IDBR: Development of a time-gated Raman/fluorescence micro-spectroscopy instrument for biological applications

拉曼光谱是光子通过分子键的非弹性散射，因此可以在不使用外部标记的情况下在样品内进行特定化学测量。这一独特的特性使基于拉曼的技术具有在各种生物研究应用中使用的潜力。然而，生物材料中的许多化学成分是荧光的，作为背景，因此严重限制了拉曼光谱在这些应用中的使用。尽管已经尝试减少这种背景，但只有光学时间门控方法才能足够有效。由于拉曼信号与激发光几乎是瞬间发生的，而荧光则延迟了几纳秒，因此可以使用脉冲在几皮秒范围内的激光来实现他的目的。通过使用非线性中等功率和高功率激光脉冲，可以建立仅在拉曼信号持续时间内打开并在背景荧光产生之前关闭的时间门。这一结合使得能够在强荧光背景下测量拉曼光谱，并开辟了拉曼光谱在生物学研究中的新的潜在应用，如：(I)非侵入性地表征细菌、细胞和组织中内源性荧光团的化学成分，(Ii)通过使用天然标志物了解细胞过程和疾病，如癌症、血管或神经退行性疾病，(Iii)开发既可用作荧光标记又可用作拉曼标记的新型探针，(Iv)用于血液分析的非侵入性医学传感器，如用于糖尿病治疗的葡萄糖传感器。这项技术将对分析化学、生物医学、药理学、法医学、食品安全、农业、生物燃料研究、环境监测和生物防御等相关领域产生重大影响。推广将涉及从中学到博士后的各级学生和教育工作者，以及生物技术行业的代表和参与者。

Raman spectroscopy is the inelastic scattering of photons by molecular bonds, and therefore enables chemically-specific measurements within a sample without the use of external labels. This unique characteristic gives Raman-based techniques the potential to be used in a variety of biological research applications. However, many of the chemical components within the biological material are fluorescent, acting as a background and thus severely limiting the use of Raman spectroscopy for these applications. Although there have been attempts to reduce this background, only an optical time-gated approach can be sufficiently effective. Since the Raman signal occurs almost instantaneously with the excitation light while the fluorescence is delayed by several nanoseconds, lasers with pulses in the range of several picoseconds can be used for his purpose. A time gate that opens only for the duration of the Raman signal and closes before the background fluorescence is produced can be built by using a nonlinear medium and highpower laser pulses. This combination allows for the measurement of Raman spectra in the presence of a strong fluorescence background and opens up new potential applications of Raman spectroscopy for biological research such as (i) non-invasive characterization of the chemical composition of the endogenous fluorophores in bacteria, cells and tissues, (ii) understanding cellular processes and diseases such as cancer, vascular or neurodegenerative diseases by using natural markers, (iii) developing new probes that can be used both as fluorescence and Raman labels, (iv) noninvasive medical sensors for blood analytes such as glucose sensors for diabetes treatment. This technique will have a significant impact on related fields such as analytical chemistry, biomedicine, pharmacology, forensics, food safety, agriculture, biofuel research, environmental monitoring, and bio-defense.Outreach will involve students and educators at all levels, from middle school to postdoctoral, as well as representatives and participants from the biotechnology industry.Further information can be requested from the PI.