Müller M, Squier J, Wolleschensky R, Simon U, Brakenhoff GJ
BioCentrum Amsterdam, University of Amsterdam, Institute of Molecular Cell Biology, Kruislaan 316, 1098 SM Amsterdam, The Netherlands.
J Microsc. 1998 Aug;191(2):141-150. doi: 10.1046/j.1365-2818.1998.00357.x.
The excitation efficiency in two-photon absorption (TPA) microscopy depends strongly - owing to the square dependence of the TPA fluorescence on the excitation intensity - on the temporal width of the excitation pulse. Because of their inherently large frequency bandwidth, ultrashort optical pulses tend to broaden substantially because of dispersion from propagation through the dispersive elements in the microscope. In this paper, the dispersion characteristics of a wide range of microscope objectives are investigated. It is shown that the induced dispersion can be pre-compensated in all cases for pulses as short as 15 fs. Because of the excellent agreement between the results from theoretical modelling and the experimental data, predictions of the possibility of dispersion control for microscope objectives in general, as well as for even shorter pulses, can be inferred. Since for TPA imaging the background due to single photon absorption processes and scattering is independent of the pulse width, proper dispersion pre-compensation - which minimizes the pulse duration at the focal point and hence maximizes the excitation efficiency - provides optimal image contrast in TPA microscopy.
在双光子吸收（TPA）显微镜中，由于TPA荧光与激发强度的平方关系，激发效率强烈依赖于激发脉冲的时间宽度。由于其固有的大频率带宽，超短光脉冲在通过显微镜中的色散元件传播时，往往会因色散而大幅展宽。本文研究了多种显微镜物镜的色散特性。结果表明，对于短至15飞秒的脉冲，在所有情况下都可以对诱导色散进行预补偿。由于理论建模结果与实验数据之间的良好一致性，可以推断出一般显微镜物镜以及更短脉冲的色散控制可能性。由于对于TPA成像，单光子吸收过程和散射引起的背景与脉冲宽度无关，适当的色散预补偿——可使焦点处的脉冲持续时间最小化，从而使激发效率最大化——能在TPA显微镜中提供最佳的图像对比度。
