Functional sample comprises a protocol for preparation of the TEM grids coated with the Cu(OH)2 nanowires. Such TEM grids are specifically suitable for preparation of the macromolecular samples for cryo-electron microscopy using spray of piezo-dispenzing devices.Funkční vzorek poskytuje protokol pro přípravu TEM mřížek potažených vrstvou Cu(OH)2 nanovláken. Takové TEM mřížky jsou specificky vhodné pro přípravu komplexů makromolekul pro účely kryo-elektronové mikroskopie pomocí sprejovacího zařízení.Functional sample comprises a protocol for preparation of the TEM grids coated with the Cu(OH)2 nanowires. Such TEM grids are specifically suitable for preparation of the macromolecular samples for cryo-electron microscopy using spray of piezo-dispenzing devices

Citterbardová Karolína

Nováček Jiří

Vykoukal Vít

English

Univerzitní repozitář Masarykovy univerzity

Functional sample VV001/2020The protocol for robust and efficient preparation of the self-blottingnanowire TEM gridsAuthors:Vít VykoukalKarolína CitterbardováJiří NováčekCEITEC, Masaryk UniversityThe functional sample was developed within the frame of Technological Agency ofthe Czech Republic project  TN01000008Content1. Introduction …………………………………………………………………………………….. 22. The goal of the functional sample …………………………………………………………...  23. Functional sample description ……………………………………………………………….  34. Functional sample optimization procedure …………………………………………………. 45. Novelty of the functional sample …………………………………………………………….. 76. Acknowledgements ………………………………………………………………..………….. 77. References …………………………………………………………………………………….. 711. IntroductionTransmission electron microscopy of vitreous biological specimens (cryo-EM)  has becomea powerful tool in Structural Biology research over the last decade and is nowadays beingrecognized as one of the primary techniques in biology [Kuhlbrandt 2014; Nogales 2015].The increased recognition is primarily due to technological developments which helped toovercome some of the limitations of earlier cryo-EM. These technological developmentsprimarily  comprise  utilization  of  direct  electron  detection  cameras  (DEDs)[McMullan,Faruqi,Clare & Henderson 2014], development of stable transmission electronmicroscopes capable of long unsupervised data acquisition, and software tools for dataanalysis  [Lyumkis,Brilot,Theobald & Grigorieff 2013; Punjani,Rubinstein,Fleet & Brubaker2017; Zivanov et al. 2018]. In contrast, the mainstream technique for sample preparationhas remained the same for  more than 30 years  [Dubochet  et  al.  1988].  The standardprotocol applies 2.5 – 4 μl of the specimen to a TEM grid, followed by blotting 99.9% of thesample  away  with  a  filter  paper,  and  plunge  freezing  the  grid  into  the  liquid  ethane.Besides the fact that the majority of the specimen is wasted during preparation, it is verydifficult to fully control the blotting process. Therefore, large numbers of grids are usuallyprepared for single followed by costly screening in order to find single grid which is suitablefor cryo-EM data acquisition. The sample preparation is thus nowadays identified as themain step limiting throughput of cryo-EM. Recent developments of the instruments whichdeposit  nl  -  pl  amounts of the sample directly to the TEM grid without  a need furthersample  blotting  decreases  sample  amount  and  increases  the  reproducibility  of  theprepared cryo-EM samples [Razinkov et al. 2016; Arnold et al. 2017; Ravelli et al. 2020].One of these devices is called “Spotiton” and its principle based on piezo-dispensing of thepl sample droplets to the specifically treated “nanowired” TEM grids [Razinkov et al. 2016].The surface of the grid is coated with Cu(OH)2 nanowires which wick away the excess ofthe liquid. Here, we present a protocol for robust and reproducible preparation of the TEMgrids coated with Cu(OH)2 nanowires.2. The goal of the functional sampleThe document  provides  a  protocol  for  preparation  of  the  TEM grids  coated with  the Cu(OH)2nanowires. Such TEM grids are specifically suitable for preparation of the macromolecular samplesfor cryo-electron microscopy using spray of piezo-dispenzing devices.23. Functional sample descriptionThe  200-mesh  or  300-mesh  Au  TEM  grids  were  purchased  from  SPI(https://www.2spi.com/category/grids-au-square/grids-au/).  The  grids  were  washed  for10 minutes in acetone, 10 minutes in iso-propyl alcohol (IPA), and air-dried under ambientconditions on the glass slide for another 30 minutes. The grids were positioned such thatthe  flat  (“shiny”)  side  was  facing  the  glass  slide.  The  glass  slide  with  the  grids  wastransferred to the Quorum Technologies Q150T ES sputter coater.  The 57 x 0.25 mmCopper  target  (99.99% Cu)  was  mounted  into  the  instrument  and  Cu  sputtering  wasperformed  at  60 mA current  under  the  Argon  atmosphere  for  30  minutes  resulting  in700 nm of Cu on the top of the Au TEM grids. The sputter coater was equipped with thefilm  thickness  monitor  which  was  used  to  calibrate  the  proper  sputtering  time.Subsequently,  the  TEM  grids  were  transferred  to  the  glow  discharge  unit  (QuorumTechnologies SC7620),  an plasma cleaned in Ar:O2 (75:25) atmosphere for 2 minutes(7 mA, 7 Pa). The grids were then floated on the surface of a reaction mixture comprising0.13 M ammonium persulfate and 2.5 M sodium hydroxide at pH 13.8 with the Copper sidefacing towards the liquid. The ammonium persulfate solution was always freshly prior tothe experiment.  The reaction was carried out  for  20 s.  Cu(OH)2 nanowire synthesis  isdescribed by the equation Cu+4 NaOH+ ( NH 4 )2 S2O8 →Cu (OH )2+2Na2 SO 4+2NH 3↑+2 H2O                 (1)The reaction was stopped by addition of high access of water. Subsequently, the gridswere washed with ethanol for 10 minutes, another 10 minutes with 1:1 IPA:acetone, anddried under ambient conditions. The overall grid quality, the nanowire morphology and sizewas determined using FIB/SEM microscopy (Fig. 1).The final TEM grids were uniformly covered with the 2.0 ± 0.3 um Cu(OH)2 nanowires.3Fig. 1: Detail of the Cu(OH)2 nanowires on the TEM grid. The cross-section through the grid bar wasprepared using focused ion beam to facilitate nanowire size analysis. The layers of Au, Cu and Cu(OH)2nanowires can be clearly separated.4. Functional sample optimization procedureThe final protocol for the Cu(OH)2 nanowire grid preparation resulted from an optimizationprocess where various parameters were systematically changed and its effect on the finalproduct was monitored. Here, we summarize the major parameters which influence thequality of the nanowire grid preparation.4.1 The reaction time determines nanowire length and characteristicsThe reaction time (eq. 1) was incrementally increased for the TEM grids sputter coatedwith  700 nm Cu layer,  and the morphology of  the nanowires was studied in  scanningelectron microscope (SEM, Fig. 2). In order to determine the nanowire size distribution, apart of the nanowire coated grid bar was removed by a focused ion beam (FIB) whichfacilitated measurement of the nanowire length. The results are summarized in Table 1.4Interaction time [s] Nanowire size range [nm]10 720 – 111020 1790 – 230030 1100 – 235040 860 – 150050 840 – 160060 630 – 1510Table 1: Dependence between the reaction time and nanowire size distribution for 700 nm Cu layer.The  results  in  Table  1  reveal  that  there  is  an  optimum  reaction  time  for  which  thenanowires reach the maximum length and provide relatively narrow size distribution. Thenanowire length distribution broadens with additional prolongation of the reaction time overthe optimum (here 20 s) and the maximum size gradually decreases. The major reason forthe  decreasing  nanowire  size  is  the  sintering  of  individual  nanowires  at  the  top  sideresulting in formation of aggregated compact layer at the grid surface (Fig. 2E,F). Thesintering  artifacts  significantly  decrease  the  surface  of  the  grid  and  consequently  itsblotting capacity.4.2 The thickness of the Cu layer influences distribution of the nanowire lengthThe effect of the Cu layer thickness on the nanowire characteristics was studied. We havefound out that the nanowire growth is not initiated when the thickness of the sputteredlayer is smaller than 200 nm. The maximal length of the nanowires increases with theincreasing thickness of the sputter Cu layer. However, the variability in the nanowire sizedistribution also increases for the thicker Cu layers. This effect is summarized in Table 2which shows the size distribution of the nanowires grown from the 1.4 μm thick Cu layer.Interaction time [s] Nanowire size range [nm]30 600 – 210060 1060 – 462090 730 – 5240Table 1: Dependence between the reaction time and nanowire size distribution for 1.4 μm Cu layer.5The comparison with the data in Table 1 documents that even though the nanowires aresignificantly longer when grown from the 1.4 μm instead of 700 nm sputtered Cu layer. Onthe other hand, the distribution of the nanowire sizes also dramatically increases for the1.4 μm layer and may diminish the reproducibility of the blotting capacity.Fig. 2: Detail of the TEM grid with the Cu(OH)2 nanowires developed after 10 s (A), 20 s (B), 30 s (C), 40 s (D),50 s (E), 60 s (F) reaction. The scale bar represents 20 μm.65. Novelty of the functional sampleThe  functional  sample  provides  a  protocol  for  reproducible  preparation  of  TEM  gridscoated with a uniform layer of Cu(OH)2 nanowires. The novelty of the protocol stems insputtering of Cu layer on Au TEM grids instead of utilization of TEM grids directly made ofCu  which  significantly  improves  the  reproducibility  of  the  results.  In  addition,  we  alsoprovide a description of  the main nanowire parameters which are determinant for  the blotting capacity of the grid and describe conditions under which sintering artifacts appear.6. AcknowledgementsThis work was financially supported by TA CR (project TN01000008).7. ReferencesArnold, S. A.; Albiez, S.; Bieri, A.; Syntychaki, A.; Adaixo, R.; McLeod, R. A.; Goldie, K. N.; Stahlberg, H. and Braun, T. (2017). Blotting-free and lossless cryo-electron microscopy grid preparation from nanoliter-sized protein samples and single-cell extracts, Journal of Structural Biology 197 : 220-226.Dubochet, J.; Adrian, M.; Chang, J.-J.; Homo, J.-C.; Lepault, J.; McDowall, A. W. and Schultz, P. (1988). Cryo-electron microscopy of vitrified specimens, Quarterly Reviews of Biophysics 21 : 129-228.Kuhlbrandt, W. (2014). The Resolution Revolution, Science 343 : 1443-1444.Lyumkis, D.; Brilot, A. F.; Theobald, D. L. and Grigorieff, N. (2013). Likelihood-based classification of cryo-EM images using FREALIGN, Journal of Structural Biology 183 : 377-388.McMullan, G.; Faruqi, A.; Clare, D. and Henderson, R. (2014). Comparison of optimal performance at 300keV of three direct electron detectors for use in low dose electron microscopy, Ultramicroscopy 147 : 156-163.Nogales, E. (2015). The development of cryo-EM into a mainstream structural biology technique, Nature Methods 13 : 24-27.Punjani, A.; Rubinstein, J. L.; Fleet, D. J. and Brubaker, M. A. (2017). cryoSPARC: algorithms for rapid unsupervised cryo-EM structure determination, Nature Methods 14 : 290-296.Ravelli, R. B. G.; Nijpels, F. J. T.; Henderikx, R. J. M.; Weissenberger, G.; Thewessem, S.; Gijsbers, A.; Beulen, B. W. A. M. M.; López-Iglesias, C. and Peters, P. J. (2020). Cryo-EM structures from sub-nl volumes using pin-printing and jet vitrification, Nature Communications 11.7Razinkov, I.; Dandey, V. P.; Wei, H.; Zhang, Z.; Melnekoff, D.; Rice, W. J.; Wigge, C.; Potter, C. S. and Carragher, B. (2016). A new method for vitrifying samples for cryoEM, Journal of Structural Biology 195 : 190-198.Zivanov, J.; Nakane, T.; Forsberg, B. O.; Kimanius, D.; Hagen, W. J.; Lindahl, E. and Scheres, S. H. (2018). New tools for automated high-resolution cryo-EM structure determination inRELION-3, eLife 7.8

Protocol for robust and efficient preparation of the self-blotting nanowire TEM grids

https://is.muni.cz/publication/1728419/Vykoukal_nanowires_Gfunc_20.pdf

Protocol for robust and efficient preparation of the self-blotting nanowire TEM grids

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