BioSense在SCI材料方法里可直接复制的标准句式。
1 仪器通用基础描述
中文
本研究采用丹麦BioSense Solutions公司oCelloScope全自动微生物成像动力学监测系统开展时序动态检测。设备置于恒温培养箱预热2 h并完成光路灰度校准，依托FluidScope倾斜Z轴堆叠扫描采集微孔全深度图像；通过UniExplorer软件完成数据采集与分析，利用SESA静态杂质分割算法、BCA背景校正消除药物沉淀、结晶、菌体沉降带来的干扰，同步输出菌体像素总面积与等效TA浊度两组动力学数据，留存Z轴时序图像用于菌体形态定性定量分析。96孔板全部使用疏水透气封板膜密封，实现全程无开盖、无扰动长时间培养监测。
English
Time-lapse dynamic monitoring of microorganisms was performed using the oCelloScope system (BioSense Solutions, Farum, Denmark). The instrument was pre-incubated in a constant-temperature incubator for 2 hours for thermal stabilization and full optical path grayscale calibration before detection. FluidScope tilted Z-stack scanning technology was applied to acquire full-depth stacked images of each well. Data collection and analysis were processed by UniExplorer software. The SESA static impurity segmentation algorithm and BCA background correction algorithm were adopted to eliminate interferences such as drug precipitation, crystal particles and bacterial sedimentation. Two quantitative readouts, total bacterial pixel area and equivalent TA turbidity, were exported simultaneously. Time-series Z-stack images were stored for qualitative and quantitative analysis of bacterial morphology. All 96-well plates were sealed with hydrophobic breathable sealing films to achieve long-term undisturbed incubation without repeated plate opening.
2 基础药敏动力学 & 生长曲线
中文
受试菌株于MH肉汤连续传代2次活化至对数生长期，菌悬液标准化至终浓度5×10⁵ CFU/mL。在96孔平底微孔板设置药物2倍倍比浓度梯度，同步设立阳性生长对照、阴性空白对照、溶剂对照、无菌药物基质对照，每孔终体积100 μL。加样后300 g低速离心1 min去除微小气泡，温度平衡后上机时序扫描，扫描间隔30 min，总时长16–20 h。计算延滞期、比生长速率、曲线下AUC面积并判定动力学MIC，结合原位图像观察药物诱导菌体丝状化、裂解、团聚等表型变化。
English
Tested strains were subcultured twice in Mueller-Hinton broth to logarithmic phase and standardized to a final inoculum density of 5×10⁵ CFU/mL. Two-fold serial dilutions of test compounds were prepared in 96-well flat-bottom microplates, with positive growth controls, negative blank controls, solvent controls and sterile drug-only matrix controls included. The final volume per well was 100 μL. Plates were centrifuged at 300 g for 1 min to remove microbubbles, temperature-equilibrated and loaded for time-lapse scanning at 30 min intervals over a total duration of 16–20 h. Kinetic parameters including lag phase, maximum specific growth rate and area under the curve (AUC) were calculated to determine the kinetic MIC. In situ images were captured to record drug-triggered phenotypes such as filamentation, cell lysis and bacterial aggregation.
3 耐药株 vs 野生株药敏对比
中文
同源野生敏感株与耐药突变株在培养基、药物梯度、培养温度、扫描参数完全一致条件下平行上机检测，仅菌株耐药表型为单一变量。通过AUC生长抑制面积量化两组整体受抑制程度差异，对比延滞期与最大增殖生物量评价耐药株恢复能力；相同药物浓度下调取时序原位图像，直观区分野生株不可逆裂解与耐药株形态完整持续增殖特征，定量表征耐药菌株动力学耐受特点。
English
Isogenic wild-type susceptible strains and resistant mutants were tested in parallel under identical medium, drug gradients, incubation temperature and scanning parameters, with bacterial resistance phenotype as the sole variable. The AUC growth inhibition area was used to quantify the overall inhibitory difference between the two groups. Lag phase length and maximum proliferative biomass were compared to evaluate the recovery capacity of resistant strains. Time-series in situ images at equivalent drug concentrations visually distinguished irreversible lysis in susceptible strains and intact continuous proliferation in resistant populations, so as to quantitatively characterize the kinetic tolerance of resistant bacteria.
4 生物膜药物渗透动态监测
中文
96孔板静态培养构建细菌成熟生物膜，轻柔漂洗去除浮游菌后加入梯度受试药物，利用oCelloScope Z轴分层时序扫描连续追踪药物从生物膜表层向内层包裹菌渗透抑制全过程。借助SESA算法排除药物析出假阳性，以菌体像素总面积时序曲线反映生物膜内活菌动态，通过生长延滞时长判定药物渗透滞后效应；堆叠图像观察生物膜厚度、胞外基质降解及底层细菌存活状态，平行设置浮游菌组作为参照，计算生物膜屏障介导的药物耐受倍数。
English
Mature bacterial biofilms were formed via static incubation in 96-well microplates. After gentle washing to remove loosely attached planktonic cells, serially diluted test agents were added. Continuous Z-stack time-lapse scanning on the oCelloScope platform tracked the whole penetration and inhibition process of drugs from the biofilm surface to embedded inner bacteria. The SESA algorithm eliminated false signals caused by drug precipitation. Time-series curves of total bacterial pixel area reflected the survival dynamics of biofilm-embedded bacteria, and lag phase delay was calculated to evaluate drug penetration retardation. Stacked images were analyzed for biofilm thickness variation, extracellular matrix degradation and viability of bottom-layer bacteria. Parallel planktonic bacterial groups were set as references to calculate the drug tolerance multiple mediated by biofilm physical barriers.
5 碳点/纳米材料 生长+形态双维度评价
中文
碳点及纳米抗菌材料经超声充分分散配制梯度工作液，单独设置无菌纳米颗粒对照组校正颗粒沉降造成的光学假阳性。依托oCelloScope时序成像体系完成生长动力学与菌体形态双指标评价，SESA算法区分静态纳米颗粒与动态增殖活菌；以AUC生长抑制率量化抑菌活性，选取对数生长期关键图像统计菌体长度、长宽比、破损细胞占比等形态参数，区分单纯抑菌作用与膜损伤介导的裂解杀菌模式。
English
Carbon dots and nano-antibacterial materials were fully dispersed by sonication and serially diluted. Independent sterile nanoparticle control groups were established to calibrate false-positive optical signals from particle sedimentation. The oCelloScope time-lapse imaging platform was adopted for dual evaluation of growth kinetics and bacterial morphology. The SESA algorithm discriminated static nanoparticles from viable proliferative bacteria. The AUC growth inhibition rate quantified antibacterial potency. Morphological parameters including average cell length, aspect ratio and the percentage of damaged cells were quantified from key time-point images to differentiate bacteriostatic activity and membrane-damaging bactericidal lysis.
6 亚抑菌浓度 生长延滞+形态畸变定量
中文
预先测定菌株动力学MIC，设置1/2 MIC、1/4 MIC、1/8 MIC等亚抑菌浓度梯度，通过oCelloScope密闭不间断时序扫描同步量化生长动力学与细胞形态指标。从动力学曲线提取延滞期延长幅度、比生长速率下降比例表征生长阻滞效应；利用Z轴堆叠图像统计菌体长宽比、丝状化比例等畸变参数，联合解析亚抑菌药物对细菌分裂周期与细胞形态建成的双重干扰作用。
English
The kinetic MIC of tested strains was determined in advance, and serial sub-inhibitory concentrations including 1/2 MIC, 1/4 MIC and 1/8 MIC were designed. Undisturbed continuous time-lapse scanning on the oCelloScope system realized synchronous quantification of growth kinetics and cellular morphology under sub-inhibitory stress. Extended lag phase duration and reduced specific growth rate were extracted from kinetic curves to quantify growth retardation. Z-stack images were used to calculate morphological deformation indicators such as cell aspect ratio and filamentation ratio. Combined analysis clarified the dual interference of sub-inhibitory drugs on bacterial cell division cycle and morphological development.
7 复合抗菌制剂 正交试验高通量筛选
中文
采用正交试验设计排布多组分复合制剂不同复配比例，大幅缩减实验组数量，96孔板高通量上板后通过oCelloScope快速时序扫描完成全部配比药效评价。以各组AUC生长抑制率作为核心评价指标，经正交极差分析明确各组分对整体药效贡献权重，筛选最优复配方案；通过原位成像甄别组分相互反应析出沉淀导致的假阳性，并验证最优配比是否具备协同杀菌与诱导菌体裂解增效效果。
English
Orthogonal experimental design was applied to arrange different ratios of multi-component composite antibacterial formulations to greatly reduce the number of experimental groups. All combinations were loaded on 96-well plates and subjected to high-throughput efficacy evaluation via rapid time-lapse scanning on the oCelloScope system. The AUC growth inhibition rate was taken as the core efficacy index. Range analysis of orthogonal tests confirmed the contribution weight of each component and screened the optimal compound formula. In situ imaging identified false-positive results caused by precipitation from component interaction, and verified the synergistic bactericidal effect and cell-lysis capacity of the optimized proportion.
8 消毒剂杀菌动力学 & 长效防腐定量评价
中文
梯度稀释消毒剂体系与标准化菌悬液混匀后密封微孔板上机，oCelloScope连续时序监测完整记录菌体生物量下降全过程。通过曲线最大下降速率、灭活延迟时间量化杀菌起效速度，长时间持续观测判断菌体复生长能力以评估长效防腐性能；原位堆叠图像区分单纯抑菌、缓慢杀菌、快速裂解三种作用表型，最终将成像动力学定量数据与终点平板CFU涂布计数交叉验证，完成消毒与防腐效果系统性表征。
English
Serially diluted disinfectants were mixed with standardized bacterial suspensions, and sealed microplates were loaded for continuous time-lapse monitoring on the oCelloScope platform to fully record the declining dynamics of bacterial biomass. The maximum decline rate and inactivation lag time of kinetic curves quantified the speed of bactericidal action. Long-term continuous observation judged regrowth potential to evaluate long-term preservative efficacy. In situ stacked images differentiated three functional phenotypes: simple bacteriostasis, slow bactericidal activity and rapid bacterial lysis. Finally, kinetic quantitative results were cross-verified with endpoint agar plate CFU counting for systematic characterization of disinfection and preservation performance.
9 连续传代 细菌耐药适应性演化追踪
中文
在亚抑菌药物持续选择压力下对目标菌株进行多代连续传代驯化，每一代均使用oCelloScope完成完整动力学监测与形态时序追踪。跨代统计延滞期、AUC抑制面积、动力学MIC变化趋势反映耐药适应性提升规律；通过时序图像记录长期药物胁迫下菌体丝状化比例逐步下降、形态向野生型恢复的演化特征。同步设置无药平行传代对照组，区分药物诱导适应性耐受与菌株自发突变，并增设停药复壮实验判定耐药表型是否为可稳定遗传突变。
English
Target strains were domesticated via serial passages under continuous sub-inhibitory drug selective pressure. The oCelloScope platform was used for full kinetic monitoring and morphological time-lapse tracking for each generation. Variation trends of lag phase, AUC inhibition area and kinetic MIC across passages were statistically analyzed to reflect the progression of adaptive resistance. Time-series images recorded the evolutionary feature of gradually decreased filamentation ratio and morphological recovery to wild-type phenotype under long-term drug stress. Parallel drug-free passage control groups were set to distinguish drug-induced adaptive tolerance from spontaneous mutation. Additional drug withdrawal rejuvenation assays were performed to confirm whether the resistant phenotype belonged to stably heritable mutation.
10 体外动力学支撑细胞、动物体内药效关联性分析
中文
借助oCelloScope体外动力学平台系统测定候选药物动力学MIC、MBC、抗生素后效应PAE、杀菌速率及菌体形态改变等多维参数，依据PAE时长确定动物给药间隔，根据抑菌/杀菌表型设计细胞感染模型给药浓度与作用时长。在结果讨论中利用动力学特征解释体内外药效差异：体外抑菌但体内效果优良的药物，通过药物诱导菌体丝状化更易被巨噬细胞清除的形态证据阐明免疫协同机制；体外有效体内偏弱的样品，依靠短PAE数据说明体内代谢过快导致有效抑菌窗口不足，为细胞与动物实验结果提供动力学机制支撑。
English
Multiple kinetic parameters including kinetic MIC, MBC, post-antibiotic effect (PAE), bactericidal rate and drug-induced morphological changes were systematically determined on the oCelloScope in vitro platform. The administration interval for animal studies was designed according to PAE duration, while dosage and treatment duration for cellular infection models were determined based on bacteriostatic or bactericidal phenotypes. Kinetic characteristics were applied to interpret inconsistent efficacy between in vitro and in vivo outcomes in the discussion section. For agents with weak in vitro activity but satisfactory in vivo performance, the mechanism of host immune synergy was illustrated by the evidence that drug-induced filamentous bacteria are more susceptible to macrophage clearance. For compounds with effective in vitro but poor in vivo efficacy, short PAE data explained insufficient antibacterial window caused by rapid in vivo metabolism, providing solid kinetic evidence to support cellular and animal experimental results.
11 单日百级化合物高通量初筛方案
中文
采用单浓度初筛模式开展大批量化合物抗菌活性高通量筛选，微孔板分批轮换连续上机，oCelloScope启用FluidScope快速扫描参数压缩单板检测时长，监测周期缩短至6 h实现快速初筛。以AUC生长抑制率作为活性判定阈值，结合SESA算法与原位显微图像剔除色素、药物沉淀造成的假阳性样品，仅将初筛阳性候选物转入后续梯度动力学MIC精筛，实现单日大批量化合物快速活性分级与药效排序。
English
A single-concentration screening strategy was adopted for high-throughput antibacterial activity evaluation of large compound libraries. Microplates were loaded cyclically and continuously. The FluidScope rapid scanning mode was activated on the oCelloScope system to shorten single-plate detection time, and the monitoring period was reduced to 6 hours for rapid primary screening. The AUC growth inhibition rate was set as the activity threshold. Combined with the SESA algorithm and in situ microscopic images, false-positive samples caused by pigment interference and drug precipitation were eliminated. Only primary positive hits were forwarded to subsequent gradient kinetic MIC fine screening, realizing rapid activity grading and potency ranking of hundreds of compounds within a single working day.