Photothermal and Photoionization Spectroscopies of Chemical Reactions in a Single Cell

氨基酸、核苷酸等生物重要化学物质大多是天然非荧光物质，其中很大一部分仅在紫外波长处有光吸收。光热和光电离光谱方法具有很好的潜力，这些化学品的原位检测灵敏度。为了发展对生物细胞中非荧光分子的超灵敏检测和成像方法，设计了一种多色激发下的光热信号检测器，利用瞬态吸收增强信号，实现了对生物化学物质的超灵敏检测。建立了紫外激发-可见光增强光热检测系统，并与微型高效液相色谱系统联用进行分离分析。紫外吸收化学品的混合物，成功地分离和检测，没有任何标记程序。利用钛蓝宝石激光器的三次谐波发射和基波发射的探测激光，研制了紫外激发光热显微镜。获得了酵母细胞的光热图像。在细胞的光热振幅图像中，可以看到与细胞大小相对应的几微米大小的特征。观察到261 nm激光束激发的强光热信号伴随着784 nm的小强度透射探测光，使用220、266、355、532或1064 nm的激光定量评价激光照射对酵母细胞生理功能的影响。结果发现，在相同的能量密度条件下，532 nm光照射12小时后酵母细胞的存活概率是355 nm光照射的10倍，是266 nm光照射的104倍。

Most of biologically important chemicals such as amino acids, nucleotides and so on, are natively nonfluorescent, and a large part of them have optical absorption only at ultraviolet wavelengths. Photothermal and photoionization spectroscopic methods have a good potential for ultrahigh sensitive in-situ detection of these chemicals. We aim to develop ultrasensitive detection and imaging methods for nonfluorescent molecules in living cells.A photothermal signal detector under multi-color excitation has been designed for ultrasensitive detection of biological chemicals, in which transient absorption is utilized for signal enhancement. Ultraviolet excitation-visible enhancement photothermal detection system has been constructed and applied to separation analysis in combination with a micro-HPLC system. A mixture of ultraviolet-absorbing chemicals were successfully separated and detected without any labeling procedure. Two-color excitation-induced enhancement of photothermal signal was successfully observed.An ultraviolet-excitation photothermal microscope has been developed with the excitation of the third harmonics emission of a Ti-sapphire laser and the probe laser of its fundamental emission. Photothermal images of the yeast cells were obtained. In a photothermal amplitude image of the cells, features with the size of several micrometers are seen, corresponding to the size of the cells. It was observed that a strong photothermal signal excited with the 261-nm laser beam accompanied with a small intensity of the transmitted probe light at 784-nm.Influences of laser irradiation on a physiological function of yeast cells were quantitatively evaluated using laser light at 220, 266, 355, 532, or 1064nm. It was found that the survival probability of yeast cells after 12 hours from irradiation by 532-nm light is 10 times larger than that by 355-nm light and 104 times larger than that by 266-nm light under the same energy density condition.