Heisterkamp Alexander, Baumgart Judith, Maxwell Iva Z, Ngezahayo Anaclet, Mazur Eric, Lubatschowski Holger
Laser Zentrum Hannover, Hollerithallee 8, D-30419 Hannover, Germany.
Methods Cell Biol. 2007;82:293-307. doi: 10.1016/S0091-679X(06)82009-1.
The use of ultrashort laser pulses for microscopy has steadily increased over the past years. In this so-called multiphoton microscopy, laser pulses with pulse duration around 100 femtoseconds (fs) are used to excite fluorescence within the samples. Due to the high peak powers of fs lasers, the absorption mechanism of the laser light is based on nonlinear absorption. Therefore, the fluorescence signal is highly localized within the bulk of biological materials, similar to a confocal microscope. However, this nonlinear absorption mechanism can not only be used for imaging but for selective alteration of the material at the laser focus: The absorption can on one hand lead to the excitation of fluorescent molecules of fluorescently tagged cells by the simultaneous absorption of two or three photons or on the other hand, in case of higher order processes, to the creation of free-electron plasmas and, consequently, plasma-mediated ablation. Typical imaging powers are in the range of tens of milliwatts using 100-fs pulses at a repetition rate of 80-90 MHz, while pulse energies needed for ablation powers are as low as a few nanojoules when using high numerical aperture microscope objectives for focusing the laser radiation into the sample. Since the first demonstration of this technique, numerous applications of fs lasers have emerged within the field of cellular biology and microscopy. As the typical wavelengths of ultrashort laser systems lie in the near infrared between 800 and 1000 nm, high penetration depth can be achieved and can provide the possibility of imaging and manipulating the biological samples with one single laser system.
在过去几年中，超短激光脉冲在显微镜领域的应用稳步增加。在这种所谓的多光子显微镜中，脉冲持续时间约为100飞秒（fs）的激光脉冲被用于激发样品内的荧光。由于飞秒激光的高峰值功率，激光的吸收机制基于非线性吸收。因此，荧光信号在生物材料内部高度局域化，类似于共聚焦显微镜。然而，这种非线性吸收机制不仅可用于成像，还可用于在激光焦点处对材料进行选择性改变：一方面，吸收可通过同时吸收两个或三个光子导致荧光标记细胞的荧光分子被激发；另一方面，在高阶过程中，可导致自由电子等离子体的产生，进而导致等离子体介导的烧蚀。使用100飞秒脉冲、重复频率为80 - 90MHz时，典型的成像功率在几十毫瓦范围内，而当使用高数值孔径显微镜物镜将激光辐射聚焦到样品中时，烧蚀功率所需的脉冲能量低至几纳焦。自该技术首次被证明以来，飞秒激光在细胞生物学和显微镜领域出现了众多应用。由于超短激光系统的典型波长位于800至1000nm的近红外区域，可实现高穿透深度，并能提供使用单个激光系统对生物样品进行成像和操作的可能性。
