波谱与自发拉曼显微荧光背景的抑制
Summary
我们讨论一个复杂的非线性光学系统，采用超快全光开关，隔离荧光信号的拉曼的建设和运营。使用这个系统，我们能够成功地分离的拉曼光谱和荧光信号，利用脉冲能量和平均的权力，保持生物安全。
Abstract
拉曼光谱仪是常常困扰着一个强烈的荧光背景，特别是对生物样品。如果样品是兴奋的超短脉冲，一个系统，可以暂时分离频谱重叠信号皮秒时间尺度可以隔离的火车，及时到达迟到荧光光的拉曼散射光。在这里，我们讨论一个复杂的非线性光学系统，采用全光开关在一个低功率光学克尔门的形式，隔离拉曼光谱和荧光信号的建设和运营。一个单一的808 nm的激光2.4 W的平均功率和80 MHz的重复率是分裂的，与约200毫瓦808纳米的光被转换为<5兆瓦404 nm的光发送到的样本，以激发拉曼散射。其余未转换的808纳米的光，然后发送到非线性介质泵全光快门行为。快门打开和关闭了约5％的峰值效率在800 FS。使用这个系统，我们能够成功地分离拉曼光谱和荧光信号，在80 MHz的重复使用的脉冲能量和平均的权力，保持生物安全。由于该系统具有光功率没有余力，我们几个细节设计和调整的考虑，有助于最大限度地提高系统的吞吐量。我们还讨论了克尔介质内的信号和泵光束的空间和时间上的重叠，以及详细的协议光谱采集获得的协议。最后，我们报告了几个有代表性的拉曼光谱中存在强荧光，用我们的时间浇注系统取得的成果。
Protocol
1。在准备，并把这个系统内的拉曼样品，必须采取一定的照顾。 由于该系统通常使用非常高数值孔径物镜，工作距离很短，样品置于盖玻片上。生物样品通常是放置在1号安装盖玻片厚度Attofluor细胞室（Invitrogen公司，卡尔斯巴德，加利福尼亚州）。 液体样品，特别是那些对人体无毒，被放置在一个小玻璃瓶，巩固有机硅环氧开幕盖玻片。瓶子，然后倒测量。 因为我们?…
Discussion
拉曼光谱生物医学领域已经看到在过去的几年中，作为其表现出的潜力解决一些困难的挑战，在生物诊断的越来越大的兴趣。例如，拉曼光谱已经证明，在癌症的检测3，4，5，6诊断价值。拉曼光谱技术也已用于细菌定量7，8和细菌对药物的反应9。它也发现在其他医学领域的应用范围从骨骼健康10 biofluid分析，11，12的广泛应用。
然而，…
Disclosures
The authors have nothing to disclose.
Acknowledgements
这项工作是由美国国家科学基金会奖DBI的0852891。号PHY0120999根据合作协议，由加利福尼亚大学戴维斯分校生物光子科学与技术，一个指定的美国国家科学基金会科学技术中心管理中心还资助了这项工作的一部分。
Materials
| Name | Company | Catalog Number | Comments |
|---|---|---|---|
| Lenses | ThorLabs | Various | All lenses coated to have maximum transmission losses of 1% each |
| Tunable Ti:Sapph laser | Coherent | Chameleon | 30 nJ, 200 fs, 80 MHz |
| 40X oil immersion objective | Olympus | UApo/340 | NA = 1.35 |
| Inverted microscope | Olympus | IX-71 | Modified to remove all lenses in side port |
| Half wave plate | Thorlabs | AHWP05M-600 | |
| Glan-Thompson polarizer | Thorlabs | GTH10M | ˜10% transmission loss |
| Spectrometer | PI Acton | SP2300i | |
| CCD | PI Acton | Pixis 100B | |
| Mathmatical software | The MathWorks | MATLAB | version 2008a |
| Faraday isolator | EOT | BB8-5I | |
| Piezo-electric mirror | Newport | AG-M100 | |
| BBO crystal | CASIX | custom | 1 mm thickness |
| Bandpass filter 1 | Andover | 008FC14 | 808 ± 0.4 nm |
| Dichroic mirror | Semrock | FF662-FDI01 | band edge at 662 nm |
| Long-pass filter | Semrock | BLP01-405R | band edge at 417 nm |
| Bandpass filter 2 | Semrock | FF02-447/60 | 417-447 nm |
| CS2 | Sigma-Aldrich | 335266 | 99% purity |
| Coumarin 30 | Sigma-Aldrich | 546127 | 99% purity |
| Immersion oil | Cargille | 16242 | Type DF |
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Tags
Fluorescence BackgroundRaman SpectroscopyBiological SamplesUltrafast PulsesPicosecond TimescaleRaman Scattered LightFluorescence SignalsNonlinear Optical SystemOptical Kerr GateLaser PowerRepetition RateNonlinear MediumAll-optical ShutterPeak EfficiencyPulse EnergiesAverage PowersBiologically SafeOptical Power
Cite This Article
Smith, Z. J., Knorr, F., Pagba, C. V., Wachsmann-Hogiu, S. Rejection of Fluorescence Background in Resonance and Spontaneous Raman Microspectroscopy. J. Vis. Exp. (51), e2592, doi:10.3791/2592 (2011).