Coaligned dual-channel STED nanoscopy and molecular diffusion analysis at 20 nm resolution

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1 Supplementary Materials to Coaligned dual-channel STED nanoscopy and molecular diffusion analysis at 20 nm resolution Fabian Göttfert*, Christian A. Wurm*, Veronika Mueller*, Sebastian Berning*, Volker C. Cordes, Alf Honigmann* and Stefan W. Hell* *Department of NanoBiophotonics, Department of Cellular Logistics, Max Planck Institute for Biophysical Chemistry, Am Fassberg 11, Göttingen, Germany Address reprint requests and inquiries to SWH: Fabian Göttfert and Christian A. Wurm contributed equally to this work Determination of the absorption and emission spectra. The spectra were measured using a Varian Cary 4000 UV-VIS spectrophotometer and a Varian Cary Eclipse fluorescence spectrophotometer (Agilent Technologies, Santa Clara, CA, USA). Preparation of mammalian cell samples for confocal and STED microscopy. The cultivation of cells, antibody and immunolabelling was based on established protocols 1,2. Cultured Xenopus X177 and A6 cells were seeded on cover slips one day before fixation with formaldehyde (2.4%/ RT/ 5 min). After extraction in 0.3 % TritonX 100 in PBS and blocking in 5 % bovine serum albumin in PBS, the cells were incubated with a mouse monoclonal antibody specific for Xenopus gp210 3, novel rabbit antibodies selected for being pan-specific for most Xenopus FG repeat nucleoporins and especially targeting Nup214, Ganp, CG1, Nup98 und Nup153 in situ, and mouse monoclonal antibody QE5, targeting Nup153, Nup214 and Nup62 (Abcam, Cambridge, UK). The detection of these primary antibodies was performed using secondary antibodies (Dianova, Hamburg, Germany) custom labelled with the dyes Atto590, Atto594, Atto647N (Atto-Tec, Siegen, Germany), STAR580 and STAR635P (Abberior, Göttingen, Germany) and KK Finally, the samples were mounted in Mowiol containing DABCO. Preparation of samples for STED-FCS measurements. Glass supported lipid bilayers were prepared by spin-coating 20 µl of 2 g/l DOPC (1,2-dioleoyl-sn-glycero-3-phosphocholine, Avanti Polar Lipids) dissolved in 1 : 1 methanol/ chloroform containing ~ 0.01 mol % DSPE-PEG-KK114 onto piranha solution cleaned cover slides at 2000 rpm. After solvent evaporation under vacuum for 20 min, the bilayer was hydrated in 150 mm NaCl, 20 mm HEPES, ph 7.2 and rinsed several times until a single clean membrane remained (as determined by confocal imaging). The diffusion constant of fluorescent lipid analogues in defect-free glass supported membranes was D ~4 µm²/ s. The plasma membrane of adherent Ptk2 cells was labelled with DSPE-PEG-KK114 using bovine serum albumin (BSA) as lipid carrier. A lipid-bsa stock solution was prepared by dissolving 100 nmol of DSPE-PEG-KK114 in 10 µl ethanol and adding 1 ml of 7 g/ l fat free BSA (Sigma-Aldrich). The cells were incubated with a 1:20 dilution of the stock solution in growth medium (HDMEM) for 20 min at room temperature and then briefly washed with medium. 1

2 STED and confocal imaging. STED and the corresponding confocal microscopy were carried out by piezostage scanning (dwell times: 30 µs; pixel size: 5.9 nm) (Nano-PDQ, Mad City Labs, Madison, WI, USA) (Fig. S1). Excitation of the fluorophores was performed with two pulsed diode lasers emitting 70 ps pulses, one at 595 nm and the other one at 640 nm (both from PicoQuant, Berlin, Germany). To clean the wavefronts, the excitation beams were coupled into single mode polarization maintaining fibers. STED was performed using a frequency-doubled fiber laser (ELP DG, IPG Photonics Corporation, Oxford, MA, USA) emitting 1.2 ns-pulses at 775 nm and 20 MHz repetition rate with a maximum average power of 5 W. [Lasers with similar pulse specifications are also available from MPB Communications Inc (Montreal, Quebec, Canada) and OneFive GmbH (Zürich, Switzerland).] The average power of the STED beam in the focal plane was mw, corresponding to mw at the back aperture of the objective lens. The same STED beam was used for inhibiting fluorescence from both color channels (Fig. S1B). Triggering and synchronisation of the excitation pulses to the STED pulses was performed with a photodiode connected to a constant fraction discriminator (CFD) positioned to intercept a small reflection of the STED beam (before coupling the STED beam into a 30 m fiber) and custom-built electronics. Since the pulses of our STED laser exhibit temporal jitter, it is important to trigger each excitation pulse to the corresponding STED pulse. (This may not apply to lasers by other manufacturers due to improved pulseto-pulse stability.) In the pulse-interleaved excitation scheme, the signals from the CFD are electronically distributed to the excitation diodes, so that only every second STED pulse triggers the same excitation diode (Fig. S1C). While the coarse pulse timing of the STED and excitation lasers is set by the lengths of the fibers, the beam paths and the cables, the fine tuning is realized by electronic delay. After the fiber, the STED beam passes a polymeric phase plate (Vortex pattern, RPC Photonics, Rochester, NY, USA). In combination with a λ/4 waveplate, the phase plate is responsible for the doughnut shaped STED-focus. The excitation and the STED beams were co-aligned and coupled into a 1.4 numerical aperture oil immersion lens (NA 1.4 HCX PL APO, 100x, Leica Microsystems, Wetzlar, Germany). The fluorescence was collected by the same lens, spectrally separated and filtered into two ranges: nm and nm. The fluorescence was detected by fiber-coupled single-photon-counting modules (SPCM- AQRH13, Perkin Elmer), with their fiber cores acting as confocal pinholes. The signal from each detection channel is then passed through a home-built electronic gating unit that is triggered by the same (delayed) pulses as the excitation lasers. The time gating pipes the signal within a 10 ns window to a data acquisition card (NI PCI-6259, National Instruments, Austin, Texas, USA) (Fig. S1C). The time gating not only limits the detected fluorescence of each channel to the corresponding excitation pulse, but also suppresses the detection of signals in the first nanosecond after the excitation pulse, reducing the detected fluorescence signal to 70 % for the applied dyes ( F 3.5 ns). The loss in signal is compensated by the increased resolution as spontaneously emitted photons during the action of the STED pulse are neglected 5. [As an alternative to home-built time gating electronics we recommend time correlated single photon counting modules (e.g. by PicoQuant or Becker & Hickl, Berlin, Germany) which offer the possibility of adjusting the time window in post processing. An implementation for National Instruments 2

3 data acquisition cards with FPGA chip is an alternative option.] For better visibility, the images shown in Fig. 1, Fig. 2, and Suppl. Fig. 3 a, b were smoothed by convolution with a 2D Gaussian function (σ = 1 pixel and σ = 4 pixel for Fig. 2c). Note that stage scanning has been used here for convenience only. Coupling a (commercially available) beam-scanning device between the objective lens and the light sources, which is technically straightforward, reduces both the recording time and bleaching further. STED-FCS measurements and data analysis The 640 nm excitation laser was precisely focused into the membrane by the z-piezo-actuator of the stage. The optimal z-position was found by observing the fluorescence intensity in real-time and maximizing the amplitude of the single molecule fluctuations. The fluctuating intensities were autocorrelated with a hardware correlator card (Flex02-01D, Correlator.com, NJ), with typical measurement times of s. The measurement was repeated for increasing STED power. The resulting correlation curves were fitted using MATLAB according to a two-dimensional anomalous diffusion model 6 : G( ) 1/ N * (1 ( T (1 T )) * exp( / )) *1/(1 ( / ) ) T D where the particle number N denotes the average number of fluorescent molecules in the detection volume, T the fraction of molecules that are on average in the dark triplet state, T the triplet dwell time, D the average transit time of the molecules diffusing through the observation area, and the anomalous diffusion exponent, which is 1 for unhindered diffusion and <1 for anomalous hindered diffusion. Previous analysis 7,8 of STED-FCS curves in cell membranes have shown that anomaly exponents are < 1 in most cases. However, significant hindered diffusion with a clear non-linear relation of diffusion time vs. detection area is indicated by α < 0.7. In the case of free diffusion, the recorded diffusion time D is proportional to the observation area. Thus the FWHM of the observation area (corresponding to the resolution of the STED microscope) can be extracted for each STED power by: FWHM * /. Here, FWHM Conf D Conf Conf is given by the average diffusion time in a confocal recording, whereas the confocal FWHM conf is precisely gained by imaging 20 nm large crimson fluorescent beads (Life Technologies, Paisley, UK) in the confocal mode. For this purpose the beads are immobilized on a coverslip, treated with poly-l-lysine (0.1 % in water, Sigma-Aldrich, Steinheim, Germany) and embedded in Mowiol. Note that our measurement confirms that the STED microscopy resolution scales as FWHM FWHM 1 P / P conf STED S. The dye- and wavelength specific threshold power is determined by fitting to P s = 1.4 mw. 3

4 SUPPORTING REFERENCES 1. Hase, M. E., N. V. Kuznetsov and V. C. Cordes Amino acid substitutions of coiled-coil protein Tpr abrogate anchorage to the nuclear pore complex but not parallel, in-register homodimerization. Mol. Biol. Cell 12, Wurm, C. A., D. Neumann,, S. Jakobs Sample preparation for STED microscopy. Methods Mol. Biol. 591, Cordes, V. C., A. Gajewski, S. Stumpp and G. Krohne Immunocytochemistry of annulate lamellae: potential cell biological markers for studies of cell differentiation and pathology. Differentiation 58, Wurm, C. A., K. Kolmakov,, S. W. Hell Novel red fluorophores with superior performance in STED microscopy. Opt. Nano. 1, Moffitt, J. R., C. Osseforth and J. Michaelis Time-gating improves the spatial resolution of STED microscopy. Opt. Express 19, Schwille, P., F. J. MeyerAlmes and R. Rigler Dual-color fluorescence cross-correlation spectroscopy for multicomponent diffusional analysis in solution. Biophys. J. 72, Eggeling, C., C. Ringemann,..., S. W. Hell Direct observation of the nanoscale dynamics of membrane lipids in a living cell. Nature. 457:1159-U Mueller V, C. Ringemann,, C. Eggeling STED Nanoscopy Reveals Molecular Details of Cholesterol- and Cytoskeleton- Modulated Lipid Interactions in Living Cells. Biophys. J. 101,

5 FIGURE S1 STED setup and spectral rationale for the used fluorophores. (A) The setup uses two pulsed laser diodes (595 nm and 640 nm) for excitation and a single compact fiber laser for STED. Fluorescence is detected in two distinct wavelength ranges: 620±20 nm and 670±20 nm. The use of a single STED doughnut at 775 nm for both dyes and interleaved pulsed excitation and detection ensures nearly perfect colocalization accuracy, because the doughnut minimum predetermines the coordinate of both emitting dyes. Dichroic mirrors: DM1: z760sprdc; DM2: z585rdc; DM3: z635rdc; Notch: stop line 658 nm (Chroma, Bellows Falls, VT & Semrock, Rochester, NY). The mirrors were tuned in angle to match the laser lines. (B) The spectra of the used dyes (Atto594 and KK114) are closely overlapping. Yet the dyes can be well separated due to the distinct excitation wavelengths and the selection of the detection channels. Note that detection sensitivity can be further enhanced by replacing the 670±20 nm emission filter2 with a 650 nm long pass filter extending up to 775 nm. Detection is accomplished with avalanche photodiodes (APD). (C) Interleaved pulsed excitation and detection ensures temporal succession of the two excitation pulses in the nanosecond range, thus eliminating drift between the recordings of the two color channels, which is important when recording protein colocalization. For further details see the Supplementary Methods and Materials section. 5

6 FIGURE S2 Spectral crosstalk. Xenopus A6 cells were labeled with primary antibodies against NUP subunits in the central channel of the nuclear pore complex (NUP153, NUP214, and NUP62); fluorophore tagged secondary antibodies were appended as indicated. The crosstalk for both channels was measured under conditions similar to the recordings of Fig. 1. The conditions were kept constant for the individual recordings. The images show that crosstalk is negligible. Therefore, no data postprocessing is necessary to separate the data recorded by the two color channels. 6

7 FIGURE S3 Colocalization accuracy. To analyze the colocalization accuracy of the setup, Xenopus A6 cells were labeled with an antiserum detecting multiple NUP subunits in the central channel of the nuclear pore complex (NUP153, NUP214, and NUP62). This antiserum was in turn detected by two secondary antibody species tagged with Abberior STAR635P and Atto594, respectively. STED microscopy was performed as for the other samples. (A) Already upon overlaying the individual channels, a high degree of colocalization of the signals is visible. (B) To quantify the colocalization accuracy, we cross correlated larger images of the two channels. The correlation is maximal for no displacement of the images, a Gaussian fit confirms that the mismatch of the channels is less than 3 nm. To further demonstrate the tolerance of the colocalization accuracy to misalignment we imaged 20 nm fluorescent beads (Crimson FluoSpheres, Life Technologies) with two misaligned excitation beams (detecting at 670±20 nm). The displacement is obvious in the confocal image (C) while the STED image shows good colocalization (D). The cross correlation of the two channels shows a shift of approximately 100 nm for the confocal image and subpixel accuracy in the STED-image. Scale bars, 200 nm (A), 1000 nm (B-D) 7

8 FIGURE S4 STED imaging resolution. The resolution was determined by measuring the size of the smallest discernible features (A) as an upper bound for the resolution. (B) Line profiles across these features were extracted and fitted by Gaussians. (C) The histograms indicate a resolution of ~20 nm and ~30 nm in the raw data provided by the 670±20 nm and 620±20 nm channels respectively. 8

9 FIGURE S5 Dual color STED overview image. Overview image (smoothed raw data) of about half a nucleus labeled with the indicated antibodies. The STED image shown in Fig. 1 is an excerpt (boxed) of this overview. 9

10 Figure S6 Dual color confocal overview image. Overview image (smoothed raw data) of about half a nucleus labeled with the indicated antibodies. The confocal image shown in Fig. 1 is an excerpt (boxed) of this overview. 10

11 FIGURE S7 Anomaly exponents from FCS analysis in model and cell membranes. The anomaly exponent α from the FCS analysis presented in figure 3 A, B. In model membranes no anomaly was detected (α ~ 1). In the plasma membrane a weak anomaly was detected (α > 0.85). However, in combination with the linear relation of transit time versus detection area we conclude that diffusion of the lipid analogue was mainly free. 11