Loss of image quality in photobleaching during microscopic imaging of fluorescent probes bound to chromatin
Loss of image quality in photobleaching during microscopic imaging of fluorescent probes bound to chromatin
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DOI：
10.1117/1.2136313
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发表时间：
2005-11-01
影响因子：
3.5
通讯作者：
Rajwa, B
中科院分区：
文献类型：
--
作者：
Bernas, T;Robinson, JP;Rajwa, B
荧光探针的长时间激发最终导致其发光能力的丧失。检测到的光子数量的减少随后降低了荧光显微镜的分辨能力。荧光强度损失对生物标本显微图像质量的不利影响已被认识到，但尚未定量确定。我们提出了三种不依赖于人的质量测定方法。这些技术不需要参考图像，基于实际分辨率距离、信息熵和信噪比（SNR）的计算。我们应用这三种方法研究了碘化丙啶（PI）和色霉素A3 （CA(3)）染色和荧光共聚焦显微镜成像对细胞核光漂白的影响。我们得出结论，图像质量的相对损失小于相应的荧光强度的下降。此外，质量损失的程度与成像系统的光学特性和检测器的噪声特性有关。我们讨论了这些发现对生物图像的最佳配准和压缩的重要性。(C) 2005光学仪器工程师学会。
Prolonged excitation of fluorescent probes leads eventually to loss of their capacity to emit light. A decrease in the number of detected photons reduces subsequently the resolving power of a fluorescence microscope. Adverse effects of fluorescence intensity loss on the quality of microscopic images of biological specimens have been recognized, but not determined quantitatively. We propose three human-independent methods of quality determination. These techniques require no reference images and are based on calculation of the actual resolution distance, information entropy, and signal-to-noise ratio (SNR). We apply the three measures to study the effect of photobleaching in cell nuclei stained with propidium iodide (PI) and chromomycin A3 (CA(3)) and imaged with fluorescence confocal microscopy. We conclude that the relative loss of image quality is smaller than the corresponding decrease in fluorescence intensity. Furthermore, the extent of quality loss is related to the optical properties of the imaging system and the noise characteristics of the detector. We discuss the importance of these findings for optimal registration and compression of biological images. (C) 2005 Society of Photo-Optical Instrumentation Engineers.