Photothermal and Photoionization Spectroscopies of Chemical Reactions in a Single Cell

大多数重要的生物化学物质，如氨基酸、核苷酸等，都是天然的非荧光物质，其中很大一部分只有在紫外光波长下才有光吸收。光热和光电离光谱方法在这些化学物质的超高灵敏度原位检测方面具有很好的潜力。我们的目标是发展对活细胞中非荧光分子的超灵敏检测和成像方法。为了对生物化学物质进行超灵敏检测，设计了一种多色激发下的光热信号检测器，其中利用瞬时吸收来增强信号。建立了紫外光激发-可见增强光热检测系统，并与微型高效液相色谱系统联用，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一种紫外线吸收化学品的混合物。成功地观察到了双色激发诱导的光热信号增强。利用钛宝石激光器的三次谐波发射及其基波发射的探测激光器，研制了紫外光激发光热显微镜。获得了酵母细胞的光热图像。在细胞的光热幅度图像中，可以看到几个微米大小的特征，对应于细胞的大小。观察到由261 nm激光激发的强光热信号伴随着784 nm处较小的透射光强度，利用220、266、355、532或1064 nm的激光定量评价了激光照射对酵母细胞生理功能的影响。结果表明，在相同的能量密度条件下，酵母细胞经532 nm光照射12h后的存活概率是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.