SCI真菌生长文章，成像法材料方法必备说明。
1 菌株与孢子悬液制备 / Strains and spore suspension preparation
中文
丝状真菌接种于PDA固体培养基，25–28 ℃恒温培养7 d获得成熟分生孢子。使用含0.05%吐温80无菌生理盐水洗脱孢子，四层无菌纱布过滤去除菌丝碎片，血球计数板计数后梯度稀释至工作浓度1×10³～1×10⁴ 个/mL。孢子悬液冰浴保存，30 min内完成96孔板接种，避免孢子提前萌发。酵母菌在YPD培养基活化，调节至0.5麦氏浊度后稀释为1×10³ CFU/mL备用。
English
Filamentous fungi were cultured on PDA solid medium at 25–28 °C for 7 days to produce mature conidia. Conidia were washed with 0.05% Tween 80 sterile saline, filtered through four layers of sterile gauze to remove mycelial debris, counted with a hemocytometer and serially diluted to 1×10³–1×10⁴ spores/mL. The spore suspension was stored on ice and inoculated into 96-well plates within 30 min to prevent premature germination. Yeast strains were activated on YPD medium, adjusted to 0.5 McFarland turbidity and diluted to 1×10³ CFU/mL before use.
2 培养基与试验体系设置 / Culture medium and experimental setup
中文
采用PDB、察氏液体培养基或RPMI 1640液体培养基，121 ℃高压灭菌20 min冷却使用，全部试验采用纯液体培养，不添加琼脂凝固剂。抗真菌药物、不同碳源、渗透压、水分活度、温度胁迫、次生代谢自抑制等处理采用二倍梯度稀释，母液经0.22 μm滤器除菌，有机溶剂终浓度控制在1%以内。
设置空白培养基对照组、溶剂对照组、正常生长阳性对照组，每组不少于4个生物学复孔，单孔终体积固定100 μL。96孔板外圈孔加入对应液体培养基作为蒸发补偿孔；超过5天长周期监测使用透气疏水封板膜密封，保证通气并减少水分挥发与杂菌污染。
English
Liquid media including PDB, Czapek’s broth and RPMI 1640 were autoclaved at 121 °C for 20 min. All assays were performed in liquid culture without agar gelling agent. Treatments including antifungal agents, carbon sources, osmotic pressure, water activity, temperature stress and autotoxic metabolites were prepared via two-fold serial dilution. Stock solutions were sterilized by 0.22 μm filtration, and the final solvent proportion was limited below 1%.
Blank medium control, solvent control and untreated positive growth control were established, with at least four biological replicates per group and a final volume of 100 μL per well. The outermost wells of 96-well plates were filled with medium as evaporation compensation. Plates for incubation longer than 5 days were sealed with breathable hydrophobic film to maintain aeration and avoid water loss and contamination.
3 仪器设备与扫描参数（SCI强制必填）/ Instrument and scanning parameters (required for SCI)
中文
真菌生长动态成像与形态定量采用丹麦BioSense公司oCelloScope全自动微生物生长动态监测系统完成。设备置于高精度恒温培养箱内，开机预热2 h并完成光路校准与空白培养基基线校正。
丝状真菌选用FluidScope倾斜光路液体深层Z轴堆栈扫描模式，酵母菌采用常规透射扫描模式；全程开启SESA静态杂质过滤算法，消除培养基沉淀、药物析出结晶、不溶性颗粒物带来的假阳性信号。Z轴步长10 μm，覆盖微孔底部至液面全部深度；整块板单次扫描时长≤3 min。萌发延滞期定量扫描间隔30 min，长期动力学监测间隔1–2 h，总监测时长48 h至14 d。培养箱温度波动控制在±0.5 ℃以内，配套UniExplorer软件完成图像存储、动力学拟合与形态参数自动量化。
English
Fungal growth kinetics and morphological quantification were detected using the oCelloScope automated microbial growth monitoring system (BioSense, Denmark). The device was placed in a high-precision incubator, preheated for 2 h, and calibrated for optical path and blank medium baseline.
FluidScope tilted-light liquid deep Z-stack scanning mode was used for filamentous fungi, and conventional transmission mode for yeasts. The SESA static impurity filtering algorithm was activated to eliminate false-positive signals from precipitates, drug crystals and insoluble particles. The Z-axis step was set as 10 μm to cover the full liquid depth of each well, and one full-plate scan took less than 3 min. Time-lapse intervals were set as 30 min for lag-phase measurement and 1–2 h for long-term kinetic recording, with total monitoring duration ranging from 48 h to 14 days. Incubation temperature fluctuation was controlled within ±0.5 °C. Image storage, kinetic curve fitting and morphological analysis were processed by the supporting UniExplorer software.
4 微孔板装载与时序监测 / Plate loading and time-lapse monitoring
中文
所有加样操作在二级生物安全柜内无菌完成。加样完毕后微孔板300 g水平离心1 min，使孢子沉降至孔底并消除微小气泡。密封后室温静置60 min待孢子沉降稳定，将微孔板固定于仪器板架防止扫描位移。在UniExplorer软件设置板型、温度、扫描间隔与总时长，启动全自动无人值守时序扫描，整个周期全程不开盖、不挪动板体。
English
All liquid dispensing was operated aseptically in a Class II biosafety cabinet. Plates were centrifuged horizontally at 300 g for 1 min to settle spores uniformly and remove microbubbles. After sealing and 60 min static placement at room temperature, plates were fixed firmly on the instrument rack to avoid displacement during continuous scanning. Parameters including plate type, temperature, scanning interval and total duration were set in UniExplorer software to start automatic unattended time-lapse scanning without plate opening or movement during the whole experiment.
5 数据导出与量化指标定义 / Data export and quantitative indicators
中文
监测结束后微孔板高压灭菌无害化处理。软件导出两类数据：
1 动力学定量数据：BCA算法计算菌丝累积面积时序曲线、萌发延滞期、生长曲线下面积AUC、最大生长速率、生长抑制率；
2 成像原始数据：各时间点最佳焦平面图片、Z轴三维堆叠重建图，用于统计菌丝长度、分枝数、菌丝球等效直径、畸形菌丝（肿胀、空泡化、断裂扭曲）占比。
试验有效性判定：空白对照组无真菌信号；阳性对照组生长曲线连续稳定；平行复孔动力学数据相对偏差＜5%，数据方可纳入统计分析。
English
Plates were autoclaved for biosafety disposal after the experiment. Two types of data were exported:
1 Kinetic parameters: time-course mycelial area calculated by BCA algorithm, germination lag phase, area under curve (AUC), maximum growth rate and inhibition rate;
2 Original imaging data: in-focus images and Z-stack 3D reconstruction images for morphological analysis, including hyphal length, branch number, pellet diameter and the proportion of abnormal hyphae (swelling, vacuolation, fragmentation and distortion).
Validity criteria: no fungal signals in blank controls, continuous stable growth curves in positive controls, and relative deviation less than 5% among biological replicates before statistical analysis.
6 方法学补充备注（可放讨论部分）/ Methodological notes (for Discussion)
中文
高盐、高糖、难溶性药物、植物提取物易在液体培养基析出颗粒，必须依靠SESA过滤算法结合空白基线扣除背景干扰，避免颗粒被误计为菌丝生物量。7–14天长周期密闭培养必须使用外圈蒸发补偿孔与透气密封膜，维持孔内药物浓度、渗透压稳定。萌发延滞期精准测定需采用高频扫描间隔，不可超过1 h。
English
High concentrations of salts, sugars, poorly soluble fungicides and plant extracts easily produce precipitates, which must be eliminated by SESA filtering and blank baseline correction to avoid overestimation of fungal biomass. Evaporation compensation wells and breathable sealing films are required for 7–14 days closed incubation to stabilize drug concentration and osmotic pressure. High-frequency scanning intervals (≤1 h) are necessary for accurate lag-phase quantification.
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