这项研究得到了伦德贝克基金会、诺和诺德基金会、丹麦独立研究理事会的支持|医学和NORDEA基金会向健康老龄化中心提供资助。

丹麦国家伦理委员会根据欧洲理事会《保护用于实验和其他科学目的的脊椎动物公约》中规定的准则,批准了涉及动物的所有程序,符合《到达准则》。这是一个终端程序,小鼠在麻醉恢复前被安乐死。
1. 手术前准备
<ol><li>用70%乙醇清洁手术台和周围所有区域。手术前彻底清洁和干燥手术工具。</li>
	<li>麻醉NG2DsRed小鼠(Tg(Cspg4-DsRed.T1)1Akik/J;两性;4-8个月大)通过排前注射氯胺酮+木黄素(60毫克/千克=10毫克/千克)溶解在消毒水中,pH 7.4。每25分钟服用补充剂量的氯胺酮(30毫克/千克),直到手术完成。定期检查尾巴和脚趾捏反射,以验证麻醉的深度。</li>
	<li>在小鼠背面的四个不同位置注射盐水,总体积为 1 mL,以防止实验期间脱水。为防止眼睛干燥,请涂抹眼部润滑剂。使用直肠温度探头和加热毯将体温保持在37°C。</li>
</ol>2. 外科手术
<ol><li>气管 切开
	<ol>
<li>将鼠标放在鼠标背面。在计划的切口下注射0.04 mL 0.5%的利多卡因,等待2分钟。分离下颌腺和甲状腺肌,然后暴露气管。</li>
		<li>用剪刀在两个气管环之间进行气管切开术。将长度为 16.2 mm、直径为 1 mm 的小金属管插入气管。将管子连接到机械呼吸机和气盖仪,以监控末端呼气CO2(图1A)。</li>
	</ol></li>
	<li>导管插入
	<ol>
<li>在计划切口下下注射0.5%利多卡因(0.04 mL),等待2分钟。</li>
		<li>使用细尖钳轻轻将动脉与静脉分开。用微血管夹停止血液流向上游。用10-0尼龙缝合两条血管来收缩下游的血流。</li>
		<li>使用微解剖剪刀在动脉和静脉中做一个小切口。将塑料导管插入两个血管。通过拧紧缝合线(8-0)固定导管或10-0尼龙缝合线取决于容器大小),而不损害导管流明。拆下微血管夹并关闭皮肤。</li>
		<li>通过动脉导管监测血压。使用血气分析仪测量每个实验前后动脉血样(50 μL)中的血气水平(正常值:pO2,90-120 mmHg;pCO 2,35-40 mmHg;pH,7.25-7.45)。使用静脉导管输液和麻醉。</li>
	</ol></li>
	<li>开颅 术
	<ol>
<li>把毛毛从老鼠头上的耳之间去掉。在计划切口下注射0.04 mL 0.5%利多卡因皮下,等待2分钟。</li>
		<li>用10%的氯化铁溶液擦拭头骨,去除骨膜。用盐水彻底冲洗头骨。用氰丙烯酸酯胶水和活化剂将金属头杆粘到头骨上(图1A)。 注:金属头杆长75毫米,宽13.5毫米,中心有一个直径为5毫米的圆孔。</li>
		<li>钻一个4毫米直径的颅骨切除术,中心在0.5毫米的后面,3毫米在右感觉桶皮层的右脑膜。用细尖容器扩张器拆下杜拉。</li>
		<li>用0.75%的角胶覆盖暴露的皮层,溶解在人工脑脊液中(aCSF;pH = 7.4),冷却至35°C。用10-15°角的玻璃盖玻片覆盖80-90%的颅骨,以10-15°为角度覆盖金属头杆(图1B),允许插入和放置玻璃微移液器。</li>
		<li>为了尽量减少脑脉动,用氰丙烯酸酯胶和活化剂将玻璃盖板的两个角粘附在金属头杆上。小心地应用激活剂,以防止进入暴露的大脑。之后用盐水彻底冲洗粘合的玻璃盖玻片。</li>
	</ol></li>
	<li>执行胡须垫刺激探头的插入。将一组定制的双极电极(8 mm 长度和 0.25 mm 厚度)切入面部左侧,以刺激三叉神经对侧到颅骨的副轨道。将阴极靠近间断位轨道,并将阳极插入粘胶肌肉7。在 20 s 的列车中,在 20 s 的 2 Hz 下,以 1.5 mA 的强度执行 1 ms 的电刺激。</li>
	<li>手术后,将0.05 mL荧光素异异体-dextran(FITC-dextran,4%w/v,分子量[MW] 50,000)放入股骨静脉,为血浆贴上标签。停止氯胺酮,将麻醉切换到连续静脉输注β-氯洛(17%w/v,最终浓度),与FITC-dextran(2%w/v,最终浓度)混合在0.02 mL/10 g/h。等待约半小时,以稳定新的麻醉状态。 注:FITC-dextran和β-氯酮均溶解在0.9%盐碱中。</li>
</ol>3. 第一次双光子成像会话
<ol><li>将鼠标转移到商用双光子显微镜(材料表)的舞台上。在红色荧光蛋白(RFP,图1C)和绿色荧光蛋白(GFP,图1C)模式下,以5倍目标拍摄暴露皮层的照片。使用 RFP 图片作为"地图",用于在下一个会话中插入玻璃微移液器。 注: GFP"地图"用于区分静脉/静脉与动脉/动脉。</li>
	<li>使用双光子显微镜和 25x 1.0 数值孔径 (NA) 水浸物物用压电电机执行双光子成像。将激发波长设置为 900 nm。搜索皮层,跟随每个穿透动脉,并找到它的水平分支(第1阶毛细血管)。</li>
	<li>图像穿透动脉及其1阶毛细血管在休息和胡须垫刺激期间。请参阅第 5 节中的详细成像参数。</li>
	<li>在 RFP"地图"上用 >5% 的血管扩张标记 1阶毛细血管的位置(图1C)。具有 <5% 血管扩张的位置被视为在胡须桶皮层区域之外。 注:如果在实验中使用野生型小鼠而不是 NG2DsRed 小鼠,则 GFP 图像将用作"地图"。</li>
</ol>4. 插入玻璃微移液器
<ol><li>使用移液器拉拔器(材料表)准备玻璃微移液器,用于膨胀。玻璃微移液器的电阻为3-3.5 MΩ,片长为4.5-5 mm。装载混合1mM ATP和10μM Alexa 594的移液器,以便在荧光灯和双光子显微镜下可视化移液器尖端。</li>
	<li>将玻璃微移液器安装到贴片夹支架上,并将其连接到空气泵。将泵保持压力设置为 0 psi。</li>
	<li>使用红色外观荧光照明,用 5x 目标聚焦移液器尖端。将玻璃微移液器放入甘蔗上方的 ACSF 中。将空气泵的保持压力调节到 0.2 psi。可以看到一个小的红色云从ACSF的移液器尖端弹出。这是为了防止在移液器插入过程中堵塞。</li>
	<li>选择 RFP"地图"中标记的位置之一作为目标。将移液器尖端移到覆盖玻璃边缘的水平平面上,其距离为 30 μm,大致指向 x-y 平面中的目的地。</li>
	<li>小心地将移液器尖端以 z 轴在盖玻璃滑片下下。然后开始将玻璃移液器推进到目的地,直到在大脑表面上方 +500 μm。经常在盖玻璃边缘、移液器尖端和大脑表面之间切换对焦,确保有足够的可用空间来移动移液器。</li>
	<li>将目标切换为 25 倍。重新居心移液器尖端,并在 RFP"地图"上进一步向目的地推进移液器尖端。将移液器尖端保持在甘蔗色层中。</li>
	<li>当移液器尖端在大脑表面上方 ±100 μm 时,将成像模式切换到双光子显微镜。专注于要吹嘘的目标位置。记下其 x、y 坐标(x 0, y0) 及其深度低于曲面 (z0)。计算大脑表面插入点的坐标 (xi, yi) 如下: xi = x0 = z0 / 晒黑 | yi = y0 其中*是移液器支架和水平平面之间的角度。</li>
	<li>将移液器尖端置于大脑表面的坐标(xi,yi)。轻轻地慢慢插入移液器,直到它达到 (x0, y0, z0)。如果容器位于插入路径上,则拔下移液器并重新计算其他插入坐标和路径;或稍微转动舞台板(如图 1 A所示)。</li>
	<li>将泵保持压力设置为 0 psi,将喷射压力设置为 10-15 psi。在双光子成像期间,在目标位置将 200-400 ms 的浮松 ATP。如讨论部分所述,调整每个移液器的膨胀压力和持续时间,并随时间延长。</li>
</ol>5. 超堆栈双光子成像
<ol><li>使用双光子显微镜和 25 x 1.0 NA 水浸物使用压电电机执行实验。将激发波长设置为 900 nm。过滤发出的光,以收集DsRed(围生)/四甲基二甲胺异氰酸二聚氰胺(TRITC-dextran)染色血浆的红光和FITC-dextran(血浆)/GCaMP6(GFP,星化钙)的绿光。</li>
	<li>在感兴趣位置产生高分辨率 z 堆栈,其中包含穿透性动脉、一阶毛细管、第二阶毛细管和可能相邻的荧光细胞(例如,圆环状细胞、星形细胞)。通过尽可能包括血管的 3D 结构,确定超堆栈成像的 x_y_z 体积。每个堆栈的总高度可能从 30-50 μm 不等,而 x-y 平面大致覆盖 60 μm x 40 μm 的区域。</li>
	<li>在成像软件中设置图像堆栈,由 8-10 个平面组成,平面间距离为 4-5 μm。压电电机目标在 z 轴上的每一个电平停止,以获取每个平面的图像。采样速率为每堆栈 1 s,x-y 平面中的像素分辨率为 0.2-0.3 μm(图 2A)。一个记录通常包括 10 个预浮图栈和 150 个后吹捧图像堆栈。</li>
</ol>6. 数据处理
<ol><li>使用定制的分析软件处理数据。通过最大强度投影将每个图像堆栈拼合到一个图像中(图 2B)。绘制一个矩形感兴趣区域 (ROI),宽度垂直于 3 μm,垂直穿过感兴趣容器(图 2C)。</li>
	<li>为了尽量减少红血球阴影和皮层轻微振动的干扰,通过将矩形 ROI 投影到一条线中,从而平均矩形 ROI,然后表示血管段的平均轮廓。为每个最大强度图像执行此操作。</li>
	<li>将轮廓线绘制为 2D 图像,x 轴按时间顺序表示最大强度图像(图2D,上面板)。使用有源轮廓算法(Chan-Vese分割)查找容器8和9的边缘(以红色曲线表示;图 2D,上面板)。</li>
	<li>计算直径的时间过程随着血管边缘之间的差值而变化(图 2D中上下红色曲线之间的垂直距离,下面板)。将直径标准化更改为预膨胀直径基线,直径曲线的图曲线随时间而变化。为每个 ROI 执行此操作 (图 2E)</li>
	<li>容器响应振幅定义为膨胀后的最高/最低峰值振幅。响应延迟定义为半最大振幅的延迟(图2G)。通过沿血管放置节点(图2F)并测量每个 ROI 和穿透动脉之间的地理距离,手动跟踪骨架血管结构。通过将距离除以延迟差异来计算 CVR 速度。</li>
</ol>7. 病毒载体注射
<ol><li>为了同时研究星形钙反应,按照标准的立体定向病毒载体注射程序,将0.6μL病毒载体(pZac2.1 gfaABC1D-lck-gCaMP6f,Addgene)注入NG2DsRed小鼠的体感皮层10.</li>
	<li>三周后,小鼠躯体感觉皮层中的星形细胞表达基因编码的钙指标GCaMP6f。遵循上述外科手术和小鼠双光子成像。为了区分星形细胞和血管亮度,TRITC-dextran代替FITC-dextran用于染色血管氧化铝红色。</li>
</ol>

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作者没有什么可透露的。

血管研究的一个挑战是精确测量血管直径。我们在这里描述的方法使用电动压电物镜,通过双光子显微镜进行快速和重复的超堆栈成像。首先,这种方法允许反复检查穿透性动脉、1阶和第二阶毛细血管,而不会失去焦点,导致在体内毛细血管中发现缓慢进行的血管反应。其次,结合病毒载体注射技术,使我们能够研究星形钙在三维的反应,这是研究血流调节所必需的。
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大脑有高能量消耗率。人体消耗的氧气和葡萄糖的20%专门用于大脑功能,而大脑只占身体总量的2%。维持正常的大脑功能需要在复杂的血管网络中通过血液流动提供充足和高效的氧气和营养。局部大脑活动和脑血流(CBF)是强强耦合的,取决于神经元、星形细胞、坐体、平滑肌细胞(SMC)和内皮细胞(ECs)1的功能特性。最近,从穿透动脉中分枝的毛细血管的前几条订单已经出现为"热点"2,显示出毛细血管血流的积极调节。在用玻璃微移剂3的ATP的胡须刺激和局部弹出(喷出)期间,在小鼠躯体感觉皮层的这个"热点"上发现了缓慢的血管反应(CVR)。
虽然通过双光子激光扫描荧光显微镜在体内成像已被广泛用于研究大脑皮层的神经血管反应,但大多数研究测量了血管直径,并研究了其在二维 (2D) x-y 平面。挑战是:首先,脑血管及其拥抱的星形细胞、围细胞和SMC在三维(3D)中构建分支。因此,在 3D 中研究它们的交互作用至关重要。其次,即使是少量的聚焦漂移也会影响容器直径和细胞荧光信号的精确测量。最后,CVR 在三个方面是快速而深远的。3D 体积扫描是检测 CVR 和揭示其机制的最佳选择。我们在双光子显微镜中实现了压电运动目标,以研究小鼠体内的躯体感觉皮层,通过对获得的图像进行最大强度投影,从而进行精确的直径测量。
玻璃微移液器经常用于体内脑研究,例如,散装有机染料4,记录EEGs5和贴片夹紧6。然而,限制仍然存在。通常,玻璃微移液器的尖端放置不精确,或者微移液器不用于局部干预。在这里,我们优化了微移液器插入和局部弹出的过程。
此外,3D 双光子显微镜和基因编码荧光指标的结合为在 3D 范围内研究神经血管耦合提供了前所未有的机会。在这项研究中,我们利用了这一点,将携带星形细胞特定基因编码钙指标的病毒载体注射到小鼠躯体感觉皮层中。通过组合不同的荧光标记,同时成像星形细胞和容器直径。
总体而言,我们提出了通过玻璃微移液器和快速双光子超堆栈成像进行局部喷射(浮肿)的优化方法,从而能够精确测量毛细管直径变化。此外,我们的方法提供了一个新的工具,可以同时研究星形细胞和血管直径反应中的Ca2+反应的三维轮廓。

<table><tbody><tr><td>Agarose</td><td>Sigma&amp;ndash;Aldrich</td><td>A6138</td><td>Apply upon exposed cortex for protection</td></tr><tr><td>Alexa 594</td><td>Life Technologies</td><td>A-10438</td><td>Stain puffing compound to red fluorescent color</td></tr><tr><td>ATP</td><td>Sigma-Aldrich</td><td>A9187</td><td>Vasodilator and vasoconstrictor, puffing compound</td></tr><tr><td>Cyanoacrylate glue and activator</td><td>Loctite</td><td>Adhesives and SF7452</td><td>Glue for metal piece and coverglass</td></tr><tr><td>Eye lubricant</td><td>Neutral Ophtha, Ophtha A/S, Denmark</td><td></td><td>Keep the mouse eyes moisterized</td></tr><tr><td>FITC-dextran</td><td>Sigma-Aldrich</td><td>FD500S</td><td>Blood serum dye, green fluorescent color</td></tr><tr><td>NG2DsRed mice</td><td>Jackson Laboratory</td><td>8241</td><td>These transgenic mice express an red fluorescent protein variant (DsRed) under the control of the mouse NG2 (Cspg4) promoter</td></tr><tr><td>pZac2.1 gfaABC1D-lck-GCaMP6f</td><td>Addgene</td><td>52924-AAV5</td><td>Astrocyte specific viral vectors carrying genetically encoded calcium indicators</td></tr><tr><td>TRITC-dextran</td><td>Sigma-Aldrich</td><td>52194</td><td>Blood serum dye, red fluorescent color</td></tr><tr><td>&lt;strong&gt;List of Equipments&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Air pump</td><td>WPI</td><td>PV830</td><td>Give air pressure to pipette puffing</td></tr><tr><td>Blood gas analyzer</td><td>Radiometer</td><td>ABL 700</td><td>Measure levels of blood gases&amp;nbsp;</td></tr><tr><td>Blood pressure monitor</td><td>World Precision Instruments</td><td>BP-1</td><td>Monitor aterial blood pressure</td></tr><tr><td>Body temperature controller</td><td>CWE</td><td>Model TC-1000</td><td>Keep the mouse body temperature in physiological range</td></tr><tr><td>Capnograph</td><td>Harvard Apparatus</td><td>Type 340</td><td>Monitor the end-expiratory CO&lt;sub&gt;2&lt;/sub&gt; from the mouse</td></tr><tr><td>Electrical stimulator</td><td>A.M.P.I.</td><td>ISO-flex</td><td>Apply whisker pad stimulation</td></tr><tr><td>Mechanical ventilator</td><td>Harvard Apparatus</td><td>D-79232</td><td>Mechanically ventilate the mouse in physiological range</td></tr><tr><td>Micropipette puller</td><td>Sutter Instrument</td><td>P-97</td><td></td></tr><tr><td>Two-photon microscope</td><td>Femtonics Ltd</td><td>Femto3D-RC</td><td></td></tr><tr><td>&lt;strong&gt;List of Surgical Instruments&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Anatomical tweezer&amp;nbsp;</td><td>Lawton</td><td>09-0007</td><td></td></tr><tr><td>Angled and balanced tweezer</td><td>S&amp;amp;T AG</td><td>00595 FRAS-18 RM-8</td><td></td></tr><tr><td>Iris scissor</td><td>Lawton</td><td>05-1450</td><td></td></tr><tr><td>Micro vascular clamp</td><td>S&amp;amp;T AG</td><td>462</td><td></td></tr><tr><td>Mouse vascular catheters</td><td>Verutech</td><td>100828</td><td></td></tr></tbody></table>

in vivo three-dimensional two-photon microscopy to study conducted vascular responses by local atp ejection using a glass micro-pipette

维持正常的大脑功能需要由复杂的血管网络充分而有效地供应氧气和营养。然而,大脑血流(CBF)的调节不完全理解,特别是在毛细管水平。双光子显微镜是一种强大的工具,广泛用于研究CBF及其调控。目前,由于缺乏体内双光子显微镜研究(1)三维CBF反应,(2)血管反应,以及(3)血管网络内的局部干预,这一领域受到限制。在这里,我们描述了一种使用双光子显微镜的3D体内方法,用于研究通过玻璃微移液器局部弹出ATP引起的血管反应。我们的方法使用快速和重复的超堆栈双光子成像,通过获得的图像的最大强度投影提供精确的直径测量。此外,我们还表明,该方法也可用于研究3D星形钙反应。我们还讨论了玻璃微移液器插入和双光子超堆栈成像的优点和局限性。

手术后,小鼠被运送到双光子显微镜(图1A)。在目标位置的目标血管附近插入了一个含有1 mM ATP的玻璃微移液器(图1B)。
我们执行超堆栈成像,同时给出 1 mM ATP (图 2A,补充视频 1) 的浮肿。每个图像堆栈通过最大强度投影拼合到一个图像(图 2<s...

Watch this Scientific Journal Video about In Vivo Three-Dimensional Two-Photon Microscopy to Study Conducted Vascular Responses by Local ATP Ejection Using a Glass Micro-Pipette at JoVE.com

In Vivo Three-Dimensional Two-Photon Microscopy to Study Conducted Vascular Responses by Local ATP Ejection Using a Glass Micro-Pipette

我们使用玻璃微移液器和快速的双光子超堆成像方法提供优化的局部弹出程序,从而能够精确测量毛细管直径变化,并在三维中调查其调节。

在 Vivo 三维双光子显微镜中,利用玻璃微移器研究本地 ATP 喷射的血管反应

Maintenance of normal brain function requires a sufficient and efficient supply of oxygen and nutrition by a complex network of vessels. However, the ...

该协议旨在研究由局部弹出ATP引起的脑毛细血管反应缓慢。该技术的优点是通过局部移液器弹出来模拟局部突触活动，通过 3D 双光子显微镜保持聚焦的微量容器。该方法还可用于研究脑神经瘤和胶质活动在休息和刺激三个维度。演示程序将是迈克尔 L

in-vivo-three-dimensional-two-photon-microscopy-to-study-conducted

Universidad de Cartagena UDEC

Research

JoVE Journal

Neuroscience

7.4K 次观看.  University of Copenhagen. 该协议旨在研究由局部弹出ATP引起的脑毛细血管反应缓慢。该技术的优点是通过局部移液器弹出来模拟局部突触活动，通过 3D 双光子显微镜保持聚焦的微量容器。该方法还可用于研究脑神经瘤和胶质活动在休息和刺激三个维度。演示程序将是迈克尔 L

视频: In Vivo Three-Dimensional Two-Photon Microscopy to Study Conducted Vascular Responses by Local ATP Ejection Using a Glass Micro-Pipette

In vivo Micro-circulation Measurement in Skeletal Muscle by Intra-vital Microscopy

BACKGROUND: Regulatory factors and detailed physiology of in vivo microcirculation have remained not fully clarified after many different modalities of imaging had invented. While many macroscopic parameters of blood flow reflect flow velocity, changes in blood flow velocity and red blood cell (RBC) flux does not hold linear relationship in the microscopic observations. There are reports of discrepancy between RBC velocity and RBC flux, RBC flux and plasma flow volume, and of spatial and temporal heterogeneity of flow regulation in the peripheral tissues in microscopic observations, a scientific basis for the requirement of more detailed studies in microcirculatory regulation using intravital microscopy. METHODS: We modified Jeff Lichtman's method of in vivo microscopic observation of mouse sternomastoid muscles. Mice are anesthetized,
ventilated, and injected with PKH26L-fluorescently labeled RBCs for microscopic observation.RESULT &amp; CONCLUSIONS: Fluorescently labeled RBCs are detected and distinguished well by a wide-field microscope. Muscle contraction evoked by electrical stimulation induced increase in RBC flux. Quantification of other parameters including RBC velocity and capillary density were feasible. Mice tolerated well the surgery, injection of stained RBCs, microscopic observation, and electrical stimulation. No muscle or blood vessel damage was observed, suggesting that our method is relatively less invasive and suited for long-term observations.

A new versatile method for observation of microcirculation is presented. It is considered suitable for long-term observation, and for combination with pharmacophysiological or molecular biological interventions.

在体内微循环在骨骼肌内重要的显微镜测量

BACKGROUND: Regulatory factors and detailed physiology of in vivo microcirculation have remained not fully clarified after many different modalities of ...

科研

生物学

In Vivo 2-Photon Calcium Imaging in Layer 2/3 of Mice

To understand network dynamics of microcircuits in the neocortex, it is essential to simultaneously record the activity of a large number of neurons . In-vivo two-photon calcium imaging is the only method that allows one to record the activity of a dense neuronal population with single-cell resolution . The method consists in implanting a cranial imaging window, injecting a fluorescent calcium indicator dye that can be taken up by large numbers of neurons and finally recording the activity of neurons with time lapse calcium imaging using an in-vivo two photon microscope. Co-injection of astrocyte-specific dyes allows one to differentiate neurons from astrocytes. The technique can be performed in mice expressing fluorescent molecules in specific subpopulations of neurons to better understand the network interactions of different groups of cells.

To understand network dynamics of microcircuits in the neocortex, it is essential to simultaneously record the activity of a large number of neurons . In-vivo two-photon calcium imaging is the only method that allows one to record the activity of a dense neuronal population with single-cell resolution .

在体内2光子层钙成像对小鼠的2 / 3

To understand network dynamics of microcircuits in the neocortex, it is essential to simultaneously record the activity of a large number of neurons .  ...

Long-term Intravital Immunofluorescence Imaging of Tissue Matrix Components with Epifluorescence and Two-photon Microscopy

Besides being a physical scaffold to maintain tissue morphology, the extracellular matrix (ECM) is actively involved in regulating cell and tissue function during development and organ homeostasis. It does so by acting via biochemical, biomechanical, and biophysical signaling pathways, such as through the release of bioactive ECM protein fragments, regulating tissue tension, and providing pathways for cell migration. The extracellular matrix of the tumor microenvironment undergoes substantial remodeling, characterized by the degradation, deposition and organization of fibrillar and non-fibrillar matrix proteins. Stromal stiffening of the tumor microenvironment can promote tumor growth and invasion, and cause remodeling of blood and lymphatic vessels. Live imaging of matrix proteins, however, to this point is limited to fibrillar collagens that can be detected by second harmonic generation using multi-photon microscopy, leaving the majority of matrix components largely invisible. Here we describe procedures for tumor inoculation in the thin dorsal ear skin, immunolabeling of extracellular matrix proteins and intravital imaging of the exposed tissue in live mice using epifluorescence and two-photon microscopy. Our intravital imaging method allows for the direct detection of both fibrillar and non-fibrillar matrix proteins in the context of a growing dermal tumor. We show examples of vessel remodeling caused by local matrix contraction. We also found that fibrillar matrix of the tumor detected with the second harmonic generation is spatially distinct from newly deposited matrix components such as tenascin C. We also showed long-term (12 hours) imaging of T-cell interaction with tumor cells and tumor cells migration along the collagen IV of basement membrane. Taken together, this method uniquely allows for the simultaneous detection of tumor cells, their physical microenvironment and the endogenous tissue immune response over time, which may provide important insights into the mechanisms underlying tumor progression and ultimate success or resistance to therapy.

The extracellular matrix undergoes substantial remodeling during wound healing, inflammation and tumorigenesis. We present a novel intravital immunofluorescence microscopy approach to visualize the dynamics of fibrillar as well as mesh-like matrix components with high spatial and temporal resolution using epifluorescence or two-photon microscopy.

与落射荧光组织基质成分和双光子显微镜的长期活体免疫荧光成像

Besides being a physical scaffold to maintain tissue morphology, the extracellular matrix (ECM) is actively involved in regulating cell and tissue ...

生物工程

In vivo Imaging of Tumor Angiogenesis using Fluorescence Confocal Videomicroscopy

Fibered confocal fluorescence in vivo imaging with a fiber optic bundle uses the same principle as fluorescent confocal microscopy. It can excite fluorescent in situ elements through the optical fibers, and then record some of the emitted photons, via the same optical fibers. The light source is a laser that sends the exciting light through an element within the fiber bundle and as it scans over the sample, recreates an image pixel by pixel. As this scan is very fast, by combining it with dedicated image processing software, images in real time with a frequency of 12 frames/sec can be obtained.
We developed a technique to quantitatively characterize capillary morphology and function, using a confocal fluorescence videomicroscopy device. The first step in our experiment was to record 5 sec movies in the four quadrants of the tumor to visualize the capillary network. All movies were processed using software (ImageCell, Mauna Kea Technology, Paris France) that performs an automated segmentation of vessels around a chosen diameter (10 &mu;m in our case). Thus, we could quantify the 'functional capillary density', which is the ratio between the total vessel area and the total area of the image. This parameter was a surrogate marker for microvascular density, usually measured using pathology tools.
The second step was to record movies of the tumor over 20 min to quantify leakage of the macromolecular contrast agent through the capillary wall into the interstitium. By measuring the ratio of signal intensity in the interstitium over that in the vessels, an 'index leakage' was obtained, acting as a surrogate marker for capillary permeability.

In vivo Imaging of Tumor Angiogenesis using Fluorescence Confocal Videomicroscopy

In this paper, we present a method to analyze tumor microvessels in vivo using dynamic contrast-enhanced fluorescence videomicroscopy. Two quantitative parameters were acquired: functional capillary density reflecting the vascularity of the tumor, and index leakage reflecting the leakiness of the endothelial wall.

在肿瘤血管生成中使用荧光共聚焦电视显微镜活体成像

Fibered confocal fluorescence in vivo imaging with a fiber optic bundle uses the same principle as fluorescent confocal microscopy. It can excite ...

医学

Microiontophoresis and Micromanipulation for Intravital Fluorescence Imaging of the Microcirculation

Microiontophoresis entails passage of current through a micropipette tip to deliver a solute at a designated site within an experimental preparation. Microiontophoresis can simulate synaptic transmission1 by delivering neurotransmitters and neuropeptides onto neurons reproducibly2. Negligible volume (fluid) displacement avoids mechanical disturbance to the experimental preparation. Adapting these techniques to the microcirculation3 has enabled mechanisms of vasodilation and vasoconstriction to be studied at the microscopic level in vivo4,5. A key advantage of such localized delivery is enabling vasomotor responses to be studied at defined sites within a microvascular network without evoking systemic or reflexive changes in blood pressure and tissue blood flow, thereby revealing intrinsic properties of microvessels.
A limitation of microiontophoresis is that the precise concentration of agent delivered to the site of interest is difficult to ascertain6. Nevertheless, its release from the micropipette tip is proportional to the intensity and duration of the ejection current2,7, such that reproducible stimulus-response relationships can be readily determined under defined experimental conditions (described below). Additional factors affecting microiontophoretic delivery include solute concentration and its ionization in solution. The internal diameter of the micropipette tip should be &tilde; 1 &mu;m or less to minimize diffusional 'leak', which can be counteracted with a retaining current. Thus an outward (positive) current is used to eject a cation and a negative current used to retain it within the micropipette.
Fabrication of micropipettes is facilitated with sophisticated electronic pullers8. Micropipettes are pulled from glass capillary tubes containing a filament that 'wicks' solution into the tip of the micropipette when filled from the back end ("backfilled"). This is done by inserting a microcapillary tube connected to a syringe containing the solution of interest and ejecting the solution into the lumen of the micropipette. Micromanipulators enable desired placement of micropipettes within the experimental preparation. Micromanipulators mounted on a movable base can be positioned around the preparation according to the topography of microvascular networks (developed below). 
The present protocol demonstrates microiontophoresis of acetylcholine (ACh+ Cl-) onto an arteriole of the mouse cremaster muscle preparation (See associated protocol: JoVE ID#2874) to produce endothelium-dependent vasodilation. Stimulus delivery is synchronized with digitized image acquisition using an electronic trigger. The use of Cx40BAC-GCaMP2 transgenic mice9 enables visualization of intracellular calcium responses underlying vasodilation in arteriolar endothelial cells in the living microcirculation.

Microiontophoresis entails movement of ions from a micropipette in response to a difference in electrical potential between the inside and outside of the micropipette. Biologically active molecules are thereby delivered in proportion to electrical current. We illustrate acetylcholine microiontophoresis in conjunction with micromanipulation to study endothelium-dependent vasodilation in the microcirculation.

Microiontophoresis和显微操作的微循环活体荧光成像

Microiontophoresis entails passage of current through a micropipette tip to deliver a solute at a designated site within an experimental  preparation. ...

Extended Time-lapse Intravital Imaging of Real-time Multicellular Dynamics in the Tumor Microenvironment

In the tumor microenvironment, host stromal cells interact with tumor cells to promote tumor progression, angiogenesis, tumor cell dissemination and metastasis. Multicellular interactions in the tumor microenvironment can lead to transient events including directional tumor cell motility and vascular permeability. Quantification of tumor vascular permeability has frequently used end-point experiments to measure extravasation of vascular dyes. However, due to the transient nature of multicellular interactions and vascular permeability, the kinetics of these dynamic events cannot be discerned. By labeling cells and vasculature with injectable dyes or fluorescent proteins, high-resolution time-lapse intravital microscopy has allowed the direct, real-time visualization of transient events in the tumor microenvironment. Here we describe a method for using multiphoton microscopy to perform extended intravital imaging in live mice to directly visualize multicellular dynamics in the tumor microenvironment. This method details cellular labeling strategies, the surgical preparation of a mammary skin flap, the administration of injectable dyes or proteins by tail vein catheter and the acquisition of time-lapse images. The time-lapse sequences obtained from this method facilitate the visualization and quantitation of the kinetics of cellular events of motility and vascular permeability in the tumor microenvironment.

This protocol describes the use of multiphoton microscopy to perform extended time-lapse imaging of multicellular interactions in real time, in vivo at single cell resolution.

实时多细胞动力学延长时间推移活体成像在肿瘤微环境

In the tumor microenvironment, host stromal cells interact with tumor cells to promote tumor progression, angiogenesis, tumor cell dissemination and ...

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.

应用先进的背皮窗室对转基因荧光小鼠肿瘤相关血管的活体显微镜观察

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

Brain Pericyte Calcium and Hemodynamic Imaging in Transgenic Mice In Vivo

Recent advances in protein biology and mouse genetics have made it possible to measure intracellular calcium fluctuations of brain cells in vivo and to correlate this with local hemodynamics. This protocol uses transgenic mice that have been prepared with a chronic cranial window and express the genetically encoded calcium indicator, RCaMP1.07, under the &#945;-smooth muscle actin promoter to specifically label mural cells, such as vascular smooth muscle cells and ensheathing pericytes. Steps are outlined on how to prepare a tail vein catheter for intravenous injection of fluorescent dyes to trace blood flow, as well as how to measure brain pericyte calcium and local blood vessel hemodynamics (diameter, red blood cell velocity, etc.) by two photon microscopy in vivo through the cranial window in ketamine/xylazine anesthetized mice. Finally, details are provided for the analysis of calcium fluctuations and blood flow movies via the image processing algorithms developed by Barrett et al. 2018, with an emphasis on how these processes can be adapted to other cellular imaging data.

Brain Pericyte Calcium and Hemodynamic Imaging in Transgenic Mice In Vivo

This protocol presents steps to acquire and analyze fluorescent calcium images from brain ensheathing pericytes and blood flow data from nearby blood vessels in anesthetized mice. These techniques are useful for studies of mural cell physiology and can be adapted to investigate calcium transients in any cell type.

活体转基因小鼠脑围产层钙和血液学成像

Recent advances in protein biology and mouse genetics have made it possible to measure intracellular calcium fluctuations of brain cells in vivo and to ...

神经科学

Two-photon Imaging of Intracellular Ca2+ Handling and Nitric Oxide Production in Endothelial and Smooth Muscle Cells of an Isolated Rat Aorta

Calcium is a very important regulator of many physiological processes in vascular tissues. Most endothelial and smooth muscle functions highly depend on changes in intracellular calcium ([Ca2+]i) and nitric oxide (NO). In order to understand how [Ca2+]i, NO and downstream molecules are handled by a blood vessel in response to vasoconstrictors and vasodilators, we developed a novel technique that applies calcium-labeling (or NO-labeling) dyes with two photon microscopy to measure calcium handling (or NO production) in isolated blood vessels. Described here is a detailed step-by-step procedure that demonstrates how to isolate an aorta from a rat, label calcium or NO within the endothelial or smooth muscle cells, and image calcium transients (or NO production) using a two photon microscope following physiological or pharmacological stimuli. The benefits of using the method are multi-fold: 1) it is possible to simultaneously measure calcium transients in both endothelial cells and smooth muscle cells in response to different stimuli; 2) it allows one to image endothelial cells and smooth muscle cells in their native setting; 3) this method is very sensitive to intracellular calcium or NO changes and generates high resolution images for precise measurements; and 4) described approach can be applied to the measurement of other molecules, such as reactive oxygen species. In summary, application of two photon laser emission microscopy to monitor calcium transients and NO production in the endothelial and smooth muscle cells of an isolated blood vessel has provided high quality quantitative data and promoted our understanding of the mechanisms regulating vascular function.

Two-photon Imaging of Intracellular Ca2+ Handling and Nitric Oxide Production in Endothelial and Smooth Muscle Cells of an Isolated Rat Aorta

Vascular cell functiondepends on activity of intracellular messengers. Described here is an ex vivo two photon imaging method that allows the measurement of intracellular calcium and nitric oxide levels in response to physiological and pharmacological stimuli in individual endothelial and smooth muscle cells of an isolated aorta.

细胞内钙的双光子成像<sup&gt; 2+</sup&gt;的离体大鼠主动脉的处理和一氧化氮的内皮细胞和平滑肌细胞

Calcium is a very important regulator of many physiological processes in vascular tissues. Most endothelial and smooth muscle functions highly depend on ...

Multiphoton Intravital Imaging for Monitoring Leukocyte Recruitment during Arteriogenesis in a Murine Hindlimb Model

Arteriogenesis strongly depends on leukocyte and platelet recruitment to the perivascular space of growing collateral vessels. The standard approach for analyzing collateral arteries and leukocytes in arteriogenesis is ex vivo (immuno-) histological methodology. However, this technique does not allow the measurement of dynamic processes such as blood flow, shear stress, cell-cell interactions, and particle velocity. This paper presents a protocol to monitor in vivo processes in growing collateral arteries during arteriogenesis utilizing intravital imaging. The method described here is a reliable tool for dynamics measurement and offers a high-contrast analysis with minimal photo-cytotoxicity, provided by multiphoton excitation microscopy. Prior to analyzing growing collateral arteries, arteriogenesis was induced in the adductor muscle of mice by unilateral ligation of the femoral artery.
After the ligation, the preexisting collateral arteries started to grow due to increased shear stress. Twenty-four hours after surgery, the skin and subcutaneous fat above the collateral arteries were removed, constructing a pocket for further analyses. To visualize blood flow and immune cells during in vivo imaging, CD41-fluorescein isothiocyanate (FITC) (platelets) and CD45-phycoerythrin (PE) (leukocytes) antibodies were injected intravenously (i.v.) via a catheter placed in the tail vein of a mouse. This article introduces intravital multiphoton imaging as an alternative or in vivo complementation to the commonly used static ex vivo (immuno-) histological analyses to study processes relevant for arteriogenesis. In summary, this paper describes a novel and dynamic in vivo method to investigate immune cell trafficking, blood flow, and shear stress in a hindlimb model of arteriogenesis, which enhances evaluation possibilities notably.

The recruitment of leukocytes and platelets constitutes an essential component necessary for the effective growth of collateral arteries during arteriogenesis. Multiphoton microscopy is an efficient tool for tracking cell dynamics with high spatio-temporal resolution in vivo and less photo-toxicity to study leukocyte recruitment and extravasation during arteriogenesis.

多光子活体成像用于监测小鼠后肢模型中动脉生成过程中白细胞募集

Arteriogenesis strongly depends on leukocyte and platelet recruitment to the perivascular space of growing collateral vessels. The standard approach for ...