Frequency-domain fluorescence spectroscopy using 280-nm and 300-nm light-emitting diodes: Measurement of proteins and protein-related fluorophores
Frequency-domain fluorescence spectroscopy using 280-nm and 300-nm light-emitting diodes: Measurement of proteins and protein-related fluorophores
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DOI：
10.1016/j.ab.2005.04.044
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发表时间：
2005-09-15
影响因子：
2.9
通讯作者：
Jameson, DM
中科院分区：
文献类型：
--
作者：
Barbieri, B;Terpetschnig, E;Jameson, DM
在过去的几十年里，荧光方法在许多科学和技术领域变得极其重要，包括分析化学、生物物理学、细胞生物学、临床化学和生物技术，仅举几例。荧光分子激发态寿命的测定通常是对感兴趣的过程或反应的定量评价的关键。荧光寿命的测量传统上是使用脉冲响应法（通过非常短的光脉冲来实现激发，然后观察荧光的直接衰减）或频域法（通过正弦调制激发光的强度并确定荧光相对于激发的相移）[1-3]。除了荧光寿命的测量，频域方法允许表征荧光团[3]的旋转模式。当然，用于寿命测量的激发光源的选择取决于目标荧光团的吸收特性。事实上，大多数常用的荧光团吸收波长在中紫外线或更长，即350纳米以上的光。对于这些探针，氩离子、氪离子、氦镉、氦氖等激光已被广泛应用[1-3]。最近，发光二极管（led） 1被用于时间分辨测量[4,5]。然而，为了研究内在的蛋白质荧光（来自色氨酸或酪氨酸残基），需要较深的紫外线光源，特别是270- 300纳米区域的光源。通常，这些波长是通过利用氙气弧灯与单色器（用于波长隔离）和波克尔斯电池（用于光调制）耦合获得的频域测量。另一种方法是利用锁模激光器、同步辐射源或高功率连续波氩离子激光器与波克尔斯电池耦合的谐波含量来实现这一目标。最近，McGuinness等人描述了一种新型发光二极管的使用，其发光波长接近280nm，作为激发源，用于对蛋白质进行时域荧光寿命测量。在本报告中，我们评估了这类led作为频域时间分辨测量的激励源的使用。
In the past few decades, fluorescence methods have become extremely important in many areas of science and technology including analytical chemistry, biophysics, cell biology, clinical chemistry, and biotechnology, to name but a few. Determinations of the excited-state lifetimes of fluorescent molecules are often critical for quantitative evaluation of the processes or reactions of interest. Fluorescence lifetime measurements have traditionally been realized using either the impulse-response method (in which excitation is achieved by a very brief pulse of light after which the direct decay of the fluorescence is observed) or the frequency-domain method (in which the intensity of the excitation light is modulated sinusoidally and the phase shift of the fluorescence, relative to the excitation, is determined)[1–3]. In addition to the measurement of the fluorescence lifetimes, the frequency-domain method permits characterization of the rotational modes of fluorophores [3]. The choice of excitation light sources for lifetime measurements depends, of course, on the absorption properties of the target fluorophore. In fact, most of the commonly used fluorophores absorb light at wavelengths in the mid-UV or longer, ie, above 350 nm. For these probes, lasers such as argon–ion, krypton–ion, helium–cadmium, and helium–neon have been widely utilized [1–3]. More recently, light-emitting diodes (LEDs) 1 have been used for time-resolved measurements [4, 5]. However, to study intrinsic protein fluorescence (from tryptophan or tyrosine residues), light sources in the deeper UV, specifically in the 270-to 300-nm region, are required. Often these wavelengths are obtained for frequency-domain measurements by utilizing a xenon–arc lamp coupled with a monochromator (for wavelength isolation) and a Pockels cell (for light modulation). Alternatively, this goal has been realized using the harmonic content of a mode-locked laser, a synchrotron radiation source, or a high-power continuous wave argon–ion laser coupled with a Pockels cell.Recently, McGuinness et al.[6] described the use of a new class of LEDs that emit near 280nm as the excitation source for time-domain fluorescence lifetime measurements on proteins. In this report we evaluate the use of this class of LEDs as an excitation source for frequency-domain time-resolved measurements.