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Where in the cell is my protein?

Published online by Cambridge University Press:  21 June 2021

David J. DeRosier Open the ORCID record for David J. DeRosier [Opens in a new window]
David J. DeRosier*
Affiliation:
Brandeis University, Waltham, MA, USA
*
Author for correspondence: David J. DeRosier, E-mail: derosier@brandeis.edu
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Abstract

The application of cryo-correlative light and cryo-electron microscopy (cryo-CLEM) gives us a way to locate structures of interest in the electron microscope. In brief, the structures of interest are fluorescently tagged, and images from the cryo-fluorescent microscope (cryo-FM) maps are superimposed on those from the cryo-electron microscope (cryo-EM). By enhancing cryo-FM to include single-molecule localization microscopy (SMLM), we can achieve much better localization. The introduction of cryo-SMLM increased the yield of photons from fluorophores, which can benefit localization efforts. Dahlberg and Moerner (2021, Annual Review of Physical Chemistry, 72, 253–278) have a recent broad and elegant review of super-resolution cryo-CLEM. This paper focuses on cryo(F)PALM/STORM for the cryo-electron tomography community. I explore the current challenges to increase the accuracy of localization by SMLM and the mapping of those positions onto cryo-EM images and maps. There is much to consider: we need to know if the excitation of fluorophores damages the structures we seek to visualize. We need to determine if higher numerical aperture (NA) objectives, which add complexity to image analysis but increase resolution and the efficiency of photon collection, are better than lower NA objectives, which pose fewer problems. We need to figure out the best way to determine the axial position of fluorophores. We need to have better ways of aligning maps determined by FM with those determined by EM. We need to improve the instrumentation to be easier to use, more accurate, and ice-contamination free. The bottom line is that we have more work to do.

Keywords

Cryo-CLEMcryo-electron microscopycryo-ETcryo-superresolution fluorescence microscopyFIB-SEM microscopylocalization of structures
Type
Review
Information
Quarterly Reviews of Biophysics , Volume 54 , 2021 , e9
Copyright
Copyright © The Author(s), 2021. Published by Cambridge University Press

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References

Adrian, M, Dubochet, J, Lepault, J and McDowall, AW (1984) Cryo-electron microscopy of viruses. Nature 308, 32–36.CrossRefGoogle ScholarPubMed
Backlund, MP, Lew, MD, Backer, AS, Sahl, SJ and Moerner, WE (2014) The role of molecular dipole orientation in single-molecule fluorescence microscopy and implications for super-resolution imaging. ChemPhysChem 15, 587–599.CrossRefGoogle ScholarPubMed
Backlund, MP, Arbabi, A, Petrov, PN, Arbabi, E, Saurabh, S, Faraon, A and Moerner, WE (2016) Removing orientation-induced localization biases in single-molecule microscopy using a broadband metasurface mask. Nature Photonics 10, 459–462.CrossRefGoogle ScholarPubMed
Bates, M, Dempsey, GT, Chen, KH and Zhuang, X (2012) Multicolor super-resolution fluorescence imaging via multi-parameter fluorophore detection. ChemPhysChem 13, 99–107.CrossRefGoogle ScholarPubMed
Baumeister, W, Grimm, R and Walz, J (1999) Electron tomography of molecules and cells. Trends in Cell Biology 9, 81–85.CrossRefGoogle ScholarPubMed
Betzig, E, Patterson, GH, Sougrat, R, Lindwasser, OW, Olenych, S, Bonifacino, JS, Davidson, MW, Lippincott- Schwartz, J and Hess, HF (2006) Imaging intracellular fluorescent proteins at nanometer resolution. Science 313, 1642–1645.CrossRefGoogle ScholarPubMed
Bohm, J, Frangakis, AS, Hegerl, R, Nickell, S, Typke, D and Baumeister, W (2000) Toward detecting and identifying macromolecules in a cellular context: template matching applied to electron tomograms. Proceedings of the National Academy of Sciences of the United States of America 97, 14245–14250.CrossRefGoogle Scholar
Bonora, S, Jian, Y, Zhang, P, Zam, A, Pugh, EN, Zawadzki, RJ and Sarunic, MV (2015) Wavefront correction and high-resolution in vivo OCT imaging with an objective integrated multi-actuator adaptive lens. Optics Express 23, 21931–21941.CrossRefGoogle ScholarPubMed
Booth, MJ (2014) Adaptive optical microscopy: the ongoing quest for a perfect image. Light: Science & Applications 3, e165.CrossRefGoogle Scholar
Brilot, AF, Chen, JZ, Cheng, A, Pan, J, Harrison, SC, Potter, CS, Carragher, B, Henderson, R and Grigorieff, N (2012) Beam-induced motion of vitrified specimen on holey carbon film. Journal of Structural Biology 177, 630–637.CrossRefGoogle ScholarPubMed
Buckley, G, Gervinskas, G, Taveneau, C, Venugopal, H, Whisstock, JC and de Marco, A (2020) Automated cryo-lamella preparation for high-throughput in-situ structural biology. Journal of Structural Biology 210, 107488.CrossRefGoogle ScholarPubMed
Bulina, ME, Chudakov, DM, Britanova, OV, Yanushevich, YG, Staroverov, DB, Chepurnykh, TV, Merzlyak, EM, Shkrob, MA, Lukyanov, S and Lukyanov, KA (2006) A genetically encoded photosensitizer. Nature Biotechnology 24, 95–99.CrossRefGoogle ScholarPubMed
Campbell, MG, Cheng, A, Brilot, AF, Moeller, A, Lyumkis, D, Veesler, D, Pan, J, Harrison, SC, Potter, CS, Carragher, B and Grigorieff, N (2012) Movies of ice-embedded particles enhance resolution in electron cryo-microscopy. Structure 20, 1823–1828.CrossRefGoogle ScholarPubMed
Chakraborty, S, Jasnin, M and Baumeister, W. Three-dimensional organization of the cytoskeleton. (2020) A cryo-electron tomography perspective. Protein Science 29, 1302–1320, Erratum in: Protein Sci. 29, 2132–2133.CrossRefGoogle ScholarPubMed
Chang, YW, Chen, S, Tocheva, EI, Treuner-Lange, A, Löbach, S, Søgaard-Andersen, L and Jensen, GJ (2014) Correlated cryogenic photoactivated localization microscopy and cryo-electron tomography. Nature Methods 11, 737–739.CrossRefGoogle ScholarPubMed
Chien, FC, Lien, CH and Dai, YH (2015) Dual-color dynamic tracking of GM-CSF receptors/JAK2 kinases signaling activation using temporal focusing multiphoton fluorescence excitation and astigmatic imaging. Optics Express 23, 30943–30955.CrossRefGoogle ScholarPubMed
Dahlberg, PD and Moerner, WE (2021) Cryogenic super-resolution fluorescence and electron microscopy correlated at the nanoscale. Annual Review of Physical Chemistry 72, 253–278.CrossRefGoogle Scholar
Dahlberg, PD, Sartor, AM, Wang, J, Saurabh, S, Shapiro, L and Moerner, WE (2018) Identification of PAmKate as a red photoactivatable fluorescent protein for cryogenic super-resolution imaging. Journal of the American Chemical Society 140, 12310–12313.CrossRefGoogle ScholarPubMed
Dahlberg, PD, Saurabh, S, Sartor, AM, Wang, J, Mitchell, PG, Chiu, W, Shapiro, L and Moerner, WE (2020) Cryogenic single-molecule fluorescence annotations for electron tomography reveal in situ organization of key proteins in Caulobacter. Proceedings of the National Academy of Sciences of the United States of America 117, 13937–13944.Google ScholarPubMed
Downing, KH (1991) Spot-scan imaging in transmission electron microscopy. Science (New York, N.Y.) 251, 53–59.CrossRefGoogle ScholarPubMed
Engelhardt, J, Keller, J, Hoyer, P, Reuss, M, Staudt, T and Hell, SW (2011) Molecular orientation affects localization accuracy in superresolution far-field fluorescence microscopy. Nano Letters 11, 209–213.CrossRefGoogle ScholarPubMed
Faoro, R, Bassu, M, Mejia, YX, Stephan, T, Dudani, N, Boeker, C, Jakobs, S and Burg, TP (2018) Aberration- corrected cryoimmersion light microscopy. Proceedings of the National Academy of Sciences of the United States of America 115, 1204–1209.CrossRefGoogle ScholarPubMed
Fernandez, JJ, Li, S and Agard, DA (2019) Consideration of sample motion in cryo-tomography based on alignment residual interpolation. Journal of Structural Biology 205, 1–6.CrossRefGoogle ScholarPubMed
Ghesquière, P, Mineva, T, Talbi, D, Theulé, P, Noble, JA and Chiavassa, T (2015) Diffusion of molecules in the bulk of a low density amorphous ice from molecular dynamics simulations. Physical Chemistry Chemical Physics 17, 11455–11468.CrossRefGoogle ScholarPubMed
Gordon, MP, Ha, T and Selvin, PR (2004) Single-molecule high-resolution imaging with photobleaching. Proceedings of the National Academy of Sciences of the United States of America 101, 6462–6465.CrossRefGoogle ScholarPubMed
Gorelick, S, Buckley, G, Gervinskas, G, Johnson, TK, Handley, A, Caggiano, MP, Whisstock, JC, Pocock, R and de Marco, A (2019) PIE-scope, integrated cryo-correlative light and FIB/SEM microscopy. Elife 8, e45919.CrossRefGoogle ScholarPubMed
Hassani, H and Kreysing, E (2019) Noninvasive measurement of the refractive index of cell organelles using surface plasmon resonance microscopy. Optics Letters 44, 1359–1362.CrossRefGoogle ScholarPubMed
Hess, ST, Girirajan, TP and Mason, MD (2006) Ultra-high resolution imaging by fluorescence photoactivation localization microscopy. Biophysical Journal 91, 4258–4272.CrossRefGoogle ScholarPubMed
Hoffman, DP, Shtengel, G, Xu, CS, Campbell, KR, Freeman, M, Wang, L, Milkie, DE, Pasolli, HA, Iyer, N, Bogovic, JA, Stabley, DR, Shirinifard, A, Pang, S, Peale, D, Schaefer, K, Pomp, W, Chang, CL, Lippincott- Schwartz, J, Kirchhausen, T, Solecki, DJ, Betzig, E and Hess, HF (2020) Correlative three-dimensional super-resolution and block-face electron microscopy of whole vitreously frozen cells. Science 367, eaaz5357.CrossRefGoogle ScholarPubMed
Huang, B, Wang, W, Bates, M and Zhuang, X (2008) Three-dimensional super-resolution imaging by stochastic optical reconstruction microscopy. Science 319, 810–813.CrossRefGoogle ScholarPubMed
Juette, MF, Gould, TJ, Lessard, MD, Mlodzianoski, MJ, Nagpure, BS, Bennett, BT, Hess, ST and Bewersdorf, J (2008) Three-dimensional sub-100 nm resolution fluorescence microscopy of thick samples. Nature Methods 5, 527–529.CrossRefGoogle ScholarPubMed
Kao, HP and Verkman, AS (1994) Tracking of single fluorescent particles in three dimensions: use of cylindrical optics to encode particle position. Biophysical Journal 67, 1291–1300.CrossRefGoogle ScholarPubMed
Ke, Z, Oton, J, Qu, K, Cortese, M, Zila, V, McKeane, L, Nakane, T, Zivanov, J, Neufeldt, CJ, Cerikan, B, Lu, JM, Peukes, J, Xiong, X, Kräusslich, HG, Scheres, SHW, Bartenschlager, R and Briggs, JAG (2020) Structures and distributions of SARS-CoV-2 spike proteins on intact virions. Nature 588, 498–502.CrossRefGoogle ScholarPubMed
Kühlbrandt, W (2014) Biochemistry. The resolution revolution. Science 343, 1443–1444.CrossRefGoogle ScholarPubMed
Lambert, TJ (2019) FPBase: a community-editable fluorescent protein database. Nature Methods 16, 277–278.CrossRefGoogle ScholarPubMed
Le Gros, MA, McDermott, G, Uchida, M, Knoechel, CG and Larabell, CA (2009) High-aperture cryogenic light microscopy. Journal of Microscopy 235, 1–8.CrossRefGoogle ScholarPubMed
Lew, MD and Moerner, WE (2014) Azimuthal polarization filtering for accurate, precise, and robust single- molecule localization microscopy. Nano Letters 14, 6407–6413.CrossRefGoogle ScholarPubMed
Lew, MD, Thompson, MA, Badieirostami, M and Moerner, WE (2010) In vivo Three-Dimensional Superresolution Fluorescence Tracking using a Double-Helix Point Spread Function. 2010 Proc SPIE Int Soc Opt Eng. 7571, 75710Z.CrossRefGoogle Scholar
Li, H and Vaughan, JC (2018) Switchable fluorophores for single-molecule localization microscopy. Chemical Reviews 118, 9412–9454.CrossRefGoogle ScholarPubMed
Liu, B, Xue, Y, Zhao, W, Chen, Y, Fan, C, Gu, L, Zhang, Y, Zhang, X, Sun, L, Huang, X, Ding, W, Sun, F, Ji, W and Xu, T (2015) Three-dimensional super-resolution protein localization correlated with vitrified cellular context. Scientific Reports 5, 13017–13028.CrossRefGoogle ScholarPubMed
Mailfert, S, Touvier, J, Benyoussef, L, Fabre, R, Rabaoui, A, Blache, MC, Hamon, Y, Brustlein, S, Monneret, S, Marguet, D and Bertaux, N (2018) A theoretical high-density nanoscopy study leads to the design of UNLOC, a parameter-free algorithm. Biophysical Journal 115, 565–576.CrossRefGoogle ScholarPubMed
Marko, M, Hsieh, C, Moberlychan, W, Mannella, CA and Frank, J (2006) Focused ion beam milling of vitreous water: prospects for an alternative to cryo-ultramicrotomy of frozen-hydrated biological samples. Journal of Microscopy 222, 42–47.CrossRefGoogle ScholarPubMed
Marko, M, Hsieh, C, Schalek, R, Frank, J and Mannella, C (2007) Focused-ion-beam thinning of frozen- hydrated biological specimens for cryo-electron microscopy. Nature Methods 4, 215–217.CrossRefGoogle ScholarPubMed
Metlagel, Z, Krey, JF, Song, J, Swift, MF, Tivol, WJ, Dumont, RA, Thai, J, Chang, A, Seifikar, H, Volkmann, N, Hanein, D, Barr-Gillespie, PG and Auer, M (2019) Electron cryo-tomography of vestibular hair-cell stereocilia. Journal of Structural Biology 206, 149–155.CrossRefGoogle ScholarPubMed
Mortensen, KI, Churchman, LS, Spudich, JA and Flyvbjerg, H (2010) Optimized localization analysis for single-molecule tracking and super-resolution microscopy. Nature Methods 7, 377–381.CrossRefGoogle ScholarPubMed
Nahmani, M, Lanahan, C, DeRosier, D and Turrigiano, GG (2017) High-numerical-aperture cryogenic light microscopy for increased precision of superresolution reconstructions. Proceedings of the National Academy of Sciences of the United States of America 114, 3832–3836.CrossRefGoogle ScholarPubMed
Pennacchietti, F, Gould, TJ and Hess, ST (2017) The role of probe photophysics in localization-based superresolution microscopy. Biophysical Journal 113, 2037–2054.CrossRefGoogle ScholarPubMed
Rickgauer, JP, Grigorieff, N and Denk, W (2017) Single-protein detection in crowded molecular environments in cryo-EM images. Elife 6, e25648.CrossRefGoogle ScholarPubMed
Rigort, A and Plitzko, JM (2015) Cryo-focused-ion-beam applications in structural biology. Archives of Biochemistry and Biophysics 581, 122–130.CrossRefGoogle ScholarPubMed
Rust, MJ, Bates, M and Zhuang, X (2006) Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM). Nature Methods 3, 793–795.CrossRefGoogle Scholar
Sartori, A, Gatz, R, Beck, F, Rigort, A, Baumeister, W and Plitzko, JM (2007) Correlative microscopy: bridging the gap between fluorescence light microscopy and cryo-electron tomography. Journal of Structural Biology 160, 135–145.CrossRefGoogle ScholarPubMed
Schorb, M and Briggs, JA (2014) Correlated cryo-fluorescence and cryo-electron microscopy with high spatial precision and improved sensitivity. Ultramicroscopy 143, 24–32.CrossRefGoogle ScholarPubMed
Schorb, M, Gaechter, L, Avinoam, O, Sieckmann, F, Clarke, M, Bebeacua, C, Bykov, YS, Sonnen, AF, Lihl, R and Briggs, JAG (2017) New hardware and workflows for semi-automated correlative cryo-fluorescence and cryo-electron microscopy/tomography. Journal of Structural Biology 197, 83–93.CrossRefGoogle ScholarPubMed
Schur, FK (2019) Toward high-resolution in situ structural biology with cryo-electron tomography and subtomogram averaging. Current Opinion in Structural Biology 58, 1–9.CrossRefGoogle ScholarPubMed
Schwartz, CL, Sarbash, VI, Ataullakhanov, FI, McIntosh, JR and Nicastro, D (2007) Cryo-fluorescence microscopy facilitates correlations between light and cryo-electron microscopy and reduces the rate of photobleaching. Journal of Microscopy 227, 98–109.CrossRefGoogle ScholarPubMed
Shu, X, Lev-Ram, V, Deerinck, TJ, Qi, Y, Ramko, EB, Davidson, MW, Jin, Y, Ellisman, MH and Tsien, RY (2011) A genetically encoded tag for correlated light and electron microscopy of intact cells, tissues, and organisms. PLoS Biology 9, e1001041.CrossRefGoogle ScholarPubMed
Siemons, M, Cloin, BMC, Salas, DM, Nijenhuis, W, Katrukha, EA and Kapitein, LC (2020) Comparing strategies for deep astigmatism-based single-molecule localization microscopy. Biomedical Optics Express 11, 735–751.CrossRefGoogle ScholarPubMed
Stallinga, S and Rieger, B (2010) Accuracy of the Gaussian point spread function model in 2D localization microscopy. Optics Express 18, 24461–24476.CrossRefGoogle ScholarPubMed
Tacke, S, Erdmann, P, Wang, Z, Klumpe, S, Grange, M, Plitsko, J and Raunser, S. (2020) A streamlined workflow for automated cryo focused ion beam milling. bioRxiv 2020.02.24.963033.CrossRefGoogle Scholar
Thompson, RE, Larson, DR and Webb, WW (2002) Precise nanometer localization analysis for individual fluorescent probes. Biophysical Journal 82, 2775–2783.CrossRefGoogle ScholarPubMed
Tuijtel, MW, Koster, AJ, Jakobs, S, Faas, FGA and Sharp, TH (2019) Correlative cryo super-resolution light and electron microscopy on mammalian cells using fluorescent proteins. Scientific Reports 9, 1369.CrossRefGoogle ScholarPubMed
Valles, M and Hess, ST (2017) A cross beam excitation geometry for localization microscopy. Isci Notes 2017, 1.Google ScholarPubMed
Wang, L, Bateman, B, Zanetti-Domingues, LC, Moores, AN, Astbury, S, Spindloe, C, Darrow, MC, Romano, M, Needham, SR, Beis, K, Rolfe, DJ, Clarke, DT and Martin-Fernandez, ML (2019) Solid immersion microscopy images cells under cryogenic conditions with 12 nm resolution. Communications Biology 2, 74.CrossRefGoogle ScholarPubMed
Watanabe, R, Buschauer, R, Böhning, J, Audagnotto, M, Lasker, K, Lu, TW, Boassa, D, Taylor, S and Villa, E (2020) The in situ structure of Parkinson's disease-linked LRRK2. Cell 182, 1508–1518.CrossRefGoogle ScholarPubMed
Weisenburger, S, Jing, B, Hänni, D, Reymond, L, Schuler, B, Renn, A and Sandoghdar, V (2014) Cryogenic colocalization microscopy for nanometer-distance measurements. ChemPhysChem 15, 763–770.CrossRefGoogle ScholarPubMed
Weisenburger, S, Boening, D, Schomburg, B, Giller, K, Becker, S, Griesinger, C and Sandoghdar, V (2017) Cryogenic optical localization provides 3D protein structure data with angstrom resolution. Nature Methods 14, 141–144.CrossRefGoogle ScholarPubMed
Zachs, T, Schertel, A, Medeiros, J, Weiss, GL, Hugener, J, Matos, J and Pilhofer, M (2020) Fully automated, sequential focused ion beam milling for cryo-electron tomography. Elife 9, e52286.CrossRefGoogle ScholarPubMed
Zhang, Q, Zhong, L, Tang, P, Yuan, Y, Liu, S, Tian, J and Lu, X (2017) Quantitative refractive index distribution of single cell by combining phase-shifting interferometry and AFM imaging. Scientific Reports 7, 2532.CrossRefGoogle ScholarPubMed