【Small】脑类器官成像突破：无标记紫外光声显微技术首次实现活体三维细胞核级毒性评估
文章标题：A Label-Free Ultraviolet Photoacoustic Microscopy Enables Nuclear Imaging and Toxicity Assessment in an Intact Brain Organoid
通讯作者：Lihong V. Wang, Jeesu Kim, Jong-Chan Park, Byullee Park
文章概要
本研究开发了一种创新的压电轴向扫描式紫外光声显微成像系统（UV-PAM-Z），首次在不进行任何化学染色或物理切片的前提下，实现了对三维完整活体脑类器官的超高分辨率细胞核三维成像。该技术不仅突破了传统光学成像的深度与损伤极限，更成功应用于产前酒精暴露导致的精神神经发育障碍模型中。实验结果表明，在酒精毒性刺激下，脑类器官的细胞核面积、最大直径和圆度分别显著下降了46.1%、20.8%和6.0%。本研究为三维生物样本的药物筛选、毒理学评估以及疾病模型的建立提供了一种完全无标记、无损伤且高保真的定量表征新平台。
引言
产前酒精暴露是导致胎儿酒精谱系障碍（FASDs）的主要诱因，常伴随严重的神经发生受阻、细胞凋亡以及长期认知功能障碍。为了克服传统动物模型存在的物种差异与伦理限制，诱导多能干细胞（hiPSCs）分化形成的三维皮质类器官已成为模拟人类大脑发育及药物毒性反应的重要体外平台。然而，对这些结构极其复杂的类器官进行病理学评估并非易事。细胞核形态学变化是反映细胞应激、凋亡和神经发育受损的最核心指标。传统的高分辨荧光显微镜成像高度依赖于外源性荧光染料（如DAPI）标记以及耗时的组织物理切片或化学透明化处理。这些繁琐的操作极易改变组织天然结构、引入实验误差，且难以对活体样本实施实时、长期的无毒性监测。如何在保留生物样本最原生状态的前提下，对完整三维类器官进行亚微米级的细胞核解析，是生物医学成像领域长期面临的重大科学挑战。
Schematic illustration of organoid generation and UV-PAM-Z imaging workflow. (a) Protocol for forebrain organoid generation and its potential applications. (b) Comparison between traditional fluorescence imaging, which requires sectioning and labeling, and label-free UV-PAM-Z imaging of intact organoids. (c) Working principle of UV-PAM-Z imaging based on pulsed laser excitation and ultrasound detection. (d) Alcoholic challenge experiments with control and EtOH-treated organoids, showing nuclear morphological differences using UV-PAM-Z. PBMC, peripheral blood mononuclear cell; hiPSC, human induced pluripotent stem cell; EB, embryoid body; EBM, embryoid body formation medium; Dor, dorsomorphin; SB, SB431542; NB, Neurobasal medium; EGF, epidermal growth factor; FGF-2, basic fibroblast growth factor; BDNF, brain-derived neurotrophic factor; NT3, neurotrophin-3; D, day; FLI, fluorescence imaging; PAI, photoacoustic imaging; UV-PAM-Z, z-scanning UV photoacoustic microscopy; EtOH, ethanol.
主要实验及结论
为了解决上述瓶颈，研究团队构建了基于266纳米脉冲激光激发的无标记压电扫描式紫外光声显微成像系统（UV-PAM-Z），其工作原理与成像工作流如图1所示。该系统巧妙地利用了细胞核内天然核酸对266纳米紫外光的强吸光特性。当脉冲激光照射核酸分子时，核酸由于热弹性膨胀激发出宽带超声信号，由同轴配置的高频超声换能器接收并重构为图像。通过集成步进精度达0.5微米的压电位移台，系统能够对未切片的完整活体类器官进行高精度的深度解析扫描。为了验证该成像系统在三维脑类器官中的分辨率性能，研究团队利用金纳米颗粒和碳纤维进行了精细的基准测试，如图2所示。结果证实系统达到了278纳米的超高侧向分辨率和25.6微米的纵向分辨率，这一性能足以在单细胞尺度上清晰区分相邻的独立细胞核。此外，深度扫描实验中，光声信号焦点位置与压电位移台位移之间展现出极强的线性关系，这极大地保障了三维深度重构的几何保真度。
Schematic of the UV-PAM-Z system and performance of the system, including resolution and z-axis scanning. (a) Schematic of the UV-PAM-Z system and enlarged view of the scanning region. The ND filter was used to control the laser power, while the PD was used to monitor the relative laser power. (b) Lateral resolution measurement using a 100 nm gold nanoparticle target. The measured intensity profile and corresponding Gaussian fit are shown. (c) Axial resolution measurement using an isolated carbon-fiber target suspended approximately 2 mm above the substrate. The acquired A-line signal, envelope signal, and corresponding Gaussian fit are shown. (d) Frequency spectrum of the PA signal generated from (c). (e) Schematic of the phantom used to validate the z-axis piezo stage, composed of an angled carbon fiber for depth-dependent imaging. (f) PA MAP images of the carbon fiber acquired at different z-stage positions (0, 50, and 100 µm; scale bar = 50 µm). (g) Linear correlation between the z-axis piezo stage position and the PA signal peak position, confirming accurate depth scanning performance. PC, personal computer; DAQ, digital acquisition; PD, photodetector; ND, neutral density filter; L, lens; P, pinhole; M, mirror; OL, objective lens; UST, ultrasound transducer; MAP, maximum amplitude projection; PA, photoacoustic; UV-PAM-Z, z-scanning UV photoacoustic microscopy; FWHM, full width at half maximum.
Validation of UV-PAM-Z system reliability by quantitative comparison with DAPI-stained nuclear imaging. (a–c) Brightfield, DAPI, and UV-PAM-Z images of the same organoid section region. The UV-PAM-Z image was acquired prior to staining. (d,e) DAPI and high-precision step-size UV-PAM-Z images in the same region. (f) Histograms of nuclear area and maximum diameter analyzed from images in (d,e). (g,h) Magnified DAPI and UV-PAM-Z images of the yellow boxed regions in (d,e), respectively. (i) Normalized intensity profiles along the yellow lines in (g,h). (j) Overlayed image of (g,h), showing co-registered nuclear signals from UV-PAM-Z and DAPI in the same location. (k) Spatial centroid positions of corresponding nuclei from UV-PAM-Z and DAPI images. UV-PAM-Z, z-scanning UV photoacoustic microscopy; norm, normalized.
在验证了硬件系统的技术参数后，研究人员对95天龄的脑类器官切片进行了平行对照实验，以评估定量成像的可靠性，如图3所示。在同一片组织区域中，无标记的紫外光声成像不仅在视觉上呈现出比传统明场显微镜更高的对比度，而且其细胞核检测结果与随后进行的经典DAPI荧光染色重合度极高。统计直方图表明，光声成像测得的平均细胞核面积与平均核直径与荧光成像的数据高度吻合，且两者的二维信号剖面相关系数高达0.843，细胞核质心位置的平均偏差仅为0.86微米。这一系列极其严谨的定量对比证明，无标记紫外光声显微技术在提取细胞核形态学特征方面，具有完全媲美经典免疫荧光染色的高精度和高保真度，同时完全避免了洗涤和封片过程可能导致的样本微观形变。
Intact organoid imaging and 3D visualization using UV-PAM-Z. (a) Macroscopic image of the organoid and corresponding intact organoid PA MAP image acquired with UV-PAM-Z. (b) Enlarged image (yellow box), showing segmented grid (3 × 3) and individual nucleus-level visualization. Edge-detected result of a representative region selected from the enlarged image. (c) 3D histogram showing the regional distribution of nuclear area quantified from the nine segmented regions. (d) 3D histogram showing the regional distribution of nuclear diameter quantified from the nine segmented regions. (e) 3D-rendered image of the entire organoid reconstructed from three z-stack images. (f) Orthogonal cross-sectional B-mode images extracted from the dataset shown in (e): xz plane (top, along the red line) and yz plane (bottom, along the green line). Representative nuclear PA signals are highlighted by yellow circles. (g) Height-encoded MAP image representing the height distribution of nuclei, derived from (e). (h) Height-resolved images obtained by stepwise slicing from the bottom surface in 50 µm intervals. PA, photoacoustic; MAP, maximum amplitude projection; UV-PAM-Z, z-scanning UV photoacoustic microscopy.
基于此，研究团队进一步向最具挑战性的三维完整活体脑类器官成像发起尝试，具体成像及三维可视化效果如图4所示。研究人员将未做任何切片和染色的完整类器官置于定制的透明石英底片夹具中进行原位成像。在一项大视野无损多深度切片扫描中，系统成功在完全不破坏组织的前提下，获取了类器官表层向内延伸超过100微米深度的细胞核立体空间分布，并重构出高精度的三维渲染视图。高度编码的最大振幅投影清晰展现了细胞核在类器官不同深度层面的空间堆叠规律。为了排除紫外光激发的潜在光毒性，研究团队还在成像后进行了活死细胞染色和MTT代谢活性检测，结果证实，在当前精心优化的低能量激光照射参数下，脑类器官整体依然保持了与对照组无异的高存活率和代谢活性。
Comparison of morphological changes and statistical analysis of nuclei in organoids following control and alcoholic challenge. (a) Representative UV-PAM-Z MAP images of nuclei from control (I–III) and EtOH-treated (IV–VI) organoids. (b) Enlarged nuclear morphologies extracted from selected yellow boxes in a (CTL, i–iv). (c) Bar graphs with scatter overlay showing the statistical distributions of nuclear area, maximum diameter, and circularity for control and EtOH-treated groups. Each data point represents one organoid (n = 14 total organoids; n = 7 per group). (*, p < 0.05; **, p < 0.01; ****, p < 0.0001; unpaired t-test). CTL, control; PA, photoacoustic; EtOH, ethanol; UV-PAM-Z, z-scanning UV photoacoustic microscopy; MAP, maximum amplitude projection.
最后，该平台被直接应用于急性酒精暴露导致的细胞神经毒性实时评估。研究人员将脑类器官暴露于2%浓度的乙醇溶液中处理6小时，随后利用紫外光声成像观察其细胞核微观形态的变化，并配合支持向量机（SVM）机器学习算法进行了分类评估，如图5和图6所示。在酒精刺激下，类器官表层的细胞核呈现出明显的结构不规则、固缩及碎片化形态，这与细胞凋亡的典型病理学表征完全一致。定量统计分析表明，酒精处理组的细胞核面积和最大 caliper 直径呈现极显著的萎缩，核圆度也有所降低。通过提取这些多维度的细胞核形态学特征并输入分类器，机器学习算法在留一器官对交叉验证中，实现了高达83.6%的均值分类准确率，曲线下面积（AUC）达到0.85以上。这一结果表明，无标记紫外光声显微成像不仅能敏感地捕捉到单个细胞核尺度的早期毒性形变，还能结合智能算法实现高度自动化的药物响应判别。
Machine learning-based classification of control and ethanol-treated nuclei using nuclear morphological features. (a) 2D feature space-based classification using area-diameter and area-circularity feature pairs (left: scatter plot, middle: confusion matrix, right: ROC curve). (b) 3D feature space-based classification integrating area, diameter, and circularity. The displayed scatter plots, confusion matrices, and ROC curves represent pooled results obtained from seven iterations of leave-one-organoid-pair-out cross-validation.
总结及展望
本研究成功开发并应用压电扫描式紫外光声显微成像系统（UV-PAM-Z），首次在不使用任何荧光染料和切片技术的情况下，完成了对活体完整脑类器官内部细胞核形貌的三维无标记高分辨解析。该技术巧妙利用了生物体核酸分子的天然紫外吸收特性，不仅在分辨率上达到了单细胞核分析的标准，更通过精密的深度控制实现了高达100微米的组织穿透成像。在酒精诱导的脑神经毒性实验中，该系统对细胞核固缩与碎片化病理过程的定量捕获，充分展示了其作为高通量、无损伤毒理学评估平台的巨大潜力。
尽管目前的系统受限于机械扫描速度而难以进行超快速的实时动态追踪，但研究团队指出，未来通过引入振镜光学扫描器（Galvo-scanner）或微机电系统（MEMS）微镜，成像速率有望得到数量级的提升。此外，结合具有超大焦深的超构透镜设计或先进的人工智能算法，能够进一步拓展有效的成像深度并降低局部紫外光照带来的光毒性风险。该技术不仅为人类大脑发育和退行性疾病的研究开辟了全新的活体观测窗口，也将在未来的新型药物筛选、类器官病理学诊断和转化医学研究中发挥举足轻重的作用。# 【Small】脑类器官成像突破：无标记紫外光声显微技术首次实现活体三维细胞核级毒性评估
文章标题：A Label-Free Ultraviolet Photoacoustic Microscopy Enables Nuclear Imaging and Toxicity Assessment in an Intact Brain Organoid
通讯作者：Lihong V. Wang, Jeesu Kim, Jong-Chan Park, Byullee Park
文章概要
本研究开发了一种创新的压电轴向扫描式紫外光声显微成像系统（UV-PAM-Z），首次在不进行任何化学染色或物理切片的前提下，实现了对三维完整活体脑类器官的超高分辨率细胞核三维成像。该技术不仅突破了传统光学成像的深度与损伤极限，更成功应用于产前酒精暴露导致的精神神经发育障碍模型中。实验结果表明，在酒精毒性刺激下，脑类器官的细胞核面积、最大直径和圆度分别显著下降了46.1%、20.8%和6.0%。本研究为三维生物样本的药物筛选、毒理学评估以及疾病模型的建立提供了一种完全无标记、无损伤且高保真的定量表征新平台。
引言
产前酒精暴露是导致胎儿酒精谱系障碍（FASDs）的主要诱因，常伴随严重的神经发生受阻、细胞凋亡以及长期认知功能障碍。为了克服传统动物模型存在的物种差异与伦理限制，诱导多能干细胞（hiPSCs）分化形成的三维皮质类器官已成为模拟人类大脑发育及药物毒性反应的重要体外平台。然而，对这些结构极其复杂的类器官进行病理学评估并非易事。细胞核形态学变化是反映细胞应激、凋亡和神经发育受损的最核心指标。传统的高分辨荧光显微镜成像高度依赖于外源性荧光染料（如DAPI）标记以及耗时的组织物理切片或化学透明化处理。这些繁琐的操作极易改变组织天然结构、引入实验误差，且难以对活体样本实施实时、长期的无毒性监测。如何在保留生物样本最原生状态的前提下，对完整三维类器官进行亚微米级的细胞核解析，是生物医学成像领域长期面临的重大科学挑战。
Schematic illustration of organoid generation and UV-PAM-Z imaging workflow. (a) Protocol for forebrain organoid generation and its potential applications. (b) Comparison between traditional fluorescence imaging, which requires sectioning and labeling, and label-free UV-PAM-Z imaging of intact organoids. (c) Working principle of UV-PAM-Z imaging based on pulsed laser excitation and ultrasound detection. (d) Alcoholic challenge experiments with control and EtOH-treated organoids, showing nuclear morphological differences using UV-PAM-Z. PBMC, peripheral blood mononuclear cell; hiPSC, human induced pluripotent stem cell; EB, embryoid body; EBM, embryoid body formation medium; Dor, dorsomorphin; SB, SB431542; NB, Neurobasal medium; EGF, epidermal growth factor; FGF-2, basic fibroblast growth factor; BDNF, brain-derived neurotrophic factor; NT3, neurotrophin-3; D, day; FLI, fluorescence imaging; PAI, photoacoustic imaging; UV-PAM-Z, z-scanning UV photoacoustic microscopy; EtOH, ethanol.
主要实验及结论
为了解决上述瓶颈，研究团队构建了基于266纳米脉冲激光激发的无标记压电扫描式紫外光声显微成像系统（UV-PAM-Z），其工作原理与成像工作流如图1所示。该系统巧妙地利用了细胞核内天然核酸对266纳米紫外光的强吸光特性。当脉冲激光照射核酸分子时，核酸由于热弹性膨胀激发出宽带超声信号，由同轴配置的高频超声换能器接收并重构为图像。通过集成步进精度达0.5微米的压电位移台，系统能够对未切片的完整活体类器官进行高精度的深度解析扫描。为了验证该成像系统在三维脑类器官中的分辨率性能，研究团队利用金纳米颗粒和碳纤维进行了精细的基准测试，如图2所示。结果证实系统达到了278纳米的超高侧向分辨率和25.6微米的纵向分辨率，这一性能足以在单细胞尺度上清晰区分相邻的独立细胞核。此外，深度扫描实验中，光声信号焦点位置与压电位移台位移之间展现出极强的线性关系，这极大地保障了三维深度重构的几何保真度。
Schematic of the UV-PAM-Z system and performance of the system, including resolution and z-axis scanning. (a) Schematic of the UV-PAM-Z system and enlarged view of the scanning region. The ND filter was used to control the laser power, while the PD was used to monitor the relative laser power. (b) Lateral resolution measurement using a 100 nm gold nanoparticle target. The measured intensity profile and corresponding Gaussian fit are shown. (c) Axial resolution measurement using an isolated carbon-fiber target suspended approximately 2 mm above the substrate. The acquired A-line signal, envelope signal, and corresponding Gaussian fit are shown. (d) Frequency spectrum of the PA signal generated from (c). (e) Schematic of the phantom used to validate the z-axis piezo stage, composed of an angled carbon fiber for depth-dependent imaging. (f) PA MAP images of the carbon fiber acquired at different z-stage positions (0, 50, and 100 µm; scale bar = 50 µm). (g) Linear correlation between the z-axis piezo stage position and the PA signal peak position, confirming accurate depth scanning performance. PC, personal computer; DAQ, digital acquisition; PD, photodetector; ND, neutral density filter; L, lens; P, pinhole; M, mirror; OL, objective lens; UST, ultrasound transducer; MAP, maximum amplitude projection; PA, photoacoustic; UV-PAM-Z, z-scanning UV photoacoustic microscopy; FWHM, full width at half maximum.
Validation of UV-PAM-Z system reliability by quantitative comparison with DAPI-stained nuclear imaging. (a–c) Brightfield, DAPI, and UV-PAM-Z images of the same organoid section region. The UV-PAM-Z image was acquired prior to staining. (d,e) DAPI and high-precision step-size UV-PAM-Z images in the same region. (f) Histograms of nuclear area and maximum diameter analyzed from images in (d,e). (g,h) Magnified DAPI and UV-PAM-Z images of the yellow boxed regions in (d,e), respectively. (i) Normalized intensity profiles along the yellow lines in (g,h). (j) Overlayed image of (g,h), showing co-registered nuclear signals from UV-PAM-Z and DAPI in the same location. (k) Spatial centroid positions of corresponding nuclei from UV-PAM-Z and DAPI images. UV-PAM-Z, z-scanning UV photoacoustic microscopy; norm, normalized.
在验证了硬件系统的技术参数后，研究人员对95天龄的脑类器官切片进行了平行对照实验，以评估定量成像的可靠性，如图3所示。在同一片组织区域中，无标记的紫外光声成像不仅在视觉上呈现出比传统明场显微镜更高的对比度，而且其细胞核检测结果与随后进行的经典DAPI荧光染色重合度极高。统计直方图表明，光声成像测得的平均细胞核面积与平均核直径与荧光成像的数据高度吻合，且两者的二维信号剖面相关系数高达0.843，细胞核质心位置的平均偏差仅为0.86微米。这一系列极其严谨的定量对比证明，无标记紫外光声显微技术在提取细胞核形态学特征方面，具有完全媲美经典免疫荧光染色的高精度和高保真度，同时完全避免了洗涤和封片过程可能导致的样本微观形变。
Intact organoid imaging and 3D visualization using UV-PAM-Z. (a) Macroscopic image of the organoid and corresponding intact organoid PA MAP image acquired with UV-PAM-Z. (b) Enlarged image (yellow box), showing segmented grid (3 × 3) and individual nucleus-level visualization. Edge-detected result of a representative region selected from the enlarged image. (c) 3D histogram showing the regional distribution of nuclear area quantified from the nine segmented regions. (d) 3D histogram showing the regional distribution of nuclear diameter quantified from the nine segmented regions. (e) 3D-rendered image of the entire organoid reconstructed from three z-stack images. (f) Orthogonal cross-sectional B-mode images extracted from the dataset shown in (e): xz plane (top, along the red line) and yz plane (bottom, along the green line). Representative nuclear PA signals are highlighted by yellow circles. (g) Height-encoded MAP image representing the height distribution of nuclei, derived from (e). (h) Height-resolved images obtained by stepwise slicing from the bottom surface in 50 µm intervals. PA, photoacoustic; MAP, maximum amplitude projection; UV-PAM-Z, z-scanning UV photoacoustic microscopy.
基于此，研究团队进一步向最具挑战性的三维完整活体脑类器官成像发起尝试，具体成像及三维可视化效果如图4所示。研究人员将未做任何切片和染色的完整类器官置于定制的透明石英底片夹具中进行原位成像。在一项大视野无损多深度切片扫描中，系统成功在完全不破坏组织的前提下，获取了类器官表层向内延伸超过100微米深度的细胞核立体空间分布，并重构出高精度的三维渲染视图。高度编码的最大振幅投影清晰展现了细胞核在类器官不同深度层面的空间堆叠规律。为了排除紫外光激发的潜在光毒性，研究团队还在成像后进行了活死细胞染色和MTT代谢活性检测，结果证实，在当前精心优化的低能量激光照射参数下，脑类器官整体依然保持了与对照组无异的高存活率和代谢活性。
Comparison of morphological changes and statistical analysis of nuclei in organoids following control and alcoholic challenge. (a) Representative UV-PAM-Z MAP images of nuclei from control (I–III) and EtOH-treated (IV–VI) organoids. (b) Enlarged nuclear morphologies extracted from selected yellow boxes in a (CTL, i–iv). (c) Bar graphs with scatter overlay showing the statistical distributions of nuclear area, maximum diameter, and circularity for control and EtOH-treated groups. Each data point represents one organoid (n = 14 total organoids; n = 7 per group). (*, p < 0.05; **, p < 0.01; ****, p < 0.0001; unpaired t-test). CTL, control; PA, photoacoustic; EtOH, ethanol; UV-PAM-Z, z-scanning UV photoacoustic microscopy; MAP, maximum amplitude projection.
最后，该平台被直接应用于急性酒精暴露导致的细胞神经毒性实时评估。研究人员将脑类器官暴露于2%浓度的乙醇溶液中处理6小时，随后利用紫外光声成像观察其细胞核微观形态的变化，并配合支持向量机（SVM）机器学习算法进行了分类评估，如图5和图6所示。在酒精刺激下，类器官表层的细胞核呈现出明显的结构不规则、固缩及碎片化形态，这与细胞凋亡的典型病理学表征完全一致。定量统计分析表明，酒精处理组的细胞核面积和最大 caliper 直径呈现极显著的萎缩，核圆度也有所降低。通过提取这些多维度的细胞核形态学特征并输入分类器，机器学习算法在留一器官对交叉验证中，实现了高达83.6%的均值分类准确率，曲线下面积（AUC）达到0.85以上。这一结果表明，无标记紫外光声显微成像不仅能敏感地捕捉到单个细胞核尺度的早期毒性形变，还能结合智能算法实现高度自动化的药物响应判别。
Machine learning-based classification of control and ethanol-treated nuclei using nuclear morphological features. (a) 2D feature space-based classification using area-diameter and area-circularity feature pairs (left: scatter plot, middle: confusion matrix, right: ROC curve). (b) 3D feature space-based classification integrating area, diameter, and circularity. The displayed scatter plots, confusion matrices, and ROC curves represent pooled results obtained from seven iterations of leave-one-organoid-pair-out cross-validation.
总结及展望
本研究成功开发并应用压电扫描式紫外光声显微成像系统（UV-PAM-Z），首次在不使用任何荧光染料和切片技术的情况下，完成了对活体完整脑类器官内部细胞核形貌的三维无标记高分辨解析。该技术巧妙利用了生物体核酸分子的天然紫外吸收特性，不仅在分辨率上达到了单细胞核分析的标准，更通过精密的深度控制实现了高达100微米的组织穿透成像。在酒精诱导的脑神经毒性实验中，该系统对细胞核固缩与碎片化病理过程的定量捕获，充分展示了其作为高通量、无损伤毒理学评估平台的巨大潜力。