Significance: Complementary absorption and fluorescence contrast could prove useful for a wide range of biomedical applications. However, current absorption-based photoacoustic microscopy systems require the ultrasound transducers to physically touch the samples, thereby increasing contamination and limiting strong optical focusing in reflection mode. Aim: We sought to develop an all-optical system for imaging cells and tissues using the three combined imaging modalities: photoacoustic remote sensing (PARS), epifluorescence, and confocal laser scanning microscopy (CLSM). Approach: A PARS subsystem with ultraviolet excitation was used to obtain label-free absorption-contrast images of nucleic acids in ex vivo tissue samples. Co-integrated epifluorescence and CLSM subsystems were used to verify the 2D and 3D nuclei distribution. Results: Complementary absorption and fluorescence contrast were demonstrated in phantom imaging experiments and subsequent cell and tissue imaging experiments. Lateral and axial resolution of ultraviolet-PARS (UV-PARS) is shown to be 0.39 and 1.6 μm, respectively, with 266-nm light. CLSM lateral and axial resolution was measured as 0.97 and 2.0 μm, respectively. This resolution is sufficient to image individual cell layers with fine optical sectioning. UV-PARS images of cell nuclei are validated in thick tissue using CLSM. Conclusions: Multimodal absorption and fluorescence contrast are obtained with a non-contact all-optical microscopy system for the first time and utilized to obtain images of cells and tissues with subcellular resolution.

中文翻译：

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多模态 3D 光声遥感和共聚焦荧光显微成像

意义：互补吸收和荧光对比可以证明对广泛的生物医学应用有用。然而，当前基于吸收的光声显微镜系统需要超声换能器物理接触样品，从而增加污染并限制反射模式下的强光学聚焦。目标：我们试图开发一种使用三种组合成像方式对细胞和组织进行成像的全光学系统：光声遥感 (PARS)、落射荧光和共聚焦激光扫描显微镜 (CLSM)。方法：使用具有紫外线激发的 PARS 子系统获得离体组织样本中核酸的无标记吸收对比图像。共集成落射荧光和 CLSM 子系统用于验证 2D 和 3D 核分布。结果：在体模成像实验和随后的细胞和组织成像实验中证明了互补吸收和荧光对比度。紫外线-PARS (UV-PARS) 的横向和轴向分辨率分别为 0.39 和 1.6 μm，266 nm 光。CLSM 横向和轴向分辨率分别测量为 0.97 和 2.0 μm。该分辨率足以对具有精细光学切片的单个细胞层进行成像。使用 CLSM 在厚组织中验证细胞核的 UV-PARS 图像。结论：首次使用非接触式全光学显微镜系统获得多模态吸收和荧光对比度，并用于获得亚细胞分辨率的细胞和组织图像。