Abstract
A microscopy platform that leverages the arrival time of individual photons to enable 3D single-particle tracking of fast-moving (translational diffusion coefficient of ≃3.3 µm2/s) particles in high-background environments is reported here. It combines a hardware-based time-gating module, which enables the rate of photon processing to be as high as 100MHz, with a two-photon-excited 3D single-particle tracking confocal microscope to enable high sample penetration depth. Proof-of-principle experiments where single quantum dots are tracked in solutions containing dye-stained cellulose, are shown with tracking performance markedly improved using the hardware-based time-gating module. Such a microscope designis anticipated to be of use to a variety of communities who wish to track single particles in cellular environments, which commonly have high fluorescence and scattering background.
Highlights
There is increasing recognition that molecular actions are difficult to divorce from the surrounding context, as exemplified by those of biological macromolecules.[1,2,3] This, in a sense, is not surprising; a significant and ongoing effort of physical chemistry/chemical physics is to fully comprehend the effect of the solvent on a variety of different unimolecular[4] and bimolecular[5,6] reactions
This was achieved as follows: The emission from a two-photon-excited probe was split in a 1:9 ratio, with 10% of the emission going to an Electron Multiplying Charge-Coupled Device (EMCCD) camera (Roper Scientific) and 90% going to the tracking optics
When ∼single nm deviations in the particle position occur, the detectors will have an imbalance in their signal that is used for feedback control, with the stage counteracting this motion to return the detectors to balance by moving the particle back to the center of the microscope focus
Summary
There is increasing recognition that molecular actions are difficult to divorce from the surrounding context, as exemplified by those of biological macromolecules.[1,2,3] This, in a sense, is not surprising; a significant and ongoing effort of physical chemistry/chemical physics is to fully comprehend the effect of the solvent on a variety of different unimolecular[4] and bimolecular[5,6] reactions. 1. Cartoon illustrating an example of a time-gated real-time 3D single-particle tracking experiment using an enzyme-biomaterial system, where the gQD-labeled enzymes provide a long lifetime signal and the dye-stained (DS) biomaterial generates a short lifetime signal. DeVore et al.[47,48] first used gQDs and time gating simultaneously in real-time 3D single-particle tracking to improve the signal-to-background ratio (SBR). With this improvement in SBR, they were able to track gQDs with a translational diffusion coefficient of ∼0.1 μm2/s in dye-labeled rat mast cells. We use two-photon excitation because it improves the penetration depth into cellular and tissue or plant-cell samples, as well as further increasing the SBR by reducing both laser[52] and Raman scatterings[53] and excluding out-of-focus light.[27,54]
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