The University of Arizona, Department of Biomedical Engineering, Tucson, Arizona, 85721-0240, USA.
J Biomed Opt. 2012 Jul;17(7):076003. doi: 10.1117/1.JBO.17.7.076003.
The use of upconverting lanthanide nanoparticles in fast-scanning microscopy is hindered by a long luminescence decay time, which greatly blurs images acquired in a nondescanned mode. We demonstrate herein an image processing method based on Richardson-Lucy deconvolution that mitigates the detrimental effects of their luminescence lifetime. This technique generates images with lateral resolution on par with the system's performance, ∼1.2 μm, while maintaining an axial resolution of 5 μm or better at a scan rate comparable with traditional two-photon microscopy. Remarkably, this can be accomplished with near infrared excitation power densities of 850 W/cm(2), several orders of magnitude below those used in two-photon imaging with molecular fluorophores. By way of illustration, we introduce the use of lipids to coat and functionalize these nanoparticles, rendering them water dispersible and readily conjugated to biologically relevant ligands, in this case epidermal growth factor receptor antibody. This deconvolution technique combined with the functionalized nanoparticles will enable three-dimensional functional tissue imaging at exceptionally low excitation power densities.
上转换镧系纳米粒子在快速扫描显微镜中的应用受到其长荧光衰减时间的阻碍，这极大地模糊了在非扫描模式下获得的图像。本文展示了一种基于 Richardson-Lucy 反卷积的图像处理方法，该方法减轻了其荧光寿命的不利影响。该技术生成的图像具有与系统性能相当的横向分辨率，约为 1.2μm，同时在与传统双光子显微镜相当的扫描速度下保持 5μm 或更好的轴向分辨率。值得注意的是，这可以在近红外激发功率密度为 850 W/cm²的条件下实现，比使用分子荧光团进行双光子成像低几个数量级。作为说明，我们引入了使用脂质来包覆和功能化这些纳米粒子，使它们在水中可分散，并容易与生物相关配体结合，在这种情况下是表皮生长因子受体抗体。这种反卷积技术与功能化纳米粒子相结合，将能够以极低的激发功率密度进行三维功能组织成像。
