Sample Preparation for Correlative Light and Electron Microscopy (CLEM) Analyses in Cellular Microbiology

Materials and Reagents

1. General lab equipment: Gloves, lab coat, pipettes, 15 and 50 ml centrifuge tubes (e.g., BD Biosciences, Falcon^®), microcentrifuge tubes (e.g., Eppendorf), plastic Pasteur pipettes, beakers
   
2. 10 cm plastic Petri dish or a comparable vessel
   
3. 3.5 cm plastic Petri dish or a comparable vessel (Acetone-resistant)
   
4. Eukaryotic cells as biological sample (here: transfected HeLa cells expressing LAMP1-GFP)
   
5. Cell-specific culture medium: DMEM (Biochrom AG, catalog number: FG 0445 ) + 10% Fetal Bovine Serum (FCS) (Thermo Fisher Scientific, Gibco^TM, catalog number: 10270 )
   
6. Cell-specific imaging medium: MEM (Biochrom AG, catalog number: F 0475 ) + 30 mM HEPES
   
7. HEPES = 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (Carl Roth GmbH, catalog number: HN77.5 )
   
8. Glutaraldehyde [CH₂(CH₂CHO)₂], 25% in H₂O (Electron Microscopy Sciences, catalog number: E16221 )
   
9. Glycine, ultra pure (Biomol GmbH, catalog number: 04943.1 )
10. Large gelatin capsules, size 13 (Electron Microscopy Sciences, catalog number: 70114 )
    
11. Calcium chloride (CaCl₂) (Sigma-Aldrich, catalog number: C 5670 )
    
12. Osmium tetroxide (OsO₄), 0.1 g ampules (Electron Microscopy Sciences, catalog number: 19134 )
    
13. Ruthenium red [[(NH₃)₅RuORu(NH₃)₄ORu(NH₃)₅]Cl₆] (AppliChem GmbH , catalog number: A34880001 )
    
14. Potassium hexacyanoferrate(III) [K₃[Fe(CN)₆]] (Sigma-Aldrich, catalog number: 244023 )
    
15. Ethanol p.a. grade (very pure chemical)
    
16. Acetone p.a. grade (very pure chemical)
    
17. EPON 812 (SERVA Electrophoresis GmbH, catalog number: 21045.02 )
    
18. DDSA = Dodecenylsuccinic anhydride (SERVA Electrophoresis GmbH, catalog number: 20755 )
    
19. MNA = Methylnadic anhydride (SERVA Electrophoresis GmbH, catalog number: 29452.03 )
    
20. DMP-30 = 2, 4, 6-Tris(dimethylamino-methyl)phenol (SERVA Electrophoresis GmbH, catalog number: 36975 )
    
21. Uranyl acetate dihydrate [UO₂(CH₃COO)₂^.2H₂O] (Electron Microscopy Sciences, catalog number: 22400 )
    
22. Lead(II) nitrate [Pb(NO₃)₂] (Sigma-Aldrich, catalog number: 31137 )
    
23. Trisodium citrate dihydrate (Na₃C₆H₅O₇^.2H₂O) (Carl Roth, catalog number: 3580.1 )
    
24. 1 N Sodium hydroxide (NaOH), carbonate-free (Electron Microscopy Sciences, catalog number: 21170-01 )
    
25. 65% Nitric acid (HNO₃)
    
26. Autoclaved ultrapure H₂O (MilliQ)
    
27. Liquid nitrogen
    
28. Graded ethanol series (50%, 70%, 80%, 90%, 95%, 100%)
    
29. Mixes of acetone and EPON (3:1 and 1:3 mix)
    
30. 1 M HEPES buffer (see Recipes)
    
31. 2 M CaCl₂ in H₂O (see Recipes)
    
32. 10% glycine in 0.2 M HEPES buffer (see Recipes)
    
33. 1% ruthenium red in H₂O (see Recipes)
    
34. 15% potassium hexacyanoferrate(III) in H₂O (see Recipes)
    
35. 2x fixative (see Recipes)
    
36. Post-fixative (see Recipes)
    
37. EPON resin (see Recipes)
    
38. 2% uranyl acetate in H₂O (see Recipes)
    
39. Lead citrate according to Reynolds (see Recipes)
    
40. 3% nitric acid (HNO₃) (see Recipes)
    

Equipment

1. Cell culture equipment: Cell culture incubator (37 °C, 5% CO₂, 90% humidity) and clean bench
   
2. MatTek Glass Bottom Culture Dishes, 35 mm, uncoated, Glass No. 2, gridded (Glass Bottom Dishes, MatTek Corporation, catalog number: P35G-2-14-CGRD )
   Attention: Glass No. 2 may be too thick for some objectives with high numerical aperture (NA), but this is the only format available and worked in our application.
   
3. Confocal laser-scanning microscope (CLSM) Leica SP5 equipped with an incubation chamber (homemade) maintaining 37 °C and humidity during live cell imaging (Attention: CO₂-containing atmosphere can be omitted if using imaging medium buffered with 30 mM HEPES. For carbonate-buffered cell culture media, the atmosphere should contain 5% CO₂). The microscope is operated with software package LAS-AF for setting adjustment, image acquisition and image processing
   
   1. Several objectives, such as 10x (HC PL FL 10x, NA 0.3, DIC, dry), 20x (HC PL APO CS 20x, NA 0.7, DIC, dry), 40x (HCX PL APO CS 40x, NA 1.25-0.75, DIC, oil immersion) and 100x objective (HCX PL APO CS 100x, NA 1.4-0.7, DIC, oil immersion)
      
   2. The polychroic mirror TD 488/543/633 for the three channels GFP/RFP/DIC (Leica Microsystems)
4. Fume hood
   
5. Ice or cold metal block
   
6. Scalpel
   
7. Oven, heatable to 60 °C
   
8. Dewar vessel for liquid nitrogen
   
9. Stereo microscope
   
10. Dark permanent marker
    
11. Vice
    
12. Jigsaw
    
13. Universal specimen holder for EPON blocks (Leica Microsystems, catalog number: 16701761 )
    
14. Double edge carbon steel blades (Plano, catalog number: 121-9 )
    
15. Ultramicrotome (Leica EM UC6)
    
16. Diamant knife ultra 45 °C, 2 mm (DIATOME, catalog number: DU4520 )
    
17. Formvar-coated EM cooper slit grids 2 x 1 mm (homemade coating) (Electron Microscopy Sciences, catalog number: G2010-Cu )
    
18. Forceps for EM grids, type 7 stainless steel (Plano GmbH, catalog number: T5039 )
19. Storage box for grids (Electron Microscopy Sciences, catalog number: G71138 )
    
20. Large forceps for liquid nitrogen and small forceps for embedding procedure
    
21. Automated staining device (nanofilm surface analysis ultrastainer)
    
22. TEM (ZEISS, model: EFTEM 902 A ), operated at 80 kV and equipped with a 2K wide-angle slow-scan CCD camera (Teacher Retirement System of Texas) with software ImageSP (Teacher Retirement System of Texas, model: image SysProg)
    

Software

1. ImageJ, http://rsbweb.nih.gov/ij/, Photoshop 5.5 (Adobe), or higher or similar software for stitching and overlay of images
   
2. Imaris (Bitplane), ImageJ, ZEN (ZEISS), LASAF (Leica Microsystems), or similar software for processing fluorescence images
   

Procedure