这项工作由瑞士国家基金会 (SNF) 项目赠款31003A_135649、31003A_153457 和 31003A_172994 (JVS) 提供资金, Leopoldina 2011-16 (至 BS)。这项工作得益于在伯尔尼大学的 "显微成像中心" (MIC) 的光学设置。

所有动物的工作都已得到州动物试验委员会的批准, 并根据联邦准则进行。
1. 麻醉鼠标
<ol><li>佩戴个人防护用品, 包括实验室外套和手套。</li>
	<li>将氯胺酮、甲苯噻嗪和生理盐水混合在一起, 其工作浓度分别为20毫克/毫升和1毫克/毫升。将工作库存腹腔 (ip) 注入8-10 µL/克鼠标。把鼠标放回笼子里。 注: 本协议已测试6至40周的男性和女性小鼠的 C57BL/6 背景。</li>
	<li>准备乙酰丙嗪溶液在2.5 毫克/毫升, 并注入30µL, 15 分钟后, 氯胺酮-甲苯噻嗪注射 (75 µg/小鼠)。</li>
	<li>5-15 分钟后, 检查是否有足够的麻醉和镇痛通过控制反射,例如, 撤回反射的爪子或角膜反射。</li>
	<li>控制的反射大约每30分钟在整个实验期间。皮下注射氯胺酮/甲苯噻嗪在1.5 µL/克鼠, 每60分钟或更早, 如果反射是阳性的。 注意: 由于老鼠的固定, 吸入性麻醉 (如异氟醚) 不能与此协议一起使用。</li>
</ol>2. 从操作区取出毛皮
<ol><li>使用电动剃刀刮胡子的区域, 大致从5毫米的嘴尾向前腿, 伸展在颌骨对两个耳朵。</li>
	<li>用棉签将脱毛膏涂抹在剃光区。经过3分钟的仔细和彻底清除,例如, 用湿纸巾擦拭。</li>
</ol>3. 将鼠标固定在舞台上
注: 本协议使用自定义阶段 (图 1), 配备两个可自由调节的盖子滑动架 (一个附在舞台上的螺钉, 另一个固定在它上);可调节的立体定向耳棒;可以用螺钉拧紧的绳子;可自由调节的金属加热环;和一个凸起的区域, 指定放置鼠标的躯干。这一阶段是从腘淋巴结阶段修改, 增加了立体定向支架和支持下盖滑动持有形象化 SMG。本阶段的详细计划或报价可根据要求提供。
<ol><li>通过拆卸加热环和可拆卸盖滑动架的支架来准备舞台。拧松耳棒支架和固定护盖滑动架的螺钉。把一块纱布贴到舞台的中心区域, 充当老鼠的床上用品。</li>
	<li>使用胶水 (请参阅材料表) 将20毫米直径的盖玻璃连接到圆柱形金属支架的顶部 (这将是下盖玻璃)。将25毫米的盖子玻璃粘附在扁金属架的底部 (这将是上盖玻璃)。确保盖眼镜与金属持有者重叠约2-3 毫米, 其余的玻璃仍然没有油脂和胶水。</li>
	<li>将鼠标放回纱布上, 垂直于伸展的绳子, 头部居中在耳棒之间。</li>
	<li>用手术胶带将鼠标的上肢固定在舞台上。</li>
	<li>接下来, 把绳子钩到前门牙上, 收紧, 直到下巴达到水平位置。使用一个有角的弯曲钳稍微拉出舌头从嘴, 这有助于呼吸。</li>
	<li>紧紧的, 但不是有力的, 拉尾巴和磁带到舞台上。</li>
	<li>将耳条对齐, 以在条形和舞台边缘之间达到近似的30°角。为了确保这个角度, 使用三角形标尺在一张纸上绘制一个右和左的30°角, 并将透明的舞台放在纸张上以相应地对齐。</li>
	<li>通过同时将左右杆向内移动, 直到下巴 immotile, 拧紧螺钉, 固定耳条之间的颚部。在固定下颚之前、期间和之后观察胸部运动。呼吸减少或缺席表明, 酒吧是固定在颈部, 而不是颚骨;在这种情况下, 请立即重复安装过程。</li>
	<li>给鼠标加热,例如, 在舞台下面放置一盏暖气灯和/或加热垫。</li>
</ol>4. 用显微镜手术暴露涎腺
注意: 以下步骤需要使用显微镜、细钳和手术剪刀。由于过程是终端, 没有必要保持无菌的操作条件。乙醇清洗仪器足够了。
<ol><li>找到右侧的 SMG 叶的近似位置, 寻找一个轻微的突起在皮肤大约15毫米尾骶部和5毫米内侧的颚线。抬起一个小的皮肤褶皱的中心隆起和削减它创造和开放约5毫米长度。</li>
	<li>立即在伤口周围的皮肤上涂上4毫米高的真空油脂环。(使用真空油脂填充注射器与钝25克皮下针)。</li>
	<li>用盐水填充油脂环, 以确保组织在手术过程中保持湿润。</li>
	<li>在显微镜下, 使用以下程序扰乱结缔组织: 用一个细钳牢牢地握住直接附着在 SMG 上的结缔组织, 然后用第二个钳把远端结缔组织拉到破裂。确保某些结缔组织仍然附着在 SMG 上;这是可取的, 因为它允许处理。不要用镊子触摸腺体本身, 因为这会导致出血。</li>
	<li>重复这种扰乱结缔组织的运动首先在腺体的顶端, 然后在内侧 (在 SMG 的右和左叶之间)。请勿将舌下与颌下腺分开。</li>
	<li>继续逆时针移动外侧结缔组织 (内侧至尾部, 到远端, 延髓)。</li>
	<li>确保血液供应, 传出淋巴管和涎腺导管, 这些都位于腺体的颅骨部位的捆绑, 没有破裂。</li>
	<li>一旦外侧结缔组织被破坏, 使用镊子把连接到 smg 尾端的结缔组织向上提起, 以解除 smg 的尾端。从尾部开始, 并向延髓端移动, 扰乱了 SMG 叶下方的结缔组织。</li>
	<li>继续, 直到所有的连接组织的所有地点的 SMG 被删除 (不包括延髓网站与主要喂养/排泄血管和 WD)。</li>
</ol>5. 固定腺体
<ol><li>确认含生理盐水的润滑脂环至少有4毫米高且未泄漏 (见步骤 4.2)。</li>
	<li>安装下盖玻璃 (见步骤 3.2), 通过附加金属棒到其高度可调的持有人。确保盖板滑动架调整到最高位置, 并在连接到舞台时不接触润滑脂环。将持有者从尾部方向定位到小鼠的角度, 其盖子滑动覆盖了露出的裂片的大约两个 (尾部和远端部分)。把盖子玻璃放低, 直到它与腺体接触。</li>
	<li>完成盖玻璃顶部的润滑脂环, 形成新的盐水库。</li>
	<li>小心地拉上的 SMG 瓣顶部的盖子滑与镊子, 只接触连接到它的结缔组织 (器官可以翻转与底部的网站面对向上)。</li>
	<li>确保润滑脂环的均匀高度至少为4毫米, 不泄漏。如有必要, 用30克填充生理盐水的注射器重新填充盐水水库。</li>
	<li>确保上盖玻璃的盖玻璃架处于其最高位置。使用螺钉将金属支架与 25 mm 上盖玻璃连接在支架上。使用可调节螺丝的持有人定位的封面玻璃, 使 SMG 在中间居中。</li>
	<li>使用可调支架螺钉, 将顶盖玻璃降低至直接与 SMG 接触。在此步骤中观察腺体的形状和着色。如果腺体表面积较大, 或者着色改变 (例如从粉红色到白色), 立即将盖玻璃置于更高的位置, 以减轻 SMG 的压力。</li>
	<li>在水库外寻找气泡和盐水, 检查盐水水库是否漏水。如果检测到泄漏或气泡, 请卸下盖板和所有油脂, 然后从步骤2重新启动。</li>
	<li>螺丝关闭所有固定螺钉。</li>
</ol>6. 固定加热环和温度探头
<ol><li>将温度探头插入储层并将其放置在靠近腺体的位置, 然后将探头导线贴到舞台上。</li>
	<li>将润滑脂环应用到顶盖滑动上, 与在中心的 SMG (润滑脂环的直径应对应于加热环的直径)。将加热环放在盖子滑动顶部, 用油脂密封。将加热环的油管胶带放到舞台上。</li>
</ol>7. 成像
注: 我们使用直立双光子显微镜系统, 配备可调谐 Ti: Sa 激光器和荧光显微镜与水浸泡20X 或25X 目标。通过使用自动阶段和 VivoFollow19, 可以实现在采集过程中对组织漂移进行实时校正。这提高了图像采集的稳定性, 但不是必需的。
<ol><li>使用显微镜制造商提供的适当软件打开直立显微镜系统。将 Ti: Sa 激光器调至 780-900 nm 的激发波长。</li>
	<li>将加热管连接至加热环。蠕动式水泵循环热水 (通过在80°c 水浴中浸没部分油管加热) 通过环来温暖 SMG。</li>
	<li>将温度探头连接到数字温度计。如果温度不介于摄氏35-39 摄氏度之间, 则调整蠕动泵的速度。泵的工作速度越快, 对象得到的温度越热, 反之亦然。</li>
	<li>在开始图像采集之前, 对血液流速进行视觉控制。仔细检查白细胞通过薄毛细血管或荧光葡聚糖注射液。 注: 静脉注射的荧光葡聚糖在血液流动是生理的时候, 会立即填满整个 SMG 的血管。这在这个过程中几乎总是如此。</li>
	<li>为了遵循免疫细胞, 成像深度通常介于20-150 µm 的 2nd谐波生成信号所识别的 SMG 表面之下。记录图像栈150-300 µm 的视野, 像素分辨率约 512 x 512 或更高, z 深度20-60 µm 与间距2-4 µm 之间的图像。每20-30 秒重复一次堆栈捕获, 总工期为20-60 分钟。</li>
	<li>使用数字温度计控制在获得一个图像序列后的最低和最高温度水平。如果最小或最大值不在37到2°c 的范围内, 则丢弃图像序列。在图像序列之间重新填充水以实现目标的浸入。</li>
	<li>在成像会话结束时, 弄死在麻醉下通过 CO2窒息老鼠。 注: 这是根据瑞士联邦动物实验指南;其他有关安乐死的准则可能适用于其他地方。</li>
</ol>

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F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stabilized+imaging+of+immune+surveillance+in+the+mouse+lung.">Stabilized imaging of immune surveillance in the mouse lung.</a> Nat Methods. 8 (8), 91-96 (2011).</li><li>Liou, H. L. R., Myers, J. T., Barkauskas, D. S., Huang, A. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intravital+imaging+of+the+mouse+popliteal+lymph+node.">Intravital imaging of the mouse popliteal lymph node.</a> J Vis Exp. (60), e3720 (2012).</li><li>Sramkova, M., Masedunskas, A., Parente, L., Molinolo, A., Weigert, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+plasmid+DNA+in+the+salivary+gland+epithelium:+novel+approaches+to+study+dynamic+cellular+processes+in+live+animals.">Expression of plasmid DNA in the salivary gland epithelium: novel approaches to study dynamic cellular processes in live animals.</a> Am J Physiol Cell Physiol. 297 (6), C1347-C1357 (2009).</li><li>Masedunskas, A., Porat-shliom, N., Tora, M., Milberg, O., Weigert, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intravital+microscopy+for+imaging+subcellular+structures+in+live+mice+expressing+fluorescent+proteins.">Intravital microscopy for imaging subcellular structures in live mice expressing fluorescent proteins.</a> J Vis Exp. (79), e50558 (2013).</li><li>Masedunskas, A., Sramkova, M., Parente, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+for+the+actomyosin+complex+in+regulated+exocytosis+revealed+by+intravital+microscopy.">Role for the actomyosin complex in regulated exocytosis revealed by intravital microscopy.</a> Proc Natl Acad Sci. 108 (33), 13552-13557 (2011).</li><li>Milberg, O., Shitara, A., Ebrahim, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Concerted+actions+of+distinct+nonmuscle+myosin+II+isoforms+drive+intracellular+membrane+remodeling+in+live+animals.">Concerted actions of distinct nonmuscle myosin II isoforms drive intracellular membrane remodeling in live animals.</a> J Cell Biol. 216 (7), 1925-1936 (2017).</li><li>Kuriki, Y., Liu, Y., Xia, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cannulation+of+the+mouse+submandibular+salivary+gland+via+the+Wharton's+duct.">Cannulation of the mouse submandibular salivary gland via the Wharton's duct.</a> J Vis Exp. (51), e3074 (2011).</li><li>Chen, G. Y., Nu&#241;ez, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sterile+inflammation:+Sensing+and+reacting+to+damage.">Sterile inflammation: Sensing and reacting to damage.</a> Nat Rev Immunol. 10 (12), 826-837 (2010).</li><li>McLaren, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Some+causes+of+variation+of+body+temperature+in+mice.">Some causes of variation of body temperature in mice.</a> Q J Exp Physiol Cogn Med Sci. 46 (1), 38-45 (1961).</li><li>Baumgart, K., Wagner, F., Gr&#246;ger, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiac+and+metabolic+effects+of+hypothermia+and+inhaled+hydrogen+sulfide+in+anesthetized+and+ventilated+mice.">Cardiac and metabolic effects of hypothermia and inhaled hydrogen sulfide in anesthetized and ventilated mice.</a> Crit Care Med. 38 (2), 588-595 (2010).</li><li>Crouch, A. C., Manders, A. B., Cao, A. A., Scheven, U. M., Greve, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cross-sectional+area+of+the+murine+aorta+linearly+increases+with+increasing+core+body+temperature.">Cross-sectional area of the murine aorta linearly increases with increasing core body temperature.</a> Int J Hyperth. , 1-13 (2017).</li><li>Smith, C. J., Caldeira-Dantas, S., Turula, H., Snyder, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Murine+CMV+infection+induces+the+continuous+production+of+mucosal+resident+T+cells.">Murine CMV infection induces the continuous production of mucosal resident T cells.</a> Cell Rep. 13 (6), 1137-1148 (2015).</li><li>Lindquist, R. L., Shakhar, G., Dudziak, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualizing+dendritic+cell+networks+in+vivo.">Visualizing dendritic cell networks in vivo.</a> Nat Immunol. 5 (12), 1243-1250 (2004).</li><li>Riedl, J., Flynn, K. C., Raducanu, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lifeact+mice+for+studying+F-actin+dynamics.">Lifeact mice for studying F-actin dynamics.</a> Nat Methods. 7 (3), 168-169 (2010).</li><li>Chtanova, T., Hampton, H. R., Waterhouse, L. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Real-time+interactive+two-photon+photoconversion+of+recirculating+lymphocytes+for+discontinuous+cell+tracking+in+live+adult+mice.">Real-time interactive two-photon photoconversion of recirculating lymphocytes for discontinuous cell tracking in live adult mice.</a> J Biophotonics. 7 (6), 425-433 (2014).</li><li>Kyratsous, N. I., Bauer, I. J., Zhang, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualizing+context-dependent+calcium+signaling+in+encephalitogenic+T+cells+in+vivo+by+two-photon+microscopy.">Visualizing context-dependent calcium signaling in encephalitogenic T cells in vivo by two-photon microscopy.</a> Proc Natl Acad Sci U S A. 114 (31), E6381-E6389 (2017).</li><li>Mank, M., Reiff, D. F., Heim, N., Friedrich, M. W., Borst, A., Griesbeck, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+FRET-based+calcium+biosensor+with+fast+signal+kinetics+and+high+fluorescence+change.">A FRET-based calcium biosensor with fast signal kinetics and high fluorescence change.</a> Biophys J. 90 (5), 1790-1796 (2006).</li><li>Tsyboulski, D., Orlova, N., Saggau, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amplitude+modulation+of+femtosecond+laser+pulses+in+the+megahertz+range+for+frequency-multiplexed+two-photon+imaging.">Amplitude modulation of femtosecond laser pulses in the megahertz range for frequency-multiplexed two-photon imaging.</a> Opt Express. 25 (8), 9435 (2017).</li><li>Potma, E. O., Xie, X. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+single+lipid+bilayers+with+coherent+anti-Stokes+Raman+scattering+(CARS)+microscopy.">Detection of single lipid bilayers with coherent anti-Stokes Raman scattering (CARS) microscopy.</a> J Raman Spectrosc. 34 (9), 642-650 (2003).</li></ol>

没有宣布利益冲突。

该协议提供了一个简单的方法,在体内成像的小鼠颌下腺和舌下涎腺使用直立非线性显微镜经常使用的免疫学领域。该方法可用于头颈区其他外分泌腺体的成像。例如, 我们的实验室以类似的方式对泪腺进行了成像 (不显示)。
除去在 SMG 周围的结缔组织是这个协议最关键的步骤, 因为意外的组织损伤可能发生。由于 smg 的组织会变得缺氧, 因此对 smg 的主要血液供应造成的?...

唾液由外分泌腺体分泌来润滑食物, 保护口腔粘膜表面, 并提供消化酶和抗菌物质1,2。除了在口腔黏膜下层散置的小涎腺外, 根据其位置1、2, 还有三个双侧的主要腺体被确定为腮腺、舌下和颌下腺。金字塔状上皮细胞, 组织成烧瓶状囊 (腺泡细胞) 或 demilunes 包围的上皮细胞和基底膜, 分泌的浆液和粘液成分的唾液1。腺泡细胞狭窄的腔内空间排水成夹层导管, 直到它们最终加入一个排泄管道1。SMG 的主要排泄管称为沃顿商学院的管道 (WD), 并打开到舌下阜3,4。因此, SMG 上皮隔间是一个高度 arborized 的结构, 具有流形终端端点, 类似于一束葡萄1,5,6。SMG 间质由血液和淋巴管组成, 内含于结缔组织7中, 内含副交感神经8和细胞外基质 5. 正常的人和啮齿动物的涎腺也含有 T 细胞, 巨噬细胞和树突状干细胞9, 以及分泌免疫球蛋白 A (IgA) 到唾液9,10的血浆细胞。由于其在健康和疾病方面的多方面作用, SMG 在许多生物研究领域都有兴趣, 包括牙科4、免疫学11、肿瘤学12、生理学8和细胞生物学3。
动态细胞过程和交互的成像是生物学研究中的一个强有力的工具13,14。基于非线性光学 (NLO) 的深部组织成像和创新 inmicroscopes 的发展, 它依赖于通过样本对多个光子的散射或吸收, 从而可以直接检查复杂组织中的细胞过程13 ,15。多光子的吸收涉及用低能光子传递总励磁能量, 这限制了荧光对焦平面的激发, 从而允许更深层的组织穿透, 减少了光损伤和噪声。励磁13,15。这一原则采用双光子显微镜 (2PM), 并允许在高达1毫米15,16深度的荧光标本上进行成像。虽然商用2PM 设置已变得方便和可靠, 活体成像的主要挑战是仔细暴露和稳定麻醉小鼠的靶器官, 特别是对时间失效序列的成像。数据获取后数字漂移校正的几种方法已发布17,18和我们最近开发了 "VivoFollow", 一个自动校正系统, 它抵消缓慢的组织漂移实时使用计算机化阶段19。然而, 对于高质量的成像, 要尽量减少组织运动, 尤其是呼吸或心跳导致的快速运动, 这仍然是至关重要的19。为多个器官 (包括脊髓20、肝脏21、皮肤22、肺23和淋巴结24) 发布了准备和稳定程序。此外, 大鼠涎腺成像模型已经开发了3,25和进一步完善的高分辨率活体成像的小鼠 SMG 量身定做的倒置显微镜设置26,27,28。
在这里, 我们提出了一个实用的和适应性的协议, 活体成像的小鼠的直立非线性显微镜, 这是普遍用于活体成像在免疫学领域。为此, 我们改良了一个广泛使用的固定化阶段用于腘淋巴结准备。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Narketan 10 %&#160; (Ketamine) 20ml (100 mg/ml)</td>
			<td>Vetoquinol</td>
			<td>3605877535982</td>
			<td></td>
		</tr>
		<tr>
			<td>Rompun 2% (Xylazine) 25 ml (20 mg/ml)</td>
			<td>Bayer</td>
			<td>680538150144</td>
			<td></td>
		</tr>
		<tr>
			<td>Saline NaCl 0.9%</td>
			<td>B. Braun</td>
			<td>3535789</td>
			<td></td>
		</tr>
		<tr>
			<td>Prequillan 1% (Acepromazine) 10 ml (10 mg/ml)</td>
			<td>Fatro</td>
			<td>6805671900029</td>
			<td></td>
		</tr>
		<tr>
			<td>Electric shaver</td>
			<td>Wahl</td>
			<td>9818L</td>
			<td>or similar</td>
		</tr>
		<tr>
			<td>Hair removal cream</td>
			<td>Veet</td>
			<td>4002448090656</td>
			<td></td>
		</tr>
		<tr>
			<td>Durapore Surgical tape (2.5 cm x 9.1 m and 1.25 cm x 9.1 m)</td>
			<td>Durapore (3M)</td>
			<td>1538-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Durapore Surgical tape (2.5 cm x 9.1 m and 1.25 cm x 9.1 m)</td>
			<td>Durapore (3M)</td>
			<td>1538-0</td>
			<td></td>
		</tr>
		<tr>
			<td>Super glue Ultra gel, instantaneous glue</td>
			<td>Pattex, Henkel</td>
			<td>4015000415040</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope cover glass slides 20 mm and 22 mm</td>
			<td>Menzel-Gl&#228;ser</td>
			<td>631-1343/ 631-1344</td>
			<td></td>
		</tr>
		<tr>
			<td>Grease for laboratories 60 g glisseal N</td>
			<td>Borer (VWR supplier)</td>
			<td>DECO514215.00-CA15</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical scissors</td>
			<td>Fine Science Tools (F.S.T )</td>
			<td>14090-09</td>
			<td>or similar</td>
		</tr>
		<tr>
			<td>Fine Forceps</td>
			<td>Fine Science Tools (F.S.T )</td>
			<td>11252-20</td>
			<td>or similar</td>
		</tr>
		<tr>
			<td>Cotton swab</td>
			<td>Migros</td>
			<td>617027988254</td>
			<td>or similar</td>
		</tr>
		<tr>
			<td>Gauze Gazin 5 x 5 cm</td>
			<td>Lohmann and Rauscher</td>
			<td>18500</td>
			<td>or similar</td>
		</tr>
		<tr>
			<td>Stereomicroscope</td>
			<td>Leica</td>
			<td>MZ16</td>
			<td>or similar</td>
		</tr>
		<tr>
			<td>Texas Red dextran 70kDa</td>
			<td>&#160;Molecular Probes</td>
			<td>D1864</td>
			<td></td>
		</tr>
		<tr>
			<td>Cascade Blue dextran 10kDa</td>
			<td>invitrogen</td>
			<td>D1976</td>
			<td></td>
		</tr>
		<tr>
			<td>Two-photon system</td>
			<td>LaVision Biotec</td>
			<td>TrimScope I and II</td>
			<td>or similar</td>
		</tr>
		<tr>
			<td>XLUMPLANFL 20x/0.95 W objective</td>
			<td>Olympus</td>
			<td>n/a</td>
			<td>or other water immersion objective&#160;</td>
		</tr>
		<tr>
			<td>Digital thermometer</td>
			<td>Fluke</td>
			<td>95969077651</td>
			<td></td>
		</tr>
	</tbody>
</table>

preparation of murine submandibular salivary gland for upright intravital microscopy

颌下腺 (SMG) 是三大涎腺之一, 对许多不同领域的生物研究, 包括细胞生物学, 肿瘤学, 牙科和免疫学感兴趣。SMG 是由分泌上皮细胞、肌、内皮细胞、神经和细胞外基质组成的分泌腺。大鼠和小鼠的动态细胞过程以前已经被成像, 主要是使用倒置的多光子显微镜系统。在这里, 我们描述了一个简单的协议, 以手术准备和稳定麻醉小鼠在体内的在活体成像与直立多光子显微镜系统。我们提出了具有代表性的活体图像集内源性和过继转输转移荧光细胞, 包括标签的血管或唾液导管和第二次谐波生成, 以可视化纤维胶原。总之, 我们的协议允许手术制备小鼠涎腺在直立显微镜系统, 这是通常用于活体成像在免疫学领域。

该协议允许成像几乎所有的背部或腹侧的 SMG。视野通常也包括舌下涎腺, 这与在细胞成分中的 SMG 略有不同4。两个腺体由纤维胶原蛋白封装, 并细分成裂片。大多数2PM 系统可以通过测量 2nd谐波信号来产生纤维胶原的无标签图像, 但通常需要荧光分子来可视化细胞和亚型结构。例如, 绿色荧光蛋白 (GFP) 的无处不在的转基因表达与 2nd谐波信?...

Watch this Scientific Journal Video about Preparation of Murine Submandibular Salivary Gland for Upright Intravital Microscopy at JoVE.com

Preparation of Murine Submandibular Salivary Gland for Upright Intravital Microscopy

直立活体显微术制备小鼠颌下腺涎腺

我们描述了一个协议, 以手术揭露和稳定的小鼠颌下腺腺活体成像使用直立活体显微镜。该协议很容易适应小鼠和其他小啮齿目动物头部和颈部区域外分泌腺体。

The submandibular salivary gland (SMG) is one of the three major salivary glands, and is of interest for many different fields of biological research, ...

preparation-murine-submandibular-salivary-gland-for-upright

Research

JoVE Journal

Immunology and Infection

8.0K 次观看.  University of Bern. 我们描述了一个协议, 以手术揭露和稳定的小鼠颌下腺腺活体成像使用直立活体显微镜。该协议很容易适应小鼠和其他小啮齿目动物头部和颈部区域外分泌腺体。

视频: Preparation of Murine Submandibular Salivary Gland for Upright Intravital Microscopy

Intravital Microscopy of the Inguinal Lymph Node

Lymph nodes (LN's), located throughout the body, are an integral component of the immune system. They serve as a site for induction of adaptive immune response and therefore, the development of effector cells. As such, LNs are key to fighting invading pathogens and maintaining health. The choice of LN to study is dictated by accessibility and the desired model; the inguinal lymph node is well situated and easily supports studies of biologically relevant models of skin and genital mucosal infection.
The inguinal LN, like all LNs, has an extensive microvascular network supplying it with blood. In general, this microvascular network includes the main feed arteriole of the LN that subsequently branches and feeds high endothelial venules (HEVs). HEVs are specialized for facilitating the trafficking of immune cells into the LN during both homeostasis and infection. How HEVs regulate trafficking into the LN under both of these circumstances is an area of intense exploration. The LN feed arteriole, has direct upstream influence on the HEVs and is the main supply of nutrients and cell rich blood into the LN. Furthermore, changes in the feed arteriole are implicated in facilitating induction of adaptive immune response. The LN microvasculature has obvious importance in maintaining an optimal blood supply to the LN and regulating immune cell influx into the LN, which are crucial elements in proper LN function and subsequently immune response. 
The ability to study the LN microvasculature in vivo is key to elucidating how the immune system and the microvasculature interact and influence one another within the LN. Here, we present a method for in vivo imaging of the inguinal lymph node. We focus on imaging of the microvasculature of the LN, paying particular attention to methods that ensure the study of healthy vessels, the ability to maintain imaging of viable vessels over a number of hours, and quantification of vessel magnitude. Methods for perfusion of the microvasculature with vasoactive drugs as well as the potential to trace and quantify cellular traffic are also presented. 
Intravital microscopy of the inguinal LN allows direct evaluation of microvascular functionality and real-time interface of the direct interface between immune cells, the LN, and the microcirculation. This technique potential to be combined with many immunological techniques and fluorescent cell labelling as well as manipulated to study vasculature of other LNs.

A technique for performing intravital microscopy of the inguinal lymph node (LN) is outlined. Such technique allows for real-time, in vivo study of the lymph node microvasculature and structure both during homeostasis and infection. This technique can be adapted to cell trafficking studies and to other lymph node sites.

腹股沟淋巴结的活体显微镜

Lymph nodes (LN's), located throughout the body, are an integral component of the immune system. They serve as a site for induction of adaptive immune ...

科研

免疫与感染

Tracking Neutrophil Intraluminal Crawling, Transendothelial Migration and Chemotaxis in Tissue by Intravital Video Microscopy

The recruitment of circulating leukocytes from blood stream to the inflamed tissue is a crucial and complex process of inflammation1,2. In the postcapillary venules of inflamed tissue, leukocytes initially tether and roll on the luminal surface of venular wall. Rolling leukocytes arrest on endothelium and undergo firm adhesion in response to chemokine or other chemoattractants on the venular surface. Many adherent leukocytes relocate from the initial site of adhesion to the junctional extravasation site in endothelium, a process termed intraluminal crawling3. Following crawling, leukocytes move across endothelium (transmigration) and migrate in extravascular tissue toward the source of chemoattractant (chemotaxis)4. Intravital microscopy is a powerful tool for visualizing leukocyte-endothelial cell interactions in vivo and revealing cellular and molecular mechanisms of leukocyte recruitment2,5. In this report, we provide a comprehensive description of using brightfield intravital microscopy to visualize and determine the detailed processes of neutrophil recruitment in mouse cremaster muscle in response to the gradient of a neutrophil chemoattractant. To induce neutrophil recruitment, a small piece of agarose gel (~1-mm3 size) containing neutrophil chemoattractant MIP-2 (CXCL2, a CXC chemokine) or WKYMVm (Trp-Lys-Tyr-Val-D-Met, a synthetic analog of bacterial peptide) is placed on the muscle tissue adjacent to the observed postcapillary venule. With time-lapsed video photography and computer software ImageJ, neutrophil intraluminal crawling on endothelium, neutrophil transendothelial migration and the migration and chemotaxis in tissue are visualized and tracked. This protocol allows reliable and quantitative analysis of many neutrophil recruitment parameters such as intraluminal crawling velocity, transmigration time, detachment time, migration velocity, chemotaxis velocity and chemotaxis index in tissue. We demonstrate that using this protocol, these neutrophil recruitment parameters can be stably determined and the single cell locomotion conveniently tracked in vivo.

We describe a protocol of brightfield intravital microscopy for measuring dynamic neutrophil-endothelial cell interactions during neutrophil recruitment in response to the source of a neutrophil chemoattractant in vivo. Neutrophil intraluminal crawling, transendothelial migration and chemotaxis in mouse cremaster muscle tissue are visualized with time-lapsed video photography and tracked with ImageJ.

追踪中性粒细胞腔内的爬行，活体视频显微镜Transendothelial组织中的迁移和趋化

The recruitment of circulating leukocytes from blood stream to the inflamed tissue is a crucial and complex process of inflammation1,2. In the ...

Intravital Imaging of the Mouse Thymus using 2-Photon Microscopy

Two-photon Microscopy (TPM) provides image acquisition in deep areas inside tissues and organs. In combination with the development of new stereotactic tools and surgical procedures, TPM becomes a powerful technique to identify "niches" inside organs and to document cellular "behaviors" in live animals. While intravital imaging provides information that best resembles the real cellular behavior inside the organ, it is both more laborious and technically demanding in terms of required equipment/procedures than alternative ex vivo imaging acquisition. Thus, we describe a surgical procedure and novel "stereotactic" organ holder that allows us to follow the movements of Foxp3+ cells within the thymus.
Foxp3 is the master regulator for the generation of regulatory T cells (Tregs). Moreover, these cells can be classified according to their origin: ie. thymus-differentiated Tregs are called "naturally-occurring Tregs" (nTregs), as opposed to peripherally-converted Tregs (pTregs). Although significant amount of research has been reported in the literature concerning the phenotype and physiology of these T cells, very little is known about their in vivo interactions with other cells. This deficiency may be due to the absence of techniques that would permit such observations. The protocol described in this paper provides a remedy for this situation.
Our protocol consists of using nude mice that lack an endogenous thymus since they have a punctual mutation in the DNA sequence that compromises the differentiation of some epithelial cells, including thymic epithelial cells. Nude mice were gamma-irradiated and reconstituted with bone marrows (BM) from Foxp3-KIgfp/gfp mice. After BM recovery (6 weeks), each animal received embryonic thymus transplantation inside the kidney capsule. After thymus acceptance (6 weeks), the animals were anesthetized; the kidney containing the transplanted thymus was exposed, fixed in our organ holder, and kept under physiological conditions for in vivo imaging by TPM. We have been using this approach to study the influence of drugs in the generation of regulatory T cells.

We have developed novel laboratory tools and protocols for intravital imaging acquisition of the thymus. Our technique should help in the identification of &#x201C;niches&#x201D; within the thymus where T cell development occurs.

活体成像的鼠标使用2 - 光子显微镜的胸腺

Two-photon Microscopy (TPM) provides image acquisition in deep areas inside tissues and organs. In combination with the development of new stereotactic ...

Intravital Microscopy of the Spleen: Quantitative Analysis of Parasite Mobility and Blood Flow

The advent of intravital microscopy in experimental rodent malaria models has allowed major advances to the knowledge of parasite-host interactions 1,2. Thus, in vivo imaging of malaria parasites during pre-erythrocytic stages have revealed the active entrance of parasites into skin lymph nodes 3, the complete development of the parasite in the skin 4, and the formation of a hepatocyte-derived merosome to assure migration and release of merozoites into the blood stream 5. Moreover, the development of individual parasites in erythrocytes has been recently documented using 4D imaging and challenged our current view on protein export in malaria 6. Thus, intravital imaging has radically changed our view on key events in Plasmodium development. Unfortunately, studies of the dynamic passage of malaria parasites through the spleen, a major lymphoid organ exquisitely adapted to clear infected red blood cells are lacking due to technical constraints.
Using the murine model of malaria Plasmodium yoelii in Balb/c mice, we have implemented intravital imaging of the spleen and reported a differential remodeling of it and adherence of parasitized red blood cells (pRBCs) to barrier cells of fibroblastic origin in the red pulp during infection with the non-lethal parasite line P.yoelii 17X as opposed to infections with the P.yoelii 17XL lethal parasite line 7. To reach these conclusions, a specific methodology using ImageJ free software was developed to enable characterization of the fast three-dimensional movement of single-pRBCs. Results obtained with this protocol allow determining velocity, directionality and residence time of parasites in the spleen, all parameters addressing adherence in vivo. In addition, we report the methodology for blood flow quantification using intravital microscopy and the use of different colouring agents to gain insight into the complex microcirculatory structure of the spleen. 
Ethics statement
All the animal studies were performed at the animal facilities of University of Barcelona in accordance with guidelines and protocols approved by the Ethics Committee for Animal Experimentation of the University of Barcelona CEEA-UB (Protocol No DMAH: 5429). Female Balb/c mice of 6-8 weeks of age were obtained from Charles River Laboratories.

We show the method for performing intravital microscopy of the spleen using GFP transgenic malaria parasites and the quantification of parasite mobility and blood flow within this organ.

活体显微镜的脾:寄生虫的流动性和血流量的定量分析

The advent of intravital microscopy in experimental rodent malaria models has allowed major advances to the knowledge of parasite-host interactions 1,2. ...

Saliva, Salivary Gland, and Hemolymph Collection from Ixodes scapularis Ticks

Ticks are found worldwide and afflict humans with many tick-borne illnesses. Ticks are vectors for pathogens that cause Lyme disease and tick-borne relapsing fever (Borrelia spp.), Rocky Mountain Spotted fever (Rickettsia rickettsii), ehrlichiosis (Ehrlichia chaffeensis and E. equi), anaplasmosis (Anaplasma phagocytophilum), encephalitis (tick-borne encephalitis virus), babesiosis (Babesia spp.), Colorado tick fever (Coltivirus), and tularemia (Francisella tularensis) 1-8. To be properly transmitted into the host these infectious agents differentially regulate gene expression, interact with tick proteins, and migrate through the tick 3,9-13. For example, the Lyme disease agent, Borrelia burgdorferi, adapts through differential gene expression to the feast and famine stages of the tick's enzootic cycle 14,15. Furthermore, as an Ixodes tick consumes a bloodmeal Borrelia replicate and migrate from the midgut into the hemocoel, where they travel to the salivary glands and are transmitted into the host with the expelled saliva 9,16-19.
As a tick feeds the host typically responds with a strong hemostatic and innate immune response 11,13,20-22. Despite these host responses, I. scapularis can feed for several days because tick saliva contains proteins that are immunomodulatory, lytic agents, anticoagulants, and fibrinolysins to aid the tick feeding 3,11,20,21,23. The immunomodulatory activities possessed by tick saliva or salivary gland extract (SGE) facilitate transmission, proliferation, and dissemination of numerous tick-borne pathogens 3,20,24-27. To further understand how tick-borne infectious agents cause disease it is essential to dissect actively feeding ticks and collect tick saliva. This video protocol demonstrates dissection techniques for the collection of hemolymph and the removal of salivary glands from actively feeding I. scapularis nymphs after 48 and 72 hours post mouse placement. We also demonstrate saliva collection from an adult female I. scapularis tick.

Saliva, Salivary Gland, and Hemolymph Collection from Ixodes scapularis Ticks

The collection of infected tick hemolymph, salivary glands, and saliva is important to study how tick-borne pathogens cause disease. In this protocol we demonstrate how to collect hemolymph and salivary glands from feeding Ixodes scapularis nymphs. We also demonstrate saliva collection from female I. scapularis adults.

唾液，唾液腺，淋巴收集肩突硬蜱蜱

Ticks are found worldwide and afflict humans with many tick-borne illnesses. Ticks are vectors for pathogens that cause Lyme disease and tick-borne ...

Highly Resolved Intravital Striped-illumination Microscopy of Germinal Centers

Monitoring cellular communication by intravital deep-tissue multi-photon microscopy is the key for understanding the fate of immune cells within thick tissue samples and organs in health and disease. By controlling the scanning pattern in multi-photon microscopy and applying appropriate numerical algorithms, we developed a striped-illumination approach, which enabled us to achieve 3-fold better axial resolution and improved signal-to-noise ratio, i.e. contrast, in more than 100 &#181;m tissue depth within highly scattering tissue of lymphoid organs as compared to standard multi-photon microscopy. The acquisition speed as well as photobleaching and photodamage effects were similar to standard photo-multiplier-based technique, whereas the imaging depth was slightly lower due to the use of field detectors. By using the striped-illumination approach, we are able to observe the dynamics of immune complex deposits on secondary follicular dendritic cells &#8211; on the level of a few protein molecules in germinal centers.

High-resolution intravital imaging with enhanced contrast up to 120 &#181;m depth in lymph nodes of adult mice is achieved by spatially modulating the excitation pattern of a multi-focal two-photon microscope. In 100 &#181;m depth we measured resolutions of 487 nm (lateral) and 551 nm (axial), thus circumventing scattering and diffraction limits.

生发中心的高度已解决活体条纹照明显微镜

Monitoring cellular communication by intravital deep-tissue multi-photon microscopy is the key for understanding the fate of immune cells within thick ...

Intravital Microscopy Imaging of the Liver following Leishmania Infection: An Assessment of Hepatic Hemodynamics

Intravital microscopy (IVM) is a powerful optical imaging technique that has made possible the visualization, monitoring and quantification of various biological events in real time and in live animals. This technology has greatly advanced our understanding of physiological processes and pathogen-mediated phenomena in specific organs.
In this study, IVM is applied to the mouse liver and protocols are designed to image in vivo the circulatory system of the liver and measure red blood cell (RBC) velocity in individual hepatic vessels. To visualize the different vessel subtypes that characterize the hepatic organ and perform blood flow speed measurements, C57Bl/6 mice are intravenously injected with a fluorescent plasma reagent that labels the liver-associated vasculature. IVM enables in vivo, real time, measurement of RBC velocity in a specific vessel of interest. Establishing this methodology will make it possible to investigate liver hemodynamics under physiological and pathological conditions. Ultimately, this imaging-based methodology will be important for studying the influence of L. donovani infection&#160;on hepatic hemodynamics.
This method can be applied to other infectious models and mouse organs and might be further extended to pre-clinical testing of a drug&#8217;s effect on inflammation by quantifying its effect on blood flow.

Intravital Microscopy Imaging of the Liver following Leishmania Infection: An Assessment of Hepatic Hemodynamics

This article reports on a detailed method for the dynamic measurement and quantification of blood flow velocity within individual blood vessels of the mouse liver vasculature using intravital microscopy imaging in combination with a specific methodology for image acquisition and analysis.

肝脏活体如下显微成像<em&gt;利什曼原虫</em&gt;感染:肝脏血流动力学的评估

Intravital microscopy (IVM) is a powerful optical imaging technique that has made possible the visualization, monitoring and quantification of various ...

Long Term Intravital Multiphoton Microscopy Imaging of Immune Cells in Healthy and Diseased Liver Using CXCR6.Gfp Reporter Mice

Liver inflammation as a response to injury is a highly dynamic process involving the infiltration of distinct subtypes of leukocytes including monocytes, neutrophils, T cell subsets, B cells, natural killer (NK) and NKT cells. Intravital microscopy of the liver for monitoring immune cell migration is particularly challenging due to the high requirements regarding sample preparation and fixation, optical resolution and long-term animal survival. Yet, the dynamics of inflammatory processes as well as cellular interaction studies could provide critical information to better understand the initiation, progression and regression of inflammatory liver disease. Therefore, a highly sensitive and reliable method was established to study migration and cell-cell-interactions of different immune cells in mouse liver over long periods (about 6 hr) by intravital two-photon laser scanning microscopy (TPLSM) in combination with intensive care monitoring.
The method provided includes a gentle preparation and stable fixation of the liver with minimal perturbation of the organ; long term intravital imaging using multicolor multiphoton microscopy with virtually no photobleaching or phototoxic effects over a time period of up to 6 hr, allowing tracking of specific leukocyte subsets; and stable imaging conditions due to extensive monitoring of mouse vital parameters and stabilization of circulation, temperature and gas exchange.
To investigate lymphocyte migration upon liver inflammation CXCR6.gfp knock-in mice were subjected to intravital liver imaging under baseline conditions and after acute and chronic liver damage induced by intraperitoneal injection(s) of carbon tetrachloride (CCl4).
CXCR6 is a chemokine receptor expressed on lymphocytes, mainly on Natural Killer T (NKT)-, Natural Killer (NK)- and subsets of T lymphocytes such as CD4 T cells but also mucosal associated invariant (MAIT) T cells1. Following the migratory pattern and positioning of CXCR6.gfp+ immune cells allowed a detailed insight into their altered behavior upon liver injury and therefore their potential involvement in disease progression.

Stable intravital high-resolution imaging of immune cells in the liver is challenging. Here we provide a highly sensitive and reliable method to study migration and cell-cell-interactions of immune cells in mouse liver over long periods (about 6 hours) by intravital multiphoton laser scanning microscopy in combination with intensive care monitoring.

免疫细胞健康和疾病肝脏使用CXCR6.Gfp记者小鼠长期活体多光子显微镜成像

Liver inflammation as a response to injury is a highly dynamic process involving the infiltration of distinct subtypes of leukocytes including monocytes, ...

Intravital Microscopy of Leukocyte-endothelial and Platelet-leukocyte Interactions in Mesenterial Veins in Mice

Intravital microscopy is a method that can be used to investigate different processes in different regions and vessels in living animals. In this protocol, we describe intravital microscopy of mesentery veins. This can be performed in a short period of time with reproducible results showing leukocyte-endothelial interactions in vivo. We describe an inflammatory setting after LPS challenge of the endothelium. But in this model one can apply many different types of inflammatory conditions, like bacterial, chemical or biological and investigate the administration of drugs and their direct effects on the living animal and its impact on leukocyte recruitment. This protocol has been applied successfully to a number of different treatments of mice and their effects on inflammatory response in vessels. Herein, we describe the visualization of leukocytes and platelets by fluorescently labeling these with rhodamine 6G. Additionally, any specific imaging can be performed using targeted fluorescently labeled molecules.

This simplified application of intravital microscopy of mesentery veins in mice can be used in different models of inflammation to observe leukocyte-endothelial and platelet-leukocyte interactions.

在Mesenterial静脉的活体显微镜白细胞，内皮细胞和血小板白细胞相互作用小鼠

Intravital microscopy is a method that can be used to investigate different processes in different regions and vessels in living animals. In this ...

A Method for the Measurement of Salivary Gland Function in Mice

Patients with Sj&#246;gren&#39;s syndrome, an autoimmune disease affecting the exocrine glands, develop salivary gland inflammation and have reduced saliva production. Similarly, saliva production is severely compromised in patients receiving radiation treatment for head and neck cancers. Rodent models, developed to mimic these clinical conditions, facilitate an understanding of the disease pathogenesis and allow for the development of new therapeutic strategies. Therefore, the ability to accurately, reproducibly, and repeatedly measure salivary gland function in animal models is critical. Building on procedures previously described in the literature, a method was developed that meets these criteria and was used to evaluate salivary gland function in mice. An additional advantage of this new method is that it is easily mastered, and has little inter-operator variation. Salivary gland function is evaluated as the amount (weight or volume) or rate (mL/min) of saliva produced in response to pilocarpine stimulation. The collected saliva is a good source for the analyses of protein content, immunoglobulin concentrations, and other biomolecules.

Salivary gland hypofunction is a frequent consequence of autoimmune disease and radiation therapy. Reproducible evaluation of salivary gland function in mouse models of these diseases is a technical challenge. Here, a simple method for accurate and reproducible measurement of saliva production in mice is described.

小鼠涎腺功能的测定方法

Patients with Sj&#246;gren&#39;s syndrome, an autoimmune disease affecting the exocrine glands, develop salivary gland inflammation and have reduced ...