Nous remercions Marek Weiler pour effectuer les expériences de fantômes. Ce travail a été soutenu par le Conseil européen de la recherche (ERC Starting Grant de 309 495: NeoNaNo), l&#39;État fédéral allemand de Rhénanie du Nord Westphalie (NRW; High-Tech.NRW/EU-Ziel 2-Programm (EFRE); ForSaTum), l&#39;allemand Ministère de l&#39;Education et de la Recherche (BMBF) (programmes de financement du foie virtuel (0.315.743), LungSys (0315415C), LungSys2 (0316042F), Photonik Forschung Deutschland (13N13355)), l&#39;Université RWTH Aachen (I 3 Fonds de stimulation de TM), et de recherche de Philips (Aachen, Allemagne).

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A, Optics, Image Science, and Vision. 26 (4), 919-923 (2009).</li><li>Jacques, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+properties+of+biological+tissues:+a+review.">Optical properties of biological tissues: a review.</a> Physics in Medicine and Biology. 58 (11), R37 (2013).</li><li>Loening, A. M., Gambhir, S. 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Felix Gremse est fondateur et propriétaire de Gremse-IT, une entreprise en démarrage qui offre des logiciels et services d&#39;analyse d&#39;images médicales en collaboration avec Philips et le ministère des fins expérimentales d&#39;imagerie moléculaire de l&#39;Université RWTH Aachen.

Nous décrivons et appliquons un protocole d&#39;imagerie multimodale μCT-FMT. Nous utilisons FMT disponible dans le commerce et largement utilisé et dispositifs μCT 3,11,15 - 17,21. Bien que le protocole nécessite une FMT spécifique, le μCT peut être remplacé par un autre μCT avec des fonctionnalités similaires et les paramètres de numérisation comparables, par exemple, le champ de vision doit être suffisamment large pour couvrir le lit de la souris, y compris les marqueurs. La FMT a été utilisé pour l&#39;analyse de biodistribution sans le combiner avec μCT IRM ou 21, cependant, les données anatomiques est bénéfique pour augmenter la reproductibilité puisque la segmentation peut être calculée sur la base des limites d&#39;organes qui sont visibles dans les données μCT 10. Alors que les appareils μCT-FMT intégrés ont été développés 2,7, ceux-ci ne sont pas encore disponibles dans le commerce. En outre, l&#39;utilisation de deux dispositifs distincts permet tuyauterie, à savoir., La souris suivant can imager dans la μCT souris tandis que le premier est toujours dans la FMT, pour augmenter le débit. Pour réduire la charge de travail manuel, nous effectuons la détection du marqueur automatisé et de la fusion. En outre, la forme de la souris est automatiquement cette information segmenté et améliore de façon significative la 11,13,22 de reconstruction de fluorescence. Pour la reconstruction quantitative de fluorescence, cartes d&#39;absorption et de diffusion sont nécessaires 13,23. Nous tirons la carte de la diffusion par la segmentation automatique des données μCT et en attribuant des coefficients de diffusion connus de plusieurs types de tissus (poumon, os, la peau, la graisse, et le reste des tissus mous) 24. Par la suite, nous reconstruisons une carte d&#39;absorption à partir des données brutes optique qui est particulièrement important pour les organes bien irrigués comme le cœur et le foie 13,20. Numérisation plusieurs souris à de multiples points de temps se traduit rapidement par un grand nombre d&#39;ensembles de données à analyser. Pour Biodisétudes de distri-, plusieurs organes doivent être segmenté pour chaque balayage μCT-FMT. Malheureusement, les segmentations ne peuvent pas être réutilisés, parce que la souris est nouvellement positionnée dans le lit de la souris à plusieurs reprises. Nous utilisons un outil de segmentation interactif, développé dans notre institut, cependant, d&#39;autres outils peuvent aussi être approprié 25. Nous générons des segmentations de voxels-sage, parce que ceux-ci correspondent mieux aux organes complexes de formes simples telles que des ellipses et des cubes 26. Automatisée segmentation animal entier serait utile de réduire davantage la charge de travail manuel 27, mais un outil de segmentation interactive serait encore nécessaire pour corriger les erreurs de segmentation. En outre, des outils de segmentation automatique peuvent difficilement anticiper les cas particuliers tels que les pathologies correctement. Puisque nous utilisons des analyses de μCT indigènes, certains organes tels que la rate sont très difficiles à segmenter même manuellement. Les agents de contraste aideraient, mais il ya des problèmes avec la tolérabilité et il est difficile d&#39;entretenir cetna distribution de l&#39;agent de contraste régulière tout au long de l&#39;imagerie longitudinale. Notre étude fantôme montre que la localisation du signal est améliorée lors de l&#39;utilisation des informations de forme pour la reconstruction de fluorescence. In vivo, une amélioration similaire est évidente pour le point de contrôle d&#39;avance (15 minutes après l&#39;injection), quand une grande quantité de la sonde est déjà dans le la vessie urinaire. La sonde d&#39;hydroxyapatite liaison accumule à os et des articulations. Il est remarquable de voir comment cela se produit rapidement, à savoir, le signal est déjà clairement visible à la colonne vertébrale 15 minutes après l&#39;injection. Ceci est probablement dû à la faible poids moléculaire de la sonde, ce qui permet l&#39;extravasation et rapide diffusion dans les régions cibles. La sonde se lie de manière covalente à son hydroxyapatite cible et la sonde non liée est excrété. Pour les points de temps plus tard, entre 6 h et 24 h après l&#39;injection, l&#39;intensité du signal dans la colonne vertébrale reste relativement stable, sans doute, parce que presque toute la lumière recourbatures profondément dans la souris pour blanchir la fluorescence. Pour notre étude, nous avons utilisé le canal 750 nm, ce qui en résulte une faible fluorescence de fond aussi évident pour les scans acquis avant l&#39;injection. Aux longueurs d&#39;onde plus faibles, plus le signal de fond peut être 28 prévu. En résumé, nous décrivons un protocole d&#39;imagerie multimodale pour dispositifs disponibles dans le commerce et FMT μCT. Nous montrons que la combinaison offre des avantages pour la reconstruction de fluorescence. Nous illustrons la façon dont les courbes de biodistribution sont extraits à partir de la grande quantité de données d&#39;image au moyen de segmentation interactive d&#39;organes et de traitement par lots. Nous croyons que ce flux de travail standardisé peut être utile pour le développement de médicaments et d&#39;autres études d&#39;imagerie utilisant des sondes marquées par fluorescence.

tomographie de fluorescence à médiation par, tomographie moléculaire par fluorescence aussi appelée (FMT), est une technique prometteuse pour évaluer quantitativement la distribution de la fluorescence diffuse dans les tissus, tels que les souris anesthésiées, voire des tissus du corps humain, par exemple les seins ou les articulations des doigts. Contrairement aux techniques de microscopie non invasives, ce qui permet l&#39;imagerie des cibles superficielles à une résolution subcellulaire, FMT permet la reconstruction tridimensionnelle de sources fluorescentes à des profondeurs de plusieurs centimètres, mais à plus basse résolution 2. De nombreuses sondes fluorescentes ciblés sont disponibles pour l&#39;image de l&#39;angiogenèse, l&#39;apoptose, l&#39;inflammation et d&#39;autres 2-5. Certaines sondes sont activable, par exemple,. Par clivage enzymatique spécifique conduisant à unquenching de fluorochromes. En outre, des gènes rapporteurs exprimant des protéines fluorescentes peuvent être visualisés, par exemple, pour suivre la migration des cellules tumorales 6. FMT bénéficie fortement de la combinaison avec une modalité anatomique, par exemple, μCT 2,7 ou 8 IRM. Alors que les dispositifs de FMT autonomes sont disponibles dans le commerce 9, les images de fluorescence sont difficiles à interpréter sans information de référence anatomique. Récemment, nous avons pu montrer que les données fondu d&#39;image anatomique permet une analyse plus robuste 10. Les données anatomiques peuvent également être utilisés pour fournir des connaissances antérieures, telles que la forme extérieure de la souris, ce qui est important pour la modélisation optique précis et reconstruction 11 fluorescence. En outre, les cartes de diffusion et d&#39;absorption optiques peuvent être estimées en utilisant la segmentation des types de tissus et en attribuant des coefficients spécifiques de classe 12,13. Pour la lumière proche infrarouge, l&#39;hémoglobine est l&#39;absorbeur principal chez la souris, outre la mélanine et de la fourrure 14. Depuis le volume relatif de sang varie selon les régions par ordre de grandeur, une carte d&#39;absorption est particulièrement important pour les quanquanti- 13 reconstruction de fluorescence. Un avantage de l&#39;utilisation de dispositifs d&#39;imagerie non invasifs est que les souris peuvent être imagés longitudinalement, soit en de multiples points de temps. Ceci est important pour évaluer le comportement dynamique des sondes, à savoir, leur accumulation cible, biodistribution et l&#39;excrétion 10,15, ou pour évaluer la progression de la maladie 16. Lorsque Imagerie plusieurs souris à de multiples points dans le temps, une grande quantité d&#39;ensembles de données d&#39;image se produit. Pour permettre la comparabilité, ceux-ci devraient être acquis d&#39;une manière systématique, à savoir, avec un protocole bien défini et documenté. Le grand nombre de scans pose un défi pour l&#39;analyse de l&#39;image, qui est nécessaire pour extraire des mesures quantitatives à partir des données d&#39;image. Le but de notre étude est de fournir une description détaillée d&#39;un protocole d&#39;imagerie μCT-FMT que nous avons utilisé et optimisé à travers plusieurs études 10,13,15,17,18. Nous décrivonsla manière dont les ensembles de données sont générées, traitées, visualisées et analysées. Cela est démontré en utilisant une sonde moléculaire établi, OsteoSense, qui se lie à l&#39;hydroxyapatite 19, et peut être utilisé pour des maladies osseuses et l&#39;image 2 remodelage. Toutes les procédures impliquant des animaux ont été approuvés par le comité d&#39;examen gouvernemental sur les soins aux animaux.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>FMT (Fluorescence molecular tomography) FMT2500 LX</td>
			<td>PerkinElmer</td>
			<td>FMT2000</td>
			<td>Device for fluorescence molecular tomography</td>
		</tr>
		<tr>
			<td>&#181;CT (micro computed tomography) Tomoscope Duo</td>
			<td>CT Imaging GmbH</td>
			<td>Tomoscope Duo</td>
			<td>Device for micro computed tomography</td>
		</tr>
		<tr>
			<td>Multimodal Mouse Bed</td>
			<td>CT Imaging GmbH</td>
			<td>Experimental builder</td>
			<td>Partially transparent animal holder</td>
		</tr>
		<tr>
			<td>IsoFlo (isoflurane, USP)</td>
			<td>Abbott</td>
			<td>05260-05</td>
			<td>Isoflurane Inhalation anesthesia</td>
		</tr>
		<tr>
			<td>Small animal anesthesia system</td>
			<td>Harvard apparatus</td>
			<td>726419</td>
			<td>Complete Isoflurane Table-Top System</td>
		</tr>
		<tr>
			<td>Chlorophyll-free mouse food</td>
			<td>Ssniff</td>
			<td>E15051</td>
			<td>low chlorophyll / low fluorescence food</td>
		</tr>
		<tr>
			<td>OsteoSense 750EX</td>
			<td>PerkinElmer</td>
			<td>NEV10053EX</td>
			<td>Animal FMT contrast agent</td>
		</tr>
		<tr>
			<td>Portex Fine Bore Polythene Tubing</td>
			<td>Smith medical</td>
			<td>800/100/120</td>
			<td>Tube for injection catheter</td>
		</tr>
		<tr>
			<td>Sterican 30g</td>
			<td>BBraun</td>
			<td>4656300</td>
			<td>Hypodermic needle for catheter</td>
		</tr>
		<tr>
			<td>Imeron</td>
			<td>Altana pharma</td>
			<td>INLA F.1/0203/3.5337.69</td>
			<td>CT contrast agent for the phantom inclusions</td>
		</tr>
		<tr>
			<td>Agarose</td>
			<td>Sigma</td>
			<td>90-12-36-6</td>
			<td>Agarose for phantom production</td>
		</tr>
		<tr>
			<td>TiO2</td>
			<td>Applichem</td>
			<td>A1900,1000</td>
			<td>Titanium oxyde as phantom scattering agent</td>
		</tr>
		<tr>
			<td>Trypan blue</td>
			<td>Fluka</td>
			<td>93595</td>
			<td>Trypan blue to adjust phantom light propagation</td>
		</tr>
		<tr>
			<td>Cy7</td>
			<td>Lumiprobe</td>
			<td>15020</td>
			<td>Fluorochrome for the phantom inclusions</td>
		</tr>
		<tr>
			<td>Lipovenoes 20%</td>
			<td>Fresenius Kabi</td>
			<td>3094740</td>
			<td>Lipid emulsion, scattering agent for FMT contrast agents</td>
		</tr>
		<tr>
			<td>Definiens Developer XD Server</td>
			<td>Definiens AG</td>
			<td>Server XD</td>
			<td>Software platform for automated segmentation</td>
		</tr>
		<tr>
			<td>Imalytics Preclinical</td>
			<td>ExMI/Gremse-IT</td>
			<td>Version 2.0.1</td>
			<td>Software for image fusion, reconstruction and analysis</td>
		</tr>
		<tr>
			<td>NVIDIA Geforce Titan</td>
			<td>Asus</td>
			<td>GTXTITAN6GD5</td>
			<td>High end computer graphics card, 6GB Memory</td>
		</tr>
	</tbody>
</table>

hybrid &#181;ct-fmt imaging and image analysis

La fluorescence induite par tomographie (FMT) permet de déterminer longitudinal et quantitative de la distribution de la fluorescence in vivo et peut être utilisé pour évaluer la biodistribution de nouvelles sondes et pour évaluer la progression de la maladie en utilisant des sondes moléculaires établies ou des gènes rapporteurs. La combinaison avec une modalité anatomique, par exemple, micro tomodensitométrie (μCT), est bénéfique pour l&#39;analyse d&#39;image et de reconstruction de fluorescence. Nous décrivons un protocole d&#39;imagerie multimodal μCT-FMT, comprenant les étapes de traitement d&#39;image nécessaires pour extraire des mesures quantitatives. Après avoir préparé les souris et effectuer la formation d&#39;image, les ensembles de données sont des marques multimodales. Par la suite, une amélioration de la reconstruction de la fluorescence est effectuée, qui tient compte de la forme de la souris. Pour l&#39;analyse quantitative, segmentations d&#39;organes sont générés sur la base des données anatomiques en utilisant notre outil de segmentation interactive. Enfin, le cu de biodistributionrves sont générées en utilisant une fonction de traitement par lots. Nous montrons l&#39;applicabilité de la méthode en évaluant la biodistribution d&#39;une sonde bien connu qui se lie à des os et des articulations.

Nous avons appliqué le protocole décrit pour évaluer la biodistribution d&#39;une sonde, OsteoSense ciblée, qui se lie à l&#39;hydroxyapatite. 3 souris (C57BL / 6 Apoe - / - AHSG - / - souris à double inactivation, 10 semaines) ont été imagées avant et 15 min, 2 h, 4 h, 6 h, et 24 h après l&#39;injection intraveineuse de 2 nmol OsteoSense. Notre logiciel détecte automatiquement les marqueurs intégrés dans le lit de la souris multimodal (figure 1, figure 2A, B), ce qui a permis la fusion des données de μCT anatomiques avec la reconstruction de fluorescence effectuée par le FMT (figure 2C, D). Depuis OsteoSense est une sonde avec un faible poids moléculaire, une excrétion rénale rapide et donc grand signal dans la vessie est attendu. Fusion de la reconstruction de la fluorescence de la FMT a révélé des problèmes tels que le signal déplacée à l&#39;extérieur de la vessie (figure 2C, D). Ces problèmes se produisent parce que la FMT ne connaît pas la vraie forme de la souris et assume une forme de bloc. Oureconstruction r détermine la forme exacte des données et génère μCT diffusion et l&#39;absorption des cartes 13 afin de permettre une reconstruction plus précise de la fluorescence avec une meilleure localisation de signal, ce qui est particulièrement évident pour la vessie (figure 2E, F). Pour affecter la fluorescence reconstruit les régions appropriées, nous interactive segmenté plusieurs organes en utilisant notre logiciel (Figure 3). Pour chacun des 18 scans, 7 régions ont été segmentés basés sur les données μCT, ie., Cœur, poumon, foie, les reins, la colonne vertébrale, de l&#39;intestin et de la vessie. Par la suite, le logiciel a été utilisé pour calculer la concentration moyenne de fluorescence pour chacun des 126 régions. Heureusement, le logiciel fournit un mode de traitement par lots, qui calcule toutes les valeurs et les enregistre dans une seule feuille de calcul. Pour visualiser la distribution de la fluorescence, rendus 3D ont été générées pour chaque point de temps,Avec le réglage de fenêtrage comparables (figure 4A-F). En utilisant les valeurs d&#39;organes quantifiés, la biodistribution a été calculé en faisant la moyenne des valeurs d&#39;organes au cours des trois souris (figure 4G). Les scans pré, acquises avant l&#39;injection, ont montré négligeable signal de fond. 15 min après l&#39;injection, le signal le plus fort est apparu dans la vessie, en raison de l&#39;excrétion rénale rapide. Aux points de temps ultérieurs, la sonde restante avait accumulé à os et des articulations. <img alt="Figure 1" src="/files/ftp_upload/52770/52770fig1.jpg" /> Figure 1. multimodal Souris Chambres. (A) Le lit de la souris multimodal contient deux plaques de verre acrylique qui maintiennent fermement la souris. Le serrage est ajustée à l&#39;aide de deux vis. Le lit de la souris contient des marqueurs (trous vides) pour la fusion d&#39;images. Gaz anesthésique est fournie au moyen d&#39;un tube flexible qui est obsédé avec tsinge. (B) Le lit de la souris est fixé à un support de métal et son maintien dans le centre du portique rotatif μCT. (C) Eviter un écart entre le lit de la souris et le support de métal, parce que sinon, les marqueurs peuvent être incorrectement assignés conduisant à la fusion incorrecte. Le tube de gaz anesthésique doit être attaché au connecteur de tube. (D) Le lit de la souris doit être inséré dans le FMT avec la première face pour permettre une fusion automatique correct. (E) Les marqueurs sont visibles à la caméra FMT, qui est utilisé pour la détection du marqueur automatisé et de la fusion. <a href="https://www.jove.com/files/ftp_upload/52770/52770fig1large.jpg" target="_blank">S&#39;il vous plaît, cliquez ici pour voir une version plus grande de cette figure.</a> <img alt="Figure 2" src="/files/ftp_upload/52770/52770fig2.jpg" /> Figure 2. Fusion d&#39;image et de la reconstruction. (A, B) Marqueurs et la forme extérieure du mouse sont déterminées par l&#39;algorithme de segmentation automatique. (C, D) 15 min après l&#39;injection de OsteoSense, une quantité considérable de la sonde a déjà été excrétée dans la vessie urinaire. Après la fusion de la reconstruction fournie par des vendeurs avec les données μCT, les problèmes deviennent visibles. La plupart du signal apparaît autour de la vessie, mais pas à l&#39;intérieur de la vessie et quelque signal apparaît même dans l&#39;air. Cela arrive parce que la FMT assume une souris en forme de bloc. (E, F) Notre meilleure reconstruction de fluorescence, en utilisant la forme de la souris dérivée des données μCT, résultats dans une meilleure localisation de la fluorescence à l&#39;intérieur de la vessie. <a href="https://www.jove.com/files/ftp_upload/52770/52770fig2large.jpg" target="_blank">S&#39;il vous plaît cliquer ici pour voir une version plus grande de cette figure.</a> <img alt="Figure 3" src="/files/ftp_upload/52770/52770fig3.jpg" /> Figure 3. Organ Segmentat Interactive. ion (A) pour quantifier la distribution de fluorescence, plusieurs organes sont segmentés: coeur (rouge), du poumon (rose), le foie (marron), de l&#39;estomac (beige), la colonne vertébrale (violet), les reins (jaune), l&#39;intestin (vert) et la vessie urinaire (or). (B) le poumon, ce qui est fortement contrasté par rapport au tissu environnant, est segmenté en utilisant le seuillage et de la région de remplissage. (C) organes rondes, comme la vessie, les reins et le cœur sont segmentés en utilisant &quot;gribouillis&quot;. (D) Organes avec une forme plus complexe, par exemple, le foie et l&#39;estomac sont segmentés par incréments utilisent gribouillis. Pour le segment de la colonne vertébrale, un seuil élevé est appliqué à tous les segments os. Puis quelques os, par exemple., Les côtes, sont coupées, jusqu&#39;à ce que la colonne vertébrale reste. <a href="https://www.jove.com/files/ftp_upload/52770/52770fig3large.jpg" target="_blank">S&#39;il vous plaît cliquer ici pour voir une version plus grande de cette figure.</a> Figure 4. biodistribution. Pour évaluer la biodistribution, les souris sont scannées à plusieurs points de temps (AF). (A) L&#39;analyse préalable, avant l&#39;injection, montre peu de signal de fond dans le canal 750 nm. (B) 15 min après l&#39;injection, une quantité considérable de la sonde est déjà dans la vessie urinaire. (C) à la 2 h point de temps, la souris avait uriné, qui se traduit par une certaine fluorescence en dehors de la souris. Aux points de temps plus tard (DF), le signal apparaît principalement dans les os et les articulations, ie., À la colonne vertébrale et les genoux. (G) La concentration de fluorescence quantifiée est représenté pour certains organes.

Watch this Scientific Journal Video about Hybrid ÂµCT-FMT imaging and image analysis at JoVE.com

Hybrid  &amp;#181;CT-FMT imaging and image analysis

We describe a protocol for hybrid imaging, combining fluorescence-mediated tomography (FMT) with micro computed tomography (&#181;CT). After fusion and reconstruction, we perform interactive organ segmentation to extract quantitative measurements of the fluorescence distribution.

Imagerie hybride μCT-FMT et l&#39;analyse d&#39;image

Fluorescence-mediated tomography (FMT) enables longitudinal and quantitative determination of the fluorescence distribution in vivo and can be used to ...

Research

JoVE Journal

Bioengineering

Hybrid &#181;CT-FMT imaging and image analysis

13.0K vues.  RWTH Aachen University. We describe a protocol for hybrid imaging, combining fluorescence-mediated tomography (FMT) with micro computed tomography (µCT). After fusion and reconstruction, we perform interactive organ segmentation to extract quantitative measurements of the fluorescence distribution.

Vidéo: Hybrid  &#181;CT-FMT imaging and image analysis

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.

Protocoles de préparation des échantillons, d&#39;imagerie et d&#39;analyse pour la microscopie à balayage en lame de couteau

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

Recherche

Bio-ingénierie

Optical Frequency Domain Imaging of Ex vivo Pulmonary Resection Specimens: Obtaining One to One Image to Histopathology Correlation

Lung cancer is the leading cause of cancer-related deaths1. Squamous cell and small cell cancers typically arise in association with the conducting airways, whereas adenocarcinomas are typically more peripheral in location. Lung malignancy detection early in the disease process may be difficult due to several limitations: radiological resolution, bronchoscopic limitations in evaluating tissue underlying the airway mucosa and identifying early pathologic changes, and small sample size and/or incomplete sampling in histology biopsies. High resolution imaging modalities, such as optical frequency domain imaging (OFDI), provide non-destructive, large area 3-dimensional views of tissue microstructure to depths approaching 2 mm in real time (Figure 1)2-6. OFDI has been utilized in a variety of applications, including evaluation of coronary artery atherosclerosis6,7 and esophageal intestinal metaplasia and dysplasia6,8-10.
Bronchoscopic OCT/OFDI has been demonstrated as a safe in vivo imaging tool for evaluating the pulmonary airways11-23 (Animation). OCT has been assessed in pulmonary airways16,23 and parenchyma17,22 of animal models and in vivo human airway14,15. OCT imaging of normal airway has demonstrated visualization of airway layering and alveolar attachments, and evaluation of dysplastic lesions has been found useful in distinguishing grades of dysplasia in the bronchial mucosa11,12,20,21. OFDI imaging of bronchial mucosa has been demonstrated in a short bronchial segment (0.8 cm)18. Additionally, volumetric OFDI spanning multiple airway generations in swine and human pulmonary airways in vivo has been described19. Endobronchial OCT/OFDI is typically performed using thin, flexible catheters, which are compatible with standard bronchoscopic access ports. Additionally, OCT and OFDI needle-based probes have recently been developed, which may be used to image regions of the lung beyond the airway wall or pleural surface17.
While OCT/OFDI has been utilized and demonstrated as feasible for in vivo pulmonary imaging, no studies with precisely matched one-to-one OFDI:histology have been performed. Therefore, specific imaging criteria for various pulmonary pathologies have yet to be developed. Histopathological counterparts obtained in vivo consist of only small biopsy fragments, which are difficult to correlate with large OFDI datasets. Additionally, they do not provide the comprehensive histology needed for registration with large volume OFDI. As a result, specific imaging features of pulmonary pathology cannot be developed in the in vivo setting. Precisely matched, one-to-one OFDI and histology correlation is vital to accurately evaluate features seen in OFDI against histology as a gold standard in order to derive specific image interpretation criteria for pulmonary neoplasms and other pulmonary pathologies. Once specific imaging criteria have been developed and validated ex vivo with matched one-to-one histology, the criteria may then be applied to in vivo imaging studies. Here, we present a method for precise, one to one correlation between high resolution optical imaging and histology in ex vivo lung resection specimens. Throughout this manuscript, we describe the techniques used to match OFDI images to histology. However, this method is not specific to OFDI and can be used to obtain histology-registered images for any optical imaging technique. We performed airway centered OFDI with a specialized custom built bronchoscopic 2.4 French (0.8 mm diameter) catheter. Tissue samples were marked with tissue dye, visible in both OFDI and histology. Careful orientation procedures were used to precisely correlate imaging and histological sampling locations. The techniques outlined in this manuscript were used to conduct the first demonstration of volumetric OFDI with precise correlation to tissue-based diagnosis for evaluating pulmonary pathology24. This straightforward, effective technique may be extended to other tissue types to provide precise imaging to histology correlation needed to determine fine imaging features of both normal and diseased tissues.

Optical Frequency Domain Imaging of Ex vivo Pulmonary Resection Specimens: Obtaining One to One Image to Histopathology Correlation

A method to image ex vivo pulmonary resection specimens with optical frequency domain imaging (OFDI) and obtain precise correlation to histology is described, which is essential to developing specific OFDI interpretation criteria for pulmonary pathology. This method is applicable to other tissue types and imaging techniques to obtain precise imaging to histology correlation for accurate image interpretation and assessment. Imaging criteria established with this technique would then be applicable to image assessment in future in vivo studies.

D&#39;imagerie optique du domaine de fréquence<em&gt; Ex vivo</em&gt; Les spécimens résection pulmonaire: Obtention One to One image à l&#39;histopathologie Corrélation

Lung cancer is the leading cause of cancer-related deaths1. Squamous cell and small cell cancers typically arise in association with the conducting ...

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

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

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

Haute résolution spatio-temporelle d&#39;analyse de la dynamique des récepteurs par simple molécule microscopie de fluorescence

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

Magnetic Levitation Coupled with Portable Imaging and Analysis for Disease Diagnostics

Currently, many clinical diagnostic procedures are complex, costly, inefficient, and inaccessible to a large population in the world. The requirements for specialized equipment and trained personnel require that many diagnostic tests be performed at remote, centralized clinical laboratories. Magnetic levitation is a simple yet powerful technique and can be applied to levitate cells, which are suspended in a paramagnetic solution and placed in a magnetic field, at a position determined by equilibrium between a magnetic force and a buoyancy force. Here, we present a versatile platform technology designed for point-of-care diagnostics which uses magnetic levitation coupled to microscopic imaging and automated analysis to determine the density distribution of a patient's cells as a useful diagnostic indicator. We present two platforms operating on this principle: (i) a smartphone-compatible version of the technology, where the built-in smartphone camera is used to image cells in the magnetic field and a smartphone application processes the images and to measures the density distribution of the cells and (ii) a self-contained version where a camera board is used to capture images and an embedded processing unit with attached thin-film-transistor (TFT) screen measures and displays the results. Demonstrated applications include: (i) measuring the altered distribution of a cell population with a disease phenotype compared to a healthy phenotype, which is applied to sickle cell disease diagnosis, and (ii) separation of different cell types based on their characteristic densities, which is applied to separate white blood cells from red blood cells for white blood cell cytometry. These applications, as well as future extensions of the essential density-based measurements enabled by this portable, user-friendly platform technology, will significantly enhance disease diagnostic capabilities at the point of care.

We present a magnetic levitation technique coupled with automated imaging and analysis in both a smartphone-compatible device and a device with embedded imaging and processing. This is applied to measure the density distribution of cells with two demonstrated biomedical applications: sickle cell disease diagnosis and separating white and red blood cells.

Lévitation magnétique Couplé avec imagerie portable et d&#39;analyse pour le diagnostic de la maladie

Currently, many clinical diagnostic procedures are complex, costly, inefficient, and inaccessible to a large population in the world. The requirements for ...

Quantifying Intermembrane Distances with Serial Image Dilations

A recently-described extracellular nanodomain, termed the perinexus, has been implicated in ephaptic coupling, which is an alternative mechanism for electrical conduction between cardiomyocytes. The current method for quantifying this space by manual segmentation is slow and has low spatial resolution.We developed an algorithm that uses serial image dilations of a binary outline to count the number of pixels between two opposing 2 dimensional edges.This algorithm requires fewer man hours and has a higher spatial resolution than the manual method while preserving the reproducibility of the manual process.In fact, experienced and novice investigators were able to recapitulate the results of a previous study with this new algorithm.The algorithm is limited by the human input needed to manually outline the perinexus and computational power mainly encumbered by a pre-existing pathfinding algorithm.However, the algorithm&#39;s high-throughput capabilities, high spatial resolution and reproducibility make it a versatile and robust measurement tool for use across a variety of applications requiring the measurement of the distance between any 2-dimensional (2D) edges.

The purpose of this algorithm is to continuously measure the distance between two 2-dimensional edges using serial image dilations and pathfinding. This algorithm can be applied to a variety of fields such as cardiac structural biology, vascular biology, and civil engineering.

Quantification des Distances intermembranaire avec Serial Image dilatations

A recently-described extracellular nanodomain, termed the perinexus, has been implicated in ephaptic coupling, which is an alternative mechanism for ...

Substructure Analyzer: A User-Friendly Workflow for Rapid Exploration and Accurate Analysis of Cellular Bodies in Fluorescence Microscopy Images

The last decade has been characterized by breakthroughs in fluorescence microscopy techniques illustrated by spatial resolution improvement but also in live-cell imaging and high-throughput microscopy techniques. This led to a constant increase in the amount and complexity of the microscopy data for a single experiment. Because manual analysis of microscopy data is very time consuming, subjective, and prohibits quantitative analyses, automation of bioimage analysis is becoming almost unavoidable. We built an informatics workflow called Substructure Analyzer to fully automate signal analysis in bioimages from fluorescent microscopy. This workflow is developed on the user-friendly open-source platform Icy and is completed by functionalities from ImageJ. It includes the pre-processing of images to improve the signal to noise ratio, the individual segmentation of cells (detection of cell boundaries) and the detection/quantification of cell bodies enriched in specific cell compartments. The main advantage of this workflow is to propose complex bio-imaging functionalities to users without image analysis expertise through a user-friendly interface. Moreover, it is highly modular and adapted to several issues from the characterization of nuclear/cytoplasmic translocation to the comparative analysis of different cell bodies in different cellular sub-structures. The functionality of this workflow is illustrated through the study of the Cajal (coiled) Bodies under oxidative stress (OS) conditions. Data from fluorescence microscopy show that their integrity in human cells is impacted a few hours after the induction of OS. This effect is characterized by a decrease of coilin nucleation into characteristic Cajal Bodies, associated with a nucleoplasmic redistribution of coilin into an increased number of smaller foci. The central role of coilin in the exchange between CB components and the surrounding nucleoplasm suggests that OS induced redistribution of coilin could affect the composition and the functionality of Cajal Bodies.

We present a freely available workflow built for rapid exploration and accurate analysis of cellular bodies in specific cell compartments in fluorescence microscopy images. This user-friendly workflow is designed on the open-source software Icy and also uses ImageJ functionalities. The pipeline is affordable without knowledge in image analysis.

Analyseur de sous-structure : Un flux de travail convivial pour l’exploration rapide et l’analyse précise des corps cellulaires dans les images de microscopie de fluorescence

The last decade has been characterized by breakthroughs in fluorescence microscopy techniques illustrated by spatial resolution improvement but also in ...

Imaging and Quantification of the Area of Fast-Moving Microbubbles Using a High-Speed Camera and Image Analysis

An experimental and image analysis technique is presented for imaging cavitation bubbles and calculating their area. The high-speed imaging experimental technique and image analysis protocol presented here can also be applied for imaging microscopic bubbles in other fields of research; therefore, it has a wide range of applications. We apply this to image cavitation around dental ultrasonic scalers. It is important to image cavitation to characterize it and to understand how it can be exploited for various applications. Cavitation occurring around dental ultrasonic scalers can be used as a novel method of dental plaque removal, which would be more effective and cause less damage than current periodontal therapy techniques. We present a method for imaging the cavitation bubble clouds occurring around dental ultrasonic scaler tips using a high-speed camera and a zoom lens. We also calculate the area of cavitation using machine learning image analysis. Open source software is used for image analysis. The image analysis presented is easy to replicate, does not require programming experience, and can be modified easily to suit the application of the user.

Cavitation microbubbles are imaged using a high-speed camera attached to a zoom lens. The experimental setup is explained, and image analysis is used to calculate the area of the cavitation. Image analysis is done using ImageJ.

Imagerie et quantification de la zone des microbulles en mouvement rapide à l’aide d’une caméra à grande vitesse et d’une analyse d’images

An experimental and image analysis technique is presented for imaging cavitation bubbles and calculating their area. The high-speed imaging experimental ...

High-Dimensionality Flow Cytometry for Immune Function Analysis of Dissected Implant Tissues

The success of implanting laboratory-grown tissue or a medical device in an individual is subject to the immune response of the recipient host. Considering an implant as a foreign body, a hostile and dysregulated immune response may result in the rejection of the implant, while a regulated response and regaining of homeostasis can lead to its acceptance. Analyzing the microenvironments of implants dissected out under in vivo or ex vivo&#160;settings can help in understanding the pattern of immune response, which can ultimately help in developing new generations of biomaterials. Flow cytometry is a well-known technique for characterizing immune cells and their subsets based on their cell surface markers. This review describes a protocol based on manual dicing, enzymatic digestion, and filtration through a cell strainer for the isolation of uniform cell suspensions from dissected implant tissue. Further, a multicolor flow cytometry staining protocol has been explained, along with steps for initial cytometer settings to characterize and quantify these isolated cells by flow cytometry.

Isolation of cells from dissected implants and their characterization by flow cytometry can significantly contribute to understanding the pattern of immune response against implants. This paper describes a precise method for the isolation of cells from dissected implants and their staining for flow cytometric analysis.

Cytométrie en flux à haute dimensionnalité pour l’analyse de la fonction immunitaire de tissus implantaires disséqués

The success of implanting laboratory-grown tissue or a medical device in an individual is subject to the immune response of the recipient host. ...

Area-based Image Analysis Algorithm for Quantification of Macrophage-fibroblast Cocultures

Quantification of cells is necessary for a wide range of biological and biochemical studies. Conventional image analysis of cells typically employs either fluorescence detection approaches, such as immunofluorescent staining or transfection with fluorescent proteins or edge detection techniques, which are often error-prone due to noise and other non-idealities in the image background. 
We designed a new algorithm that could accurately count and distinguish macrophages and fibroblasts, cells of different phenotypes that often colocalize during tissue regeneration. MATLAB was used to implement the algorithm, which differentiated distinct cell types based on differences in height from the background. A primary algorithm was developed using an area-based method to account for variations in cell size/structure and high-density seeding conditions. 
Non-idealities in cell structures were accounted for with a secondary, iterative algorithm utilizing internal parameters such as cell coverage computed using experimental data for a given cell type. Finally, an analysis of coculture environments was carried out using an isolation algorithm in which various cell types were selectively excluded based on the evaluation of relative height differences within the image. This approach was found to accurately count cells within a 5% error margin for monocultured cells and within a 10% error margin for cocultured cells.

We present a method, which utilizes a generalizable area-based image analysis approach to identify cell counts. Analysis of different cell populations exploited the significant cell height and structure differences between distinct cell types within an adaptive algorithm.

Algorithme d’analyse d’image basé sur la zone pour la quantification des cocultures macrophages-fibroblastes

Quantification of cells is necessary for a wide range of biological and biochemical studies. Conventional image analysis of cells typically employs either ...

Hybrid Cell Analysis System to Assess Structural and Contractile Changes of Human iPSC-Derived Cardiomyocytes for Preclinical Cardiac Risk Evaluation

Cardiac contractility assessment is of immense importance for the development of new therapeutics and their safe transition into clinical stages. While human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) hold promise to serve as a human-relevant model in preclinical phases of drug discovery and safety pharmacology, their maturity is still controversial in the scientific community and under constant development. We present a hybrid contractility and impedance/extracellular field potential (EFP) technology, adding significant pro-maturation features to an industry-standard 96-well platform.
The impedance/EFP system monitors cellular functionality in real-time. Besides the beat rate of contractile cells, the electrical impedance spectroscopy readouts detect compound-induced morphological changes like cell density and integrity of the cellular monolayer. In the other component of the hybrid cell analysis system, the cells are cultured on bio-compliant membranes that mimic the mechanical environment of real heart tissue. This physiological environment supports the maturation of hiPSC-CMs in vitro, leading to more adult-like contractile responses including positive inotropic effects after treatment with isoproterenol, S-Bay K8644, or omecamtiv mecarbil. Parameters such as the amplitude of contraction force (mN/mm2) and beat duration also reveal downstream effects of compounds with influence on electrophysiological properties and calcium handling.
The hybrid system provides the ideal tool for holistic cell analysis, allowing preclinical cardiac risk assessment beyond the current perspectives of human-relevant cell-based assays.

The analysis of changes in contractile function and cellular integrity of human iPSC-derived cardiomyocytes is of immense importance for nonclinical drug development. A hybrid 96-well cell analysis system addresses both parameters in a real-time and physiological manner for reliable, human-relevant results, necessary for a safe transition into clinical stages.

Système d’analyse cellulaire hybride pour évaluer les changements structurels et contractiles de cardiomyocytes humains dérivés de CSPi pour l’évaluation préclinique du risque cardiaque

Cardiac contractility assessment is of immense importance for the development of new therapeutics and their safe transition into clinical stages. While ...