Recording the Instrument Response Function of a Multiphoton FLIM System
Recording the Instrument Response Function of a Multiphoton FLIM System
复制标题
记录多光子 FLIM 系统的仪器响应函数
DOI：
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发表时间：
2008
期刊：
影响因子：
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通讯作者：
E. W. Jonsson
中科院分区：
文献类型：
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作者：
M. Arnlind;M. Nokela;C. Rehnberg;E. W. Jonsson
FLIM data analysis in presence of SHG signals or extremely fast decay components requires the correct instrument response function (IRF) to be used. This application note describes how an accurate IRF is obtained in a multiphoton microscope. Theoretical Background A measurement in the FLIM modes of the bh SPC-150 or SPC-830 TCSPC modules delivers the photon distribution over the coordinates of the scan, the time within the fluorescence decay, and, if several detector in different spectral channels are used, the wavelength [1]. The data can be considered an array of pixels, each containing a large number of time channels spread over the fluorescence decay. In other words, the FLIM measurement delivers images with a decay curve in each pixel. Several such arrays may exist if several detectors or a multi-wavelength detector are used. To obtain fluorescence lifetimes from these data the decay curves in the individual pixels must be fitted with an appropriate model. However, the time resolution of the measurement system is finite. Therefore the fitting routine has to take the ‘instrument response function’ (IRF) into account. The IRF is the pulse shape the FLIM system records for an infinitely short fluorescence lifetime. The fitting procedure convolutes the model function with the IRF and compares the result with the photon numbers in the subsequent time channels of the current pixel. Then it varies the model parameters until the best fit between the convoluted model function and the measured decay data is obtained [7]. At first glance, recording the IRF of a FLIM system may appear easy. The emission filters would be removed or replaced with bandpass filters centred at the laser wavelength, and a scattering signal would be recorded from a scattering target. Unfortunately, recording an accurate IRF in a laser scanning microscope is much more difficult. The obvious problem is that the dichroic beamsplitter of the microscope does not pass the excitation wavelength to the detectors. Moreover, the excitation beam path usually contains a laser blocking filter that cannot easily be removed. In a two-photon microscope it may even happen that the FLIM detector is not sensitive to the excitation wavelength altogether. Of course, by increasing the laser power something is detected in any PMT. However, a photocathode for the visible spectrum is virtually transparent in the NIR, and a large fraction of the photoelectrons may be emitted not from the cathode but from the first dynode. The detector response recorded in the NIR is therefore not necessarily identical with the response at the normal detection wavelength. Moreover, a signal detected at the laser wavelength is usually contaminated by reflections and scattering in the optical system, and by fluorescence from the target [2]. The bh SPCImage and Optispec FLIM data analysis software therefore allows the user to derive an estimated IRF from the fluorescence signal itself. The IRF estimation works at satisfactory accuracy for fluorescence signals with lifetimes a few times longer than the width of the IRF [3, 4, 5]. However, if the fluorescence contains extremely fast components, or if second-harmonic signals are
