Three-dimensional single particle tracking in a light sheet microscope

Abstract

Technical development in microscopy, and particularly in fluorescence microscopy, has facilitated the investigation of ever smaller details in biological specimen. The combination of specific labeling of molecular compounds, sophisticated optical setups and sensitive detectors enables observation of single molecules. Using fast video microscopy, it is now possible to directly observe the <i>cell’s molecular machinery at work</i> by tracking single molecules with high spatial and temporal resolution. Single molecule tracking can reveal detailed information about the dynamics of biological processes. However, technical requirements for single molecule detection limit the depth of field to less than 1 μm. Thus, single molecule tracking is typically limited to studying phenomena in planar membranes or, in extended specimen, often relies on two dimensional projections of short trajectory fragments.<br /> The work presented here strives to overcome these limitations by combining real-time three-dimensional localization of single particles with an active feedback loop to keep a particle of interest within the observation volume. To this end, a light sheet microscopy setup was designed and assembled around a commercial microscope body. It was equipped with a fast piezo stage for axial sample positioning. Three-dimensional spatial information was encoded in the shape of the point spread function by astigmatic detection and retrieved by real-time image analysis code developed for this purpose. A novel localization metric based on cross-correlation template matching was devised to enable tracking based on a low number of photons detected per particle. During post-processing, relative axial localizations determined from the image data were combined with the piezo stage position to obtain full three-dimensional particle trajectories. <br /> Mechanical and optical properties of the setup were thoroughly characterized using appropriate test samples. A temporal resolution down to 1,12 ms was achieved. The localization precision of the method was experimentally determined by repeated imaging of immobilized fluorescent beads. The capability to track single emitters was validated in a biochemical model system. Lipids labeled with a synthetic dye molecule were incorporated in the bilayer membrane of giant unilamellar vesicles and tracked on their spherical surface. Trajectories of more than 20 s duration could be obtained at as little as 130 photons detected per frame. An analysis of the photophysical properties revealed that observation times per particle were limited not by failure of the tracking algorithm but by photobleaching. <br /> Applicability of the method in biological specimen was proved by tracking fluorescent nanoparticles micro-injected into <i>C. tentans</i> salivary gland cell nuclei for more than 270 s in several thousand frames. <br /> Subsequently, the method was applied to track mRNA and rRNA particles in <i>C. tentans</i> salivary gland cell nuclei. Biomolecules were specifically labeled by complementary oligonucleotides carrying up to three synthetic dye molecules. It was possible to routinely acquire trajectories of particles with a diffusion coefficient of D = 1-2 μm²/s spanning ≥ 4 s and 4-5 μm in axial direction. The longest trajectories lasted more than 16 s and covered 10 μm axially. Both, observation time and axial range, were increased by more than one order of magnitude as compared to standard 2D tracking experiments. It was thus possible to investigate mobility states not on the basis of an ensemble of short observations but for individual particles.