Fibrinolysis_Data_Bead_Conc.xlsx

Data are results from experiments investigating fluoresnce labeling on fibrinolysis. Samples and analysis were prepared and performed in the following way: <br> On a glass microscope slide a small amount of optical glue (Norland Optical Adhesive 81, Norland Products, Cranbury, NJ), pressed under a PDMS stamp, was cured under ultraviolet light, resulting in a ridged surface with gaps between ridges measuring 20 μm wide and 10 μm deep. Aliquots of peak 1 fibrinogen (Enzyme Research Laboratories, South Bend, IN), ALEXA-488 fibrinogen (Invitrogen by Thermo Fisher Scientific/Life Technologies Corporation, Eugene, OR), and human alpha thrombin (Enzyme Research Laboratories, South Bend, IN) were thawed from -80°C. In a microcentrifuge tube, fibrinogen was diluted to 0.6 mg/mL with a HEPES Buffered Saline (HBS; 20 mM HEPES, 150 mM NaCl, pH 7.4) and mixed with ALEXA-488 fibrinogen at 1/65th the fibrinogen concentration. Separately, the thrombin aliquot was diluted to 2 U/mL using HBS with CaCl2. 10 µL of fibrinogen solution was pipetted onto the ridged surface and was mixed with 10 µL of thrombin solution, giving a final fibrinogen concentration of 0.3 mg/mL and final thrombin concentration of 1 U/mL on the slide. The samples were sealed inside a petri dish along with damp paper to ensure a moist atmosphere for the sample and left to polymerize for one hour at 37°C [14]. After polymerization, a pipette was used to gently remove a superficial fibrin mesh, leaving a 2-D fibrin network on the ridged surface. In experiments where beads were used, 30 µL of a 20 nm carboxy-coated microsphere (FluoSpheres™ Carboxylate-Modified Microspheres, Invitrogen by Thermo Fisher Scientific/Life Technologies Corporation, Eugene, OR) (interchangeably referred to as beads) solution, at the specified dilution, was sonicated for 5 minutes and added to the sample. The bead solution was incubated with the fibrin for 1 minute at room temperature and then gently pipetted off. The sample was then washed three times with 30 µL of HBS buffer to ensure removal of any excess bead solution. Following the washes, 20 µL of HBS buffer was placed on top of the sample prior to conducting microscopy imaging. Samples were imaged on a Lieca DMi8 epifluorescent microscope (Leica Microsystems Inc., Buffalo Grove, IL), using a 63x oil-immersion objective. Using light with a wavelength of 480 nm, corresponding to the excitation wavelength of ALEXA-488 labeled fibrinogen, an imaging area was located. Sample areas containing at least 4 individual fibers, each separated by at least 1µm within the camera frame were selected for imaging to maximize the rate of data acquisition. Once an area was found, the microscope settings were changed to a wavelength of 553 nm, the excitation wavelength of the carboxylate-coated polystyrene beads (580 nm) under investigation. We located the fibers using the 480 nm wavelength to minimize fluorescent excitation of the beads while locating the fibers, because the bead excitation spectrum has a minimum ~480 nm, per the manufacturer’s data sheet. This was done because we suspected that the beads may be sensitive to light dosage. While later experiments did not bear this hypothesis out (see results), we maintained this experimental approach. To initiate lysis, plasmin (Enzyme Research Laboratories, South Bend, IN) was added directly to the sample and then time-series acquisition began immediately afterwards. After the addition of plasmin, the attached microscope camera (Leica DFC9000GT SCMOS 4 Megapixel monochrome camera) captured an image every 30 seconds for one hour. The camera shutter was set to remain open during the entire imaging process, unless otherwise stated. When the time-lapse was completed, the file was imported into FIJI image analysis software where the singular fibers were analyzed. In the initial frame, single fibers, all of which presented as tensed structures, were counted and designated for further analysis. Cleaving times were found by analyzing each image series frame-by-frame until finding the frame in which the fiber cleaved. The number of frames prior to cleavage was multiplied by the time interval between frames to find the cleavage time. Additionally, during these intermediate frames the occurrences of bundling were quantified, categorized as two neighboring, largely disconnected fibers, joined to form a singular new fiber. Over the course of imaging, many fibers lost the tension they presented in the initial frames and became loose structures. We identified these fibers that lost their initial tension as “elongated”, and they were counted in the final frame of the timelapse, along with any fibers that remained tensed.

本数据集为探究纤维蛋白溶解（fibrinolysis）过程中荧光标记效果的实验结果。样品制备与分析流程如下：
在显微镜载玻片上滴加少量光学胶（Norland Optical Adhesive 81，Norland Products公司，美国新泽西州克兰伯里市），将聚二甲基硅氧烷（PDMS）印章按压于胶层之上，经紫外光固化后得到带有脊状结构的表面，脊间间隙宽度为20 μm，深度为10 μm。
取峰值1纤维蛋白原（Enzyme Research Laboratories公司，美国印第安纳州南本德市）、ALEXA-488标记纤维蛋白原（Invitrogen，赛默飞世尔科技/赛默飞生命科技公司，美国俄勒冈州尤金市）及人α凝血酶（Enzyme Research Laboratories公司，美国印第安纳州南本德市）的冻存样品，从-80℃冰箱中取出解冻。
在微量离心管中，用HEPES缓冲生理盐水（HBS：20 mM HEPES，150 mM NaCl，pH 7.4）将纤维蛋白原稀释至0.6 mg/mL，并按纤维蛋白原浓度的1/65比例加入ALEXA-488标记纤维蛋白原，混合均匀。另取凝血酶冻存液，使用含氯化钙的HBS缓冲液将其稀释至2 U/mL。
用移液枪取10 μL纤维蛋白原溶液滴加至脊状表面，再加入10 μL凝血酶溶液，此时载玻片上体系的最终纤维蛋白原浓度为0.3 mg/mL，凝血酶浓度为1 U/mL。将样品与湿润的滤纸一同置于培养皿中密封，以维持湿润环境，于37℃下聚合1小时[14]。
聚合完成后，用移液枪轻柔移除表层的纤维蛋白网，仅在脊状表面保留二维纤维蛋白网络。若实验中使用微球，则取30 μL特定稀释度的20 nm羧基修饰微球（FluoSpheres™ Carboxylate-Modified Microspheres，Invitrogen，赛默飞世尔科技/赛默飞生命科技公司，美国俄勒冈州尤金市，下文简称微球）溶液，经5分钟超声处理后加入样品体系。将微球溶液与纤维蛋白于室温下孵育1分钟，随后轻柔移去微球溶液，用30 μL HBS缓冲液洗涤样品三次，以去除残留的微球溶液。洗涤完成后，在样品表面滴加20 μL HBS缓冲液，以备显微成像。
使用徕卡DMi8落射荧光显微镜（Leica Microsystems公司，美国伊利诺伊州巴法罗格罗夫市）进行成像，配置63倍油浸物镜。先以480 nm波长的激发光（对应ALEXA-488标记纤维蛋白原的激发波长）定位成像区域，选取相机视野内至少包含4根独立纤维、且纤维间间距不小于1 μm的区域进行成像，以提升数据采集效率。定位目标区域后，将显微镜激发波长切换为553 nm（对应羧基修饰聚苯乙烯微球的激发波长，微球实际发射波长为580 nm）。由于根据厂商数据表，微球的激发光谱在~480 nm处存在最小值，因此在定位纤维时使用480 nm波长的光，以尽可能降低微球的荧光激发——尽管我们最初推测微球可能对光剂量敏感，后续实验并未验证这一假说（详见结果部分），但仍沿用了该实验方案。
将纤溶酶（Enzyme Research Laboratories公司，美国印第安纳州南本德市）直接加入样品体系，随后立即启动时间序列成像。加入纤溶酶后，搭载的徕卡DFC9000GT SCMOS 400万像素单色相机每30秒捕获一张图像，持续拍摄1小时。除特殊说明外，相机快门在整个成像过程中保持常开。
延时成像完成后，将图像文件导入FIJI图像分析软件，对单根纤维进行分析。在初始帧中，计数所有呈现为紧绷结构的单根纤维，并标记用于后续分析。通过逐帧分析各图像序列，确定纤维断裂的时刻：将断裂前的帧数乘以单帧间隔时间，即可得到纤维断裂时间。此外，在中间帧中还需量化纤维束化事件的发生情况——束化定义为两根相邻但基本分离的纤维融合为一根新的纤维。在成像过程中，多数纤维会失去初始状态下的紧绷状态，变为松散结构。我们将这类失去初始紧绷状态的纤维定义为“伸长型纤维”，并在延时序列的最后一帧中对其进行计数，同时计数所有仍保持紧绷状态的纤维。