de gfp sob o promotor de Cx3cr124 são vantajosos para visualização no vivo das pilhas microglial na retina normal e para a investigação de micróglia/macrófago dinâmica na doença da retina,21. Microglia são os macrófagos residentes da retina, que desempenham um papel fundamental na homeostase do tecido e reparação dos tecidos após lesão1,25,26. Ativação da microglia na retina tem sido relatada em degeneração, sugerindo um papel dessas células na doença da retina,2,3,4,5, , isquemia e lesão da retina 6.
O objectivo do presente protocolo é descrever um método relativamente simples para a imagem da retina e medição de espessura da retina usando SD-OCT e para visualização de células de gfp microglia positivo no rato retina utilizando Cx3cr1gfp/gfp SLO (sistema Heidelberg Spectralis HRA + OCT). Este protocolo pode ser utilizado para medições de imagem e espessura da retina saudável ou doente em várias linhas de rato. Além disso, as análises morfométricas podem ser realizadas para a identificação e quantificação de números microglia e ativação da microglia na retina usando SLO21. As células da micróglia são associadas com doenças degenerativas no sistema nervoso central (CNS), incluindo a retina27,28,29. Assim, combinando os dois métodos utilizados no presente protocolo, pode ser feita correlação de distribuição microglia e degeneração da retina, que pode facilitar o monitoramento severidade da doença ou a eficácia da terapêutica se aproxima na vivo.

<table>
	<tbody>
		<tr>
			<td>Spectralis Imaging system (HRA+OCT)</td>
			<td>Heidelberg Engineering, Germany</td>
			<td>N/A</td>
			<td>ophthalmic imaging platform system</td>
		</tr>
		<tr>
			<td>Heidelberg Eye Explorer</td>
			<td>Heidelberg Engineering, Germany</td>
			<td>N/A</td>
			<td>Version 1.9.13.0</td>
		</tr>
		<tr>
			<td>78D standard ophthalmic non-contact slit lamp lens</td>
			<td>Volk Optical Inc., Ohio, USA</td>
			<td>V78C</td>
			<td></td>
		</tr>
		<tr>
			<td>Spectralis wide angle 55&#176; lens</td>
			<td>Heidelberg Engineering, Germany</td>
			<td>50897-002</td>
			<td></td>
		</tr>
		<tr>
			<td>ultra widefield 102&#176; lens</td>
			<td>Heidelberg Engineering, Germany</td>
			<td>50117-001</td>
			<td></td>
		</tr>
		<tr>
			<td>medetomidine hydrochloride 1 mg/mL (Domitor)</td>
			<td>Provet AG, Lyssach, Switzerland</td>
			<td>Swissmedic Nr. 50&#39;590 - ATCvet: QN05CM91</td>
			<td>anesthetic/analgesic</td>
		</tr>
		<tr>
			<td>ketamine 50mg/ml (Ketalar)</td>
			<td>Parke-Davis, Zurich, Switzerland</td>
			<td>72276388</td>
			<td>anesthetic</td>
		</tr>
		<tr>
			<td>tropicamide 0.5% + phenylephrine HCl 2.5% (Augentropfen mix)</td>
			<td>ISPI, Bern, Switzerland</td>
			<td>N/A</td>
			<td>pupil dilation</td>
		</tr>
		<tr>
			<td>Omnican Insulin-50 0.5 ml G30 0.3 x 12mm</td>
			<td>B. Braun Mesungen AG, Carl-Braun-Stra&#223;e, Germany</td>
			<td>9151125</td>
			<td></td>
		</tr>
		<tr>
			<td>hydroxypropylmethylcellulose (Methocel 2%)</td>
			<td>OmniVision, Neuhausen, Switzerland</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>+4 dpt rigid gas permeable contact lens</td>
			<td>Quantum I, Bausch + Lomb Inc., Rochester, NY</td>
			<td>N/A</td>
			<td>Base Curve: 7.20 to 8.40 mm 
			Diameter: 9.00 / 9.60 / 10.20 mm 
			Power: -25.00 to +25.00 Diopters</td>
		</tr>
		<tr>
			<td>balanced salt solution (BSS)</td>
			<td>Inselspital, Bern, Switzerland</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>silicon forceps</td>
			<td>N/A</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>atipamezole 5 mg/mL (Antisedan)</td>
			<td>Provet AG, Lyssach, Switzerland</td>
			<td>N/A</td>
			<td>&#945;2 adrenergic receptor antagonist</td>
		</tr>
		<tr>
			<td>GraphPad Prism 7</td>
			<td>GraphPad Software, Inc, San Diego, CA, USA</td>
			<td>N/A</td>
			<td>statistical analysis software</td>
		</tr>
	</tbody>
</table>

in vivo imaging of cx3cr1gfp/gfp reporter mice with spectral-domain optical coherence tomography and scanning laser ophthalmoscopy

Tomografia de coerência óptica de domínio espectral (SD-OCT) e digitalização Oftalmoscopia do laser (SLO) são usados extensivamente em oftalmologia experimental. No presente protocolo, camundongos expressando verde proteína fluorescente (gfp) sob o promotor da Cx3cr1 (BALB/c-Cx3cr1gfp/gfp) foram usados para imagem microglia células na vivo na retina. Microglia são macrófagos residentes da retina e têm sido implicados em várias doenças da retina1,2,3,4,5,6. Este protocolo fornece uma abordagem detalhada para geração de B-retina, com SD-OCT e imagem latente de distribuição de célula microglia em camundongos Cx3cr1gfp/gfp com SLO na vivo, usando um sistema de plataforma de imagem oftálmica. O protocolo pode ser usado em várias linhas de rato de repórter. No entanto, existem algumas limitações do protocolo aqui apresentado. Em primeiro lugar, ambos SLO e SD-OCT, quando usado no modo de alta resolução, coletar dados com alta resolução axial, mas a resolução lateral são mais baixos (3,5 µm e 6 µm, respectivamente). Além disso, o nível de saturação e foco em SLO é altamente dependente da seleção do parâmetro e o alinhamento correcto do olho. Além disso, usar dispositivos projetados para pacientes humanos em ratos é desafiador, devido a maior potência óptica total do olho do rato comparado ao olho humano; Isso pode levar a lateral ampliação imprecisões7, que também são dependentes a ampliação pela lente do mouse entre outros. No entanto, apesar de que a tomografia axial posição é dependente da ampliação lateral, as medições de SD-OCT axiais são precisos8.

Usando o protocolo aqui apresentado, SD-OCT verifica e SLO imagens foram obtidas a partir de Cx3cr1gfp/gfp ratos na mesma sessão de imagens. Figura 3 inclui representante SD-OCT único exames obtidos com um 30 ° ou uma lente de 55 ° (Figura 3A) e imagens representativas de SLO obtidos com um 55 ° ou uma lente de 102 °, onde gfp positivas microglia células são visualizadas. Maior refletividade da coroid...

Watch this Scientific Journal Video about In Vivo Imaging of Cx3cr1gfp/gfp Reporter Mice with Spectral-domain Optical Coherence Tomography and Scanning Laser Ophthalmoscopy at JoVE.com

In Vivo Imaging of Cx3cr1gfp/gfp Reporter Mice with Spectral-domain Optical Coherence Tomography and Scanning Laser Ophthalmoscopy

Este protocolo descreve técnicas como de alta resolução de imagem como tomografia de coerência óptica de domínio espectral e digitalização laser Oftalmoscopia pode ser utilizado em pequenos roedores, usando um sistema de plataforma de imagem oftálmica, para obter informações sobre espessura da retina e microglial celular distribuição, respectivamente.

Na Vivo Imagem de Cx3cr1gfp/gfp repórter ratos com a tomografia de coerência óptica espectral-domínio e digitalização Laser Oftalmoscopia

Spectral domain optical coherence tomography (SD-OCT) and scanning laser ophthalmoscopy (SLO) are extensively used in experimental ophthalmology. In the ...

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Research

JoVE Journal

Medicine

In Vivo Imaging of Cx3cr1gfp/gfp Reporter Mice with Spectral-domain Optical Coherence Tomography and Scanning Laser Ophthalmoscopy

10.5K visualizações.  Bern University Hospital and University of Bern. Este protocolo descreve técnicas como de alta resolução de imagem como tomografia de coerência óptica de domínio espectral e digitalização laser Oftalmoscopia pode ser utilizado em pequenos roedores, usando um sistema de plataforma de imagem oftálmica, para obter informações sobre espessura da retina e microglial celular distribuição, respectivamente.

Vídeo: In Vivo Imaging of Cx3cr1gfp/gfp Reporter Mice with Spectral-domain Optical Coherence Tomography and Scanning Laser Ophthalmoscopy

Oral Biofilm Analysis of Palatal Expanders by Fluorescence In-Situ Hybridization and Confocal Laser Scanning Microscopy

Confocal laser scanning microscopy (CLSM) of natural heterogeneous biofilm is today facilitated by a comprehensive range of staining techniques, one of them being fluorescence in situ hybridization (FISH).1,2 We performed a pilot study in which oral biofilm samples collected from fixed orthodontic appliances (palatal expanders) were stained by FISH, the objective being to assess the three-dimensional organization of natural biofilm and plaque accumulation.3,4 FISH creates an opportunity to stain cells in their native biofilm environment by the use of fluorescently labeled 16S rRNA-targeting probes.4-7,19 Compared to alternative techniques like immunofluorescent labeling, this is an inexpensive, precise and straightforward labeling technique to investigate different bacterial groups in mixed biofilm consortia.18,20 General probes were used that bind to Eubacteria (EUB338 + EUB338II + EUB338III; hereafter EUBmix),8-10 Firmicutes (LGC354 A-C; hereafter LGCmix),9,10 and Bacteroidetes (Bac303).11 In addition, specific probes binding to Streptococcus mutans (MUT590)12,13 and Porphyromonas gingivalis (POGI)13,14 were used. The extreme hardness of the surface materials involved (stainless steel and acrylic resin) compelled us to find new ways of preparing the biofilm. As these surface materials could not be readily cut with a cryotome, various sampling methods were explored to obtain intact oral biofilm. The most workable of these approaches is presented in this communication. Small flakes of the biofilm-carrying acrylic resin were scraped off with a sterile scalpel, taking care not to damage the biofilm structure. Forceps were used to collect biofilm from the steel surfaces. Once collected, the samples were fixed and placed directly on polysine coated glass slides. FISH was performed directly on these slides with the probes mentioned above. Various FISH protocols were combined and modified to create a new protocol that was easy to handle.5,10,14,15 Subsequently the samples were analyzed by confocal laser scanning microscopy. Well-known configurations3,4,16,17 could be visualized, including mushroom-style formations and clusters of coccoid bacteria pervaded by channels. In addition, the bacterial composition of these typical biofilm structures were analyzed and 2D and 3D images created.

We present a protocol for structural and compositional analysis of natural oral biofilm from orthodontic appliances with in situ hybridization (FISH) and confocal laser scanning microscopy (CLSM). Oral biofilm samples were collected from palatal expanders, scraping acrylic-resin flakes off their surface and referring them for molecular processing.

Análise de biofilme oral de Palatal Expansores por fluorescência de hibridização in situ e microscopia confocal a laser Scanning

Confocal laser scanning microscopy (CLSM) of natural heterogeneous biofilm is today facilitated by a comprehensive range of staining techniques, one of ...

Medicina

Intracranial Implantation with Subsequent 3D In Vivo Bioluminescent Imaging of Murine Gliomas

The mouse glioma 261 (GL261) is recognized as an in vivo model system that recapitulates many of the features of human glioblastoma multiforme (GBM). The cell line was originally induced by intracranial injection of 3-methyl-cholantrene into a C57BL/6 syngeneic mouse strain 1; therefore, immunologically competent C57BL/6 mice can be used. While we use GL261, the following protocol can be used for the implantation and monitoring of any intracranial mouse tumor model. GL261 cells were engineered to stably express firefly luciferase (GL261-luc). We also created the brighter GL261-luc2 cell line by stable transfection of the luc2 gene expressed from the CMV promoter. C57BL/6-cBrd/cBrd/Cr mice (albino variant of C57BL/6) from the National Cancer Institute, Frederick, MD were used to eliminate the light attenuation caused by black skin and fur. With the use of albino C57BL/6 mice; in vivo imaging using the IVIS Spectrum in vivo imaging system is possible from the day of implantation (Caliper Life Sciences, Hopkinton, MA). The GL261-luc and GL261-luc2 cell lines showed the same in vivo behavior as the parental GL261 cells. Some of the shared histological features present in human GBMs and this mouse model include: tumor necrosis, pseudopalisades, neovascularization, invasion, hypercellularity, and inflammation 1.
Prior to implantation animals were anesthetized by an intraperitoneal injection of ketamine (50 mg/kg), xylazine (5 mg/kg) and buprenorphine (0.05 mg/kg), placed in a stereotactic apparatus and an incision was made with a scalpel over the cranial midline. A burrhole was made 0.1mm posterior to the bregma and 2.3mm to the right of the midline. A needle was inserted to a depth of 3mm and withdrawn 0.4mm to a depth of 2.6mm. Two &mu;l of GL261-luc or GL261-luc2 cells (107 cells/ml) were infused over the course of 3 minutes. The burrhole was closed with bonewax and the incision was sutured.
Following stereotactic implantation the bioluminescent cells are detectable from the day of implantation and the tumor can be analyzed using the 3D image reconstruction feature of the IVIS Spectrum instrument. Animals receive a subcutaneous injection of 150&mu;g luciferin /kg body weight 20 min prior to imaging. Tumor burden is quantified using mean tumor bioluminescence over time. Tumor-bearing mice were observed daily to assess morbidity and were euthanized when one or more of the following symptoms are present: lethargy, failure to ambulate, hunched posture, failure to groom, anorexia resulting in &gt;10% loss of weight. Tumors were evident in all of the animals on necropsy.

Intracranial Implantation with Subsequent 3D In Vivo Bioluminescent Imaging of Murine Gliomas

Intracranial implantation of GL261 cells into C57BL/6 mice produces malignant gliomas that recapitulate many of the hallmarks of human glioblastoma multiforme. We used GL261 cells stably expressing luciferase to allow us to use in vivo imaging to follow tumor progression. The surgery and 3D in vivo imaging are demonstrated.

Implantação intracraniana com 3D subseqüentes In Vivo Bioluminescent imagem de Gliomas murino

The mouse glioma 261 (GL261) is recognized as an in vivo model system that recapitulates many of the features of human glioblastoma multiforme (GBM). The ...

Doppler Optical Coherence Tomography of Retinal Circulation

Noncontact retinal blood flow measurements are performed with a Fourier domain optical coherence tomography (OCT) system using a circumpapillary double circular scan (CDCS) that scans around the optic nerve head at 3.40 mm and 3.75 mm diameters. The double concentric circles are performed 6 times consecutively over 2 sec. The CDCS scan is saved with Doppler shift information from which flow can be calculated. The standard clinical protocol calls for 3 CDCS scans made with the OCT beam passing through the superonasal edge of the pupil and 3 CDCS scan through the inferonal pupil. This double-angle protocol ensures that acceptable Doppler angle is obtained on each retinal branch vessel in at least 1 scan. The CDCS scan data, a 3-dimensional volumetric OCT scan of the optic disc scan, and a color photograph of the optic disc are used together to obtain retinal blood flow measurement on an eye. We have developed a blood flow measurement software called "Doppler optical coherence tomography of retinal circulation" (DOCTORC). This semi-automated software is used to measure total retinal blood flow, vessel cross section area, and average blood velocity. The flow of each vessel is calculated from the Doppler shift in the vessel cross-sectional area and the Doppler angle between the vessel and the OCT beam. Total retinal blood flow measurement is summed from the veins around the optic disc. The results obtained at our Doppler OCT reading center showed good reproducibility between graders and methods (&lt;10%). Total retinal blood flow could be useful in the management of glaucoma, other retinal diseases, and retinal diseases. In glaucoma patients, OCT retinal blood flow measurement was highly correlated with visual field loss (R2&gt;0.57 with visual field pattern deviation). Doppler OCT is a new method to perform rapid, noncontact, and repeatable measurement of total retinal blood flow using widely available Fourier-domain OCT instrumentation. This new technology may improve the practicality of making these measurements in clinical studies and routine clinical practice.

Total retinal blood flow is measured by Doppler optical coherence tomography and semi-automated grading software.

Doppler Tomografia de Coerência Óptica de circulação retiniana

Noncontact retinal blood flow measurements are performed with a Fourier domain optical coherence tomography (OCT) system using a circumpapillary double ...

Segmentation and Measurement of Fat Volumes in Murine Obesity Models Using X-ray Computed Tomography

Obesity is associated with increased morbidity and mortality as well as reduced metrics in quality of life.1 Both environmental and genetic factors are associated with obesity, though the precise underlying mechanisms that contribute to the disease are currently being delineated.2,3 Several small animal models of obesity have been developed and are employed in a variety of studies.4 A critical component to these experiments involves the collection of regional and/or total animal fat content data under varied conditions.
Traditional experimental methods available for measuring fat content in small animal models of obesity include invasive (e.g. ex vivo measurement of fat deposits) and non-invasive (e.g. Dual Energy X-ray Absorptiometry (DEXA), or Magnetic Resonance (MR)) protocols, each of which presents relative trade-offs. Current invasive methods for measuring fat content may provide details for organ and region specific fat distribution, but sacrificing the subjects will preclude longitudinal assessments. Conversely, current non-invasive strategies provide limited details for organ and region specific fat distribution, but enable valuable longitudinal assessment. With the advent of dedicated small animal X-ray computed tomography (CT) systems and customized analytical procedures, both organ and region specific analysis of fat distribution and longitudinal profiling may be possible. Recent reports have validated the use of CT for in vivo longitudinal imaging of adiposity in living mice.5,6 Here we provide a modified method that allows for fat/total volume measurement, analysis and visualization utilizing the Carestream Molecular Imaging Albira CT system in conjunction with PMOD and Volview software packages.

Fat content analysis is routinely conducted in studies utilizing murine obesity models. Emerging methods in small animal CT imaging and analysis are providing for longitudinal detail rich fat content analysis. Here we detail step by step procedures for performing small animal CT imaging, analysis, and visualization.

Segmentação e medição de volumes de gordura em modelos de obesidade em roedores usando raios-X Tomografia Computadorizada

Obesity is associated with increased morbidity and mortality as well as reduced metrics in quality of life.1 Both environmental and genetic factors are ...

Generation and 3-Dimensional Quantitation of Arterial Lesions in Mice Using Optical Projection Tomography

The generation and analysis of vascular lesions in appropriate animal models is a cornerstone of research into cardiovascular disease, generating important information on the pathogenesis of lesion formation and the action of novel therapies. Use of atherosclerosis-prone mice, surgical methods of lesion induction, and dietary modification has dramatically improved understanding of the mechanisms that contribute to disease development and the potential of new treatments.
Classically, analysis of lesions is performed ex vivo using 2-dimensional histological techniques. This article describes application of optical projection tomography (OPT) to 3-dimensional quantitation of arterial lesions. As this technique is non-destructive, it can be used as an adjunct to standard histological and immunohistochemical analyses.
Neointimal lesions were induced by wire-insertion or ligation of the mouse femoral artery whilst atherosclerotic lesions were generated by administration of an atherogenic diet to apoE-deficient mice.
Lesions were examined using OPT imaging of autofluorescent emission followed by complementary histological and immunohistochemical analysis. OPT clearly distinguished lesions from the underlying vascular wall. Lesion size was calculated in 2-dimensional sections using planimetry, enabling calculation of lesion volume and maximal cross-sectional area. Data generated using OPT were consistent with measurements obtained using histology, confirming the accuracy of the technique and its potential as a complement (rather than alternative) to traditional methods of analysis.
This work demonstrates the potential of OPT for imaging atherosclerotic and neointimal lesions. It provides a rapid, much needed ex vivo technique for the routine 3-dimensional quantification of vascular remodelling.

Ex vivo analysis of arterial lesions from animal models of cardiovascular disease classically relies on histological and immunohistochemical techniques. These provide 2-dimensional measurements in 3-dimensional lesions. This manuscript describes the generation of arterial lesions for quantitative analysis in 3-dimensions using optical projection tomography.

Geração e quantificação 3-Dimensional de Arterial lesões em ratos por meio de tomografia óptica de Projeção

The generation and analysis of vascular lesions in appropriate animal models is a cornerstone of research into cardiovascular disease, generating ...

In Vivo Optical Imaging of Brain Tumors and Arthritis Using Fluorescent SapC-DOPS Nanovesicles

We describe a multi-angle rotational optical imaging (MAROI) system for in vivo monitoring of physiopathological processes labeled with a fluorescent marker. Mouse models (brain tumor and arthritis) were used to evaluate the usefulness of this method. Saposin C (SapC)-dioleoylphosphatidylserine (DOPS) nanovesicles tagged with CellVue Maroon (CVM) fluorophore were administered intravenously. Animals were then placed in the rotational holder (MARS) of the in vivo imaging system. Images were acquired in 10&#176; steps over 380&#176;. A rectangular&#160;region of interest (ROI) was placed across the full image width at the model disease site. Within the ROI, and for every image, mean fluorescence intensity was computed after background subtraction. In the mouse models studied, the labeled nanovesicles were taken up in both the orthotopic and transgenic brain tumors, and in the arthritic sites (toes and ankles). Curve analysis of the multi angle image ROIs determined the angle with the highest signal. Thus, the optimal angle for imaging each disease site was characterized. The MAROI method applied to imaging of fluorescent compounds is a noninvasive, economical, and precise tool for in vivo quantitative analysis of the disease states in the described mouse models.

In Vivo Optical Imaging of Brain Tumors and Arthritis Using Fluorescent SapC-DOPS Nanovesicles

We describe a multi-angle rotational optical imaging (MAROI) system for in vivo quantitation of a fluorescent marker delivered by saposin C (SapC)-dioleoylphosphatidylserine (DOPS) nanovesicles. Employing mouse models of cancer and arthritis, we demonstrate how the MAROI signal curve analysis can be used for the precise mapping and biological characterization of disease processes.

In Vivo Imagem Óptico de tumores cerebrais e artrite Usando fluorescente SAPC-DOPS nanovesículas

We describe a multi-angle rotational optical imaging (MAROI) system for in vivo monitoring of physiopathological processes labeled with a fluorescent ...

Combined In vivo Optical and &#181;CT Imaging to Monitor Infection, Inflammation, and Bone Anatomy in an Orthopaedic Implant Infection in Mice

Multimodality imaging has emerged as a common technological approach used in both preclinical and clinical research. Advanced techniques that combine in vivo optical and &#956;CT imaging allow the visualization of biological phenomena in an anatomical context. These imaging modalities may be especially useful to study conditions that impact bone. In particular, orthopaedic implant infections are an important problem in clinical orthopaedic surgery. These infections are difficult to treat because bacterial biofilms form on the foreign surgically implanted materials, leading to persistent inflammation, osteomyelitis and eventual osteolysis of the bone surrounding the implant, which ultimately results in implant loosening and failure. Here, a mouse model of an infected orthopaedic prosthetic implant was used that involved the surgical placement of a Kirschner-wire implant into an intramedullary canal in the femur in such a way that the end of the implant extended into the knee joint. In this model, LysEGFP mice, a mouse strain that has EGFP-fluorescent neutrophils, were employed in conjunction with a bioluminescent Staphylococcus aureus strain, which naturally emits light. The bacteria were inoculated into the knee joints of the mice prior to closing the surgical site. In vivo bioluminescent and fluorescent imaging was used to quantify the bacterial burden and neutrophil inflammatory response, respectively. In addition, &#956;CT imaging was performed on the same mice so that the 3D location of the bioluminescent and fluorescent optical signals could be co-registered with the anatomical &#956;CT images. To quantify the changes in the bone over time, the outer bone volume of the distal femurs were measured at specific time points using a semi-automated contour based segmentation process. Taken together, the combination of in vivo bioluminescent/fluorescent imaging with &#956;CT imaging may be especially useful for the noninvasive monitoring of the infection, inflammatory response and anatomical changes in bone over time.

Combined In vivo Optical and  &#181;CT Imaging to Monitor Infection, Inflammation, and Bone Anatomy in an Orthopaedic Implant Infection in Mice

Combined optical and &#956;CT imaging in a mouse model of orthopaedic implant infection, utilizing a bioluminescent engineered strain of Staphylococcus aureus, provided the capability to noninvasively and longitudinally monitor the dynamics of the bacterial infection, as well as the corresponding inflammatory response and anatomical changes in the bone.

Combinado<em&gt; In vivo</em&gt; Optical e μCT imagem para monitorar infecções, inflamações, and Bone Anatomy em uma infecção Orthopaedic Implant em Ratos

Multimodality imaging has emerged as a common technological approach used in both preclinical and clinical research. Advanced techniques that combine in ...

Assessment of Viability of Human Fat Injection into Nude Mice with Micro-Computed Tomography

Lipotransfer is a vital tool in the surgeon&#8217;s armamentarium for the treatment of soft tissue deficits of throughout the body. Fat is the ideal soft tissue filler as it is readily available, easily obtained, inexpensive, and inherently biocompatible.1 However, despite its burgeoning popularity, fat grafting is hampered by unpredictable results and variable graft survival, with published retention rates ranging anywhere from 10-80%. 1-3
To facilitate investigations on fat grafting, we have therefore developed an animal model that allows for real-time analysis of injected fat volume retention. Briefly, a small cut is made in the scalp of a CD-1 nude mouse and 200-400 &#181;l&#160;of processed lipoaspirate is placed over the skull. The scalp is chosen as the recipient site because of its absence of native subcutaneous fat, and because of the excellent background contrast provided by the calvarium, which aids in the analysis process. Micro-computed tomography (micro-CT) is used to scan the graft at baseline and every two weeks thereafter. The CT images are reconstructed, and an imaging software is used to quantify graft volumes.
Traditionally, techniques to assess fat graft volume have necessitated euthanizing the study animal to provide just a single assessment of graft weight and volume by physical measurement ex vivo. Biochemical and histological comparisons have likewise required the study animal to be euthanized. This described imaging technique offers the advantage of visualizing and objectively quantifying volume at multiple time points after initial grafting without having to sacrifice the study animal. The technique is limited by the size of the graft able to be injected as larger grafts risk skin and fat necrosis. This method has utility for all studies evaluating fat graft viability and volume retention. It is particularly well-suited to providing a visual representation of fat grafts and following changes in volume over time.

Fat grafting is an essential technique for reconstructing soft tissue deficits. However, it remains an unpredictable procedure characterized by variable graft survival. Our goal was to devise a mouse model that utilizes a novel imaging method to compare volume retention between differing techniques of fat graft preparation and delivery.

Avaliação da viabilidade de gordura humana injecção em ratinhos nu com Micro-Tomografia Computadorizada

Lipotransfer is a vital tool in the surgeon&#8217;s armamentarium for the treatment of soft tissue deficits of throughout the body. Fat is the ideal soft ...

Validation of Nanobody and Antibody Based In Vivo Tumor Xenograft NIRF-imaging Experiments in Mice Using Ex Vivo Flow Cytometry and Microscopy

This protocol outlines the steps required to perform ex vivo validation of in vivo near-infrared fluorescence (NIRF) xenograft imaging experiments in mice using fluorophore labelled nanobodies and conventional antibodies.
First we describe how to generate subcutaneous tumors in mice, using antigen-negative cell lines as negative controls and antigen-positive cells as positive controls in the same mice for intraindividual comparison. We outline how to administer intravenously near-infrared fluorophore labelled (AlexaFluor680) antigen-specific nanobodies and conventional antibodies. In vivo imaging was performed with a small-animal NIRF-Imaging system. After the in vivo imaging experiments the mice were sacrificed. We then describe how to prepare the tumors for parallel ex vivo analyses by flow cytometry and fluorescence microscopy to validate in vivo imaging results.
The use of the near-infrared fluorophore labelled nanobodies allows for non-invasive same day imaging in vivo. Our protocols describe the ex vivo quantification of the specific labeling efficiency of tumor cells by flow cytometry and analysis of the distribution of the antibody constructs within the tumors by fluorescence microscopy. Using near-infrared fluorophore labelled probes allows for non-invasive, economical in vivo imaging with the unique ability to exploit the same probe without further secondary labelling for ex vivo validation experiments using flow cytometry and fluorescence microscopy.

Validation of Nanobody and Antibody Based In Vivo Tumor Xenograft NIRF-imaging Experiments in Mice Using Ex Vivo Flow Cytometry and Microscopy

This protocol outlines the steps required to perform ex vivo validation of in vivo near-infrared fluorescence xenograft imaging experiments in mice using fluorophore labelled nanobodies and conventional antibodies.

Validação de nanocorpo e Antibody Based<em&gt; In Vivo</em&gt; Experimentos Tumor Xenoenxerto NIRF-imagem em ratos usando<em&gt; Ex Vivo</em&gt; Citometria de fluxo e microscopia

This protocol outlines the steps required to perform ex vivo validation of in vivo near-infrared fluorescence (NIRF) xenograft imaging experiments in mice ...

In Vivo, Percutaneous, Needle Based, Optical Coherence Tomography of Renal Masses

Optical coherence tomography (OCT) is the optical equivalent of ultrasound imaging, based on the backscattering of near infrared light. OCT provides real time images with a 15 &#181;m axial resolution at an effective tissue penetration of 2-3 mm. Within the OCT images the loss of signal intensity per millimeter of tissue penetration, the attenuation coefficient, is calculated. The attenuation coefficient is a tissue specific property, providing a quantitative parameter for tissue differentiation. 
Until now, renal mass treatment decisions have been made primarily on the basis of MRI and CT imaging characteristics, age and comorbidity. However these parameters and diagnostic methods lack the finesse to truly detect the malignant potential of a renal mass. A successful core biopsy or fine needle aspiration provides objective tumor differentiation with both sensitivity and specificity in the range of 95-100%. However, a non-diagnostic rate of 10-20% overall, and even up to 30% in SRMs, is to be expected, delaying the diagnostic process due to the frequent necessity for additional biopsy procedures. 
We aim to develop OCT into an optical biopsy, providing real-time imaging combined with on-the-spot tumor differentiation. This publication provides a detailed step-by-step approach for percutaneous, needle based, OCT of renal masses.

In Vivo, Percutaneous, Needle Based, Optical Coherence Tomography of Renal Masses

Optical coherence tomography (OCT) is a high resolution imaging technique that allows analysis of tissue specific optical properties providing the means for tissue differentiation. We developed needle based OCT, providing real-time imaging combined with on-the-spot tumor differentiation. This publication describes a method for percutaneous, needle based OCT of renal masses.

Na Vivo, percutânea, Agulha Based, Tomografia de Coerência Óptica de massas renais

Optical coherence tomography (OCT) is the optical equivalent of ultrasound imaging, based on the backscattering of near infrared light. OCT provides real ...