Three-dimensional, single-molecule fluorescence imaging beyond the diffraction limit by using a double-helix point spread function

Abstract

We demonstrate single-molecule fluorescence imaging beyond the optical diffraction limit in 3 dimensions with a wide-field microscope that exhibits a double-helix point spread function (DH-PSF). The DH-PSF design features high and uniform Fisher information and has 2 dominant lobes in the image plane whose angular orientation rotates with the axial (z) position of the emitter. Single fluorescent molecules in a thick polymer sample are localized in single 500-ms acquisitions with 10- to 20-nm precision over a large depth of field (2 microm) by finding the center of the 2 DH-PSF lobes. By using a photoactivatable fluorophore, repeated imaging of sparse subsets with a DH-PSF microscope provides superresolution imaging of high concentrations of molecules in all 3 dimensions. The combination of optical PSF design and digital postprocessing with photoactivatable fluorophores opens up avenues for improving 3D imaging resolution beyond the Rayleigh diffraction limit.

Fig. 1.

DH-PSF imaging system and z-calibration. (A) Collection path of the single-molecule DH-PSF setup. IL is the imaging (tube) lens of the microscope, L1 and L2 are focal-length-matched achromatic lenses, and SLM is a liquid crystal spatial light modulator. (B) Typical calibration curve of angle between 2 lobes with respect to the horizontal versus axial position measured with a piezo-controlled objective. (Inset) 3D plot of the DH-PSF intensity profile (Scale bar: 400 nm.) (C) Images of a fluorescent bead used for the calibration curve at different axial positions, with 0 being in focus.

Fig. 2.

3D localization of a single molecule. (A) Histogram of 44 localizations of one single photoactivated DCDHF-V-PF₄ molecule in x, y, and z in a layer of PMMA. The standard deviations of the measurements in x, y, and z are 12.8, 12.1, and 19.5 nm, respectively. The smooth curve is a Gaussian fit in each case. An average of 9,300 photons were detected per estimation on top of background noise fluctuations of 48 photons per pixel. (B) Representative single-molecule image with DH-PSF acquired in one 500-ms frame. (C) Localizations plotted in 3 dimensions.

Fig. 3.

3D superlocalizations of a low concentration of DCDHF-P molecules in a thick PMMA sample. (A) Comparison of the standard PSF (i.e., Upper, SLM off) to the DH-PSF image of 2 molecules (Lower, SLM on). (Scale bar: 1 μm.) (B) Representative image of many single molecules at different x, y, and z positions. (Scale bar: 2 μm.) (C) 4D (x, y, and z, time) representation of single-molecule position determinations during a sequence of 97 frames, with a color map showing the time of acquisition.

Fig. 4.

3D superresolution imaging. (A) High concentrations of single molecules of DCDHF-V-PF₄-azide in a thick PMMA sample image using the PALM/STORM/F-PALM method with the DH-PSF as described in the text. Color indicates total number of photons used for estimation after background correction. (B) Zoom-in of position estimations for molecules 1 and 2 (blue and red, respectively) separated by 14 nm (x), 26 nm (y), and 21 nm (z); Euclidean distance (green): 36 nm. (Scale bar: 20 nm.) (C) Image from activation cycle 1 showing molecule 1. (D) Image from later in cycle 1 confirming that molecule 1 bleached. (E) Image from activation cycle 2 showing molecule 2. (Scale bar: C–E, 1 μm.)