Furuta T, Wang S S, Dantzker J L, Dore T M, Bybee W J, Callaway E M, Denk W, Tsien R Y
Department of Pharmacology, University of California, San Diego, CA 92093-0647, USA.
Proc Natl Acad Sci U S A. 1999 Feb 16;96(4):1193-200. doi: 10.1073/pnas.96.4.1193.
Photochemical release (uncaging) of bioactive messengers with three-dimensional spatial resolution in light-scattering media would be greatly facilitated if the photolysis could be powered by pairs of IR photons rather than the customary single UV photons. The quadratic dependence on light intensity would confine the photolysis to the focus point of the laser, and the longer wavelengths would be much less affected by scattering. However, previous caged messengers have had very small cross sections for two-photon excitation in the IR region. We now show that brominated 7-hydroxycoumarin-4-ylmethyl esters and carbamates efficiently release carboxylates and amines on photolysis, with one- and two-photon cross sections up to one or two orders of magnitude better than previously available. These advantages are demonstrated on neurons in brain slices from rat cortex and hippocampus excited by glutamate uncaged from N-(6-bromo-7-hydroxycoumarin-4-ylmethoxycarbonyl)-L-glutamate (Bhc-glu). Conventional UV photolysis of Bhc-glu requires less than one-fifth the intensities needed by one of the best previous caged glutamates, gamma-(alpha-carboxy-2-nitrobenzyl)-L-glutamate (CNB-glu). Two-photon photolysis with raster-scanned femtosecond IR pulses gives the first three-dimensionally resolved maps of the glutamate sensitivity of neurons in intact slices. Bhc-glu and analogs should allow more efficient and three-dimensionally localized uncaging and photocleavage, not only in cell biology and neurobiology but also in many technological applications.
如果光解作用能够由红外光子对而非传统的单个紫外光子驱动，那么在光散射介质中以三维空间分辨率进行生物活性信使的光化学释放（解笼）将得到极大促进。对光强的二次方依赖性会将光解作用限制在激光的焦点处，并且较长波长受散射的影响要小得多。然而，先前的笼形信使在红外区域的双光子激发截面非常小。我们现在表明，溴化的7-羟基香豆素-4-基甲酯和氨基甲酸酯在光解时能有效释放羧酸盐和胺，其一光子和双光子截面比以前可用的要好一个或两个数量级。这些优势在大鼠皮层和海马脑片的神经元上得到了证明，这些神经元由从N-(6-溴-7-羟基香豆素-4-基甲氧基羰基)-L-谷氨酸（Bhc-谷氨酸）解笼得到的谷氨酸激发。Bhc-谷氨酸的传统紫外光解所需强度不到先前最好的笼形谷氨酸之一γ-(α-羧基-2-硝基苄基)-L-谷氨酸（CNB-谷氨酸）所需强度的五分之一。用飞秒红外脉冲进行光栅扫描的双光子光解给出了完整脑片中神经元谷氨酸敏感性的首张三维分辨率图谱。Bhc-谷氨酸及其类似物不仅在细胞生物学和神经生物学中，而且在许多技术应用中都应能实现更高效和三维定位的解笼及光裂解。
