Bucci Andrea, Tortarolo Giorgio, Held Marcus Oliver, Bega Luca, Perego Eleonora, Castagnetti Francesco, Bozzoni Irene, Slenders Eli, Vicidomini Giuseppe
Molecular Microscopy and Spectroscopy, Istituto Italiano di Tecnologia, Genoa, Italy.
Dipartimento di Informatica, Bioingegneria, Robotica e Ingegneria dei Sistemi, University of Genoa, Genoa, Italy.
Nat Commun. 2024 Jul 23;15(1):6188. doi: 10.1038/s41467-024-50512-9.
Single-particle tracking techniques enable investigation of the complex functions and interactions of individual particles in biological environments. Many such techniques exist, each demonstrating trade-offs between spatiotemporal resolution, spatial and temporal range, technical complexity, and information content. To mitigate these trade-offs, we enhanced a confocal laser scanning microscope with an asynchronous read-out single-photon avalanche diode array detector. This detector provides an image of the particle's emission, precisely reflecting its position within the excitation volume. This localization is utilized in a real-time feedback system to drive the microscope scanning mechanism and ensure the particle remains centered inside the excitation volume. As each pixel is an independent single-photon detector, single-particle tracking is combined with fluorescence lifetime measurement. Our system achieves 40 nm lateral and 60 nm axial localization precision with 100 photons and sub-millisecond temporal sampling for real-time tracking. Offline tracking can refine this precision to the microsecond scale. We validated the system's spatiotemporal resolution by tracking fluorescent beads with diffusion coefficients up to 10 μm/s. Additionally, we investigated the movement of lysosomes in living SK-N-BE cells and measured the fluorescence lifetime of the marker expressed on a membrane protein. We expect that this implementation will open other correlative imaging and tracking studies.
单粒子追踪技术能够研究生物环境中单个粒子的复杂功能和相互作用。存在许多这样的技术，每种技术都在时空分辨率、空间和时间范围、技术复杂性以及信息含量之间展现出权衡。为了减轻这些权衡，我们用一个异步读出单光子雪崩二极管阵列探测器增强了一台共聚焦激光扫描显微镜。该探测器提供粒子发射的图像，精确反映其在激发体积内的位置。这种定位在一个实时反馈系统中被用于驱动显微镜扫描机制，并确保粒子始终位于激发体积的中心。由于每个像素都是一个独立的单光子探测器，单粒子追踪与荧光寿命测量相结合。我们的系统在有100个光子时实现了40纳米的横向和60纳米的轴向定位精度，以及用于实时追踪的亚毫秒级时间采样。离线追踪可以将这种精度提高到微秒尺度。我们通过追踪扩散系数高达10 μm/s的荧光珠验证了系统的时空分辨率。此外，我们研究了活的SK-N-BE细胞中溶酶体的运动，并测量了膜蛋白上表达的标记物的荧光寿命。我们期望这种实现方式将开启其他相关成像和追踪研究。
