Ultrahigh accuracy imaging modality for super-localization microscopy

Abstract

Super-localization microscopy encompasses techniques that depend on the accurate localization of individual molecules from generally low-light images. The obtainable localization accuracies, however, are ultimately limited by the image detector's pixelation and noise. We present the ultrahigh accuracy imaging modality (UAIM), which allows users to obtain accuracies approaching the accuracy that is achievable only in the absence of detector pixelation and noise, and which we found can experimentally provide a >200% accuracy improvement over conventional low-light imaging.

Figure 1. UAIM and its applications

(a) Visual comparison of a UAIM image and a conventional EMCCD image. The UAIM (conventional) image is that of an Atto647N molecule from which ~72 (~84) photons per image were detected on average, and was acquired with an effective pixel size of 16 nm (253.97 nm) using a 1000× (63×) magnification. The brightest pixel of the UAIM (conventional) image has a mean photon count of 0.22 (21.85). The mesh representation, which displays intensity as height, contrasts more conspicuously the spiky appearance of the UAIM image with the relatively smooth appearance of the conventional image. Scale bars, 0.5 µm. (b) Superresolution imaging of a LAMP1⁺ cellular structure using UAIM. Top, the widefield fluorescence image of the structure, formed by summing the 5063 UAIM (1000×) images from which single Alexa647 molecules were localized to produce a superresolution image. Bottom, the superresolution image constructed from the location estimates of individual Alexa647 molecules. The average number of photons detected per molecule is 128.94. Scale bars, 1 µm. (c) Single molecule tracking using UAIM. Top, the trajectory of an erbB2 receptor (Supplementary Video 1) determined by localizing its Atto647N label from 594 UAIM (1000×) images. On average, 102.85 photons per image were detected from the Atto647N dye. Bottom left, one of the 594 UAIM images. The red box encloses the tracked Atto647N molecule. Bottom right, compacted (10 × 10-binned) version of the same UAIM image that facilitates visualization of the tracked Atto647N molecule (indicated by red arrow). Scale bars, 1 µm.

Figure 2. Experimental and theoretical demonstration of UAIM

(a) Comparing the standard deviations of the maximum likelihood estimates of the x₀ coordinate of fluorescent beads imaged using UAIM (blue star) and conventional EMCCD imaging (blue circle). Each standard deviation corresponds to a different bead, identified by the mean number of photons detected from it per image. For each standard deviation, the corresponding limit of accuracy (UAIM: magenta star; conventional: magenta circle) is shown. Likewise, the corresponding ultimate limit of accuracy (UAIM: black star; conventional: black circle), which assumes an ideal detector that introduces neither noise nor pixelation, is shown. The UAIM (conventional) images were acquired with an effective pixel size of 16 nm (253.97 nm) using a 1000× (63×) magnification. (b) Theoretical analysis of point source localization. Decreasing the effective pixel size by increasing the magnification for EMCCD imaging at a high level of signal amplification (g = 1000) yields a limit of accuracy (star) that approaches the ultimate limit (blue line). The red markers at effective pixel sizes of 373.31 nm, 224.00 nm, and 160.00 nm (magnifications of M = 42.86, 71.43, and 100) correspond approximately to standard magnifications of 40× and 63×, and exactly to the standard magnification of 100×. For the same range of effective pixel sizes, the limit of accuracy (dot) corresponding to the common excess noise-based supposition, and the limit of accuracy (circle) corresponding to CCD imaging with a readout noise standard deviation of 2 electrons per pixel, are shown. (See Supplementary Note 9 for more details.)