Nature Methods:看清活体细胞和组织的化学组成
Jalink说,髓鞘因为紧密堆积了大量脂质,特别适合用CARS成像。非标记显微技术在其他方面的应用则可能没那么容易。他说经常使用激光器的研究人员很可能会想办法采用这样的技术,他补充道,“技术上讲,这是完全可行的,但是如果我能用另一种方式来获得同样的信息,我为什么要采用这个多少有些复杂而且昂贵的技术呢?”
技术一旦发展起来,研究人员就能把它们应用到新的方面。哥伦比亚大学的Rafael Yuste利用光学手段来测量神经电位。二次谐波发生(SHG)成像技术依赖于排列非常规则的分子产生的超散射光。这些分子具有极强的诱导偶极矩,或者特定的电荷分布。Yuste对位于神经元细胞膜这类分子非常感兴趣——因为电场贯穿其中。由于二次谐波信号和电场强度直接成比例,因而可以自动获得电压信号。
问题在于,能够很好地实现这一目标的分子非常少。为了达到好的效果,Yuste说,“你需要非常仔细地去扫描全谱,来寻找潜在的内源性二次谐波发色基团。”他说,发展这种技术需要依赖学科交叉,需要研究人员在他们研究领域的边缘工作。但是在现实中,这种工作往往在研究者们自己的系里得不到足够的资金和支持,这也是为什么能够实现这一目标的分子资源较少的原因。
Enejder等人相信,学科交叉能够帮助人们解决大量只能由非标记的非线性显微技术来观测的问题。虽然Enejder的背景的是物理学,她还是转到了生物系。因为在那里可以更容易的了解生物学家们在成像上到底遇到了什么问题,非线性光学如何才能帮得上忙。她说,那些把自己的眼光牢牢地局限在物理系内部的人可以继续优化技术,但是他们或许不了解生物学家到底希望看到什么:“我就完全没有这个问题。在我眼里,应用随处可见。”
当这样的交流变得日益重要的时候,对新实验的大胆尝试也变得重要起来——而这些实验与物理学家们以往的经验可能截然不同。在一项旨在制造弹性血管的生物工程项目中,Enejder和同事们想要监测植入纤维素基质的肌肉细胞的生长。与CARS一起,Enedjer和同事们利用SHG观察了植入的细胞。他们很高兴地发现,自己可以监测到被植入细胞是如何与纤维素网进行接触,开始生成胶原蛋白纤维的。在组织工程研究中,这种方法可以大大帮助确定最优参数。尽管纸张中的植物纤维素SHG成像看不到,但是细菌分泌的植物纤维素确实拥有一种有规律的模式,能够产生SHG信号,Enejder解释说,“仅仅依赖别人文章里说的哪些可以观测是不行的,你得自己去试才行。”(生物谷Bioon.com)
生物谷推荐原文出处:
Nature Methods doi:10.1038/nmeth0410-261
Nonlinear optical microscopy lets researchers see chemical composition in living cells and organisms.
Monya Baker1
A couple years ago, Annika Enejder confronted confusing results from her studies of lipid storage in the roundworm Caenorhabditis elegans. Fluorescence microscopy images very clearly showed a decrease in signal from lipid droplets when the worms were treated with statins, a widely prescribed class of cholesterol-lowering drugs. But simultaneous experiments using another type of microscopy, which visualizes lipids directly, showed no such thing. In fact, coherent anti-Stokes Raman scattering (CARS) microscopy identified lipid droplets where they could not be observed with fluorescence techniques.
What had happened was that worms fed with the commonly used fluorescent dye Nile red processed it as a toxin: instead of being localized to lipid droplets, the dye was sequestered in intestinal lysozome-like granules interspersed among lipid droplets. In fact, the dye can be misleading in other ways: statins themselves seem to affect either its staining or fluorescence. “There are so many artifacts that you have to take into account when using fluorophores,” says Enejder, a scientist at the Chalmers University of Technology in G?teborg, Sweden.
No one can deny the power of fluorescent probes and molecular stains to see the inner workings of cells, but such labels have considerable drawbacks. Delivering labels can be a problem, particularly for whole organisms. Some labels work only on dead cells; others damage cells or perturb the very processes they are intended to study. Label-free microscopy offers a way to investigate living cells while eliminating a slew of possible artifacts. Though some techniques rely on endogenous fluorophores (Box 1), most eschew fluorescence all together, along with the well-known problem of photobleaching. Instead of detecting photons emitted from excited fluorophores, these alternate techniques detect subtle changes in light as it is absorbed or altered by biological samples, relying on the nonlinear optical phenomena observed when high-intensity light moves through matter. In essence, pulses of laser light can be used to 'see' chemical composition: the C-H bonds of lipids, the amide bonds of proteins, the reduced or oxidized states of certain biomolecules, the regularly repeating units of microtubules or collagen.
Of course such techniques have their own limitations: whereas fluorescence labeling can often allow discrimination of single molecules, label-free techniques are less sensitive and specific. All but the most common substituents tend to be hidden in signals generated from a few abundant species. “What is nice is that you don't need any labeling; you can just start imaging,” explains Kees Jalink, a biophysicist at the Netherlands Cancer Institute, “but what is not so nice is that the signal is weak, you need a lot of power to irradiate a cell and may only get the coarsest of details.”
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