Nonlinear Optical Imaging of Integral Membrane Protein Crystals in Lipidic Mesophases
Nonlinear Optical Imaging of Integral Membrane Protein Crystals in Lipidic Mesophases
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DOI：
10.1021/ac902139w
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发表时间：
2010-01-15
影响因子：
7.4
通讯作者：
Cherezov, Vadim
中科院分区：
文献类型：
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作者：
Kissick, David J.;Gualtieri, Ellen J.;Cherezov, Vadim
探索了手性晶体的二阶非线性光学成像(SONICC)，用于选择性检测在不透明和混浊环境中生长的完整膜蛋白晶体。高浊度是膜蛋白结晶的标志，这是因为洗涤剂和/或脂类的广泛使用，这些往往形成各种中间相。用传统的光学方法(如本征LTV荧光、双折射、明场图像分析等)检测这种介质中的晶体。通常由于光学散射和常规形成的小尺寸晶体而变得复杂。SONICC因其与散射介质中成像的兼容性以及其高选择性的蛋白质晶体而被证明非常适合这一应用。观察到明亮的二次谐波(高达1800万次/S)，即使是相对较小的晶体(5针)，由于周围的脂类中间相(类似于1000次/S)具有最小的背景。由此产生的蛋白质晶体图像的低背景性质允许使用相对简单的颗粒计数分析来进行初步评分。对SONICC图像的粒子计数分析与基于人类专家对传统明视场和双折射图像的分析进行了比较。
Second-order nonlinear optical imaging of chiral crystals (SONICC) is explored for selective detection of integral membrane protein crystals grown in opaque and turbid environments. High turbidity is a hallmark of membrane protein crystallization due to the extensive use of detergent and/or lipids that often form various mesophases. Detection of crystals in such media by conventional optical methods (e.g., intrinsic LTV fluorescence, birefringence, bright-field image analysis, etc.) is often complicated by optical scattering and by the small sizes of the crystals that routinely form. SONICC is shown to be well-suited for this application, by nature of its compatibility with imaging in scattering media and its high selectivity protein crystals. Bright second harmonic generatioiri (SHG) (up to 18 million counts/s) was observed from; even relatively small crystals (5 pin) with a minimal background due to the surrounding lipid mesophase (similar to 1 thousand counts/s). The low background nature of the resulting protein crystal images permitted the use of a relatively simple, particle counting analysis for preliminary scoring. Comparisons between a particle counting analysis of SONICC images and protocols based on the human expert analysis of conventional bright-field and birefringence images were performed.