Quantifying single and bundled microtubules with the polarized light microscope.
Quantifying single and bundled microtubules with the polarized light microscope.
复制标题
用偏光显微镜定量单个和成束的微管。
DOI：
10.1086/bblv189n2p206
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发表时间：
1995
期刊：
影响因子：
--
通讯作者：
Oldenbourg,R
中科院分区：
文献类型：
--
作者：
Tran,P;Salmon,ED;Oldenbourg,R
偏光显微镜已成为直接对活细胞中精细结构进行无创成像的重要工具 (1)。我们中的一员（RO）通过开发一种由电子控制液晶器件和圆偏振器制成的精密通用补偿器改进了偏光显微镜。结合特殊的处理软件，新的“pob~~ ope”可以以高灵敏度和分辨率对细胞精细结构进行成像，无论样本方向如何 (2)。我们使用偏振镜对单个和成束微管的固有光学特性进行成像和量化。暗场、微分干涉对比 (DIC) 和荧光等光学显微镜技术已经证明了对单个微管动态进行实时成像的能力。然而，这些技术均具有局限性，使其不适合定量测量微管密度和分布。例如，暗视野和 DIC 显微镜虽然是非侵入性的，但无法对有丝分裂纺锤体中发现的微管密集区域中的微管数量进行成像和量化；荧光显微镜虽然是定量的，但具有侵入性并且会遭受光漂白。 pol-scope是非侵入性的，不会遭受光漂白，并且是定量的，因为它测量微管固有的光学特性。磷酸纤维素纯化的牛脑微管蛋白被允许自发组装成微管，然后用10~Mtaxol稳定。通过添加无活性的 KAR3，稳定的单个微管被诱导形成各种数量的束，KAR3 是一种成束微管的驱动蛋白样微管马达。然后使微管粘附到预涂有 KAR3 的载玻片室的盖玻片表面。我们在偏振镜上使用尼康 PlanApo 60X/1.4 NA 低应变物镜和配套的尼康通用 1.4 NA 聚光镜，用偏振光对单个微管进行成像。图 1A 显示了用 DIC 显微镜成像的微管束，该微管束不能轻易用于确定构成该束的微管的确切数量。为了进行比较，图 1B 显示了用 pol-scope 观察到的同一束微管的延迟图像，该图像由一个、两个和三个微管的明显不同区域组成。测量单个微管的延迟为0.07+0.02 nm [n=301。延迟慢轴平行于微管的长轴。此外，还发现延迟值是量化的，并且随着束中微管的数量线性增加（图1C）。我们将使用新的 pol-scope 无创地量化分裂细胞中纺锤体微管的分布和动态。
Polarized light microscopy has been an important tool for noninvasive imaging of fine structures directly in living cells (1). One of us (RO) has improved the polarizing microscope by developing a precision universal compensator made of electronically controlled liquid crystal devices and circular polarizers. Combined with special processing software, the new “pob~~ ope” can image cellular fine structures with high sensitivity and resolution, irrespective of specimen orientation (2). We have used the pol-scope to image and quantify the inherent optical properties of single and bundled microtubules. The ability to image the dynamics of a single microtubule in real time has been demonstrated with light microscopy techniques such as darkfield, differential interference contrast (DIC), and fluorescence. However, each of these techniques has limitations that make them non-ideal for quantitative measurements of microtubule density and distribution. For instance, darkfield and DIC microscopy, while noninvasive, cannot image and quantify the number of microtubules in a dense region of microtubules found in the mitotic spindle; and fluorescence microscopy, while quantitative, is invasive and suffers from photobleaching. The pol-scope is noninvasive, does not suffer from photobleaching, and is quantitative because it measures the inherent optical properties of microtubules.Phosphocellulose-purified bovine brain tubulin was allowed to spontaneously assemble into microtubules, and was then stabilized with 10~ Mtaxol. The stabilized single microtubules were induced to form bundles of various numbers by the addition of inactive KAR3, a kinesin-like microtubule motor that bundles microtubules. The microtubules were then allowed to adhere to the coverslip surface of a slide chamber precoated with KAR3. Using a Nikon PlanApo 60X/1.4 NA low-strain, objective and a matching Nikon Universal 1.4 NA condenser on the pol-scope, we imaged single microtubules with polarized light. Figure 1A shows a microtubule bundle imaged with DIC microscopy, which cannot easily be used to determine the exact number of microtubules making up the bundle. For comparison, Figure 1B shows the retardance image of the same bundle of microtubules consisting of clearly distinct regions of one, two, and three microtubules observed with the pol-scope. The retardance of a single microtubule was measured to be 0.07+ 0.02 nm [n= 301. The retardance slow axis is parallel to the long axis of the microtubule. In addition, the retardance values were found to be quantized and increased linearly with the number of microtubules in the bundle (Fig. 1C). We will use the new pol-scope to noninvasively quantify the distribution and dynamics of spindle microtubules in dividing cells.