Detection of bioorthogonal groups by correlative light and electron microscopy allows imaging of degraded bacteria in phagocytes

The correlative light-electron microscopy is reported showing the labels in their ultrastructural context.

(d) Similar detail from a.iii-d.iii without red and blue signal. (e) Similar detail from a.iii-d.iii EM image only. Scale bar 500 nm.

Supplementary figure 8:
Scheme for metabolic labeling of sialylated glycans. Peracetylated Nazidoacetylmannosamine (Ac4ManNAz) enters the cell, after which its acetyl groups are cleaved of. ManNAz then passes through the steps of the sialic acid biosynthetic pathway and is converted to azido sialic acid (SiaNAz). After conversion to the nucleotide sugar CMP-SiaNAz, SiaNAz is incorporated into cellsurface glycoconjugates by sialyltransferases. Azido tagged glycans were subsequently labeled with AlexaFluor-488 alkyne via a ccHc on Tokuyasu sections of the cell samples.

Supplementary figure 9:
Confocal image of on sectio n lab elin g of bioorthogonal tagged sialylated glycans in Jurkat cells. Cells were incubated for 72 h with 50 µM of Ac4ManNAc (a) or Ac4ManNAz (b). Cells were fixed in 2% PFA, subjected to Tokuyasu sample preparation and crysectioned into 150 nm sections. Sections were reacted with AlexaFluor-488 alkyne using ccHc-conditions and DAPI. Green = AlexaFluor-488, Blue = nuclear DAPI stain.
Supplementary figure 10 : Confocal image of on sectio n lab elin g of BM -DC s i n cu b ated with DCG -04 th at targets active cy stein e p rotease s. C ells were in cu b ated for 2h w ith 10 µM of DCG-04-Amine (a) or DCG-04-azide (b). Cells were fixed in 2% PFA, subjected to Tokuyasu sample preparation and crysectioned into 150 nm sections. Sections were reacted with AlexaFluor-488 alkyne using ccHcconditions, anti-LAMP1 and DAPI. Green = AlexaFluor-488, Red = LAMP1, Blue = nuclear DAPI stain and fiducial beads.

MATERIALS AND METHODS
E. coli culturing conditions and growth measurements E. coli B834 were grown overnight at 37 °C in Lysogeny Broth (LB) medium. The following day cultures were diluted 1:50 in LB medium and grown at 37 °C till an OD 600 between 0.3-0.5. Subsequently cells were collected and resuspended in Selenomet medium (Molecular Dimensions) and supplemented with different concentrations of either Azidohomoalanine (Aha) (Bachem) or Methionine (Met) (Sigma-Aldrich). After 30', 1h, 2h and 3h OD 600 were measured and cells were collected by centrifugation for further analysis and BM-DC infection experiments. To monitor the outgrowth of E. coli cells that were cultured in the presence of Aha/Met, cells were collected upon centrifugation at the indicated time points after which Aha/Met containing medium was replaced for LB medium and OD 600 measurements were performed.
E. coli B834 GFP A206K was grown overnight at 37 °C in LB medium. The following day cultures were diluted 1:50 in LB medium and grown at 37 °C till an OD 600 between 0.3-0.5. Throughout culturing, cultures were supplemented with 100 µg/ml Ampicillin. The vector pRD35 for the constitutive expression of GFP A206K , was constructed by cloning GFP into pUC21 using NsiI and MluI restriction sites. An A206K mutation was introduced by site directed mutagenesis PCR to prohibit dimerization of GFP  Inclusion body analysis At the indicated time points E.coli cells were fixed for 2h with 2% paraformaldehyde (PFA) in 0.1 M phosphate buffer. The fixed cells were harvested by centrifugation (13,000 x g for 1 min), and resuspended in PBS. Formvar-carbon coated copper grids were floated on small drops of fixed E. coli cells for 5 min at room temperature. Grids were then washed on 3 drops of PBS and 10 drops of aquadest. Cells were imaged with an Tecnai 12 transmission electron microscope (FEI) at 120 kV acceleration voltage.

Mammalian cells and culture conditions
Mouse bone marrow derived dendritic cells were generated from B57BL/6 mice bone marrow essentially as described 2 with some modifications. Briefly, bone marrow was flushed from femurs and tibia and cells were cultured in IMDM (Sigma Aldrich) supplemented with 8% heat-inactivated fetal calf serum, 2mM L-glutamine, 20μM 2-Mercaptoethanol (Life Technologies), penicillin 100 l.U./mL and streptomycin 50μg/mL in the presence of 20ng/mL GM-CSF (ImmunoTools). Medium was replaced on day 3 and 7 of culture and the cells were used between days 10 and 13.
E. coli B834 cells were added to the BM-DCs as suspensions in PBS in a ratio of approximately 25:1, respectively. After 45' of incubation unbound/non-internalized E.coli cells were washed off (2x PBS) and medium was replaced. At the indicated time points cells were subjected to confocal microscopy or Tokuyasu sample preparation.
DCG-04-azide or DCG-04 amine 3 were added in a final concentration of 10 µM to the BM-DCs for 2 hours after which the cells were washed with PBS and kept for 16 hours in fresh medium. Subsequently cells were subjected to Tokuyasu sample preparation.
Jurkat cells were grown in RPMI 1640 medium supplemented with 10% heat-inactivated fetal calf serum, 2mM L-glutamine, penicillin 100 l.U./mL and streptomycin 50μg/mL. Jurkat cells were incubated for 3 days with 50 µM of N-azidoacetylmannosamine (MannAz)(Invitrogen) or N-acetylmannosamine (MannAc)(Sigma-Aldrich) from stock solutions in DMSO. Bioorthogonal labeling on cryo sections Samples were prepared for cryo sectioning as described elsewhere 4 . Briefly, E. coli cells, BM-DCs and Jurkat cells were fixed for 24h in freshly prepared 2% PFA in 0.1 M phosphate buffer. Fixed cells were embedded in 12% gelatin (type A, bloom 300, Sigma) and cut with a razor blade into 0.5 mm 3 cubes. The sample blocks were infiltrated in phosphate buffer containing 2.3 M sucrose for 3h. Sucrose-infiltrated sample blocks were mounted on aluminum pins and plunged in liquid nitrogen. The frozen samples were stored under liquid nitrogen.

SDS-PAGE Analysis
Ultrathin E.coli cell sections of 75 nm were obtained as described elsewhere. Briefly, the frozen sample was mounted in a cryo-ultramicrotome (Leica). The sample was trimmed to yield a squared block with a front face of about 300 x 250 μm (Diatome trimming tool). Using a diamond knife (Diatome) and antistatic devise (Leica) a ribbon of 75 nm thick sections was produced that was retrieved from the cryo-chamber with a droplet of 1.15 M sucrose containing 1% methylcellulose. Obtained sections were transferred to a specimen grid previously coated with formvar and carbon Grids were additionally coated as indicated with either 100 nm TetraSpeck beads or 100 nm FluoroSpheres (blue) carboxylate-modified (350/440) (Life Technologies).
Samples of t h e BM-DCs and Jurkat cells were sectioned into a ribbon of 150 nm thick sections using similar methods as described above. Thicker sections showed to improve the section integrity, leading to improved morphology results. To further improve the structural integrity of the ultrathin cryo sections we used a novel micromanipulator (Manip, Diatome) that was mounted on the cryo chamber of the ultramicrotome. This device facilitated section retrieval from the cryochamber and resulted in less overstretching of the sections during thawing In case of additional immune-labeling against LAMP-1 grids were subjected to the following steps directly after the ccHc reaction. Grids were washed 5 times with PBS/glycine and blocked again with PBS/Glycine containing 1 % BSA after which the grids were incubated for 1h with PBS/Glycine 1 % BSA supplemented with a rat anti mouse LAMP-1 (Biolegend). Sections were subsequently washed 6 times with PBS, labeled with DAPI (1:5000 in PBS for 5 min), were after washed again with PBS and aquadest.
Microscopy and correlation Grids containing the sample sections were washed with 50% glycerol and placed on glass slides (pre-cleaned with 100% ethanol). Grids were then covered with a small drop of 50% glycerol after which a coverslip was mounted over the grid. Coverslips were fixed using Scotch Pressure Sensitive Tape. Samples were imaged with a Leica TCS SP8 confocal microscope (63x oil lens, N.A.=1.4). Confocal microscopy was used as it allowed to make image stacks from the sections at different focus planes, this was convenient as the sections were found to be in different focus planes whilst placed between the glass slides and coverslip. Confocal stacks were deconvolved with theoretical point spread functions using Huygens Essential deconvolution software (SVI, Hilversum, Netherlands). After fluorescence microscopy the EM grid with the sections was remove from the glass slide, rinsed in distilled water and incubated for 5' on droplets of unranylacetate/methylcellulose. Excess of uranylacetate/methylcellulose was blotted away and grids were air-dried. EM imaging was performed with an Tecnai 12 Biotwin transmission electron microscope (FEI) at 120 kV acceleration voltage. Tilt series for electron tomography were collected using Xplore3D (FEI Company) software. The angular tilt range was set from -60˚ to 60˚ with 2˚ increments, and an objective lens defocus of -2 µm at a magnification of 20 K (pixel size is 1 nm). Alignments of the tilt series and weighted-back projection reconstructions for tomography were performed using IMOD 6 .
Correlation of confocal and EM images was performed in Adobe Photoshop CS6. In Adobe Photoshop, the LM image was copied as a layer into the EM image and made 50 % transparent. Transformation of the LM image was necessary to match it to the larger scale of the EM image. This was performed via isotropic scaling and rotation. Interpolation settings; bicubic smoother. Alignment at low magnification was carried out with the aid of nuclear DAPI staining in combination with the shape of the cells, at high magnification alignment was performed using the fiducial beads 7 .