SECOM

application note

SECOM for Giants application note SECOM

Image courtesy of JP Baudoin (IHU–Médit- erannée Infection, APHM, Marseille, France)

SECOM for Giants

Jean-Pierre Baudoin*1, Sangeetha Hari*2, Dehia Sahmi-Bounsiar1, Sory Ibrahima1, Jacques Bou-Khalil1, Julien Andréani1, Bernard Lascola1.

Giant (GVs) are astonishing microbes that most complete translational apparatus of the differ from other viruses and from prokaryotic or known virosphere [2]. Tupanviruses present a eukaryotic cells [1,3,6,7,8,15,19]. Compared to similar to that of amoebal in classically described viruses, GVs are firstly larger size (~450 nm) and structure [15], including a as far as the is concerned, as well as stargate vertex and fibrils. However, the Tupanvi- larger in size [6,7,8]. Indeed classical sizes rus virion presents a large cylindrical tail (~550 X are generally below 200 nm in diameter. GVs sizes ~450 nm, including fibrils) attached to the base of range from 200 nm in diameter (Marseillevirus, the capsid. This tail is the longest described in the Pacmanvirus) up to 1.5 μm in length and 0.5 μm in virosphere. The average length of a complete diameter ( sibericum, siberi- virion is ~1.2 μm, although some particles can cum) [1]. GVs particles and/or sequences can be reach lengths of up to 2.3 μm because of the found in environmental, animal, as well as human variation in the tail’s size; this makes them one of samples. If GVs have been associated with clinical the longest viral particles described to date. cases of pneumonia and lymphoma [3,6], to date there is no clear etiological link between human Bright-field transmission electron microscopy pathologies and the presence of GVs particles/se- (TEM) has been widely used to obtain nanometer quences. GVs are of special interest compared to scale information about GVs [1,8]. The TEM nega- classical viruses because they can infect a broad- tive staining technique has been used to picture er host range than other viruses, and unlike many whole GVs morphology [1,13,14,15,24], while viruses which use specific receptors to target cells resin-embedding and ultramicrotomy have been [11], GVs can enter cells through phagocytosis used to study GVs ultrastructure and to describe that does not require specific viral–host interac- infection cycles over time [1,2,4,10,13,15,16,17]. tions, probably owing to their large size [10,22]. Scanning Transmission Electron Microscopy Tupanvirus is a tailed GV, which possesses the

* authors contributed equally. 1 APHM, Aix-Marseille Université, IRD, MEPHI, IHU Méditerranée Infection, Marseille, France. 2 Delmic BV, Kanaalweg 4, 2628EB Delft, The Netherlands.

DELMIC BV | +31 (0)15 744 0158 | [email protected] | www.delmic.com application note SECOM

(STEM) has also been used to study sections of user-independent. The scope of this work was to GVs infected cells [17], and Scanning Electron study cells infected with GVs using integrated Microscopy (SEM) [2,4,5,21,28] and Atomic Force CLEM with the goal of determining whether GVs Microscopy (AFM) [26] have been used for char- such as Tupanvirus [2] could be detected using acterizing GVs morphology. Cryoelectron micros- the SECOM, and to study their unique ultrastruc- copy (Cryo-EM) has been a popular technique as ture. it enables the preservation of GVs ultrastructure to a greater extent [4,18,25,26,27,28]. Sample preparation

Because their diameter is larger than the optical For Tupanvirus production, Acanthamoeba resolution limit, GVs are readily visible in Castelanii cells were infected for 24h at 28°C. bright-field (BF) transmitted light or fluorescence Cells were mechanically detached from Petri light microscopy (LM) [2,12,15]. But despite this dishes, and the culture medium was ultracentrif- unique property, GVs are rarely observed by LM, ugated at 2000 rpm for 10 minutes. The pellet due to the lack of access to their ultrastructure (cell debris) was discarded and the Tupanvirus [1,15]. There is thus a gap between LM and EM of particles were isolated after ultracentrifugation GVs infected cells. Correlative Light and Electron of the supernanant at 25 000 rpm for 30 minutes. Microscopy (CLEM) is a technique which helps to Acanthamoeba castellanii cells in PAS medium bridge this gap. The combination of light and were infected at 30°C for 18 hours and stained electron microscopy enables visualization of the with FM4-64FX (aldehyde-fixable; Ref. F34653, ultrastructure of fluorescently labeled cells/cellu- ThermoFischer) for 30 minutes at 30°C in dark. lar compartments and can provide a link between Stained infected cells were fixed over-night at 4° the cellular dynamics and the ultrastructure of with paraformaldehyde 4% in sodium cacodylate any object of interest. The combination of the 0.1M buffer. After rinsing 2 times for 15 minutes specificity of fluorescent labeling and the each with a cacodylate 0.1M/saccharose 0.2M in high-resolution structural information of EM water solution, cells were gradually dehydrated makes CLEM the perfect tool to study the com- with ethanol 50%, 70%, 96%, for 15, 30 and 30 plex relationship between form and function in minutes, respectively. Cells were then placed for biology. Conventional CLEM consists of sequen- 1 hour in a mix of LR-White resin 100% (Poly- tial acquisition of light and electron microscopy sciences, Ref. 17411 MUNC-500) and ethanol images in two separate microscopes. While it is a 96% in a 2:1 ratio. After 30 minutes in pure 100% powerful technique which can be applied to living LR-White resin, cells were placed in 100% or fixed cells, in ambient or cryo-conditions, there LR-White resin overnight at room temperature. are significant challenges. It is time-consuming The day after, cells were placed for 1 hour in and labour-intensive, requiring intermediate 100% resin at room temperature. For polymeriza- staining steps. There is a significant risk of sample tion, pure 100% LR-White resin was added on top contamination and damage due to the transfer of the cell pellet until the 1.5 mL Eppendorf tube between the two imaging systems. And finally, the was completely filled, and the tube cap closed, overlay between the images is performed manu- excess resin being cleaned. This method prevent- ally and is based on visual inspection of the ed contact of the resin with air as needed for images themselves. It is therefore prone to bias. thermal curing of LR-White. Polymerization was The SECOM is a system for integrated CLEM achieved at 60°C for 3 days. Between all the wherein light and electron imaging are above steps, the samples were ultracentrifugated performed in one system without the need for at 5 000 RPM and the supernatant was discarded. sample transfer [9, 23]. It is fast and accurate, The inclusion bloc was trimmed with a razor with an automated overlay procedure that is blade to fit in the ultramicrotome. Ultrathin

DELMIC BV | +31 (0)15 744 0158 | [email protected] | www.delmic.com application note SECOM

(a)

Figure 1. Fluorescence image of a 100 nm thick ultra-thin section containing whole infected cells acquired using a low magnifica- tion (40X) air objective lens on the SECOM. The sections was mounted on an ITO/glass coverslip and imaged with an excitation wavelength of 467 nm (false colour).

(b)

Figure 2 (a) False color fluorescence image of cells in a 100 nm thick ultra-thin section acquired on the SECOM using a 60X water immersion objective with an NA of 1.2 (b) Reference image of a 70 nm thick ultra-section on grid acquired on a Zeiss LSM800 confocal laser scanning microscope with a 63X objective lens (Z-maximal projection).

sections 70, 100 or 1000 nm thick were cut on a For correlative imaging with the SECOM system, UC7 (Leica) ultramicrotome. For transmission 100 nm thick uncontrasted sections were depos- electron microscopy (TEM), 70 nm thick sections ited on special glass slides coated with a conduct- were deposited on 300 mesh copper/rhodium ing layer of Indium Tin Oxide (ITO), making it grids (Maxtaform HR25, TAAB). The sections were possible to image the sections with electrons, post-stained with 5% uranyl acetate and lead while maintaining optical transparency for citrate according to the Reynolds method [20]. fluorescence imaging. The SECOM system was Electron micrographs were obtained on a Morga- mounted on a Verios 460 (Thermo Fisher) scan- nii 268D (Philips) transmission electron micro- ning electron microscope. The fluorescence scope operated at 80 keV and equipped with a images were obtained by excitation with a 467 1024x1024 pixels MegaView3 camera. For nm light source. fluorescence microscopy, 100 nm thick section- son grids were imaged with a confocal laser scan- ning AiryScan LS800 microscope (Zeiss).

DELMIC BV | +31 (0)15 744 0158 | [email protected] | www.delmic.com application note SECOM

SEM TEM

Figure 3 (a) EM image of the (uncontrasted) 100 nm thick ultra-thin section acquired on the Verios 460 SEM using the secondary electron detector with a 1 keV, 100 pA beam. The image shows sufficient contrast for the identification of high resolution features such as cell plasma membranes, mitochondria, ribososomes and tupanvirus particles in spite of the fact that no EM stain was applied. (b) A TEM image of a 70 nm thick EM-stained section is shown for comparison, acquired at 80 keV on a Morgagni 268D (Philips) transmission electron microscope.

SEM TEM

Figure 4 (a) High magnification SEM image of 100 nm thick ultra-thin section with no EM stain, showing an individual virus (b) Reference image of a 70 nm thick ultra-thin section with EM stain, acquired at 80 keV on a Morgagni 268D (Philips) transmission electron microscope.

DELMIC BV | +31 (0)15 744 0158 | [email protected] | www.delmic.com application note SECOM

LM EM *

LM/EM LM

Figure 6. An example of an image acquired on the Odemis software interface. Tupanvirus infect- ed-cells in a 100 nm thick ultra-thin section. GVs appear as LM fluorescent spots and their ultrastructural features are revealed by EM. Viral particles can be seen inside and outside the cells. *: virus factory.

Results

Thin sections containing whole infected cells with light and electrons, and images were were identified optically on the SECOM using a acquired using the Odemis interface. The work- low magnification (40X) air objective lens and 467 flow was as follows: the optical and electron nm excitation, as shown in Figure 1, followed by beam imaging parameters were optimized at a imaging with a high NA(=1.2) 60X water immer- location far away from the region of interest. sion objective (Figure 2(a)), where cells Then the fluorescence image was acquired, can clearly be identified. followed by the electron image. Finally, the over- EM images of the same (uncontrasted) sections lay procedure was performed by electron beam were acquired on the Verios 460 SEM using the exposure of a grid of points, from which the cath- in-lens secondary electron detector in immersion odoluminescence was detected by the camera mode (Figure 3). A 1 keV beam and 100 pA and used to align the objective lens with the elec- current were used to obtain the high resolution tron beam axis. The correlative imaging including EM images which have sufficient contrast to the overlay is fully automated, and a high resolu- enable the identification of virus particles and cell tion image is shown in Figure 5, with two different plasma membranes and intracellular compart- mixing ratios of optical and electron contrast, ments such as nuclei, mitochondria, ribosomes that can be varied using the slider at the bottom and tubules. Further, the high magnification SEM of the image. The Tupan virus was successfully image of the virus, shown in Figure 4 (a), shows identified and imaged on the SECOM, as shown in that it is possible to locate and image an individu- Figures 6 and 7. This also shows the optical LM, al virus. A reference image of an EM-stained EM and LM/EM correlative image in the Odemis section acquired in a TEM is shown in Figure 4 image acquisition interface. (b).Finally, correlative imaging was performed

DELMIC BV | +31 (0)15 744 0158 | [email protected] | www.delmic.com application note SECOM

Section, low-mag

LM EM

Figure 7. Another example of an image acquired on the Odemis software interface. Tupanvirus n infected-cells in a 100 nm thick ultra-thin section. Tupan viruses located outside cells detected as LM fluorescence spots (large arrows) and ultrastructural features such as capsid and tail are seen in the EM. Amoeba mitochondria can be identified by LM/EM LM EM as well (blue arrow). n : amoeba nucleus

Conclusion Acknowledgements

The conclusion of this study is that the SECOM We thank Stéphane Aguy from Eden Instruments. can successfully detect particles by We thank the whole IHU staff for providing an fluorescence LM while simultaneously providing enthusiastic research atmosphere. information on the EM ultrastructure such as the Tupanvirus capsid and tail features. Although this ultrastructural information is not as high Authors resolution as that achieved in a conventional TEM (for example, the fibrils not seen in SEM), it is Jean-Pierre Baudoin, PhD, is research engineer at sufficient for determining the gross morphology IHU–Méditerannée Infection, Assistance Publique of GVs particles and their interactions with cellu- des Hôpitaux de Marseille (APHM), Marseille, lar compartments. Further technical improve- France. ments for sample preparation may help resolve Contact: [email protected] this issue. Moreover, SECOM images would enable, for example, the study of GV cell internali- Sangeetha Hari, PhD, is the Applications Specialist zation or GV morphogenesis such as capsid-tail for the integrated CLEM system (SECOM) at assembly processes for tailed viruses. In the Delmic BV, Delft, The Netherlands. field the SECOM can be a very efficient Contact: [email protected] tool for screening suspicious samples and virus-infected cells at low and higher resolution. More broadly, the SECOM has the potential to be very useful for fundamental and clinical research in the field of .

DELMIC BV | +31 (0)15 744 0158 | [email protected] | www.delmic.com application note SECOM

LMREFERENCES EM

[1] Abergel C, Legendre M, Claverie JM. The rapidly expanding universe of giant viruses: , , Pithovirus and Mollivirus. FEMS Micr Rev. 2015 Nov; 39(6):779-96.

[2] Abrahão J, L Silva, LS Silva, JYB Khalil, R Rodrigues, T Arantes, F Assis, P Boratto, M Andrade, EG Kroon, B Ribeiro, I Bergier, H Seligmann, E Ghigo, P Colson, A Levasseur, G Kroemer, D Raoult, La Scola B. Tailed giant Tupanvirus possesses the most complete translational apparatus of the known virosphere. Nature Comm. 2018 9:749.

[3] Aherfi S, Colson P, Audoly G, Nappez C, Xerri L, Valensi A, Million M, Lepidi H, Costello R, Raoult D. Marseillevirus in lymphoma: a giant in the lymph node. Lancet Infect Dis. 2016 Oct; 16(10):e225-34.

[4] Arantes TS, Rodrigues RA, Dos Santos Silva LK, Oliveira GP, de Souza HL, Khalil JY, deOliveira DB, Torres AA, da LM/EMSilva LL, Colson P, Kroon EG, da FonsecaLM FG, Bonjardim CA, La Scola B, Abrahão JS. The Large Marseillevirus Explores Different Entry Pathways by Forming Giant Infectious Vesicles. J Virol. 2016 May 12; 90(11):5246-55.

[5] Bertelli C, Mueller L, Thomas V, Pillonel T, Jacquier N, Greub G. Cedratvirus lausannensis - digging into Pithoviri- dae diversity. Environ Microbiol. 2017 Oct; 19(10):4022-4034.

[6] Colson P, De Lamballerie X, Yutin N, Asgari S, Bigot Y, Bideshi DK, Cheng XW, Federici BA, Van Etten JL, Koonin EV, La Scola B, Raoult D. “Megavirales”, a proposed new order for eukaryotic nucleocytoplasmic large DNAviruses. 2013 Arch.Virol. 158(12), 2517–2521.

[7] Colson P, La Scola B, Levasseur A, Caetano-Anollés G, Raoult D. Mimivirus: leading the way in the discovery of giant viruses of amoebae. Nat Rev Microbiol. 2017 Apr; 15 (4):243-254.

[8] Colson P, La Scola B, Raoult D. Giant Viruses of Amoebae: A Journey Through Innovative Research and Paradigm Changes. Annu Rev Virol. 2017 Sep 29; 4(1):61-85.

[9] De Boer P, Hoogenboom JP, Giepmans BN. Correlated light and electron microscopy: ultrastructure lights up! Nat Methods. 2015 Jun; 12(6):503-13.

[10] Ghigo E, Kartenbeck J, Lien P, et al. Ameobal Pathogen Mimivirus Infects Macrophages through Phagocytosis. PLoS Pathogens. 2008; 4(6).

[11] Grove J, Marsh M. The cell biology of receptor-mediated virus entry. J Cell Biol. 2011 Dec 26; 195(7):1071-82.

[12] Jason R. Schrad, Eric J. Young, Jônatas S. Abrahão , Juliana R. Cortines , and Kristin N. Parent. Microscopic Char- acterization of the Brazilian Giant Samba Virus. Viruses 2017, 9(2), 30.

[13] Khalil JY, Andreani J, La Scola B. Updating strategies for isolating and discovering giant viruses. Curr Opin Micro- biol. 2016 Jun; 31:80-7.

[14] Khalil JY, Langlois T, Andreani J, Sorraing JM, Raoult D, Camoin L, La Scola B. Flow Cytometry Sorting to Separate Viable Giant Viruses from Amoeba Co-culture Supernatants. Front Cell Infect Microbiol. 2017 Jan 6; 6: 202.

[15] La Scola B, Audic S, Robert C, Jungang L, de Lamballerie X, Drancourt M, Birtles R, Claverie JM, Raoult D. A giant virus in amoebae. Science 299, 2033 (2003).

[16] Legendre M, Bartoli J, Shmakova L, Jeudy S, Labadie K, Adrait A, Lescot M, Poirot O, Bertaux L, Bruley C, Couté Y, Rivkina E, Abergel C, Claverie JM. Thirty-thousand-year-old distant relative of giant icosahedral DNA viruses with a pandoravirus morphology. Proc. Natl Acad. Sci. USA 111, 4274–4279 (2014).

[17] Mutsafi Y, Shimoni E, Shimon A, Minsky A. Membrane assembly during the infection cycle of the giant Mimivirus. PLoS Pathog. 2013; 9(5).

DELMIC BV | +31 (0)15 744 0158 | [email protected] | www.delmic.com application note SECOM

[18] Okamoto K, Miyazaki N, Song C, Maia FRNC, Reddy HKN, Abergel C, Claverie JM, Hajdu J, Svenda M, Murata K. Structural variability and complexity of the giant Pithovirus sibericum particle revealed by high-voltage electron cryo-tomography and energy-filtered electron cryo-microscopy. Sci Rep. 2017 Oct 16; 7(1):13291.

[19] Raoult D, Audic S, Robert C, Abergel C, Renesto P, Ogata H, La Scola B, Suzan M, Claverie JM. The 1.2 megabase genome sequence of Mimivirus. Science 306, 1344–1350 (2004).

[20] Reynolds ES. The use of lead citrate at high pH as an electron-opaque stain in electron microscopy. J Cell Biol. 1963 Apr; 17: 208-12.

[21] Rodrigues RA, dos Santos Silva LK, Dornas FP, de Oliveira DB, Magalhães TF, Santos DA, Costa AO, de Macêdo Farias L, Magalhães PP, Bonjardim CA, Kroon EG, La Scola B, Cortines JR, Abrahão JS. Mimivirus Fibrils Are Important for Viral Attachment to the Microbial World by a Diverse Glycoside Interaction Repertoire. J Virol. 2015 Dec; 89(23):11812-9.

[22] Somiya M, Liu Q, Kuroda S. Current Progress of Virus-mimicking Nanocarriers for Drug Delivery Nanotheranos- tics. 2017; 1(4): 415–429.

[23] Sueters, Di Meo, J, Liv N, Hoogenboom JP. Using Advanced Correlative Microscopy to Study Complex Biological Samples. Encyclopedia of Analytical Chemistry. 15 Jun 2016.

[24] Temmam S, Monteil-Bouchard S, Robert C, Baudoin JP, Sambou M, Aubadie-Ladrix M, Labas N, Raoult D, Median- nikov O, Desnues C. Characterization of Viral Communities of Biting Midges and Identification of Novel Thogotovirus Species and Rhabdovirus Genus. Viruses. 2016 Mar 11; 8(3):77.

[25] Xiao C, Chipman PR, Battisti AJ, Bowman VD, Renesto P, Raoult D, Rossmann MG. Cryo-electron microscopy of the giant Mimivirus. J Mol Biol. 2005 Oct 28; 353(3): 493-6.

[26] Xiao C, Kuznetsov YG, Sun S, Hafenstein SL, Kostyuchenko VA, Chipman PR, Suzan-Monti M, Raoult D, McPherson A, Rossmann MG. Structural studies of the giant mimivirus. PLoS Biol. 2009 Apr 28; 7(4):e92.

[27] Xiao C, Fischer MG, Bolotaulo DM, Ulloa-Rondeau N, Avila GA, Suttle CA. Cryo-EM reconstruction of the Cafeteria roenbergensis virus capsid suggests novel assembly pathway for giant viruses. Sci Rep. 2017 Jul 14; 7(1): 5484.

[28] Zauberman N, Mutsafi Y, Halevy DB, Shimoni E, Klein E, Xiao C, Sun S, Minsky A. Distinct DNA exit and packaging portals in the virus Acanthamoeba polyphaga mimivirus. PLoS Biol. 2008 May 13; 6(5).

DELMIC BV | +31 (0)15 744 0158 | [email protected] | www.delmic.com DELMIC B.V. is a company based in Delft, the Netherlands that produces correlative light and electron microscopy solutions. DELMIC's systems cater to a broad range of researchers in fields ranging from nanophotonics to cell biology.

The SECOM platform is a fluorescence microscope made to be integrated with a scanning electron microscope produced by DELMIC, that enables extremely fast correla- tive microscopy, with the highest optical quality and overlay accuracy.

For questions regarding this note, contact Sangeetha Hari at [email protected].

For more information on the SECOM, visit: delmic.com/secom

For more resources, visit: delmic.com/resources

delmic.com

Kanaalweg, 4 2628 EB Delft The Netherlands

+31 (0)15 744 0158 [email protected]