Gold nanoparticle printed coverslips to facilitate fluorescence-TEM correlative microscopy

Gold nanoparticle printed coverslips to facilitate fluorescence-TEM correlative microscopy Abstract Correlative light and electron microscopy (CLEM) allows combining the advantages of fluorescence microscopy and electron microscopy for cell imaging. Rare phenomenon expressing cells can be studied by specifically tagged fluorophores with fluorescence microscopy. Subsequently, cells can be fixed and ultra-structural details can be studied with transmission electron microscopy (TEM) at a higher resolution. However, precise landmarks are necessary to track the same cell throughout the CLEM process. In this technical report, we present a high contrast inkjet-printed gold nanoparticle patterns over commercial glass coverslip to facilitate cell tracking with correlative microscopy. High contrast and strong reflection from nano gold pattern can be used as a fixed landmark for cell identification with fluorescence microscopy. Nano gold printed letters over coverslips are visible in resin blocks, which can be further used to identify the cell of interest for performing sectioning of embedded cell blocks for TEM. correlative microscopy, CLEM coverslips, gold nanoparticles, CLEM methods We used a novel approach to print a customized pattern over regular microscopy coverslips using gold nanoparticles. The gold nanoparticle printing can be customized based on experimental design. In this case, we printed a set of letters and numbers (Fig. 1a and b). Inkjet printing of the gold letters on the cover glass substrates (washed with ethanol and water) was performed by using a piezoelectric DimatixTM Materials Printer (DMP-2800, FUJIFILM Dimatix, Inc., Santa Clara, USA) and a replaceable cartridge (DMC-11610) with a nominal droplet size of 10 pL. The dodecanethiol-protected gold nanoparticles (AuNPs) (3.0 ± 1.8 nm) were synthesized following the procedure reported by Hostetler et al. [1]. Fig. 1. View largeDownload slide Nano gold printed over regular coverslips. (a) Schematic representation of print pattern of gold nanoparticle letters over glass coverslip. (b) Drop view of the print design and (c) optical micrograph of the inkjet-printed gold letters. Fig. 1. View largeDownload slide Nano gold printed over regular coverslips. (a) Schematic representation of print pattern of gold nanoparticle letters over glass coverslip. (b) Drop view of the print design and (c) optical micrograph of the inkjet-printed gold letters. The ink was formulated by dispersing the AuNPs (1.5 wt %) in 80:20 wt % mixture of xylene (>98.5%, Sigma-Aldrich) and butoxyethylene (99%, Acros Organics), respectively. The printing was carried out using a single nozzle and a drop spacing of 30 μm. Firing frequency was set to 14 V and custom waveforms were used in order to gain optimal droplet formation. The gold nanoparticle-based printed pattern was clearly visible and no deformation was observed (Fig. 1c). Gold nanoparticles have shown non-toxic and inert nature for cellular studies [2–4]. Therefore, nano gold is regarded as safe material for cells to be grown on gold printed coverslips. We seeded HT1080 eGFP cells (GFP expressing) at 20–30% confluency over coverslips. Cells for correlative light and electron microscopy (CLEM) [5–7] were located by live cell imaging and their positions recorded with respect to the nano gold patterns. The live cell screening was performed with EVOS microscope. EVOS® FL cell culture microscope (Thermo Fisher Scientific Inc., USA) is a convenient microscope for quick live cell identification that allows both fluorescence and transmitted channels. The obtained images can be stitched together (Fig. 2a) to provide a better and larger field of view for rapid identification of rare cells. Fig. 2. View largeDownload slide The identification of GFP expressing cells of interest for CLEM. The cells of interest are indicated by arrows. (a) Merging of adjacent 4× images to provide a larger field of view along with nano gold printed letters. (b) Selection of one individual cell from a group of three cells for CLEM. (c) Low magnification fluorescence imaging of live GFP expressing HT1080 cells and cell of interest can be easily located with strong reflection from nano gold letters with a fluorescence microscope. Fig. 2. View largeDownload slide The identification of GFP expressing cells of interest for CLEM. The cells of interest are indicated by arrows. (a) Merging of adjacent 4× images to provide a larger field of view along with nano gold printed letters. (b) Selection of one individual cell from a group of three cells for CLEM. (c) Low magnification fluorescence imaging of live GFP expressing HT1080 cells and cell of interest can be easily located with strong reflection from nano gold letters with a fluorescence microscope. We selected a group of cells located between letters ‘R’ and ‘S’ for performing CLEM. The nano gold printed pattern has the bright contrast that was recognizable under bright field microscope with selected cells for CLEM (Fig. 2a and b). Inkjet printing of gold nanoparticles brought added the advantage of using reflection from gold particles as landmark for cells during fluorescence live cell imaging with eGFP tagged cells (Fig. 2c). Subsequently, live cell images were acquired by confocal microscope (TCS SP5 STED, Leica microsystems, Germany) using 10× objective (Fig. 3a). In order to image at high resolution, the objective was changed to 100× oil objective (Fig. 3b) using the same microscope. The process of changing objective from 10× to 100× and finding the same cell can be challenging without precise landmarks. Therefore, special attention should be paid on locating nano gold landmarks and its orientation with respect to the cell of interest for CLEM. Furthermore, the eGFP cells were imaged in confocal mode (Fig. 3c). Fig. 3. View largeDownload slide Imaging of the same cells of interest as in Fig. 2 with (a) phase contrast microscopy with 10× (low magnification) (arrow points the cells of interest), (b) fluorescence microscopy of GFP with 100× magnification and (c) confocal microscopy with 100× magnification to study cellular features. Fig. 3. View largeDownload slide Imaging of the same cells of interest as in Fig. 2 with (a) phase contrast microscopy with 10× (low magnification) (arrow points the cells of interest), (b) fluorescence microscopy of GFP with 100× magnification and (c) confocal microscopy with 100× magnification to study cellular features. The TEM sample preparation consisted of routine sample processing (Flat resin embedding). Cells were fixed with 5% glutaraldehyde s-collidine buffer (GA), postfixed with 2% OsO4 containing 3% potassium ferrocyanide, dehydrated with ethanol and flat embedded in 45359 Fluka Epoxy Embedding Medium kit. The ‘proof-of-concept’ representative images of sectioning are shown here. The cells can be seen (Fig. 4a) with nano gold letters facing outwards over a glass coverslip before embedding. The nano gold letters could serve as landmarks to identify the cells of interest over coverslips and also with cells embedded with Epoxy embedding medium (Fig. 4b and c) and further the region of interest can be located and trimmed for sectioning. Finally, cells were mounted for sectioning (Fig. 4d) with ultramicrotome using a diamond knife and 70–100 nm sections were collected. The sections were imaged with a JEOL JEM-1400 Plus transmission electron microscope operated at 80 kV acceleration voltage (Fig. 5). Fig. 4. View largeDownload slide Representation of sample processing for electron microscopy and cell identification facilitated by nano gold letters over Epon resin embedded cells. (a) Nano gold printed letters over a glass coverslip for cell identification and tracking throughout the correlation process. (b and c) Nano gold letters over Epon resin block of cells for identification. (d) Epon pyramid for 70 nm sectioning of cells and remnant of nano gold letters can be seen. Fig. 4. View largeDownload slide Representation of sample processing for electron microscopy and cell identification facilitated by nano gold letters over Epon resin embedded cells. (a) Nano gold printed letters over a glass coverslip for cell identification and tracking throughout the correlation process. (b and c) Nano gold letters over Epon resin block of cells for identification. (d) Epon pyramid for 70 nm sectioning of cells and remnant of nano gold letters can be seen. Fig. 5. View largeDownload slide The same cells of interest as in Figs. 2 and 3 were identified and imaged with TEM. Fig. 5. View largeDownload slide The same cells of interest as in Figs. 2 and 3 were identified and imaged with TEM. The contrast provided by gold printed coverslip has advantages over commercially available coverslips especially with fluorescence microscopes. The printed pattern remains detectable with fluorescence microscope and acts as orientation marker to find cells on the Epon resin block face. The nano gold pattern could be printed thick to allow cell identification with successive serial sections. Acknowledgements The Electron microscopy unit, University of Turku (Finland) and Jenni Laine are thanked for technical support and sample processing for TEM. Petri Pulkkinen, University of Helsinki, Finland, is acknowledged for gold nanoparticle synthesis. The financial contribution from the Doctoral Education Network in Materials Research at Åbo Akademi University (N.P.) is greatly acknowledged. References 1 Hostetler M J, Wingate J E, Zhong C-J, Harris J E, Vachet R W, Clark M R, Londono J D, Green S J, Stokes J J, and Wignall G D ( 1998) Alkanethiolate gold cluster molecules with core diameters from 1.5 to 5.2 Nm: core and monolayer properties as a function of core size. Langmuir.  14: 17– 30. Google Scholar CrossRef Search ADS   2 Alkilany A M, and Murphy C J ( 2010) Toxicity and cellular uptake of gold nanoparticles: what we have learned so far? J. Nanoparticle Res.  12: 2313– 2333. Google Scholar CrossRef Search ADS   3 Chithrani D B, Dunne M, Stewart J, Allen C, and Jaffray D A ( 2010) Cellular uptake and transport of gold nanoparticles incorporated in a liposomal carrier. Nanomedicine Nanotechnol. Biol. Med.  6: 161– 169. Google Scholar CrossRef Search ADS   4 Jain S, Hirst D G, and O'Sullivan J M ( 2012) Gold nanoparticles as novel agents for cancer therapy. Br. J. Radiol.  85: 101– 113. Google Scholar CrossRef Search ADS PubMed  5 Johnson E, Seiradake E, Jones E Y, Davis I, Grünewald K, and Kaufmann R ( 2015) Correlative in-resin super-resolution and electron microscopy using standard fluorescent proteins. Sci. Rep.  5: 9583. Google Scholar CrossRef Search ADS PubMed  6 de Boer P, Hoogenboom J P, and Giepmans B N G ( 2015) Correlated light and electron microscopy: ultrastructure lights up! Nat. Methods  12: 503– 513. Google Scholar CrossRef Search ADS PubMed  7 Grabenbauer M, Geerts W J C, Fernadez-Rodriguez J, Hoenger A, Koster A J, and Nilsson T ( 2005) Correlative microscopy and electron tomography of GFP through photooxidation. Nat. Methods  2: 857– 862. Google Scholar CrossRef Search ADS PubMed  © The Author 2017. Published by Oxford University Press on behalf of The Japanese Society of Microscopy. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Microscopy Oxford University Press

Gold nanoparticle printed coverslips to facilitate fluorescence-TEM correlative microscopy

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Oxford University Press
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© The Author 2017. Published by Oxford University Press on behalf of The Japanese Society of Microscopy. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com
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0022-0744
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10.1093/jmicro/dfx118
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Abstract

Abstract Correlative light and electron microscopy (CLEM) allows combining the advantages of fluorescence microscopy and electron microscopy for cell imaging. Rare phenomenon expressing cells can be studied by specifically tagged fluorophores with fluorescence microscopy. Subsequently, cells can be fixed and ultra-structural details can be studied with transmission electron microscopy (TEM) at a higher resolution. However, precise landmarks are necessary to track the same cell throughout the CLEM process. In this technical report, we present a high contrast inkjet-printed gold nanoparticle patterns over commercial glass coverslip to facilitate cell tracking with correlative microscopy. High contrast and strong reflection from nano gold pattern can be used as a fixed landmark for cell identification with fluorescence microscopy. Nano gold printed letters over coverslips are visible in resin blocks, which can be further used to identify the cell of interest for performing sectioning of embedded cell blocks for TEM. correlative microscopy, CLEM coverslips, gold nanoparticles, CLEM methods We used a novel approach to print a customized pattern over regular microscopy coverslips using gold nanoparticles. The gold nanoparticle printing can be customized based on experimental design. In this case, we printed a set of letters and numbers (Fig. 1a and b). Inkjet printing of the gold letters on the cover glass substrates (washed with ethanol and water) was performed by using a piezoelectric DimatixTM Materials Printer (DMP-2800, FUJIFILM Dimatix, Inc., Santa Clara, USA) and a replaceable cartridge (DMC-11610) with a nominal droplet size of 10 pL. The dodecanethiol-protected gold nanoparticles (AuNPs) (3.0 ± 1.8 nm) were synthesized following the procedure reported by Hostetler et al. [1]. Fig. 1. View largeDownload slide Nano gold printed over regular coverslips. (a) Schematic representation of print pattern of gold nanoparticle letters over glass coverslip. (b) Drop view of the print design and (c) optical micrograph of the inkjet-printed gold letters. Fig. 1. View largeDownload slide Nano gold printed over regular coverslips. (a) Schematic representation of print pattern of gold nanoparticle letters over glass coverslip. (b) Drop view of the print design and (c) optical micrograph of the inkjet-printed gold letters. The ink was formulated by dispersing the AuNPs (1.5 wt %) in 80:20 wt % mixture of xylene (>98.5%, Sigma-Aldrich) and butoxyethylene (99%, Acros Organics), respectively. The printing was carried out using a single nozzle and a drop spacing of 30 μm. Firing frequency was set to 14 V and custom waveforms were used in order to gain optimal droplet formation. The gold nanoparticle-based printed pattern was clearly visible and no deformation was observed (Fig. 1c). Gold nanoparticles have shown non-toxic and inert nature for cellular studies [2–4]. Therefore, nano gold is regarded as safe material for cells to be grown on gold printed coverslips. We seeded HT1080 eGFP cells (GFP expressing) at 20–30% confluency over coverslips. Cells for correlative light and electron microscopy (CLEM) [5–7] were located by live cell imaging and their positions recorded with respect to the nano gold patterns. The live cell screening was performed with EVOS microscope. EVOS® FL cell culture microscope (Thermo Fisher Scientific Inc., USA) is a convenient microscope for quick live cell identification that allows both fluorescence and transmitted channels. The obtained images can be stitched together (Fig. 2a) to provide a better and larger field of view for rapid identification of rare cells. Fig. 2. View largeDownload slide The identification of GFP expressing cells of interest for CLEM. The cells of interest are indicated by arrows. (a) Merging of adjacent 4× images to provide a larger field of view along with nano gold printed letters. (b) Selection of one individual cell from a group of three cells for CLEM. (c) Low magnification fluorescence imaging of live GFP expressing HT1080 cells and cell of interest can be easily located with strong reflection from nano gold letters with a fluorescence microscope. Fig. 2. View largeDownload slide The identification of GFP expressing cells of interest for CLEM. The cells of interest are indicated by arrows. (a) Merging of adjacent 4× images to provide a larger field of view along with nano gold printed letters. (b) Selection of one individual cell from a group of three cells for CLEM. (c) Low magnification fluorescence imaging of live GFP expressing HT1080 cells and cell of interest can be easily located with strong reflection from nano gold letters with a fluorescence microscope. We selected a group of cells located between letters ‘R’ and ‘S’ for performing CLEM. The nano gold printed pattern has the bright contrast that was recognizable under bright field microscope with selected cells for CLEM (Fig. 2a and b). Inkjet printing of gold nanoparticles brought added the advantage of using reflection from gold particles as landmark for cells during fluorescence live cell imaging with eGFP tagged cells (Fig. 2c). Subsequently, live cell images were acquired by confocal microscope (TCS SP5 STED, Leica microsystems, Germany) using 10× objective (Fig. 3a). In order to image at high resolution, the objective was changed to 100× oil objective (Fig. 3b) using the same microscope. The process of changing objective from 10× to 100× and finding the same cell can be challenging without precise landmarks. Therefore, special attention should be paid on locating nano gold landmarks and its orientation with respect to the cell of interest for CLEM. Furthermore, the eGFP cells were imaged in confocal mode (Fig. 3c). Fig. 3. View largeDownload slide Imaging of the same cells of interest as in Fig. 2 with (a) phase contrast microscopy with 10× (low magnification) (arrow points the cells of interest), (b) fluorescence microscopy of GFP with 100× magnification and (c) confocal microscopy with 100× magnification to study cellular features. Fig. 3. View largeDownload slide Imaging of the same cells of interest as in Fig. 2 with (a) phase contrast microscopy with 10× (low magnification) (arrow points the cells of interest), (b) fluorescence microscopy of GFP with 100× magnification and (c) confocal microscopy with 100× magnification to study cellular features. The TEM sample preparation consisted of routine sample processing (Flat resin embedding). Cells were fixed with 5% glutaraldehyde s-collidine buffer (GA), postfixed with 2% OsO4 containing 3% potassium ferrocyanide, dehydrated with ethanol and flat embedded in 45359 Fluka Epoxy Embedding Medium kit. The ‘proof-of-concept’ representative images of sectioning are shown here. The cells can be seen (Fig. 4a) with nano gold letters facing outwards over a glass coverslip before embedding. The nano gold letters could serve as landmarks to identify the cells of interest over coverslips and also with cells embedded with Epoxy embedding medium (Fig. 4b and c) and further the region of interest can be located and trimmed for sectioning. Finally, cells were mounted for sectioning (Fig. 4d) with ultramicrotome using a diamond knife and 70–100 nm sections were collected. The sections were imaged with a JEOL JEM-1400 Plus transmission electron microscope operated at 80 kV acceleration voltage (Fig. 5). Fig. 4. View largeDownload slide Representation of sample processing for electron microscopy and cell identification facilitated by nano gold letters over Epon resin embedded cells. (a) Nano gold printed letters over a glass coverslip for cell identification and tracking throughout the correlation process. (b and c) Nano gold letters over Epon resin block of cells for identification. (d) Epon pyramid for 70 nm sectioning of cells and remnant of nano gold letters can be seen. Fig. 4. View largeDownload slide Representation of sample processing for electron microscopy and cell identification facilitated by nano gold letters over Epon resin embedded cells. (a) Nano gold printed letters over a glass coverslip for cell identification and tracking throughout the correlation process. (b and c) Nano gold letters over Epon resin block of cells for identification. (d) Epon pyramid for 70 nm sectioning of cells and remnant of nano gold letters can be seen. Fig. 5. View largeDownload slide The same cells of interest as in Figs. 2 and 3 were identified and imaged with TEM. Fig. 5. View largeDownload slide The same cells of interest as in Figs. 2 and 3 were identified and imaged with TEM. The contrast provided by gold printed coverslip has advantages over commercially available coverslips especially with fluorescence microscopes. The printed pattern remains detectable with fluorescence microscope and acts as orientation marker to find cells on the Epon resin block face. The nano gold pattern could be printed thick to allow cell identification with successive serial sections. Acknowledgements The Electron microscopy unit, University of Turku (Finland) and Jenni Laine are thanked for technical support and sample processing for TEM. Petri Pulkkinen, University of Helsinki, Finland, is acknowledged for gold nanoparticle synthesis. The financial contribution from the Doctoral Education Network in Materials Research at Åbo Akademi University (N.P.) is greatly acknowledged. References 1 Hostetler M J, Wingate J E, Zhong C-J, Harris J E, Vachet R W, Clark M R, Londono J D, Green S J, Stokes J J, and Wignall G D ( 1998) Alkanethiolate gold cluster molecules with core diameters from 1.5 to 5.2 Nm: core and monolayer properties as a function of core size. Langmuir.  14: 17– 30. Google Scholar CrossRef Search ADS   2 Alkilany A M, and Murphy C J ( 2010) Toxicity and cellular uptake of gold nanoparticles: what we have learned so far? J. Nanoparticle Res.  12: 2313– 2333. Google Scholar CrossRef Search ADS   3 Chithrani D B, Dunne M, Stewart J, Allen C, and Jaffray D A ( 2010) Cellular uptake and transport of gold nanoparticles incorporated in a liposomal carrier. Nanomedicine Nanotechnol. Biol. Med.  6: 161– 169. Google Scholar CrossRef Search ADS   4 Jain S, Hirst D G, and O'Sullivan J M ( 2012) Gold nanoparticles as novel agents for cancer therapy. Br. J. Radiol.  85: 101– 113. Google Scholar CrossRef Search ADS PubMed  5 Johnson E, Seiradake E, Jones E Y, Davis I, Grünewald K, and Kaufmann R ( 2015) Correlative in-resin super-resolution and electron microscopy using standard fluorescent proteins. Sci. Rep.  5: 9583. Google Scholar CrossRef Search ADS PubMed  6 de Boer P, Hoogenboom J P, and Giepmans B N G ( 2015) Correlated light and electron microscopy: ultrastructure lights up! Nat. Methods  12: 503– 513. Google Scholar CrossRef Search ADS PubMed  7 Grabenbauer M, Geerts W J C, Fernadez-Rodriguez J, Hoenger A, Koster A J, and Nilsson T ( 2005) Correlative microscopy and electron tomography of GFP through photooxidation. Nat. Methods  2: 857– 862. Google Scholar CrossRef Search ADS PubMed  © The Author 2017. Published by Oxford University Press on behalf of The Japanese Society of Microscopy. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com

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MicroscopyOxford University Press

Published: Feb 1, 2018

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