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首页> 外文会议>Conference on 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：用于多光子励磁激光扫描荧光成像的脉冲宽度考虑

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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.
机译：荧光显微镜是生物学家的无处不在的，强大的工具，主要是由于丰富的高度特异性荧光探针的可用性。多光子（两个或更多个光子）激发荧光显微镜是一种光学截面技术，其在改善生活材料的可行性和渗透到标本中的能力方面提供了优于其他光学切片技术的显着优点。使用较长的激发波长（通常是荧光团的激发峰的两倍）增加了激发到样品中的渗透，但基本上消除了样品的大部分中的单光子激发。为了获得生产多光子事件所需的高峰功率密度，同时保持低于破坏性水平的平均功率水平，使用超短脉冲激励源。一些来源，如模式锁定，Ti：蓝宝石激光器，可以产生小于100 fs的脉冲。这种短暂需要预先啁啾，以便补偿显微镜光学器件的群体速度分散，使得脉冲宽度保持在样品处。在没有这种预补偿的情况下，我们表明，使用典型的显微镜光学器件产生固定水平的平均功率，从60FS到250FS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，因为可以使用变化源脉冲宽度来改变两和三光子激发的相对量。通过预啁啾（补偿）系统，如果脉冲长度是四倍的，则需要两倍的功率来获得先前的双光子激发荧光水平，但只能产生三光子激发（或吸收）的一半。通过调整预补偿，可以在补偿系统上改变脉冲宽度。这可以用于赞成三相激发荧光荧光团的三光子激发，或者，另一方面，可能希望在使用700nm-800nm辐射的实时样品的两光子成像期间减少三光子激发的水平作为内源性荧光团的有害激发或通过（并因此损伤）蛋白质和核酸的吸收。因此，可变脉冲宽度可以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和更便宜的固定脉冲长度源。

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