CIL:8927, Mus musculus, permanent cell line cell. In Cell Image Library

This is part of a triplicate data set of non-overlapping fields of NIH 3T3 fibroblasts cultured on polystyrene. Each set is indicated by the well number. Each image contains 4 images of each field as a time series. The first image is the phase contrast image. The second image channel is Tenascin-C promoter driven destabilized EGFP reporter vector. The third image is Texas Red C2-maleimide (to stain cell body), and the fourth image is Dapi (to stain nuclei).

Images were collected on a Zeiss Axiovert 100. The samples were viewed with a Zeiss A-plan 10x Ph1 0.25 NA objective and recorded with a CoolSnapFX camera using 2 x 2 binning. The filters were as follows: 1.) A custom dichroic multipass beam splitter optimized for imaging DAPI, EGFP and TxRed (part# BS51019+400dclp, Chroma Technology, VT); 2.) DAPI excitation filter-360/40 nm; 3.) DAPI emission filter-460/50 nm; 4.) EGFP excitation filter-470/40 nm; 5.) EGFP emission filter-525/50 nm; 6.) TxRed excitation filter-568/24 nm; 7.) TxRed emission filter-630/60 nm. Autofocus on the TxRed color channel was performed at each location before the series of images were collected.

Protocol: The cells were seeded on TCPS dishes at ~1000 cells/well during a passage cycle. The test cultures were incubated overnight before being rinsed with PBS and then fixed with 300 uM m-maleimidobenzoyl-N-hydroxysuccinimidyl ester (MBS) in microtubule stabilizing buffer (MTSB) composed of 4% (w/v) polyethylene glycol (PEG) 8000, 100 mM 1,4-piperazinediethanesulfonic acid (PIPES),10 mM ethylene glycol-bis(2-aminoethylether)-N,N,N',N'-tetraacetic acid (EGTA), pH 6.9 for at least 16h at RT. The fixative was removed and solution of 0.05% Triton X100 in PBS containing 10 ng/mL of Tx Red C2-maleimide and 2 ng/mL DAPI for 2h. The staining solution was removed, cells were rinsed with PBS/3% BSA, and 0.05% sodium azide and PBS. A 50% glycerol/10 mM Tris, pH 8.0 containing 2ng/mL DAPI and 0.9g/l 1,4-diazobicyclo[2,2,2]octane (DABCO) as an antifade reagent was then added to each well for imaging purposes. The bottom of the wells were wiped with 70% ethanol wipe and then a dry wipe before imaging.

Purpose: The purpose of the dataset was to measure the distribution of EGFP fluorescence intensities within individual cells in the population. Using the image sets collected, a plugin for ImageJ was used to perform the following image analysis tasks. 1.) The Txred images (which is a general purpose cell body stain) were segmented by manual thresholding. 2.) The resulting mask was used to define cell ROIs on the DAPI and EGFP images. 3.) The number of nuclei in each ROI is determined from the DAPI image and the integrated intensity of the EGFP signal in the cell is determined from ROI in the EGFP image. 4.) A local background intensity around each cell in the EGFP image is determined by dilating the ROI by 3 pixels and determining the pixel intensities in only the 3 pixel dilation area. When this data is placed in a spread sheet, you can use the results to identify cell clusters (i.e. have more than 1 nuclei), debris or partial cell (i.e. no nuclei), poor EGFP/cell measurements (i.e. high background intensity). The spreadsheet can be used to measure the distribution of EGFP cell intensities within a population of cells. The phase images were collected as quality control and validation data. The phase images provide a human validation mechanism if there are questions about the staining and/or fluorescence detection in an image.

References: 1. Langenbach, K.J., Elliott, J.T., Tona, A., and Plant, A.L. (2006) Evaluating the correlation between fibroblast morphology and promoter activity on thin films of extracellular matrix proteins. BMC-Biotechnology 6(1):14. 2.Elliott, J.T., Halter, M., Woodward, J.T., Langenbach, K.J., Tona, A., Plant, A.L. (2008) Evaluating the performance of fibrillar collagen films formed at polystyrene surfaces as cell culture substrates. Biointerphases. 3(2):19-28.

本数据集为聚苯乙烯（polystyrene）培养的NIH 3T3成纤维细胞（NIH 3T3 fibroblasts）非重叠视野的三份重复实验数据集的一部分，每套数据集以孔号标识。每个视野对应4幅时序图像：第一幅为相差显微镜图像（phase contrast image）；第二幅成像通道为腱生蛋白-C（Tenascin-C）启动子驱动的不稳定增强型绿色荧光蛋白（enhanced green fluorescent protein, EGFP）报告载体的成像结果；第三幅为Texas Red C2-马来酰亚胺（Texas Red C2-maleimide）染色（用于标记细胞体）；第四幅为4',6-二脒基-2-苯基吲哚（4',6-diamidino-2-phenylindole, DAPI）染色（用于标记细胞核）。

图像采集使用蔡司Axiovert 100显微镜（Zeiss Axiovert 100）完成。样本采用蔡司A-plan 10× Ph1 0.25数值孔径（numerical aperture, NA）物镜（Zeiss A-plan 10x Ph1 0.25 NA objective）成像，并通过CoolSnapFX相机（CoolSnapFX camera）以2×2像素合并模式采集图像。所用滤光片参数如下：1. 针对DAPI、EGFP和TxRed成像优化的定制二向色多通分色镜（dichroic multipass beam splitter，货号BS51019+400dclp，Chroma Technology公司，佛蒙特州，美国）；2. DAPI激发滤光片（excitation filter）：360/40 nm；3. DAPI发射滤光片（emission filter）：460/50 nm；4. EGFP激发滤光片：470/40 nm；5. EGFP发射滤光片：525/50 nm；6. TxRed激发滤光片：568/24 nm；7. TxRed发射滤光片：630/60 nm。在采集每一系列图像前，需在每个视野处对TxRed彩色通道执行自动对焦。

实验方案：传代培养（passage cycle）期间，将细胞以约1000个/孔的密度接种于组织培养聚苯乙烯（tissue culture polystyrene, TCPS）培养皿中。待测培养物先过夜孵育，随后用磷酸盐缓冲液（phosphate buffered saline, PBS）冲洗，再置于由4%（w/v）聚乙二醇（polyethylene glycol, PEG）8000、100 mM 1,4-哌嗪二乙磺酸（1,4-piperazinediethanesulfonic acid, PIPES）、10 mM 乙二醇双(2-氨基乙基醚)-N,N,N',N'-四乙酸（ethylene glycol-bis(2-aminoethylether)-N,N,N',N'-tetraacetic acid, EGTA）组成、pH 6.9的微管稳定缓冲液（microtubule stabilizing buffer, MTSB）中，加入300 μM 间-马来酰亚胺苯甲酰-N-羟基琥珀酰亚胺酯（m-maleimidobenzoyl-N-hydroxysuccinimidyl ester, MBS）固定至少16小时，室温（room temperature, RT）静置。移除固定液后，加入含0.05% Triton X100、10 ng/mL Texas Red C2-马来酰亚胺及2 ng/mL DAPI的PBS溶液孵育2小时。弃去染色液后，用含3%牛血清白蛋白（bovine serum albumin, BSA）的PBS、0.05%叠氮化钠（sodium azide）及PBS依次冲洗细胞。随后向每孔加入含2 ng/mL DAPI与0.9 g/L 1,4-二氮杂双环[2.2.2]辛烷（1,4-diazobicyclo[2,2,2]octane, DABCO，抗荧光淬灭试剂）的50%甘油/10 mM Tris（pH 8.0）溶液，用于后续成像。成像前需先用70%乙醇擦拭孔板底部，再用无尘擦拭布擦干。

数据集用途：本数据集旨在检测群体中单个细胞内EGFP荧光强度的分布情况。利用采集的图像集，通过ImageJ插件可执行以下图像分析任务：1. 以手动阈值分割（manual thresholding）法对TxRed图像（通用细胞体染色标记）进行分割；2. 利用生成的掩膜（mask）在DAPI和EGFP图像中定义细胞感兴趣区域（region of interest, ROI）；3. 从DAPI图像中统计每个ROI内的细胞核数量，并从EGFP图像对应ROI中计算细胞内EGFP信号的积分光密度（integrated intensity）；4. 通过将ROI膨胀3个像素，仅在该膨胀区域内提取像素强度，以此计算EGFP图像中每个细胞周围的局部背景强度。将上述数据导入电子表格（spread sheet）后，可通过分析结果识别细胞簇（含多个细胞核）、细胞碎片或部分细胞（无细胞核）以及EGFP/细胞测量质量不佳（背景强度过高）的样本。电子表格可用于分析细胞群体内EGFP细胞荧光强度的分布情况。相差图像作为质量控制（quality control）与验证数据采集，当对图像的染色或荧光检测结果存在疑问时，可通过相差图像开展人工验证。

参考文献：1. Langenbach, K.J., Elliott, J.T., Tona, A., and Plant, A.L. (2006) 评估成纤维细胞形态与细胞外基质蛋白薄膜上启动子活性的相关性。《BMC生物技术》（BMC-Biotechnology）, 6(1):14。2. Elliott, J.T., Halter, M., Woodward, J.T., Langenbach, K.J., Tona, A., Plant, A.L. (2008) 评估聚苯乙烯表面形成的纤维胶原薄膜作为细胞培养底物的性能。《生物界面》（Biointerphases）, 3(2):19-28。