Abstract
Molecular trafficking within cells, tissues and engineered three-dimensional multicellular models is critical to the understanding of the development and treatment of various diseases including cancer. However, current tracking methods are either confined to two dimensions or limited to an interrogation depth of ∼15 μm. Here we present a three-dimensional tracking method capable of quantifying rapid molecular transport dynamics in highly scattering environments at depths up to 200 μm. The system has a response time of 1 ms with a temporal resolution down to 50 μs in high signal-to-noise conditions, and a spatial localization precision as good as 35 nm. Built on spatiotemporally multiplexed two-photon excitation, this approach requires only one detector for three-dimensional particle tracking and allows for two-photon, multicolour imaging. Here we demonstrate three-dimensional tracking of epidermal growth factor receptor complexes at a depth of ∼100 μm in tumour spheroids.
Highlights
Molecular trafficking within cells, tissues and engineered three-dimensional multicellular models is critical to the understanding of the development and treatment of various diseases including cancer
We demonstrate tracking of epidermal growth factor receptor (EGFR) complexes tagged with fluorescent beads in tumour spheroids, demonstrating deep 3D Single-particle tracking (SPT) in multicellular models
Our control loop period is 1–5 ms, we emphasize that our temporal resolution can be significantly better than 1 ms by outputting the individual photon event data (Time Tag) from the time-correlated single-photon-counting (TCSPC) board
Summary
Tissues and engineered three-dimensional multicellular models is critical to the understanding of the development and treatment of various diseases including cancer. SPT with superior temporal resolution (bounded mainly by the emission rate of the fluorescent label) and simultaneous fluorescence lifetime measurements have been achieved using confocal setups with 3–5 single-element/photon-counting detectors (photomultiplier tubes (PMTs) or avalanche photodiodes) for spatial filtering[18,19,20], these methods have limited working depth (not using 2P excitation for tracking) and suffer from loss of signals due to the non-overlapping excitation and collection efficiency peaks in spatial filtering (Supplementary Fig. 3)[28]. There is no single solution to all of the above issues To address this challenge, we have developed a 2P 3D SPT method capable of tracking particles at depths up to 200 mm in scattering samples with 22/90 [xy/z]-nm spatial localization precision and 1 ms response time. We demonstrate tracking of epidermal growth factor receptor (EGFR) complexes tagged with fluorescent beads in tumour spheroids, demonstrating deep 3D SPT in multicellular models We have coined this technique tracking single particles using nonlinear and multiplexed illumination (TSUNAMI). HWP, half-wave plate; PBS, polarizing beam splitter; DM, dichroic mirror; BD, beam dump
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