Marriott G, Clegg R M, Arndt-Jovin D J, Jovin T M
Department of Molecular Biology, Max Planck Institute for Biophysical Chemistry, Göttingen, Federal Republic of Germany.
Biophys J. 1991 Dec;60(6):1374-87. doi: 10.1016/S0006-3495(91)82175-0.
An optical microscope capable of measuring time resolved luminescence (phosphorescence and delayed fluorescence) images has been developed. The technique employs two phase-locked mechanical choppers and a slow-scan scientific CCD camera attached to a normal fluorescence microscope. The sample is illuminated by a periodic train of light pulses and the image is recorded within a defined time interval after the end of each excitation period. The time resolution discriminates completely against light scattering, reflection, autofluorescence, and extraneous prompt fluorescence, which ordinarily decrease contrast in normal fluorescence microscopy measurements. Time resolved image microscopy produces a high contrast image and particular structures can be emphasized by displaying a new parameter, the ratio of the phosphorescence to fluorescence. Objects differing in luminescence decay rates are easily resolved. The lifetime of the long lived luminescence can be measured at each pixel of the microscope image by analyzing a series of images that differ by a variable time delay. The distribution of luminescence decay rates is displayed directly as an image. Several examples demonstrate the utility of the instrument and the complementarity it offers to conventional fluorescence microscopy.
已开发出一种能够测量时间分辨发光（磷光和延迟荧光）图像的光学显微镜。该技术采用两个锁相机械斩波器和一个连接到普通荧光显微镜的慢扫描科学电荷耦合器件（CCD）相机。样品由周期性的光脉冲序列照射，并在每个激发周期结束后的规定时间间隔内记录图像。时间分辨率完全排除了光散射、反射、自发荧光和外来即时荧光的干扰，这些通常会降低普通荧光显微镜测量中的对比度。时间分辨图像显微镜产生高对比度图像，通过显示一个新参数——磷光与荧光的比率，可以突出特定结构。发光衰减率不同的物体很容易分辨出来。通过分析一系列具有可变时间延迟的图像，可以在显微镜图像的每个像素处测量长寿命发光的寿命。发光衰减率的分布直接显示为一幅图像。几个例子展示了该仪器的实用性及其对传统荧光显微镜的互补性。
