CIL:8878, 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培养的NIH 3T3成纤维细胞非重叠视野的三份重复实验数据集的一部分，每组数据集以孔号标识。每个视野的图像集由4幅时序图像组成：第一幅为相差图像；第二幅为由腱生蛋白-C（Tenascin-C）启动子驱动的不稳定增强型绿色荧光蛋白（EGFP）报告载体的荧光图像；第三幅为Texas Red C2-马来酰亚胺染色图像（用于标记细胞体）；第四幅为4',6-二脒基-2-苯基吲哚（DAPI）染色图像（用于标记细胞核）。

图像采集于Zeiss Axiovert 100型显微镜。样本采用Zeiss A-plan 10× Ph1 0.25 NA物镜成像，并通过CoolSnapFX相机以2×2的像素合并模式采集图像。所用滤光组件如下：1. 针对DAPI、EGFP及TxRed成像优化的定制多通道二向色分光镜（货号BS51019+400dclp，Chroma Technology公司，佛蒙特州）；2. DAPI激发滤光片：360/40 nm；3. DAPI发射滤光片：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。在采集每个视野的图像序列前，均以TxRed通道进行自动对焦。

实验方案：细胞以约1000个/孔的密度接种于组织培养聚苯乙烯（TCPS）培养皿，处于传代周期内。待测培养物先孵育过夜，随后用磷酸盐缓冲液（PBS）冲洗，再置于含300 μM 间马来酰亚胺苯甲酰-N-羟基琥珀酰亚胺酯（MBS）的微管稳定缓冲液（MTSB）中固定；该缓冲液成分为4%（w/v）聚乙二醇（PEG）8000、100 mM 1,4-哌嗪二乙磺酸（PIPES）、10 mM 乙二醇双(2-氨基乙基醚)-N,N,N',N'-四乙酸（EGTA），pH 6.9，于室温固定至少16小时。弃去固定液后，将细胞置于含0.05% Triton X100、10 ng/mL Texas Red C2-马来酰亚胺及2 ng/mL DAPI的PBS溶液中孵育2小时。弃去染色液后，依次用含3%牛血清白蛋白（BSA）的PBS、0.05%叠氮化钠PBS及纯PBS冲洗细胞。随后向每个孔中加入含50%甘油、10 mM Tris（pH 8.0）、2 ng/mL DAPI及0.9 g/L 1,4-二氮杂双环[2.2.2]辛烷（DABCO，抗荧光淬灭剂）的溶液，用于后续成像。成像前，先用70%乙醇擦拭孔板底部，再用无尘布擦干。

研究目的：本数据集旨在检测细胞群体中单个细胞内EGFP荧光强度的分布情况。利用采集得到的图像集，通过ImageJ插件完成以下图像分析任务：1. 以手动阈值分割法对Texas Red染色的细胞图像（通用细胞体染色标记）进行分割；2. 利用生成的掩膜在DAPI及EGFP图像中划定细胞感兴趣区域（ROI）；3. 基于DAPI图像统计每个ROI内的细胞核数量，并基于EGFP图像的ROI测定细胞内EGFP信号的总荧光强度；4. 通过将ROI向外扩张3个像素，仅测定扩张区域内的像素强度，以此确定EGFP图像中每个细胞周围的局部背景荧光强度。将上述数据导入电子表格后，可通过分析结果识别细胞簇（即含多个细胞核）、细胞碎片或不完全细胞（即无细胞核）以及EGFP荧光检测质量不佳的样本（即背景荧光强度过高）。该电子表格可用于统计细胞群体内EGFP荧光强度的分布情况。相差图像作为质量控制与验证数据采集，若对图像的染色效果或荧光检测结果存在疑问，可通过相差图像进行人工验证。

参考文献：
1. Langenbach KJ, Elliott JT, Tona A, Plant AL. (2006) 细胞形态与细胞外基质蛋白薄膜上的启动子活性相关性评估. BMC生物技术, 6(1):14.
2. Elliott JT, Halter M, Woodward JT, Langenbach KJ, Tona A, Plant AL. (2008) 聚苯乙烯表面形成的纤维状胶原薄膜作为细胞培养底物的性能评估. 生物界面, 3(2):19-28.