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首页> 外文会议>Optical Investigations of Cells In Vitro and In Vivo >Pulse-width considerations for multiple-photon excitation laser scanning fluorescence imaging
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Pulse-width considerations for multiple-photon excitation laser scanning fluorescence imaging

机译：多光子激发激光扫描荧光成像的脉冲宽度注意事项

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Abstract: Fluorescence microscopy is a ubiquitous and powerful tool for the biologist mainly due to the availability of a wealth of highly specific fluorescent probes. Multiphoton (two or more photon) excitation fluorescence microscopy is an optical sectioning technique that offers significant advantages over other optical sectioning techniques in terms of improved viability of living material and the ability to penetrate deeper into specimens. The use of a longer excitation wavelength (typically twice that of the excitation peak of the fluorophore) increases the penetration of the excitation into the sample, yet essentially eliminates single-photon excitation in the bulk of the sample. In order to attain the high peak-power densities necessary for the production of multiphoton events while keeping mean power levels below damaging levels, ultrashort-pulsed excitation sources are used. Some sources, such as mode-locked, Ti:sapphire lasers, can produce pulses less than 100 fs. Pulses this short need to be pre-chirped in order to compensate for the group velocity dispersion of the microscope optics so that the pulse width is maintained at the sample. Without such pre-compensation we show that the average power required to produce a fixed level of two-photon excited signal, using typical microscope optics, is fairly constant from 60fs to 250fs. We argue that the choice of pulse width is an important consideration for a biological imaging system since varying the source pulse width may be used to change the relative amounts of two- and three- photon excitation. With a pre-chirped (compensated) system, if the pulse length is quadrupled then twice the power will be required to attain the previous level of two-photon excited fluorescence, but only half the three-photon excitation (or absorption) will be produced. Pulse widths may be varied on compensated systems by adjusting the pre-compensation. This may be used to favor three-photon excitation of UV-excited fluorophores, or, on the other hand, it may be desirable to reduce levels of three-photon excitation during two-photon imaging of live samples using 700 nm - 800 nm radiation as deleterious excitation of endogenous fluorophores or absorption by (and therefore damage to) proteins and nucleic acids could occur. Variable pulse widths may therefore prove to be an important parameter for live cell studies. Alternatively, for a given range of applications, a simpler and cheaper fixed-pulse length source with the desired characteristics may be chosen. !9
机译：摘要：荧光显微镜是生物学家无处不在且功能强大的工具，主要是因为有大量的高特异性荧光探针可供使用。多光子（两个或多个光子）激发荧光显微技术是一种光学切片技术，在改善活体材料的生存力和更深地渗透到标本方面，与其他光学切片技术相比，具有明显的优势。使用更长的激发波长（通常是荧光团激发峰波长的两倍）可以增加激发到样品中的穿透力，但实际上消除了大部分样品中的单光子激发。为了获得产生多光子事件所需的高峰值功率密度，同时将平均功率水平保持在破坏水平以下，使用了超短脉冲激发源。某些光源（例如锁模Ti：蓝宝石激光器）可以产生小于100 fs的脉冲。为了补偿显微镜光学系统的群速度色散，需要对这种短脉冲进行预调频，以便在样品处保持脉冲宽度。如果没有这种预补偿，我们将证明使用典型的显微镜光学器件产生固定水平的双光子激发信号所需的平均功率在60fs至250fs范围内相当恒定。我们认为，脉冲宽度的选择是生物成像系统的重要考虑因素，因为改变源脉冲宽度可用于改变两个和三个光子激发的相对量。对于预chi（补偿）系统，如果脉冲长度增加三倍，则将需要两倍的功率才能达到先前的两光子激发荧光水平，但仅产生三光子激发（或吸收）的一半。通过调节预补偿，可以在补偿系统上改变脉冲宽度。这可用于促进紫外线激发荧光团的三光子激发，或者，另一方面，在使用700 nm-800 nm辐射对活样品进行两光子成像期间，可能希望降低三光子激发的水平可能会引起内源性荧光团的有害激发或被蛋白质和核酸吸收（并因此受到损害）。因此，可变脉冲宽度可能被证明是活细胞研究的重要参数。或者，对于给定的应用范围，可以选择具有所需特性的更简单，更便宜的固定脉冲长度源。 ！9

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