活体、死亡小鼠及胁迫环境下植物的超弱光子发射成像 Imaging Ultraweak Photon Emission from Living and Dead Mice and from Plants under StressGB/T 7714-2015 预印本文献标准引用SALARI V, SESHAN V, FRANKLE L, et al. Imaging Ultraweak Photon Emission from Living and Dead Mice and from Plants under Stress[EB/OL]. bioRxiv, 2024-11-09. https://doi.org/10.1101/2024.11.08.622743.活体、死亡小鼠及胁迫环境下植物的超弱光子发射成像V. Salari¹,²,∗, V. Seshan¹,²,³,∗, L. Frankle⁴,⁵, D. England⁶, C. Simon¹,²,³, D. Oblak¹,²¹加拿大卡尔加里大学量子科学与技术研究所加拿大艾伯塔省卡尔加里市 T2N 1N4²加拿大卡尔加里大学物理与天文系加拿大艾伯塔省卡尔加里市³霍奇基斯脑科学研究所加拿大艾伯塔省卡尔加里市⁴加拿大国家研究委员会人类健康治疗研究中心转化生物科学部加拿大渥太华市⁵加拿大卡尔加里大学物理与天文系加拿大艾伯塔省卡尔加里市⁶加拿大国家研究委员会加拿大安大略省渥太华市苏塞克斯大道100号邮编 K1A 0R6∗ 同等贡献作者投稿日期2024年11月9日生物超弱光子发射Ultraweak Photon Emission, UPE是一种在200–1000 nm光谱区间内、光子强度极低10–10³ 光子/cm²/s的光学现象目前所有已开展检测的生命体系中均观测到该现象。本文通过多组生理相关场景实验验证了新型成像系统对超弱光子发射信号变化的检测能力。本研究采用电子倍增电荷耦合器件EMCCD与电荷耦合器件CCD相机在低噪声、量子效率高于90%的条件下捕获可见光波段单光子信号。实验结果证实活体小鼠与死亡小鼠的超弱光子发射信号存在显著成像对比度针对植物的实验表明升温与物理损伤均会提升超弱光子发射强度此外化学药剂处理可改变植物超弱光子发射特征其中在损伤部位施用麻醉剂苯佐卡因后样品发射强度在所有受试药剂中达到峰值。综上超弱光子发射成像技术可实现无标记、非侵入式成像用于判定动物生命活性、监测植物胁迫响应。一、引言超弱光子发射UPE又称生物光子发射指各类生物体系细胞培养物、细菌、植物、动物、人体等自发释放极低强度光信号的现象[1–11]。该现象最早在植物中被观测到后续在动物及人体细胞中也得到证实。历经数十年研究学界目前尚未完全阐明该发射现象的产生机理与生物学意义但已知活性氧ROS在超弱光子发射过程中起到关键调控作用[12]。当生物体遭遇环境毒素、病原体、生理失衡等胁迫刺激时体内会激活多条生化通路生成超氧阴离子、过氧化氢、羟基自由基等活性氧这类物质是细胞应对胁迫的核心信号分子[13]。活性氧过量累积会突破细胞抗氧化防御体系诱发氧化应激[14–18]。活性氧介导的脂质过氧化、蛋白质氧化过程会引发电子激发与转移进而产生超弱光子发射[19–21]。同时活性氧参与的生化反应会改变细胞内组分氧化还原状态调控自由基对反应动力学最终影响光子发射强度[22–28]。生物体系胁迫状态与活性氧生成的强关联性是细胞生理学的核心研究方向之一[13]。依托超弱光子发射检测技术有望实现各类胁迫相关病变如癌症的早期筛查[45,46,68–70]。超弱光子发射的单光子检测技术最早依托光电倍增管PMT搭建该设备虽具备开创性但检测灵敏度与空间分辨率存在局限[29]。随着成像设备技术迭代电荷耦合器件CCD[30–32,34,35]与电子倍增电荷耦合器件EMCCD成像相机[36–38]大幅提升了单光子捕获效率与成像分辨率。基于上述设备科研人员已针对多种生命体系开展大量超弱光子发射成像研究[44]例如利用CCD相机对不同生物体系的超弱光子发射成像[45–48]通过人体超弱光子发射表征生理与病理状态[49]结合成像手段解析活性氧与超弱光子发射的关联机制[22,23,50]另有多项跨体系成像研究聚焦超弱光子发射背后的生物物理机理[51–53]。据本文作者所知目前尚无文献通过成像手段探究生物死亡对超弱光子发射的影响尽管已有文献提及温度会改变超弱光子发射强度[54]但相关成像研究十分匮乏少量研究探讨了植物胁迫、物理损伤对超弱光子发射的作用[43,71]却尚未开展麻醉剂等化学药剂处理损伤组织的相关实验。本文通过活体、死亡小鼠对照实验解析死亡状态对超弱光子发射的影响同时探究温度、物理损伤、化学药剂处理等多种胁迫条件下植物超弱光子发射的变化规律完整呈现实验观测结果。二、生物自发发光现象生物自发发光指生物体无需外部光激发即可自主释放光信号的现象生物在暗环境静置一段时间后仍会持续产生该类光信号。该发光过程由细胞内特异性生化反应或温度效应驱动生命体系自发发光分为三类黑体辐射、生物发光、超弱光子发射[66]。黑体辐射BBR热辐射任意热平衡状态物体表面因自身温度产生的电磁辐射辐射强度随温度升高而增大。生物发光生物自主释放高强度光信号光子通量达到10⁵ 光子/cm²/s 及以上人眼可直接观测。该过程通常依赖荧光素酶与荧光素底物也可通过光蛋白等非酶促反应实现存在于细菌、萤火虫、水母等特定生物体内具备通讯、伪装、诱捕猎物/配偶等生物学功能。超弱光子发射UPE所有生物体系均可产生的最低强度光信号仅能依靠超高灵敏度探测器捕捉光谱覆盖紫外、可见光、近红外波段普遍认为来源于细胞内各类生化反应与代谢活动[11,56]。前文已述超弱光子发射可作为细胞代谢、氧化应激、生理健康状态的灵敏检测指标[12]。黑体辐射与超弱光子发射是所有生物均具备的发光形式但二者特征存在显著差异。黑体辐射遵循普朗克定律[57]仅由温度决定室温条件下其光谱峰值波长显著大于1000 nm。超弱光子发射特异性来源于生物体内生化过程光谱主要分布在1000 nm以下波段。温度变化虽可调控超弱光子发射强度[54]但细胞活性、生化反应、胞内化学分子等多种因素均可对其产生调控生物过程与光子发射的动态关联性是超弱光子发射区别于静态黑体辐射的核心特征。本研究通过数值积分计算两种发光形式的光谱能量密度下限黑体辐射积分区间200–2000 nm超弱光子发射积分区间200–1000 nm。依据普朗克定律分别计算室温22℃、体温37℃下黑体辐射的光谱能量密度u λ ( λ , T ) 8 π h c 2 λ 5 1 exp h c λ k B T − 1 u_{\lambda}(\lambda, T)\frac{8 \pi h c^{2}}{\lambda^{5}} \frac{1}{\exp \frac{h c}{\lambda k_{B} T}-1}uλ(λ,T)λ58πhc2expλkBThc−11式中u λ ( λ , T ) u_{\lambda}(\lambda, T)uλ(λ,T)为特定波长λ \lambdaλ、温度T TT下黑体辐射的光谱能量密度。图1 紫外-可见光-近红外波段内22℃室温、37℃体温下黑体辐射BBR光子通量同时标注超弱光子发射最低光谱能量密度最低光子通量取10 光子/(cm²·s)、生物发光估算光谱能量密度最低光子通量取10⁵ 光子/(cm²·s)生物发光光谱仅覆盖可见光区间[10,11,66]。图1结果表明本实验成像检测波段内超弱光子发射光谱能量密度比黑体辐射高出数个数量级因此黑体辐射对成像信号的干扰可忽略不计。三、实验方法高量子效率CCD与EMCCD相机是超弱光子发射成像研究中灵敏度最高的单光子检测设备之一[34,35,38]。CCD相机是数字成像设备通用图像传感器可捕获光子并转化为电信号生成数字图像高量子效率CCD相机广泛应用于科研领域[30–32]。EMCCD是CCD的特殊衍生设备搭载电子倍增寄存器可实现极微弱光信号超高灵敏检测适用于天文观测、生物发光成像等弱光场景[36,37]。EMCCD技术可在保持高量子效率的同时放大微弱光信号。图2 加拿大国家研究委员会NRC成像系统A搭载Andor iKon L系列CCD相机的IVIS Lumina成像平台配套标准硅“BV”传感器量子效率曲线C卡尔加里大学成像系统B大型避光暗箱搭载Andor iXon ultra 888 EMCCD相机配套BV传感器量子效率曲线D。BV传感器在可见光区间量子效率优异。量子效率曲线来源于相机厂商官方参数[33]。小鼠成像实验在加拿大国家研究委员会完成植物成像实验在卡尔加里大学完成。1. 成像相机本研究使用两台成像设备卡尔加里大学量子云实验室采用Andor iXon ultra 888 EMCCD相机用于胁迫环境下植物成像渥太华加拿大国家研究委员会人类健康治疗研究中心采用搭载Andor iKon L系列CCD相机的IVIS成像系统用于小鼠成像见图2。Andor iXon 888 EMCCD、iKon L系列CCD相机在可见光区间量子效率均超过95%见图2传感器最大制冷温度下暗电流分别为0.00011、0.000059 电子/像素/秒。EMCCD相机通过USB数据线连接计算机采用Solis软件完成数据采集为提升灵敏度、降低暗电流外接水冷机将传感器稳定控温至-95℃。成像参数垂直转移速度2.2 μs电子倍增增益模式2不同实验分别采用300、1000倍放大增益为提升信噪比使用2×2、4×4像素合并模式并开启宇宙射线滤除功能。两台相机均放置于暗房内全遮光密闭箱体中隔绝环境杂散光见图2。A. 实验样本1. 小鼠样本小鼠超弱光子发射成像使用珀金埃尔默IVIS Lumina III临床前动物成像仪美国马萨诸塞州沃尔瑟姆市。实验动物为查尔斯河实验室SKH1 Elite无毛小鼠加拿大魁北克省蒙特利尔市异氟烷气体麻醉后固定于成像载台全程维持麻醉状态。成像前小鼠避光适应30 min随后俯卧位开展60 min生物光子成像成像参数单只小鼠定位、无滤光片。成像结束后通过CO₂安乐处死小鼠处死完成后立即将小鼠放回成像设备避光适应30 min采用完全相同参数二次成像。利用珀金埃尔默Living Image 4.1软件定量统计总光子通量见图2。2. 植物样本卡尔加里大学采用活体拟南芥、新鲜鹅掌柴绿叶开展植物与叶片超弱光子发射成像实验探究物理损伤、不同化学药剂对植物损伤响应的调控作用。实验药剂异丙醇、过氧化氢H₂O₂、苯佐卡因。异丙醇溶剂、消毒剂可穿透细胞膜、使蛋白质变性涂抹于损伤植物组织后会破坏细胞完整性、激活防御修复信号通路改变活性氧水平调控代谢与细胞间通讯作用效果由药剂浓度、处理时长决定。苯佐卡因局部麻醉剂通过阻断电压门控钠离子通道抑制神经冲动作用于植物时可能破坏离子通道活性[39]或通过其他活性氧相关通路干扰胁迫响应信号传导[40–42]。过氧化氢典型活性氧分子含量与超弱光子发射强度直接相关同时是植物胁迫、损伤响应的核心信号分子作为第二信使激活各类防御、修复通路[43]。所有植物、叶片成像前均避光适应30 min消除延迟发光干扰。EMCCD相机控温条件下采集成像数据鹅掌柴叶片成像周期16 h拟南芥单次成像时长5 min全程观测温度、损伤、化学药剂处理引发的超弱光子发射动态变化。鹅掌柴叶片尖端切割制造物理损伤分别涂抹上述药剂模拟胁迫拟南芥仅在22℃、26℃两个控温条件下成像探究温度效应。3. 数据分析小鼠、植物成像原始数据未施加图像滤波处理其余分析工作在Andor Solis软件内完成通过直方图拉伸优化色板最大化目标区域成像对比度同一组对照实验统一使用单一色板可视化图像。温度效应实验感兴趣区域ROI选取整片叶片损伤实验同一片叶片选取两块面积相等的感兴趣区域一处为损伤位点一处为远离损伤的健康区域计算同叶片两处区域平均光子计数比值使用Python绘制变化曲线。四、实验结果1. 活体与死亡小鼠的超弱光子发射动物安乐死亡会引发体内剧烈生理变化活性氧生成模式随之改变超弱光子发射强度也会同步变化。本研究共使用4只小鼠开展对照实验验证该规律。图3 A活体小鼠上排N4、刚安乐处死小鼠下排N4避光适应30 min后60 min生物光子成像结果B活体、安乐死小鼠全身感兴趣区域总光子通量光子/秒统计柱状图*代表配对t检验p0.05。实验中活体、死亡小鼠体温均维持37℃成像载台同步控温37℃。图3A成像结果显示活体小鼠超弱光子发射强度显著高于对应死亡小鼠图3B定量统计结果验证该差异具备统计学显著性。活体小鼠持续产生高强度超弱光子发射对应体内持续进行的生化代谢与细胞活动死亡小鼠光子发射几乎完全消失仅残留少量微弱亮点该亮点位置与活体小鼠高光区域一一对应代表代谢、能量活动完全终止。2. 温度对植物超弱光子发射的调控作用温度可调控细胞内各类生化、代谢反应包含活性氧生成通路。通常升温会加快代谢速率通过线粒体呼吸、酶促反应提升活性氧产量同时高温会破坏细胞稳态、削弱抗氧化防御体系加剧氧化应激。从超弱光子发射层面分析温度改变会调控细胞内分子激发、弛豫速率最终改变光子发射模式[54]。升温可通过强化代谢、能量传递过程提升光子发射强度低温则会抑制光子释放速率。本实验使用2组活体拟南芥、4组新鲜鹅掌柴叶片开展重复对照拟南芥设置22℃、26℃两组温度鹅掌柴叶片设置24–39℃共6个温度梯度EMCCD采集成像数据并分析超弱光子发射变化。图4 A拟南芥室温实拍、22℃、26℃避光超弱光子成像图单张图像曝光时长5 minB鹅掌柴绿叶在24℃、27℃、30℃、33℃、36℃、39℃下的超弱光子成像图可见36℃前发射强度随温度上升持续升高C4组鹅掌柴叶片全叶感兴趣区域光子计数随温度变化曲线单张图像采集时长15 min。叶片放置于温控载台距离镜头小于焦距成像分辨率偏低仅分析发射强度变化温度超过36℃后超弱光子发射强度明显下降推测高温胁迫破坏细胞完整性光子发射水平降低[55]。实验结果如图4所示温度升高会显著提升植物超弱光子发射强度4组鹅掌柴叶片均呈现相同变化趋势但温度超过36℃后发射强度大幅回落该现象由高温热胁迫破坏细胞结构、抑制光子发射通路导致。3. 物理损伤与化学药剂对叶片超弱光子发射的影响植物遭受物理切割、植食动物啃食、环境胁迫损伤后会启动一系列生化、生理响应释放活性氧、茉莉酸/乙烯等激素含量改变、基因表达重编程激活修复与防御通路上述过程会造成损伤位点局部超弱光子发射强度上升[71]。本研究发现不同药剂处理损伤位点后超弱光子发射强度存在明显差异。实验材料为新鲜鹅掌柴叶片叶片尖端统一切割制造损伤见图5设置异丙醇、过氧化氢、苯佐卡因三组药剂处理组空白损伤叶片作为对照组每组4片叶片共5组生物学重复。连续16 h成像每小时采集1张图像对比不同药剂处理损伤位点的光子发射强度。图5 A所有鹅掌柴叶片尖端统一制造损伤连续16 h成像单张曝光时长1 h每组图像内左上叶片涂抹异丙醇、中间叶片涂抹过氧化氢、底部叶片涂抹苯佐卡因麻醉凝胶、右侧叶片无药剂处理作为空白对照所有药剂均匀涂抹叶片全表面B实验首小时内损伤区域/健康区域光子强度比值柱状图N5重复C16 h成像周期内各组叶片损伤位点相对健康区域光子计数比值随时间变化曲线。为单独验证苯佐卡因本身作用设置叶片单侧涂抹苯佐卡因、另一侧空白对照其余药剂均全叶涂抹。成像结果显示全部叶片损伤部位的超弱光子发射强度在16 h观测周期内始终显著高于健康区域。统计首小时损伤/健康区域光子计数比值各组药剂处理产生差异化响应20%体积分数苯佐卡因处理损伤位点的发射强度显著高于公认可增强超弱光子发射的过氧化氢同时高于异丙醇苯佐卡因以异丙醇为溶剂仅异丙醇对照组可证明麻醉剂本身具备独立调控效应。动物细胞中苯佐卡因的作用靶点为钠离子通道植物同样存在钠离子通道[39]目前暂未完全阐明苯佐卡因大幅提升损伤部位超弱光子发射的分子机制本文推测该现象与活性氧调控通路相关[40–42]仍需后续实验解析麻醉剂与植物损伤响应的互作机理。五、总结与结论本研究系统探究超弱光子发射UPE信号表征小鼠生命活性、植物各类胁迫状态的应用潜力。依托高性能EMCCD、CCD成像相机分别开展小鼠、植物多组对照实验得到以下结论活体小鼠全身超弱光子发射强度显著高于死亡小鼠直接证明超弱光子发射与生命活性高度关联植物超弱光子发射强度受温度调控升温提升发射强度该变化规律与黑体辐射存在本质区别超弱光子发射可用于表征植物物理损伤损伤位点光子释放量显著上升化学药剂处理会改变损伤部位发射强度其中苯佐卡因处理组发射强度在所有受试药剂中最高该现象的细胞分子机理仍需深入研究。超弱光子发射基础研究具备重要科研价值可无侵入式解析生物体内生化、代谢过程发射强度与生命活性直接绑定环境胁迫因素可调控超弱光子发射信号因此该技术可作为氧化应激、生理病变的灵敏检测指标为植物生物学、生物医学基础研究提供全新研究手段。应用层面植物超弱光子发射检测技术可开发为简易非侵入式监测方案用于农业、植物生物学领域实时监控不同环境下植株健康状态、生长发育情况小鼠超弱光子发射成像可拓展至生物医学、临床相关研究[58–65,67]实现无标记、非侵入式活体生化代谢成像。六、数据可用性声明实验原始数据可向通讯作者申请获取。致谢本研究得到加拿大国家研究委员会量子传感器挑战项目、加拿大自然科学与工程研究理事会发现基金、量子增强传感成像联盟量子财团基金、加拿大前沿科研基金资助。感谢Hadi Zadeh-Haghighi、Rishabh、Gabriel Bertolesi、Douglas Muench提供宝贵讨论意见。利益冲突声明本文作者声明不存在利益冲突。七、通讯作者稿件通讯、材料索取请联系Daniel Oblak邮箱doblakucalgary.ca。参考文献[1] Veselowskii, V. 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活体、死亡小鼠及胁迫环境下植物的超弱光子发射成像 Imaging Ultraweak Photon Emission from Living and Dead Mice and from Plants under StressGB/T 7714-2015 预印本文献标准引用SALARI V, SESHAN V, FRANKLE L, et al. Imaging Ultraweak Photon Emission from Living and Dead Mice and from Plants under Stress[EB/OL]. bioRxiv, 2024-11-09. https://doi.org/10.1101/2024.11.08.622743.活体、死亡小鼠及胁迫环境下植物的超弱光子发射成像V. Salari¹,²,∗, V. Seshan¹,²,³,∗, L. Frankle⁴,⁵, D. England⁶, C. Simon¹,²,³, D. Oblak¹,²¹加拿大卡尔加里大学量子科学与技术研究所加拿大艾伯塔省卡尔加里市 T2N 1N4²加拿大卡尔加里大学物理与天文系加拿大艾伯塔省卡尔加里市³霍奇基斯脑科学研究所加拿大艾伯塔省卡尔加里市⁴加拿大国家研究委员会人类健康治疗研究中心转化生物科学部加拿大渥太华市⁵加拿大卡尔加里大学物理与天文系加拿大艾伯塔省卡尔加里市⁶加拿大国家研究委员会加拿大安大略省渥太华市苏塞克斯大道100号邮编 K1A 0R6∗ 同等贡献作者投稿日期2024年11月9日生物超弱光子发射Ultraweak Photon Emission, UPE是一种在200–1000 nm光谱区间内、光子强度极低10–10³ 光子/cm²/s的光学现象目前所有已开展检测的生命体系中均观测到该现象。本文通过多组生理相关场景实验验证了新型成像系统对超弱光子发射信号变化的检测能力。本研究采用电子倍增电荷耦合器件EMCCD与电荷耦合器件CCD相机在低噪声、量子效率高于90%的条件下捕获可见光波段单光子信号。实验结果证实活体小鼠与死亡小鼠的超弱光子发射信号存在显著成像对比度针对植物的实验表明升温与物理损伤均会提升超弱光子发射强度此外化学药剂处理可改变植物超弱光子发射特征其中在损伤部位施用麻醉剂苯佐卡因后样品发射强度在所有受试药剂中达到峰值。综上超弱光子发射成像技术可实现无标记、非侵入式成像用于判定动物生命活性、监测植物胁迫响应。一、引言超弱光子发射UPE又称生物光子发射指各类生物体系细胞培养物、细菌、植物、动物、人体等自发释放极低强度光信号的现象[1–11]。该现象最早在植物中被观测到后续在动物及人体细胞中也得到证实。历经数十年研究学界目前尚未完全阐明该发射现象的产生机理与生物学意义但已知活性氧ROS在超弱光子发射过程中起到关键调控作用[12]。当生物体遭遇环境毒素、病原体、生理失衡等胁迫刺激时体内会激活多条生化通路生成超氧阴离子、过氧化氢、羟基自由基等活性氧这类物质是细胞应对胁迫的核心信号分子[13]。活性氧过量累积会突破细胞抗氧化防御体系诱发氧化应激[14–18]。活性氧介导的脂质过氧化、蛋白质氧化过程会引发电子激发与转移进而产生超弱光子发射[19–21]。同时活性氧参与的生化反应会改变细胞内组分氧化还原状态调控自由基对反应动力学最终影响光子发射强度[22–28]。生物体系胁迫状态与活性氧生成的强关联性是细胞生理学的核心研究方向之一[13]。依托超弱光子发射检测技术有望实现各类胁迫相关病变如癌症的早期筛查[45,46,68–70]。超弱光子发射的单光子检测技术最早依托光电倍增管PMT搭建该设备虽具备开创性但检测灵敏度与空间分辨率存在局限[29]。随着成像设备技术迭代电荷耦合器件CCD[30–32,34,35]与电子倍增电荷耦合器件EMCCD成像相机[36–38]大幅提升了单光子捕获效率与成像分辨率。基于上述设备科研人员已针对多种生命体系开展大量超弱光子发射成像研究[44]例如利用CCD相机对不同生物体系的超弱光子发射成像[45–48]通过人体超弱光子发射表征生理与病理状态[49]结合成像手段解析活性氧与超弱光子发射的关联机制[22,23,50]另有多项跨体系成像研究聚焦超弱光子发射背后的生物物理机理[51–53]。据本文作者所知目前尚无文献通过成像手段探究生物死亡对超弱光子发射的影响尽管已有文献提及温度会改变超弱光子发射强度[54]但相关成像研究十分匮乏少量研究探讨了植物胁迫、物理损伤对超弱光子发射的作用[43,71]却尚未开展麻醉剂等化学药剂处理损伤组织的相关实验。本文通过活体、死亡小鼠对照实验解析死亡状态对超弱光子发射的影响同时探究温度、物理损伤、化学药剂处理等多种胁迫条件下植物超弱光子发射的变化规律完整呈现实验观测结果。二、生物自发发光现象生物自发发光指生物体无需外部光激发即可自主释放光信号的现象生物在暗环境静置一段时间后仍会持续产生该类光信号。该发光过程由细胞内特异性生化反应或温度效应驱动生命体系自发发光分为三类黑体辐射、生物发光、超弱光子发射[66]。黑体辐射BBR热辐射任意热平衡状态物体表面因自身温度产生的电磁辐射辐射强度随温度升高而增大。生物发光生物自主释放高强度光信号光子通量达到10⁵ 光子/cm²/s 及以上人眼可直接观测。该过程通常依赖荧光素酶与荧光素底物也可通过光蛋白等非酶促反应实现存在于细菌、萤火虫、水母等特定生物体内具备通讯、伪装、诱捕猎物/配偶等生物学功能。超弱光子发射UPE所有生物体系均可产生的最低强度光信号仅能依靠超高灵敏度探测器捕捉光谱覆盖紫外、可见光、近红外波段普遍认为来源于细胞内各类生化反应与代谢活动[11,56]。前文已述超弱光子发射可作为细胞代谢、氧化应激、生理健康状态的灵敏检测指标[12]。黑体辐射与超弱光子发射是所有生物均具备的发光形式但二者特征存在显著差异。黑体辐射遵循普朗克定律[57]仅由温度决定室温条件下其光谱峰值波长显著大于1000 nm。超弱光子发射特异性来源于生物体内生化过程光谱主要分布在1000 nm以下波段。温度变化虽可调控超弱光子发射强度[54]但细胞活性、生化反应、胞内化学分子等多种因素均可对其产生调控生物过程与光子发射的动态关联性是超弱光子发射区别于静态黑体辐射的核心特征。本研究通过数值积分计算两种发光形式的光谱能量密度下限黑体辐射积分区间200–2000 nm超弱光子发射积分区间200–1000 nm。依据普朗克定律分别计算室温22℃、体温37℃下黑体辐射的光谱能量密度u λ ( λ , T ) 8 π h c 2 λ 5 1 exp h c λ k B T − 1 u_{\lambda}(\lambda, T)\frac{8 \pi h c^{2}}{\lambda^{5}} \frac{1}{\exp \frac{h c}{\lambda k_{B} T}-1}uλ(λ,T)λ58πhc2expλkBThc−11式中u λ ( λ , T ) u_{\lambda}(\lambda, T)uλ(λ,T)为特定波长λ \lambdaλ、温度T TT下黑体辐射的光谱能量密度。图1 紫外-可见光-近红外波段内22℃室温、37℃体温下黑体辐射BBR光子通量同时标注超弱光子发射最低光谱能量密度最低光子通量取10 光子/(cm²·s)、生物发光估算光谱能量密度最低光子通量取10⁵ 光子/(cm²·s)生物发光光谱仅覆盖可见光区间[10,11,66]。图1结果表明本实验成像检测波段内超弱光子发射光谱能量密度比黑体辐射高出数个数量级因此黑体辐射对成像信号的干扰可忽略不计。三、实验方法高量子效率CCD与EMCCD相机是超弱光子发射成像研究中灵敏度最高的单光子检测设备之一[34,35,38]。CCD相机是数字成像设备通用图像传感器可捕获光子并转化为电信号生成数字图像高量子效率CCD相机广泛应用于科研领域[30–32]。EMCCD是CCD的特殊衍生设备搭载电子倍增寄存器可实现极微弱光信号超高灵敏检测适用于天文观测、生物发光成像等弱光场景[36,37]。EMCCD技术可在保持高量子效率的同时放大微弱光信号。图2 加拿大国家研究委员会NRC成像系统A搭载Andor iKon L系列CCD相机的IVIS Lumina成像平台配套标准硅“BV”传感器量子效率曲线C卡尔加里大学成像系统B大型避光暗箱搭载Andor iXon ultra 888 EMCCD相机配套BV传感器量子效率曲线D。BV传感器在可见光区间量子效率优异。量子效率曲线来源于相机厂商官方参数[33]。小鼠成像实验在加拿大国家研究委员会完成植物成像实验在卡尔加里大学完成。1. 成像相机本研究使用两台成像设备卡尔加里大学量子云实验室采用Andor iXon ultra 888 EMCCD相机用于胁迫环境下植物成像渥太华加拿大国家研究委员会人类健康治疗研究中心采用搭载Andor iKon L系列CCD相机的IVIS成像系统用于小鼠成像见图2。Andor iXon 888 EMCCD、iKon L系列CCD相机在可见光区间量子效率均超过95%见图2传感器最大制冷温度下暗电流分别为0.00011、0.000059 电子/像素/秒。EMCCD相机通过USB数据线连接计算机采用Solis软件完成数据采集为提升灵敏度、降低暗电流外接水冷机将传感器稳定控温至-95℃。成像参数垂直转移速度2.2 μs电子倍增增益模式2不同实验分别采用300、1000倍放大增益为提升信噪比使用2×2、4×4像素合并模式并开启宇宙射线滤除功能。两台相机均放置于暗房内全遮光密闭箱体中隔绝环境杂散光见图2。A. 实验样本1. 小鼠样本小鼠超弱光子发射成像使用珀金埃尔默IVIS Lumina III临床前动物成像仪美国马萨诸塞州沃尔瑟姆市。实验动物为查尔斯河实验室SKH1 Elite无毛小鼠加拿大魁北克省蒙特利尔市异氟烷气体麻醉后固定于成像载台全程维持麻醉状态。成像前小鼠避光适应30 min随后俯卧位开展60 min生物光子成像成像参数单只小鼠定位、无滤光片。成像结束后通过CO₂安乐处死小鼠处死完成后立即将小鼠放回成像设备避光适应30 min采用完全相同参数二次成像。利用珀金埃尔默Living Image 4.1软件定量统计总光子通量见图2。2. 植物样本卡尔加里大学采用活体拟南芥、新鲜鹅掌柴绿叶开展植物与叶片超弱光子发射成像实验探究物理损伤、不同化学药剂对植物损伤响应的调控作用。实验药剂异丙醇、过氧化氢H₂O₂、苯佐卡因。异丙醇溶剂、消毒剂可穿透细胞膜、使蛋白质变性涂抹于损伤植物组织后会破坏细胞完整性、激活防御修复信号通路改变活性氧水平调控代谢与细胞间通讯作用效果由药剂浓度、处理时长决定。苯佐卡因局部麻醉剂通过阻断电压门控钠离子通道抑制神经冲动作用于植物时可能破坏离子通道活性[39]或通过其他活性氧相关通路干扰胁迫响应信号传导[40–42]。过氧化氢典型活性氧分子含量与超弱光子发射强度直接相关同时是植物胁迫、损伤响应的核心信号分子作为第二信使激活各类防御、修复通路[43]。所有植物、叶片成像前均避光适应30 min消除延迟发光干扰。EMCCD相机控温条件下采集成像数据鹅掌柴叶片成像周期16 h拟南芥单次成像时长5 min全程观测温度、损伤、化学药剂处理引发的超弱光子发射动态变化。鹅掌柴叶片尖端切割制造物理损伤分别涂抹上述药剂模拟胁迫拟南芥仅在22℃、26℃两个控温条件下成像探究温度效应。3. 数据分析小鼠、植物成像原始数据未施加图像滤波处理其余分析工作在Andor Solis软件内完成通过直方图拉伸优化色板最大化目标区域成像对比度同一组对照实验统一使用单一色板可视化图像。温度效应实验感兴趣区域ROI选取整片叶片损伤实验同一片叶片选取两块面积相等的感兴趣区域一处为损伤位点一处为远离损伤的健康区域计算同叶片两处区域平均光子计数比值使用Python绘制变化曲线。四、实验结果1. 活体与死亡小鼠的超弱光子发射动物安乐死亡会引发体内剧烈生理变化活性氧生成模式随之改变超弱光子发射强度也会同步变化。本研究共使用4只小鼠开展对照实验验证该规律。图3 A活体小鼠上排N4、刚安乐处死小鼠下排N4避光适应30 min后60 min生物光子成像结果B活体、安乐死小鼠全身感兴趣区域总光子通量光子/秒统计柱状图*代表配对t检验p0.05。实验中活体、死亡小鼠体温均维持37℃成像载台同步控温37℃。图3A成像结果显示活体小鼠超弱光子发射强度显著高于对应死亡小鼠图3B定量统计结果验证该差异具备统计学显著性。活体小鼠持续产生高强度超弱光子发射对应体内持续进行的生化代谢与细胞活动死亡小鼠光子发射几乎完全消失仅残留少量微弱亮点该亮点位置与活体小鼠高光区域一一对应代表代谢、能量活动完全终止。2. 温度对植物超弱光子发射的调控作用温度可调控细胞内各类生化、代谢反应包含活性氧生成通路。通常升温会加快代谢速率通过线粒体呼吸、酶促反应提升活性氧产量同时高温会破坏细胞稳态、削弱抗氧化防御体系加剧氧化应激。从超弱光子发射层面分析温度改变会调控细胞内分子激发、弛豫速率最终改变光子发射模式[54]。升温可通过强化代谢、能量传递过程提升光子发射强度低温则会抑制光子释放速率。本实验使用2组活体拟南芥、4组新鲜鹅掌柴叶片开展重复对照拟南芥设置22℃、26℃两组温度鹅掌柴叶片设置24–39℃共6个温度梯度EMCCD采集成像数据并分析超弱光子发射变化。图4 A拟南芥室温实拍、22℃、26℃避光超弱光子成像图单张图像曝光时长5 minB鹅掌柴绿叶在24℃、27℃、30℃、33℃、36℃、39℃下的超弱光子成像图可见36℃前发射强度随温度上升持续升高C4组鹅掌柴叶片全叶感兴趣区域光子计数随温度变化曲线单张图像采集时长15 min。叶片放置于温控载台距离镜头小于焦距成像分辨率偏低仅分析发射强度变化温度超过36℃后超弱光子发射强度明显下降推测高温胁迫破坏细胞完整性光子发射水平降低[55]。实验结果如图4所示温度升高会显著提升植物超弱光子发射强度4组鹅掌柴叶片均呈现相同变化趋势但温度超过36℃后发射强度大幅回落该现象由高温热胁迫破坏细胞结构、抑制光子发射通路导致。3. 物理损伤与化学药剂对叶片超弱光子发射的影响植物遭受物理切割、植食动物啃食、环境胁迫损伤后会启动一系列生化、生理响应释放活性氧、茉莉酸/乙烯等激素含量改变、基因表达重编程激活修复与防御通路上述过程会造成损伤位点局部超弱光子发射强度上升[71]。本研究发现不同药剂处理损伤位点后超弱光子发射强度存在明显差异。实验材料为新鲜鹅掌柴叶片叶片尖端统一切割制造损伤见图5设置异丙醇、过氧化氢、苯佐卡因三组药剂处理组空白损伤叶片作为对照组每组4片叶片共5组生物学重复。连续16 h成像每小时采集1张图像对比不同药剂处理损伤位点的光子发射强度。图5 A所有鹅掌柴叶片尖端统一制造损伤连续16 h成像单张曝光时长1 h每组图像内左上叶片涂抹异丙醇、中间叶片涂抹过氧化氢、底部叶片涂抹苯佐卡因麻醉凝胶、右侧叶片无药剂处理作为空白对照所有药剂均匀涂抹叶片全表面B实验首小时内损伤区域/健康区域光子强度比值柱状图N5重复C16 h成像周期内各组叶片损伤位点相对健康区域光子计数比值随时间变化曲线。为单独验证苯佐卡因本身作用设置叶片单侧涂抹苯佐卡因、另一侧空白对照其余药剂均全叶涂抹。成像结果显示全部叶片损伤部位的超弱光子发射强度在16 h观测周期内始终显著高于健康区域。统计首小时损伤/健康区域光子计数比值各组药剂处理产生差异化响应20%体积分数苯佐卡因处理损伤位点的发射强度显著高于公认可增强超弱光子发射的过氧化氢同时高于异丙醇苯佐卡因以异丙醇为溶剂仅异丙醇对照组可证明麻醉剂本身具备独立调控效应。动物细胞中苯佐卡因的作用靶点为钠离子通道植物同样存在钠离子通道[39]目前暂未完全阐明苯佐卡因大幅提升损伤部位超弱光子发射的分子机制本文推测该现象与活性氧调控通路相关[40–42]仍需后续实验解析麻醉剂与植物损伤响应的互作机理。五、总结与结论本研究系统探究超弱光子发射UPE信号表征小鼠生命活性、植物各类胁迫状态的应用潜力。依托高性能EMCCD、CCD成像相机分别开展小鼠、植物多组对照实验得到以下结论活体小鼠全身超弱光子发射强度显著高于死亡小鼠直接证明超弱光子发射与生命活性高度关联植物超弱光子发射强度受温度调控升温提升发射强度该变化规律与黑体辐射存在本质区别超弱光子发射可用于表征植物物理损伤损伤位点光子释放量显著上升化学药剂处理会改变损伤部位发射强度其中苯佐卡因处理组发射强度在所有受试药剂中最高该现象的细胞分子机理仍需深入研究。超弱光子发射基础研究具备重要科研价值可无侵入式解析生物体内生化、代谢过程发射强度与生命活性直接绑定环境胁迫因素可调控超弱光子发射信号因此该技术可作为氧化应激、生理病变的灵敏检测指标为植物生物学、生物医学基础研究提供全新研究手段。应用层面植物超弱光子发射检测技术可开发为简易非侵入式监测方案用于农业、植物生物学领域实时监控不同环境下植株健康状态、生长发育情况小鼠超弱光子发射成像可拓展至生物医学、临床相关研究[58–65,67]实现无标记、非侵入式活体生化代谢成像。六、数据可用性声明实验原始数据可向通讯作者申请获取。致谢本研究得到加拿大国家研究委员会量子传感器挑战项目、加拿大自然科学与工程研究理事会发现基金、量子增强传感成像联盟量子财团基金、加拿大前沿科研基金资助。感谢Hadi Zadeh-Haghighi、Rishabh、Gabriel Bertolesi、Douglas Muench提供宝贵讨论意见。利益冲突声明本文作者声明不存在利益冲突。七、通讯作者稿件通讯、材料索取请联系Daniel Oblak邮箱doblakucalgary.ca。参考文献[1] Veselowskii, V. 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双梁正问题 【 torch 实战】（Python代码实现）)
