Photothermal and Photoionization Spectroscopies of Chemical Reactions in a Single Cell

大多数生物学上重要的化学物质，如氨基酸、核苷酸等，都是非荧光的，其中很大一部分只在紫外线波长下有光学吸收。光热光谱法和光电离光谱法在这些化学物质的超高灵敏度原位检测方面具有良好的潜力。我们的目标是开发活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光热信号探测器，利用瞬态吸收增强信号，用于生物化学物质的超灵敏检测。建立了紫外激发-可见增强光热检测系统，并结合微高效液相色谱系统应用于分离分析。在没有任何标记程序的情况下，成功地分离和检测了吸收紫外线的化学物质的混合物。成功地观察到光热信号的双色激发增强。利用钛蓝宝石激光器的三次谐波激发和其基波发射的探针激光器，研制了一种紫外激发光热显微镜。获得酵母细胞的光热图像。在细胞的光热振幅图像中，可以看到几个微米大小的特征，与细胞的大小相对应。结果表明，261 nm激光束激发出强烈的光热信号，同时在784 nm处透射探针光强度很小。在220、266、355,532和1064nm激光波段定量评价激光照射对酵母细胞生理功能的影响。结果表明，在相同能量密度条件下，532 nm光照射12 h后，酵母细胞的存活率是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.