Reduced radiation damage in transmission electron microscopy of. proteins in graphene liquid cells

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1 Supplementary Information Reduced radiation damage in transmission electron microscopy of proteins in graphene liquid cells Sercan Keskin, and Niels de Jonge, INM Leibniz Institute for New Materials, D Saarbrücken, Germany Department of Physics, Saarland University, D Saarbrücken, Germany 1

2 Supplementary Figures Figure S1: Transferring a graphene sheet onto a liquid sample containing microtubules placed on a graphene substrate mounted on a holey carbon foil in a 3 mm transmission electron microscopy (TEM) grid. (a) Floating graphene-salt stack on water surface while graphene side is facing up. (b) Graphene (at arrow head) floating on water. (c) The sample is pipetted on the substrate. (d) The sample on the substrate and the graphene in the loop are brought into contact. (e) Light microscopy image of the loop with graphene and (f) graphene-liquid sample-substrate together with the schematic representation of the cross-sectional view on the right of each image. Graphene sheets are pointed to with the arrows. 2

3 Figure S2. TEM image of negatively stained microtubules on a thin carbon film. Bright dots in the image are probably clusters of unpolymerized tubulins. 3

4 Figure S3. Examination of the origins of the spatial frequencies f of a TEM image of a microtubule. (a) TEM image of negatively stained microtubule (left panel) and its computed fast Fourier transform (FFT) image (right panel). (b-e) Inverse FFT images (right panel) computed from the FFT image in panel a after masking/blocking the indicated f (left panel). Peaks were observed at f = 0.05, 0.10 and 0.14 nm -1 originated from the outline of the microtubule, and 0.20 nm -1 is from the interior lining representing protofilaments. 4

5 Figure S4. The stability of a microtubule in a graphene liquid cell (GLC) under electron beam irradiation tested via phase contrast TEM. (a left panel) Selected region of a TEM-image series of the same section of a microtubule. The electron flux was Df = 12±1 e - /A 2 s. After the first image, each image was collected at a time interval of 4.0±0.4 s of continuous exposure, so that the electron density D increased by 47±9 e - /A 2 going from one to the next image. The image acquisition time was t = 1 s, and Dz = -7µm. The right panel depicts the corresponding FFT of the section of the TEM image. The observed peaks were at f = 0.07, 0.14 and 0.18 nm -1 (b) Line profiles representing the signal intensity as function of spatial frequency f in the horizontal middle line of selected FFT images. Line profiles are given for different accumulated D. The peak at a specific f measures the structural integrity. In particular, the intensity of the 0.18 nm -1 peak (marked by blue circle in a) decreases with increasing cumulative D. The dashed line in red represents the average of the noise level (131) in the FFT image obtained at the damage limit Dmax = (7±1) 10 2 e - /A 2 (c) Intensities in the FFT plot of 0.18 nm -1 (blue triangles) as a function of D. The dashed lines represent the averaged noise level in the FFT image. The criterion for structural preservation was a signal-to-noise ratio SNR > 3. Spatial features of f = 0.18 nm -1 are thus preserved up to Dmax = (7±1) 10 2 e - /A 2 as indicated by the vertical dotted line representing the damage limit Dmax. Damaged structure is included as grey data points. 5

6 Figure S5. Dmax measured at the onset of radiation damage as function of the electron flux Df for microtubules in a GLC and for cryo-frozen microtubules on graphene substrate. The horizontal error bars represent an error margin of 10% in Df and the vertical error bars represent an error margin of 15% and 20% in cumulative D in ice and liquid, respectively. Figure S6. Radiation Damage of a microtubule in a free-standing ice layer. The sample support was a holey carbon film on a 3 mm TEM grid. (a) First TEM image in the series obtained with Df = 11±1 e - /A 2 s. (b) Second image in the series with a cumulative D = 58±9 e - /A 2. As seen, radiation damage is already visible by a significant reduction in the inner details of the microtubule. 6

7 Supplementary Experimental Methods and Materials Tubulin Polymerization Assay. Porcine brain tubulin (3 mg/ml, Cytoskeleton Inc., CO, USA) was polymerized into microtubules by following the instructions of the provider. In short, tubulin solution was supplemented with 1mM (guanosine triphosphate) GTP and 8.5 % glycerol, and was incubated at 37 C for 3.5 h. After 30 min of incubation, taxol was added to a final concentration of 20 µm. For glycerol removal, the microtubules were dialyzed against glycerol-free buffer consisting of 80 mm piperazine-n,n -bis(2-ethanesulfonic acid (PIPES) ph 6.9, 2 mm MgCl2, 0.5 mm ethylene glycol-bis(β-aminoethyl ether)-n,n,n',n'-tetra-acetic acid(egta), for 1.5 h using ZelluTrans Mini Dialyzers (Carl Roth GmbH, Germany) with a molecular cut-off of 6-8 kda. The dialysis solution was changed with a fresh one every 30 min. Nano-W (Nanoprobes Inc., Yaphank, USA) was used to negatively stain the microtubules. Graphene Transfer and Sample Preparation for electron microscopy. Multi-layer (3 to 5 layers) graphene covered with Poly-methyl-methacrylate (PMMA) on one side and a polymer layer on the other side of the graphene was used (Trivial transfer graphene, ACS Materials, CA, USA). In order to remove the PMMA layer and release the graphene easily off the substrate, it was transferred onto NaCl2 crystals (Plano GmbH, Germany) prior to use for sample preparation as described elsewhere. 1, 2 For this purpose, the polymer-graphene-pmma stack was submersed into NaCl2 saturated, deionized water solution at 45 angle to release the polymer. The floating graphene-pmma stack was then fished out with a NaCl2 crystal. After baking it in an oven at 100 C for 20 min, the stack on salt crystal was immersed into acetone for 30 min to remove the PMMA, and was subsequently air-dried. After this point, the graphene on salt was cut into the desired size as needed to cover the samples. 7

8 For transfer of graphene onto a substrate containing microtubules, the graphene on salt stack was floated on deionized water to release the salt (see Figure S1). Freely floating graphene was then scooped-up using a metal loop (Plano GmbH, Germany). Followed immediately, a 2 µl droplet of microtubule solution was pipetted onto a substrate and excess solution was blotted by a piece of filter paper. A copper grid with multi-layer graphene on one side (Micro to Nano, Netherlands) was used as sample support substrate for liquid experiments. Finally, the graphene was transferred onto the substrate covered with microtubule solution by lowering the loop slowly on the substrate. A mixture of 0.1 M tris(hydroxymethyl)aminomethane (Tris) Buffer HCl (Sigma Aldrich, MO, USA) and general tubulin buffer (Cytoskeleton Inc., CO, USA) was used for preparing graphene liquid cell. It improved the wetting of the graphene liquid cell sample with a higher number of intact microtubules observed. It was recently reported that buffer conditions significantly influence the success rate of obtaining stable liquid pockets in a graphene liquid cell. 3 Sample preparation for cryo-tem. For cryo transmission electron microscopy (TEM) experiments, the same copper grids with multi-layer graphene on one side, as used in liquid phase TEM, and a Cu 400-mesh with a carbon hole film (Plano GmbH, Germany) were used. A droplet of 3 µl microtubule solution was pipetted onto either graphene or holey carbon sample support substrate and blotted for 2 s before being flash-frozen in liquid ethane using a cryo plunge system (Gatan, CA, USA). The temperature and the humidity of the plunging chamber were 24 C and 80 %, respectively. The frozen hydrated samples were then transferred into a cryo-transfer holder (Gatan, CA, USA) for imaging. The temperature of the specimen was measured to be -177±1 C, and was monitored during the entire imaging session to ensure the temperature stability. 8

9 Negative Staining. In order to test the tubulin polymerization protocol, a control sample was prepared in which the microtubules were negatively stained. Briefly, 2 µl of microtubule solution was pipetted on holey carbon film covered copper grids (Plano GmbH, Germany) and blotted at the edge of the grid until a thin layer of sample was achieved. Then, a 2 µl of negative stain solution (methylamine tungstate, Nano-W, Nanoprobes Inc., Yaphank, USA) was pipetted onto the grid (directly on the sample solution) and completely blotted after 15 sec. The formation of the microtubules was checked with TEM (see Figure S2). Due to the staining, microtubules appear wider than what they should be, that is 30 nm instead of 23 nm, because the stain provides a layer of material around a microtubule, and a microtubule can possibly flatten due to the staining. 4 Acquisition of TEM Image Series at Room Temperature. TEM images were acquired at 200 kev beam energy using a transmission electron microscope equipped with a cold field emission gun (JEM-ARM 200F, JEOL, Japan). The samples were mounted in a standard single tilt TEM sample holder (JEOL) for imaging. A charge coupled device (CCD) camera (GIF Quantum 963, Gatan, CA, USA) was used to record images at a magnification of x30,000-60,000, an image size of 2048 x 2048 pixels, and an exposure time of 1 s. An objective lens aperture of 60 µm or 20 µm was used for imaging. The TEM images were acquired at -2 to -7 µm defocus to increase the contrast. The image series were recorded using digital imaging software (Digital Micrograph, Gatan, CA, USA). A non-exposed region was selected for the recording of a TEM image series by moving the sample stage and waiting 3 min to reduce sample drift. The beam blanker was then opened a total of ten images was recorded with continuous exposure, after which the beam was blanked for 3 min. This procedure was repeated 5-6 times to obtain a total of images. The time intervals between the images were obtained from the time stamps of the images in a TEM image series giving 4.0±0.4 s, representing the average of 18 time intervals measured from the 9

10 time stamps and their standard deviation. The relative error in the timing was added quadratically with the relative error in Df to obtain the relative error in the cumulative D. The TEM image series at Df = 40 and 70 e - /Å 2 (Figure S5) were obtained with continuous exposure and recorded with a screen capture software. We assume an error margin 10% in the time intervals of these measurements. Acquisition of TEM Image Series at Cryogenic Temperature. TEM images at low temperature were acquired at 200 kev using a transmission electron microscope equipped with a LaB6 thermionic emitter (JEM-2100, JEOL, Japan). A CCD camera (Orius, SC1000, Gatan, CA, USA) was used to record images at a magnification at x10,000-30,000, an image size of 1024 x 1024 pixels, and an exposure time of 1s. The TEM images were acquired at -7 µm defocus to increase the contrast. An objective lens aperture of 30 µm was used for imaging. The image series were recorded using Digital Micrograph imaging software (Gatan, CA, USA). A nonexposed region was selected for the recording of a TEM image series and the beam was manually blanked between each acquisition for 6 s. The time interval between beam-on and beam-off was measured 5 times and found 4.3±0.2 s from observing a visible signal on the phosphor screen of the microscope. The error margin represents variation in timing of the manually operated beam blanker. The relative error in the timing was added quadratically with the relative error in Df to obtain the relative error in the cumulative D. Calculation of the Electron Flux. In order to determine the electron flux Df, the used TEM cameras were calibrated so that the measured counts N related to the counts per electron Ce. The calibration was done by comparing by the measured counts in a camera with the measured current density on the small fluorescent screen is of the electron microscope using a conversion factor f = 2.4 provided by the microscope manufacturer (JEOL, Japan), and we estimate an error 10

11 margin of 10% in this value. For such a measurement, the TEM condenser lens was adjusted such that the entire beam was observed in the camera screen. The counts per electron Ce was calculated by using following equation: C " = N e i ( f τ (1) With τ, exposure time and e, elementary charge. We calculated Ce for four different beam intensities and found Ce = 10±1 e -1 and 3.2±0.3 e -1 for the CCD cameras used in the measurements in liquid and ice, respectively. The relative error margin in Ce was considered as the relative error margin of Df assuming the errors in other parameters are negligible. The electron flux Df was then calculated from N in a TEM image of which the exposure time was known and typically amounted to 1.0 s using the following equation and example using the parameters for the image series presented in Figure 2 (main text): D, = With A the exposed area. N C " A τ = 3.36 x ( nm) < 1s = (16 ± 2)e@ /Å < s (2) Data Analysis. ImageJ (NIH, USA) was used to analyze TEM images. A brightness-contrast adjustment was applied to aid the visualization in the presented TEM images. A region of an individual microtubule was selected (cropped) and used to obtain the corresponding fast Fourier transform (FFT) image. The maximum pixel intensity (grey value) Ipeak was measured for each observed bright spot (spatial frequencies) in the FFT images and this was repeated for each consecutive TEM image of a series. A noise level Inoise was determined for each image by averaging the pixel values over a line adjacent to the horizontal axis of the FFT images. The length and the position 11

12 of the line were identical for all the images in an experiment. In order to determine the cumulative D, where the different spatial frequencies disappear (damage threshold), the signal-tonoise ratio SNR of the bright spots was calculated using the following equation: SNR = I F"GH I JKL(" σ JKL(" (3) With σnoise, standard deviation of the pixel values of the line drawn in the background. Calculation of the Spatial Resolution. The spatial resolution achieved in TEM for a very thin sample is determined by phase contrast but since the available electron dose is limited, it is incorrect to use the standard equation for diffraction limited resolution adjusted at the Scherzer (or other) defocus. 5 Instead, one needs to calculate the signal-to-noise-limited resolution. 6, 7 As first order estimate, we considered the specific case of a carbon object in water as approximate model of protein in water or amorphous ice. The contrast is formed from an electron wave passing at the interface of the object and the surrounding medium that is water, thereby the part of the wave passing through the object experiences a slightly different phase change compared to that part passing through the medium at the side of the object. The contrast C is then a function of the scattering properties and thickness d of the object: 6 C = {N K f K "O (0) N P f P "O (0)} 2 3 d λ = C 2 d (4) 3 With electron wavelength λ, the number of atoms per unit volume for object and water No and Nw, respectively, and the zero-angle elastic scattering for the object fel(0) and water f w (0). One thus obtains the intrinsic contrast per unit length C *. The TEM pixel size was considered to be adjusted for optimal sampling under the Nyquist criterion so that d = 2 pixel size. 7 For the object to be detectable within the statistical noise of the image, the SNR needs to be larger than a value of 3 satisfying the so-called Rose criterion. 8 Assuming the object has all three dimensions equal 12

13 to d and taking the detection efficiency of the camera DQE into account, the D-limited spatial resolution dd at the maximal electron density Dmax (in e - m -2 ) it follows that: 7 d U = 1.5 C (D YGZ (5) For an electron beam of 200 kev energy and using DQE = 0.2, it is thus calculated that dd= 3.3 nm for D = 10 e - /A 2. Supplementary References 1. Textor, M.; de Jonge, N. Nano Lett 2018, 18, (6), Dahmke, I. N.; Verch, A.; Hermannsdorfer, J.; Peckys, D. B.; Weatherup, R. S.; Hofmann, S.; de Jonge, N. ACS Nano 2017, 11, (11), Hauwiller, M. R.; Ondry, J. C.; Alivisatos, A. P. J. Vis. Exp. 2018, (135) e Amos, L. A.; Hirose, K. Methods Mol. Med. 2007, 137, Reimer, L.; Kohl, H., Transmission electron microscopy: physics of image formation. Springer: New York, Rez, P. Ultramicroscopy 2003, 96, de Jonge, N. Ultramicroscopy 2018, 187, (4), Rose, A. Adv. Electron 1948, 1,