Quantitative CARS microscopic detection of analytes and their isotopomers in a two-channel microfluidic chip.
Quantitative CARS microscopic detection of analytes and their isotopomers in a two-channel microfluidic chip.
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DOI：
10.1002/smll.200900807
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发表时间：
2009-12
期刊：
影响因子：
13.3
通讯作者：
G. Bergner;Susana Chatzipapadopoulos;D. Akimov;B. Dietzek;D. Malsch;T. Henkel;S. Schlücker;J. Popp
中科院分区：
文献类型：
--
作者：
G. Bergner;Susana Chatzipapadopoulos;D. Akimov;B. Dietzek;D. Malsch;T. Henkel;S. Schlücker;J. Popp
拉曼微光谱学[1,2]在不需要样品制备的情况下提供亚微米空间分辨率的无标签振动对比，因此已成为包括分析科学、生命科学和材料科学在内的各个学科中不可或缺的表征方法。该技术特别适用于样品中化学成分浓度的空间分辨定量，以及在不可能或不希望用荧光标记标记低分子量化合物的情况下相干反斯托克斯拉曼散射（CARS）显微镜[5,6]比传统的拉曼显微光谱学具有更快的采集速率。[5-8]最近介绍了将非线性拉曼技术与微流体技术结合用于反应监测[9]和细胞测定[10]。不幸的是，CARS并不是没有背景的：信号的产生导致了分子振动（核运动）的相干激发和本征非拉曼谐振背景（电子响应）。[6,11]这种化学上的非特异性背景严重限制了car的检测和低浓度分析物的定量。多重CARS微光谱与随后的CARS波段分析允许提取化学相关的拉曼谐振CARS信号和非特异性背景。[12,13]与光谱分辨检测相结合的宽带斯托克斯激励的另一种选择是通过使用两个窄带皮秒斯托克斯光束来确定谐振信号/非谐振背景的比率：泵浦光束和一个斯托克斯光束之间的波数差被调谐到感兴趣的拉曼共振，而泵浦光束和第二个斯托克斯光束之间的波数差是非谐振的。[14]因此，使用一个二向镜和两个探测器同时记录两个CARS图像。在这里，我们提出了一种双通道微流体方法，用于分析物与它们的一种氘化同位素的定量CARS检测。微流控芯片包含两个相距200mm的通道（图1），一个通道中填充渗透氘化甲苯（C7D8），另一个通道中填充其同位素体甲苯（C7H8）作为参考。在采用该芯片的CARS显微实验中，可以在同一显微视场内同时探测两个通道。将泵浦和斯托克斯激光器之间的波数差调整为芳香C-D拉伸振动在2200左右cmÀ1（见自发）
Raman microspectroscopy [1, 2] provides label-free vibrational contrast at submicron spatial resolution without the need for sample preparation, and has therefore become an indispensable characterization method in various disciplines, including analytical, life, and materials sciences. The technique is particularly useful for spatially resolved quantification of the concentrations of chemical constituents in a sample, and in situations where labeling of low-molecular-weight compounds by ffuorescent labels [3] is not possible or not desired.[4] Coherent anti-Stokes Raman scattering (CARS) microscopy [5, 6] benefits from significantly faster acquisition rates than conventional Raman microspectroscopy.[5–8] The combination of this nonlinear Raman technique with microffuidics for reaction monitoring [9] and cytometry [10] has been introduced recently. Unfortunately, CARS is not background-free: the signal generation leads to both a coherent excitation of molecular vibrations (nuclear motions) and an intrinsic non-Ramanresonant background (electronic response).[6, 11] This chemically nonspecific background constitutes a severe limitation for CARS detection and the quantification of analytes at low concentrations. Multiplex CARS microspectroscopy with subsequent CARS band-shape analysis allows extraction of both the chemically relevant Raman-resonant CARS signal and the nonspecific background.[12, 13] An alternative to the broadband Stokes excitation in combination with spectrally resolved detection is to determine the ratio of resonant signal/nonresonant background by using two narrowband picosecond Stokes beams: the wavenumber difference between the pump beam and one Stokes beam is tuned to a Raman resonance of interest, while the wavenumber difference between the pump beam and a second Stokes beam is off-resonant.[14] Thus, two CARS images are recorded simultaneously using a dichroic mirror and two detectors.Herein, we present a two-channel microffuidic approach for quantitative CARS detection of analytes in combination with one of their deuterated isotopomers. The microffuidic chip contains two channels separated by 200mm (Figure 1): one channel is filled with perdeuterated toluene (C7D8) and the other channel contains its isotopomer toluene (C7H8) as a reference. In a CARS microscopic experiment employing this chip, both channels can be probed simultaneously within the same microscopic field of view. Tuning the wavenumber difference between pump and Stokes lasers to the aromatic C–D stretching vibration around 2200 cmÀ1 (see spontaneous