Name: real-time imaging of myeloid cells dynamics in apcmin/+ intestinal tumors by spinning disk confocal microscopy
Uploaded: 2014-10-06T14:00:00.000+00:00
Duration: 5 min 21 s
Description: Watch this Scientific Journal Video about Real-time Imaging of Myeloid Cells Dynamics in ApcMin/+ Intestinal Tumors by Spinning Disk Confocal Microscopy at JoVE.com

نود أن نشكر يو لينغ مين APC / + الفئران التنميط الجيني. وأيد هذه الدراسة من قبل أموال من INSERM والمنح (CA057621 و AI053194) من المعاهد الوطنية للصحة.

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The authors have nothing to disclose.

في هذه الورقة، يتم وصف بروتوكول مفصل عن الغزل القرص التصوير متحد البؤر ديناميات خلية الدم النخاعي في الأورام المعوية لعدة ساعات في الحيوانات الحية، التقط من الجانب المصلي للأمعاء. لتجنب الالتهاب وأن الظروف الفسيولوجية الأمثل، ?...

أدلة دامغة يوضح الآن أن المكروية الورم، وتتألف من السكان الخلية غير متجانسة، بما في ذلك الخلايا الليفية، الخلايا البطانية، الخلايا المناعية والتهابات، ومصفوفة خارج الخلية، والعوامل القابلة للذوبان، يلعب دورا حاسما في بدء وتطور الأورام الصلبة من خلال المساهمة في تقريبا جميع بصمات السرطان 1. في الواقع، خلال تطور الورم، وهناك تفاعلات ديناميكية مستمرة بين الخلايا السرطانية والخلايا تتحول انسجة أن تتطور لتوليد المكروية مواتية لخباثة 2. بين الخلايا المناعية التي تتسلل المكروية الورم، وخلايا الدم النخاعي هي الأكثر وفرة 3. تتألف من الضامة المرتبطة الورم (TAM)، المشتقة من خلايا النخاعي القامع (MDSCs)، والخلايا الجذعية (DC) والعدلات (PMNs)، يتم تجنيد خلايا الدم النخاعي من نخاع العظام والأورام التسلل تدريجيا، والإفراج عن السيتوكينات وعوامل النمو والبروتياز التي يمكن أن تعززتنتشر نمو الورم و4. الحديث المتبادل بين الخلايا السرطانية وخلايا الدم النخاعي معقد ولكن الديناميكي. وبالتالي فهم طبيعة تفاعلاتها هو أمر حاسم لتحديد سبب هذه الخلايا تعزيز تطور السرطان بدلا من المشاركة في الاستجابة المناعية المضادة للورم، ويمكن أن تساعد على إيجاد أهداف جديدة للسيطرة عليها. الملاحظة المباشرة من قبل intravital المجهري توفر معلومات عن ديناميات الخلايا داخل أنسجة الفئران الحية 5. وهناك أربعة ألوان، متعددة المنطقة، وقد صمم القرص الغزل بعدسات نظام متحد البؤر الصغيرة لدراسة خلايا انسجة داخل الأورام الثديية 6. هذا النهج يتيح التصوير المستمر على المدى الطويل، ويشمل العديد من المزايا مثل (أ) اقتناء الصور السريعة لتقليل الحركة الفنية، (ب) التخدير على المدى الطويل، (ج) أربعة اكتساب اللون لمتابعة أنواع مختلفة من الخلايا، (د) وضع العلامات الفلورية مكونات ورمي مختلفة، و (ه) مراقبة مختلف microenvironments الورم الطرافةهين نفس الماوس لتجنب الماوس لتقلب الماوس 7-9. مع هذه التكنولوجيا، وقد تم الإبلاغ عن السلوكيات مختلفة من الخلايا في فيروس الورم الثديية (MMTV) يحركها المروج التورام منتصف T الجين الورمي (PyMT) النموذج الذي يعرض مراحل متقدمة من تكون الأورام. الخلايا اللمفاوية التائية التنظيمية (Tregs، تصور من قبل التحوير Foxp3 EGFP) الهجرة تفضيلي في قربها من الأوعية الدموية في حين أن البلدان النامية (CD11c و-DTR-EGFP)، الخلايا الليفية المرتبطة سرطان (Fsp1 + / + -EGFP) وخلايا الدم النخاعي (ج-FMS -EGFP) تظهر ارتفاع الحركة في محيط الورم من داخل كتلة الورم. في حالة نقص الأكسجين الحاد النظامية، هاجرت خلايا مختلفة: Tregs وقف الهجرة على النقيض من خلايا الدم النخاعي أن مواصلة التحرك 6. بالإضافة إلى ذلك، في نموذج الفأر نفسه، وقد تبين أن التغييرات حساسية دوكسوروبيسين مع مرحلة الورم، ويرتبط توزيع المخدرات إلى الاستجابة للدواء، ودوكسوروبيسينالعلاج يؤدي إلى تجنيد تعتمد على CCR2 من خلايا الدم النخاعي للأورام. وهكذا، ويمكن أيضا أن تستخدم التصوير الحي للحصول على نظرة ثاقبة الردود المخدرات في الموقع وبيولوجيا مقاومة كيميائية 10،11. رميغدي البوليبات القولونية (APC) الطفرات الجينية تحدث عادة في أورام القولون والمستقيم وسرطان الإنسان 12 وتحور نسخة واحدة من الجين APC نتائج في المرجلات غدومي العائلية (FAP)، الذي يضفي على مخاطر عالية جدا لسرطان القولون 13. سلالة الماوس APC مين / + يحمل طفرة اقتطاع في كودون 850 من الجين APC ويتطور بشكل عفوي أورام الأمعاء متعددة في جميع أنحاء الأمعاء الدقيقة 14- 16. على المدى الطويل التصوير حيوي داخلي من الأمعاء يمثل تحديا بسبب الغزو من الإجراء، منذ فتح التجويف البريتوني من الضروري للوصول إلى الأمعاء. على المدى القصير التصوير الحي الحادي وتم udies المنشورة سابقا على الأمعاء صحي 17،18، ولكن لم يتم الإبلاغ عن الملاحظة المباشرة طويلة الأجل الأورام المعوية. وقد تم تصميم وإجراء العمليات الجراحية وصقلها لتصور الأورام من خلال سطح المصلي للأمعاء، وذلك باستخدام نظام الغزل حيوي داخلي المجهري القرص المستخدمة سابقا لأورام الثدي صورة 6،10. في هذه الورقة، وصفت البروتوكول الذي يسمح احد لاتباع سلوك خلايا الدم النخاعي داخل الأورام في الأمعاء الدقيقة باستخدام APC مين / + الفئران.

<table><tbody><tr><td>&lt;em&gt;Apc&lt;sup&gt;Min/+&lt;/sup&gt;&lt;/em&gt; mice</td><td>Jackson Laboratory</td><td>2020</td><td></td></tr><tr><td>ACTB-ECFP mice</td><td>Jackson Laboratory</td><td>3773</td><td></td></tr><tr><td>cfms-EGFP mice</td><td>Jackson Laboratory</td><td>18549</td><td></td></tr><tr><td>2,000 kDa Dextran, rhodamine-conjugated</td><td>Invitrogen</td><td>D7139</td><td></td></tr><tr><td>Isoflurane</td><td>Butler Animal Health Supply</td><td>29450</td><td></td></tr><tr><td>Nitrogen</td><td>UCSF</td><td></td><td></td></tr><tr><td>Oxygen</td><td>UCSF</td><td></td><td></td></tr><tr><td>1x PBS</td><td>UCSF cell culture facility</td><td></td><td></td></tr><tr><td>Saline Buffer</td><td>UCSF cell culture facility</td><td></td><td></td></tr><tr><td>Anti-mouse Ly-6G (GR1) antibody AF647</td><td>UCSF Monoclonal antibody core</td><td></td><td>Stock 1 mg/ml. Use at 7 &amp;mu;g/mouse</td></tr><tr><td>Atropine</td><td>LARC UCSF</td><td></td><td>Use at 1 mg/kg mouse</td></tr><tr><td>Alcohol wipes</td><td>Becton Dickinson</td><td>326895</td><td></td></tr><tr><td>28 G x 1/2 in. insulin syringe</td><td>Becton Dickinson</td><td>329465</td><td></td></tr><tr><td>Remium cover glass</td><td>Fisher Scientific</td><td>12-548-5M</td><td>24x50-1</td></tr><tr><td>Betadine</td><td>LARC UCSF</td><td></td><td></td></tr><tr><td>Heat blanket</td><td>Gaymar Industries</td><td></td><td></td></tr><tr><td>Hot bead sterilizer</td><td>Fine Science Tools</td><td>18000-45</td><td>Turn ON 30 min before use</td></tr><tr><td>Cotton tipped apllicators 6-inch</td><td>Electron Microscopy Sciences</td><td>72310-10</td><td></td></tr><tr><td>Anesthesia system</td><td>Summit Anesthesia Support</td><td></td><td></td></tr><tr><td>Inverted microscope</td><td>Carl Zeiss Inc</td><td>Zeiss Axiovert 200M</td><td></td></tr><tr><td>Stage insert</td><td>Applied Sientific Instrumentation</td><td></td><td></td></tr><tr><td>Mouse Ox oximeter, software and sensors</td><td>Starr Life Sciences</td><td>MouseOx</td><td></td></tr><tr><td>Nebulizer</td><td>Summit Anesthesia Support</td><td></td><td></td></tr><tr><td>Imaris</td><td>Bitplane</td><td></td><td></td></tr><tr><td>&amp;mu;Manager</td><td>Vale lab, UCSF</td><td></td><td>Open-source software</td></tr><tr><td>ICCD camera</td><td>Stanford Photonics</td><td>XR-Mega-10EX S-30</td><td></td></tr><tr><td>Spinning disk confocal sacan-head</td><td>Yokogawa Corporation</td><td>CSU-10b</td><td></td></tr></tbody></table>

real-time imaging of myeloid cells dynamics in apcmin/+ intestinal tumors by spinning disk confocal microscopy

خلايا الدم النخاعي هي الأكثر وفرة الخلايا المناعية داخل الأورام وثبت لتعزيز تطور الورم. تقنيات التصوير الحديثة حيوي داخلي تمكن مراقبة سلوك الخلايا الحية داخل الجهاز لكن يمكن أن يكون تحديا في بعض أنواع السرطان بسبب ورم الجهاز والوصول مثل الأمعاء. الملاحظة المباشرة من الأورام المعوية لم يتم ذكر سابقا. إجراء العمليات الجراحية الموصوفة هنا يسمح الملاحظة المباشرة للديناميات خلية الدم النخاعي داخل الأورام المعوية في الفئران الحية المعدلة وراثيا باستخدام الفئران مراسل فلوري واستشفاف طريق الحقن أو الأجسام المضادة. لهذا الغرض، تم استخدام أربعة ألوان، متعددة المنطقة،-بعدسات الصغرى القرص الغزل المجهر متحد البؤر التي تتيح التصوير المستمر طويل الأمد مع الحصول على الصور السريع. APC مين / + الفئران التي تطور أورام متعددة في الأمعاء الدقيقة وعبروا مع الفئران C-FMS-EGFP لتصور خلايا الدم النخاعي ومع الفئران ACTB-ECFPلتصور الخلايا الظهارية في الأمعاء من الخبايا. كما وصف الإجراءات لوصفها مكونات الورم مختلفة، مثل الأوعية الدموية والعدلات، وإجراءات تحديد المواقع الورم للتصوير من خلال سطح المصلي. الوقت الفاصل بين الأفلام التي تم تجميعها من عدة ساعات من التصوير تسمح للتحليل سلوك الخلية النخاعي في الموقع في المكروية المعوية.

باستخدام الغزل القرص المجهري متحد البؤر والأنسجة غير ورمي ورمي في والأمعاء الدقيقة من APC مين / +، ACTB-ECFP، الفئران C-FMS-ECFP يمكن تصور من السطح المصلي. بعد التصوير، يتم استخدام برنامج الكاميرا لتحليل وضبط اكتساب (الشكل التكميلي 1). بعد الوريدي (IV)</em...

Watch this Scientific Journal Video about Real-time Imaging of Myeloid Cells Dynamics in ApcMin/+ Intestinal Tumors by Spinning Disk Confocal Microscopy at JoVE.com

Real-time Imaging of Myeloid Cells Dynamics in ApcMin/+ Intestinal Tumors by Spinning Disk Confocal Microscopy

By using transgenic reporter mice and injectable fluorescent labels, long-term intravital spinning disk confocal microscopy enables direct visualization of myeloid cell behavior into intestinal adenoma in the ApcMin/+ colorectal cancer model.

التصوير في الوقت الحقيقي من خلايا الدم النخاعي في ديناميات<em&gt; APC<sup&gt; مين / +</sup</em&gt; الأورام المعوية التي كتبها غزل القرص متحد البؤر المجهر

Myeloid cells are the most abundant immune cells within tumors and have been shown to promote tumor progression. Modern intravital imaging techniques ...

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Immunology and Infection

Real-time Imaging of Myeloid Cells Dynamics in ApcMin/+ Intestinal Tumors by Spinning Disk Confocal Microscopy

9.6K Views.  INSERM U661, Functional Genomic Institute. By using transgenic reporter mice and injectable fluorescent labels, long-term intravital spinning disk confocal microscopy enables direct visualization of myeloid cell behavior into intestinal adenoma in the ApcMin/+ colorectal cancer model. 

Video: Real-time Imaging of Myeloid Cells Dynamics in ApcMin/+ Intestinal Tumors by Spinning Disk Confocal Microscopy

Ex Vivo Imaging of Resident CD8 T Lymphocytes in Human Lung Tumor Slices Using Confocal Microscopy

CD8 T cell are key players in the fight against cancer. In order for CD8 T cells to kill tumor cells they need to enter into the tumor, migrate within the tumor microenvironment and respond adequately to tumor antigens. The recent development of improved imaging approaches, such as 2-photon microscopy, and the use of powerful mouse tumor models have shed light on some of the mechanisms that regulate anti-tumor T cell activities. Whereas such systems have provided valuable insights, they do not always predict human responses. In human, our knowledge in the field mainly comes from a description of fixed tumor samples from human patients, as well as in vitro studies. However, in vitro models lack the complex three-dimensional tumor milieu and, therefore, are incomplete approximations of in vivo T cell activities. Fresh slices made from explanted tissue represent a complex multi-cellular tumor environment that can act as an important link between co-cultured studies and animal models. Originally set up in murine lymph nodes1 and previously described in a JoVE article2, this approach has now been transposed to human tumors to examine the dynamics of both plated3&#160;as well as resident&#160;T cells4.
Here, a protocol for the preparation of human lung tumor slices, immunostaining of resident CD8 T and tumor cells, and tracking of CD8 T lymphocytes within the tumor microenvironment using confocal microscopy is described. This system is uniquely placed to screen for novel immunotherapy agents favoring T cell migration in tumors.

Ex Vivo Imaging of Resident CD8 T Lymphocytes in Human Lung Tumor Slices Using Confocal Microscopy

This protocol describes a method to image resident tumor-infiltrating CD8 T cells labeled with fluorescently coupled antibodies within human lung tumor slices. This technique permits real-time analyses of CD8 T cell migration using confocal microscopy.

السابقين فيفو تصوير لمفاوية T CD8 المقيم في ورم الرئة البشرية شرائح باستخدام الفحص المجهري [كنفوكل]

CD8 T cell are key players in the fight against cancer. In order for CD8 T cells to kill tumor cells they need to enter into the tumor, migrate within the ...

Cancer Research

تصوير البنكرياس الفئران باستخدام نافذة مثبتة مزروعة

Stabilized Window for Intravital Imaging of the Murine Pancreas

The physiology and pathophysiology of the pancreas are complex. Diseases of the pancreas, such as pancreatitis and pancreatic adenocarcinoma (PDAC) have high morbidity and mortality. Intravital imaging (IVI) is a powerful technique enabling the high-resolution imaging of tissues in both healthy and diseased states, allowing for real-time observation of cell dynamics. IVI of the murine pancreas presents significant challenges due to the deep visceral and compliant nature of the organ, which make it highly prone to damage and motion artifacts. 
Described here is the process of implantation of the Stabilized Window for Intravital imaging of the murine Pancreas (SWIP). The SWIP allows IVI of the murine pancreas in normal healthy states, during the transformation from the healthy pancreas to acute pancreatitis induced by cerulein, and in malignant states such as pancreatic tumors. In conjunction with genetically labeled cells or the administration of fluorescent dyes, the SWIP enables the measurement of single-cell and subcellular dynamics (including single-cell and collective migration) as well as serial imaging of the same region of interest over multiple days. 
The ability to capture tumor cell migration is of particular importance as the primary cause of cancer-related mortality in PDAC is the overwhelming metastatic burden. Understanding the physiological dynamics of metastasis in PDAC is a critical unmet need and crucial for improving patient prognosis. Overall, the SWIP provides improved imaging stability and expands the application of IVI in the healthy pancreas and malignant pancreas diseases.

تسليط الضوء على المؤلف: إحداث ثورة في فهم أمراض البنكرياس من خلال التصوير المتقدم داخل الجسم 

We present a protocol for the surgical implantation of a stabilized indwelling optical window for subcellular-resolution imaging of the murine pancreas, allowing serial and longitudinal studies of the healthy and diseased pancreas.

نافذة مستقرة للتصوير داخل الجسم للبنكرياس الفئران

The physiology and pathophysiology of the pancreas are complex. Diseases of the pancreas, such as pancreatitis and pancreatic adenocarcinoma (PDAC) have ...

Imaging Neutrophils and Monocytes in Mesenteric Veins by Intravital Microscopy on Anaesthetized Mice in Real Time

Efficient immune response is dependent on rapid mobilization of blood leukocytes to the site of infection or injury. Investigating leukocyte migration in vivo is crucial for understanding the molecular basis of leukocyte transendothelial migration and interaction with vascular endothelium. One powerful approach involves intravital microscopy on transgenic mice expressing fluorescent proteins in cells of interest.
Here we present a protocol for imaging monocytes and neutrophils in the CX3CR1gfp/wt mouse i.v. injected with orange dye-labeled neutrophils with an inverted confocal microscope. Time-lapse movies gathered from 30 min&#160;to several hours of imaging allow the analysis of leukocyte behavior in mesenteric veins under both steady state and inflammatory conditions. We also describe the steps to locally induce blood vessel inflammation with TLR2/TLR1 agonist Pam3SK4 and monitor the subsequent recruitment of neutrophils and monocytes.
The presented technique can also be used to monitor other populations of leukocytes and investigate molecules implicated in leukocyte recruitment or trafficking using other stimuli or transgenic mice.

We detail a protocol to monitor the behavior of neutrophils and monocytes in mesenteric veins under steady state and inflammatory conditions using intravital confocal microscopy on anaesthetized mice.

التصوير العدلات وحيدات في مساريقي الأوردة التي كتبها حيوي داخلي المجهري على الفئران مخدرة في الوقت الحقيقي

Efficient immune response is dependent on rapid mobilization of blood leukocytes to the site of infection or injury. Investigating leukocyte migration in ...

Imaging CD4 T Cell Interstitial Migration in the Inflamed Dermis

The ability of CD4 T cells to carry out effector functions is dependent upon the rapid and efficient migration of these cells in inflamed peripheral tissues through an as-yet undefined mechanism. The application of multiphoton microscopy to the study of the immune system provides a tool to measure the dynamics of immune responses within intact tissues. Here we present a protocol for non-invasive intravital multiphoton imaging of CD4 T cells in the inflamed mouse ear dermis. Use of a custom imaging platform and a venous catheter allows for the visualization of CD4 T cell dynamics in the dermal interstitium, with the ability to interrogate these cells in real-time via the addition of blocking antibodies to key molecular components involved in motility. This system provides advantages over both in vitro models and surgically invasive imaging procedures. Understanding the pathways used by CD4 T cells for motility may ultimately provide insight into the basic function of CD4 T cells as well as the pathogenesis of both autoimmune diseases and pathology from chronic infections.

The mechanisms that govern the interstitial motility of CD4 effector T cells at sites of inflammation are relatively unknown. We present a non-invasive approach to visualize and manipulate in vitro-primed CD4 T cells in the inflamed ear dermis, allowing for study of the dynamic behavior of these cells in situ.

التصوير CD4 T خلية الخلالية الهجرة في الأدمة الملتهبة

The ability of CD4 T cells to carry out effector functions is dependent upon the rapid and efficient migration of these cells in inflamed peripheral ...

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

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

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

تصور تشكيل الالتصاق في الخلايا من خلال متقدم الغزل مجهرية Fluorescence انعكاس الداخلية القرص--المجموع

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

Bioengineering

Time-lapse 3D Imaging of Phagocytosis by Mouse Macrophages

Phagocytosis plays a key role in host defense, as well as in tissue development and maintenance, and involves rapid, receptor-mediated rearrangements of the actin cytoskeleton to capture, envelop and engulf large particles. Although phagocytic receptors, downstream signaling pathways, and effectors, such as Rho GTPases, have been identified, the dynamic cytoskeletal remodeling of specific receptor-mediated phagocytic events remain unclear. Four decades ago, two distinct mechanisms of phagocytosis, exemplified by Fc&#947; receptor (Fc&#947;R)- and complement receptor (CR)-mediated phagocytosis, were identified using scanning electron microscopy. Binding of immunoglobulin G (IgG)-opsonized particles to Fc&#947;Rs triggers the protrusion of thin membrane extensions, which initially form a so-called phagocytic cup around the particle before it becomes completely enclosed and retracted into the cell. In contrast, complement opsonized particles appear to sink into the phagocyte following binding to complement receptors. These two modes of phagocytosis, phagocytic cup formation and sinking in, have become well established in the literature. However, the distinctions between the two modes have become blurred by reports that complement receptor-mediated phagocytosis may induce various membrane protrusions. With the availability of high resolution imaging techniques, phagocytosis assays are required that allow real-time 3D (three dimensional) visualization of how specific phagocytic receptors mediate the uptake of individual particles. More commonly used approaches for the study of phagocytosis, such as end-point assays, miss the opportunity to understand what is happening at the interface of particles and phagocytes. Here we describe phagocytic assays, using time-lapse spinning disk confocal microscopy, that allow 3D imaging of single phagocytic events. In addition, we describe assays to unambiguously image Fc&#947; receptor- or complement receptor-mediated phagocytosis.

Here we describe methods using spinning disk confocal microscopy to image single phagocytic events by mouse resident peritoneal macrophages. The protocols can be extended to other phagocytic cells.

الوقت الفاصل بين التصوير 3D البلعمه التي الضامة الماوس

Phagocytosis plays a key role in host defense, as well as in tissue development and maintenance, and involves rapid, receptor-mediated rearrangements of ...

Intravital Microscopy of Tumor-associated Vasculature Using Advanced Dorsal Skinfold Window Chambers on Transgenic Fluorescent Mice

Tumor and tumor vessel development, as well as tumor response to therapeutics, are highly dynamic biological processes. Histology provides static information and is often not sufficient for a correct interpretation. Intravital evaluation, in which a process is followed in time, provides extra and often unexpected information. With the creation of transgenic animals expressing cell-specific markers and live cell tracers, improvements to imaging equipment, and the development of several imaging chambers, intravital microscopy has become an important tool to better understand biological processes. This paper describes an experimental design for the investigation of tumor vessel development and of therapeutic effects in a spatial and temporal manner. Using this setup, the stage of vessel development, tip cell and lumen formation, blood flow, extravasation, an established vascular bed, and vascular destruction can be visualized and followed. Furthermore, therapeutic effects, intratumoral fate, and the localization of chemotherapeutic compounds can also be followed.

This protocol describes the intravital imaging of transgenic mice expressing cell-specific fluorescent markers. Intravital imaging provides a non-invasive method for high-resolution observations in living animals using implantable windows through which the microscopic visualization of processes is possible. This method is especially useful to study longitudinal processes.

إينترافيتال المجهري لاستخدام المفرج المرتبطة بورم متقدم الظهرية Skinfold الدوائر النافذة في الفئران المعدلة وراثيا نيون

Tumor and tumor vessel development, as well as tumor response to therapeutics, are highly dynamic biological processes. Histology provides static ...

Medicine

Intravital Imaging of Intraepithelial Lymphocytes in Murine Small Intestine

Intraepithelial lymphocytes expressing &#947;&#948; T cell receptor (&#947;&#948; IEL) play a key role in immune surveillance of the intestinal epithelium. Due in part to the lack of a definitive ligand for the &#947;&#948; T cell receptor, our understanding of the regulation of &#947;&#948; IEL activation and their function in vivo remains limited. This necessitates the development of alternative strategies to interrogate signaling pathways involved in regulating &#947;&#948; IEL function and the responsiveness of these cells to the local microenvironment. Although &#947;&#948; IELs are widely understood to limit pathogen translocation, the use of intravital imaging has been critical to understanding the spatiotemporal dynamics of IEL/epithelial interactions at steady-state and in response to invasive pathogens. Herein, we present a protocol for visualizing IEL migratory behavior in the small intestinal mucosa of a GFP &#947;&#948; T cell reporter mouse using inverted spinning disk confocal laser microscopy. Although the maximum imaging depth of this approach is limited relative to the use of two-photon laser-scanning microscopy, spinning disk confocal laser microscopy provides the advantage of high speed image acquisition with reduced photobleaching and photodamage. Using 4D image analysis software, T cell surveillance behavior and their interactions with neighboring cells can be analyzed following experimental manipulation to provide additional insight into IEL activation and function within the intestinal mucosa.

We describe a method to visualize GFP-labeled &#947;&#948; IELs using intravital imaging of murine small intestine by inverted spinning disk confocal microscopy. This technique enables the tracking of live cells within the mucosa for up to 4 h and can be used to investigate a variety of intestinal immune-epithelial interactions. &#160;

التصوير في الوقت الحقيقي من خلايا الدم النخاعي في ديناميات

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السابقين فيفو تصوير لمفاوية T CD8 المقيم في ورم الرئة البشرية شرائح باستخدام الفحص المجهري [كنفوكل]

نافذة مستقرة للتصوير داخل الجسم للبنكرياس الفئران

نافذة مستقرة للتصوير داخل الجسم للبنكرياس الفئران

نافذة مستقرة للتصوير داخل الجسم للبنكرياس الفئران

التصوير العدلات وحيدات في مساريقي الأوردة التي كتبها حيوي داخلي المجهري على الفئران مخدرة في الوقت الحقيقي

التصوير CD4 T خلية الخلالية الهجرة في الأدمة الملتهبة

تصور تشكيل الالتصاق في الخلايا من خلال متقدم الغزل مجهرية Fluorescence انعكاس الداخلية القرص--المجموع

الوقت الفاصل بين التصوير 3D البلعمه التي الضامة الماوس

إينترافيتال المجهري لاستخدام المفرج المرتبطة بورم متقدم الظهرية Skinfold الدوائر النافذة في الفئران المعدلة وراثيا نيون

التصوير داخل الظهارة من الخلايا الليمفاوية داخل الظهارة في الميورين الأمعاء الدقيقة