Jolling Koen, Vandeven Martin, Van den Eynden Jimmy, Ameloot Marcel, Van Kerkhove Emmy
Hasselt University, Centre for Environmental Sciences, Dippenbeek, Belgium.
J Microsc. 2007 Dec;228(Pt 3):264-71. doi: 10.1111/j.1365-2818.2007.01858.x.
The SAC8.5, a low-cost Peltier-cooled black and white 8-bit CCD camera for astronomy, was evaluated for its use in imaging microscopy. Two camera-microscope configurations were used: an epifluorescence microscope (Nikon Eclipse TE2000-U) and a bottom port laser scanning confocal microscope system (Zeiss LSCM 510 META). Main advantages of the CCD camera over the currently used photomultiplier detection in the scanning setup are fast image capturing, stable background, an improved signal-to-noise ratio and good linearity. Based on DAPI-labelled Chinese Hamster Ovarian cells, the signal-to-noise ratio was estimated to be 4 times higher with respect to the currently used confocal photomultiplier detector. A linear relationship between the fluorescence signal and the FITC-inulin concentrations ranging from 0.05 to 1.8 mg mL(-1) could be established. With the SAC8.5 CCD camera and using DAPI, calcein-AM and propidium iodide we could also distinguish between viable, apoptotic and necrotic cells: exposure to CdCl(2) caused necrosis in A6 cells. Additional examples include the observation of wire-like mitochondrial networks in Mito Tracker Green-loaded Madin-Darby canine kidney cells. Furthermore, it is straightforward to interface the SAC8.5 with automated shutters to prevent rapid fluorophore photobleaching via easy to use astrovideo software. In this study, we demonstrate that the SAC8.5 black and white CCD camera is an easy-to-implement and cost-conscious addition to quantitative fluorescence microfluorimetry on living tissues and is suitable for teaching laboratories.
SAC8.5是一款用于天文学的低成本珀耳帖冷却黑白8位CCD相机，我们对其在成像显微镜中的应用进行了评估。使用了两种相机-显微镜配置：一台落射荧光显微镜（尼康Eclipse TE2000-U）和一台底部端口激光扫描共聚焦显微镜系统（蔡司LSCM 510 META）。在扫描设置中，CCD相机相对于目前使用的光电倍增管检测的主要优点是图像捕捉速度快、背景稳定、信噪比提高以及线性良好。基于用DAPI标记的中国仓鼠卵巢细胞，估计其信噪比相对于目前使用的共聚焦光电倍增管探测器高出4倍。可以建立荧光信号与浓度范围为0.05至1.8 mg mL(-1)的FITC-菊粉之间的线性关系。使用SAC8.5 CCD相机并结合DAPI、钙黄绿素-AM和碘化丙啶，我们还可以区分活细胞、凋亡细胞和坏死细胞：暴露于CdCl(2)会导致A6细胞坏死。其他例子包括观察用Mito Tracker Green标记的Madin-Darby犬肾细胞中的丝状线粒体网络。此外，通过易于使用的astrovideo软件将SAC8.5与自动快门连接起来，以防止荧光团快速光漂白也很简单。在本研究中，我们证明SAC8.5黑白CCD相机是一种易于实现且经济实惠的设备，可用于对活组织进行定量荧光显微荧光测定，适用于教学实验室。
