Multicolour localization microscopy by point-spread-function engineering.

Optical Setup

All imaging experiments were performed on the experimental system shown schematically in supplementary Fig. S1 . A 4f optical processing system was built alongside the side-port of an Olympus IX71 microscope frame, with a 100x/1.4 NA oil-immersion objective lens (UPlanSApo 100x/1.4 NA, Olympus). The 4f system consisted of two 150 mm achromat lenses (Edmund Optics), a polarizing beamsplitter (B. Halle) to reject light polarized perpendicular to the axis along which the SLM is capable of modulating phase, a 512x512 pixel SLM (XY Phase Series, Boulder Nonlinear Systems), and assorted mirrors for beam-steering. An EMCCD camera (iXon897, Andor) was used to record data. Imaging experiments were performed using simultaneous illumination by a 641 nm diode laser (CUBE, Coherent), and the 514 nm line of an Ar-ion laser (Innova 90, Coherent). Excitation light was reflected off of a multi-bandpass dichroic (FF425/532/656-Di01-25x36, Semrock), and fluorescence was transmitted through the same dichroic, and passed through an additional multi-bandpass emission filter (Em01-R442/514/647-25, Yokogawa), a notch filter (ZET635NF, Chroma), and 514 long pass filter (Semrock).

LC-SLM multicolour mask design

Given a set of N wavelengths λ i , i = 1 … N and N corresponding desired phase patterns D ( x, y ) i , i = 1 … N , an SLM voltage pattern V ( x, y ) is sought that minimizes a weighted least squared phase distance between the all N desired phase patterns and the actual corresponding phase masks. The following pixel-wise optimization is therefore performed: v x y = argmin ∑ i = 1 N w i · Dist 2 π ( P i ( v x y ) , D x y , i ) 2 , where P i ( v ) is the phase delay that wavelength λ i experiences when voltage v is set on the SLM pixel, and the phase distance function is defined as: Dist 2 π ( a , b ) = 2 π · | a − b 2 π − [ a − b 2 π ] | . In addition, since the addition of any constant phase to any desired pattern is allowed, this degree of freedom is also optimized. The optimization is performed numerically using Matlab (The MathWorks, Inc., Natick, Massachusetts, United States). See Supplementary Information for more details. Diffusion experiment Diffusion experiments were carried out in a 55% (w/v) sucrose aqueous solution. Two coverglass slides (Fisher Premium Cover Glass, no. 1) were adhered with double-sided tape (3M) forming a diffusion chamber approximately 50µm in height. A dilute concentration of fluorescent microspheres (F8803 & F8789, Invitrogen) were added to the solution and allowed to diffuse at room temperature.

Cell Labelling and Imaging

For cell imaging experiments, cultured BS-C-1 (Cercopithecus aethiops epithelial kidney, ATCC CCL-26) cells were plated onto cleaned glass coverslips (Fisher Premium Cover Glass, no. 1.5 and cultured for 48 hours in high glucose, DMEM media containing 10% (v/v) fetal bovine serum (both Gibco). Cells were then fixed in chilled 4% (w/v) paraformaldehyde (Electron Microscopy Sciences) for 20 minutes and then incubated with 10mM NH 4 Cl (Sigma) for 10 minutes. Next, cells were permeabilized with 3x washing steps containing 0.2% (v/v) Triton-X 100 in pH 7.4 PBS (both Sigma) with a 5 minute incubation between each wash and placed in blocking solution (3% w/v BSA in PBS, both Sigma) for 1 hour before labeling for 2 hours with Alexa-647-labeled monoclonal rabbit anti-alpha-tubulin primary antibodies (ab190573, Abcam) and mouse mitochondrial-marking anti-ATPB primary antibodies (ab14730, Abcam) using a 1:200 dilution in 3% w/v BSA. Cells were then washed 3x with 0.2% Triton-X 100 with a 3 minute waiting step between each wash. Goat anti-mouse Alexa 532-conjugated secondary antibody (A11002, Thermofisher) labelling was then performed with 1:500 dilution for one hour followed by 5X washing steps of 0.2% Triton-X 100. Samples were then shielded from light and kept at 4° C until imaged. Super-resolution imaging was performed 2–3 days after fixing cells. Samples were placed in a custom coverglass slide holder (Tokai Hit Co. Ltd., Japan) and warmed to room temperature and then placed in blinking media 32 (700µg/mL Glucose Oxidase, 50µg/mL Catalase, 0.55M Glucose, 140mM 2-Mercaptoethanol, all Sigma in 0.1M pH 8.0 Tris·HCl buffer, Invitrogen). The sample was scanned at low-intensity laser illumination for suitable regions and then imaged with high intensity (10kW/cm 2 ) 641 nm light and (5kW/cm 2 ) 514 nm light, with a rising intensity of the 405 nm activation laser (0-500W/cm 2 ) 32 .

Fitting algorithm

In brief, emitter localizations of Tetrapod PSF data were performed using maximum likelihood estimation with a numerical scalar imaging model, taking into account refractive index mismatch, using a custom Matlab code. The elongated PSF in the cell experiment was fit using a custom Matlab code that performs 2D Gaussian fitting, and colour discrimination was decided by ratio of Gaussian standard deviations along both axes. For more fitting details see Supplementary Information .