arXiv:2410.19210q-bio.QMeess.IV2024-10被引 9

教科研人员如何选相机拍低光荧光显微镜图像,兼顾画质与生物样本安全。

Optimising image capture for low-light widefield quantitative fluorescence microscopy

  • 对比EMCCD/qCMOS/sCMOS相机在低光下的表现差异
  • 实测显示不同相机在活体胚胎成像中噪声特性各异
  • 适合做活细胞定量荧光研究的学者参考

低光光学成像指在极低光子通量下使用相机捕获图像,广泛应用于生物研究中的光学显微镜。为减少光漂白和光毒性对生物系统的影响,需降低光照强度,但同时要保证图像质量满足定量分析需求。例如,利用自体荧光强度计算的光学氧化还原比可反映代谢状态。本文针对活体生物样本的低光荧光成像挑战,系统分析了EMCCD、qCMOS和sCMOS等专用科学相机在平台架构与噪声来源上的差异。教程涵盖用户可调参数及后处理去噪算法的应用,并以双光子光片荧光显微镜拍摄的活哺乳动物胚胎自体荧光图像为例进行说明。

原文摘要 · Abstract (English)

Low-light optical imaging refers to the use of cameras to capture images with minimal photon flux. This area has broad application to diverse fields, including optical microscopy for biological studies. In such studies, it is important to reduce the intensity of illumination to reduce adverse effects such as photobleaching and phototoxicity that may perturb the biological system under study. The challenge when minimising illumination is to maintain image quality that reflects the underlying biology and can be used for quantitative measurements. An example is the optical redox ratio which is computed from autofluorescence intensity to measure metabolism. In all such cases, it is critical for researchers to optimise selection and application of scientific cameras to their microscopes, but few resources discuss performance in the low-light regime. In this tutorial, we address the challenges in optical fluorescence imaging at low-light levels for quantitative microscopy, with an emphasis on live biological samples. We analyse the performance of specialised low-light scientific cameras such as the EMCCD, qCMOS, and sCMOS, while considering the differences in platform architecture and the contribution of various sources of noise. The tutorial covers a detailed discussion of user-controllable parameters, as well as the application of post-processing algorithms for denoising. We illustrate these concepts using autofluorescence images of live mammalian embryos captured with a two-photon light sheet fluorescence microscope.

荧光显微镜低光成像生物样本相机选型

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