A simple and cost-effective setup for super-resolution localization microscopy.

SNR definition for camera comparison The SNR is defined as follows: documentclass[12pt]{minimal} usepackage{amsmath} usepackage{wasysym} usepackage{amsfonts} usepackage{amssymb} usepackage{amsbsy} usepackage{mathrsfs} usepackage{upgreek} setlength{oddsidemargin}{-69pt} begin{document}$$begin{array}{c}SNR=Signalcdot QE/sqrt{Signalcdot QE+ExcessNoisecdot Signalcdot QE+ReadNois{e}^{2}}\ {begin{array}{l}ExcessNoise=1,EMCCD\ ExcessNoise=0,other,camerasend{array}end{array}$$end{document} S N R = S i g n a l ⋅ Q E / S i g n a l ⋅ Q E + E x c e s s N o i s e ⋅ S i g n a l ⋅ Q E + R e a d N o i s e 2 { E x c e s s N o i s e = 1 , E M C C D E x c e s s N o i s e = 0 , o t h e r c a m e r a s Please note that, only quantum efficiency (QE) and read noise are considered. The effect of dark current noise is ignored. For camera without cooling, the dark current noise can be up to several electrons per pixel per second under the room temperature, which needs to be significantly reduced via deep cooling if long exposure time (e.g., several seconds) is required. But it is not crucial in the context of SMLM imaging, because the acquisition speed is usually several tens of frames per second; and for each frame, the dark current noise is far less than 1 electron, which can be neglected. To validate this point, we measured the sum of the read noise and dark current noise of IMX265 under the experimental condition 20 , and found it is only 2.57 electrons.

Numerical simulation

To compare the performance of our low-cost SMLM setup with the standard SMLM setup, a series of image sets with different signal levels are numerically generated 21 . For each image, a single molecule is randomly distributed in the central pixel. The PSF was modeled with Gaussian function with a kernel width of 0.21 · λ / NA . The wavelength λ is set to be 700 nm to mimic the wavelength of a commonly used fluorophore (Alexa 647). The pixel size is set to be 160 nm for standard SMLM setup equipped with TIRF objective (100X oil, NA = 1.49) and EMCCD cameras (iXon 897, Andor) and 115 nm for our low-cost SMLM setup. The total photon number of the molecules is set to be from 1000 to 8000 to cover the signal range of the commonly used fluorescent dyes. The background for each pixel is set to be 1/50 th of total photon number per molecule. The noise is modeled with a Poisson model plus the excess noise for standard SMLM, and Poisson model plus the read noise with standard deviation of 2.57 electrons for our low-cost SMLM. For each signal level, 1000 images are generated and analyzed to calculate the localization precision (defined as the standard deviation of the localization error). Protocol to coat gold nanoparticles on the coverslip We first coat the glass bottom dish (FD3510-100, WPI) with poly-D-lysine (P7280, SIGMA) for 20 minutes, followed by 200 µL diluted 100 nm gold nanoparticle solution (1:60 with ddH 2 O, EM.GC100, BBI) for 3 hours. Finally, dishes are coated with another layer of poly-D-lysine for 20 minutes for better cell adherence.

Cell preparation and staining Primary mouse embryo fibroblast

(MEF) cells or MCF-10A cells were plated onto a PDL (poly-D-lysine) coated glass-bottom dish (FD3510, World Precision Instruments) at confluency of 50% and cultured overnight to let the cells attach to the dish. To stain microtubules, MEF cells were pre-extracted for 30 seconds in 0.5% Triton X-100 (Triton) in BRB80 buffer supplemented with 4 mM EGTA and fixed with Methanol (−20 °C) for 10 minutes. To stain acetylated histone H3, MCF-10A cells were fixed with 4% PFA for 15 minutes and permeabilized with 0.2% Triton for 10 minutes. After fixation, the cells were washed 3 times with PBS and blocked with 3% BSA for 1 hour, then incubated with primary antibody diluted in 3% BSA overnight at 4 °C (rabbit anti-acetyl-histone H3, EMD Milipore 06-599, 1:500). The cells were washed 3 times with PBS and incubated with lab-synthesized Alexa 647-conjugated secondary antibody (Donkey anti rabbit unconjugated antibody, Jackson ImmunoResearch, 711-005-152; Alexa 647 NHS Ester, ThermoFisher, Scientific, A20106) diluted in 3% BSA for 2 hours at room temperature, protected from light. Wash the cells with PBS 3 times and store in PBS until imaging. Immediately before imaging, the sample was switch to the STORM imaging buffer. For microtubule imaging, buffer contains 10% w/v glucose, 2% v/v β-me, 0.56 mg/mL glucose oxidase, 0.17 mg/mL catalase and 2 mM COT (cyclooctatetraene). For histone imaging, 60% TDE (2,2′-thiodiethanol) was used to dissolve the component above instead of water, and 2 mM COT was added to the buffer solution.

Protocol to coat gold nanoparticles on the coverslip We first coat the glass bottom dish (FD3510-100, WPI) with poly-D-lysine (P7280, SIGMA) for 20 minutes, followed by 200 µL diluted 100 nm gold nanoparticle solution (1:60 with ddH 2 O, EM.GC100, BBI) for 3 hours. Finally, dishes are coated with another layer of poly-D-lysine for 20 minutes for better cell adherence.

Electronic supplementary material Low cost LM supplementary