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Three dimensional multi-molecule tracking in thick samples with extended depth-of-field

Heng Li, Danni Chen, Gaixia Xu, Bin Yu, and Hanben Niu

Open Access Open Access

Abstract

We present a non-z-scanning multi-molecule tracking system with nano-resolution in all three dimensions and extended depth of field (DOF), which based on distorted grating (DG) and double-helix point spread function (DH-PSF) combination microscopy (DDCM). The critical component in DDCM is a custom designed composite phase mask (PM) combining the functions of DG and DH-PSF. The localization precision and the effective DOF of the home-built DDCM system based on the designed PM were tested. Our experimental results show that the three-dimensional (3D) localization precision for the three diffraction orders of the grating are σ-1st(x, y, z) = (6.5 nm, 9.2nm, 23.4 nm), σ0th(x, y, z) = (3.7 nm, 2.8nm, 10.3 nm), and σ+1st(x, y, z) = (5.8 nm, 6.9 nm, 18.4 nm), respectively. Furthermore, the total effective DOF of the DDCM system is extended to 14 μm. Tracking experiment demonstrated that beads separated over 12 μm along the axial direction at some instants can be localized and tracked successfully.

© 2015 Optical Society of America

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Supplementary Material (2)

Media 1: AVI (2893 KB)     
Media 2: AVI (1099 KB)     

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Figures (8)
Fig. 1
Fig. 1 Design of composite the PM. (a), (b), and (c) The image shows the simulation result of composite PM [24]. (d)The actual designed PM using micro-fabrication photoetching method.

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Fig. 2
Fig. 2 Schematic of the DDCM. The laser is expanded and collimated after an excitation filter (EF), and then sent to excite the fluorescent beads with an objective (Obj) combined with a tube lens(TL1) whose focal length is f = 180 mm. Fluorescence signal is collected by the same objective and split with laser beam by a dichromatic mirror(DM). A 4f relay system consisting of two achromatic lenses (F1 and F2, f = 200 mm) and a composite PM mounted at the Fourier plane is inserted before the detector (iXon 885, Andor).

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Fig. 3
Fig. 3 Calibration of the DDCM system by imaging a fluorescent bead. The movement of bead is controlled by a nano piezoelectric stage. The bead was moved in 100 nm steps along z axis and imaged. Three representative images of the bead are shown in (a), (b), and (c), that are three images of the bead in the −1st, 0th and + 1st diffraction order section which corresponded to −6 um, −1 um and 6 um along with the z axial position. (d) Calibration curves for the 0th and the ± 1st diffraction orders.

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Fig. 4
Fig. 4 Estimation of the localization precision in x, y (upper) and z (lower) for the −1st, 0th and the + 1st diffraction orders.

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Fig. 5
Fig. 5 Localization precision as function of the number of collected photons for the −1st(a), the 0th(b) and the + 1st(c) diffraction order.

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Fig. 6
Fig. 6 Three- dimensional tracking of three beads simultaneously. (a) One image from the movie (media 1) of the dynamic imaging of beads. (b) Trajectories viewed in 3D (top) and projected on x-z plane (bottom left) and x-y plane (bottom right), respectively.

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Fig. 7
Fig. 7 MSDs for 100nm beads in different water-glycerol mixtures with a linear fit to the slope of the data. The MSD shown in green line were derived from the data in Fig. 6.

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Fig. 8
Fig. 8 Tracking fluorescent beads in a live cell in 3D. (a) White light image of the Raw-264.7 cell. (b) Fluorescence image of the emitter (media 2). (c) Three-dimensional trajectory of the bead from the red smaller boxed region in (b), showing a variety of diffusive and linear transport characteristics. (d) The trajectory from the red smaller boxed region in (c) when viewed at higher magnification.

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