CIL:50063, Chlamydia trachomatis, Human HeLa. In Cell Image Library

In this study, we used Serial Block-Face Scanning Electron Microscopy (SBEM) to provide a comprehensive quantitative analysis of the intracellular chlamydial infection over time. We performed SBEM on monolayers of C. trachomatis-infected HeLa cells. Stacks of consecutive 60-nm-thick sections were acquired and subsequently digitally aligned, which allowed individual bacteria to be observed and analyzed in multiple successive sections. We then combined all the EM sections computationally into a 3D reconstruction of the inclusion.
Our analysis provided detailed quantitative information about the C. trachomatis
inclusion and its developmental forms.

Cell culture and Chlamydia infections
HeLa cells (ATCC CCL-2) were grown in Advanced DMEM (4.5 g glucose/L)
(Invitrogen) supplemented with 2% fetal bovine serum (FBS) (Hyclone/Thermo Fisher) and 2mM GlutaMAX-I (Invitrogen) in 5% CO2 at 37°C. Cell monolayers were infected with C. trachomatis serovar L2, strain L2/434/Bu (ATCC VR-902B) at a multiplicity of infection (MOI) of 3 in sucrose-phosphate-glutamic acid (SPG). Uninfected control experiments were performed as mock infections in SPG alone. Infections were carried out by centrifugation at 700xg in a Sorvall Legend Mach 1.6R centrifuge for 1 hour at room temperature. After centrifugation, the inoculum was replaced by fresh cell culture medium and monolayers were incubated at 37°C and 5% CO2. HeLa Cells and EBs were verified to be free of Mycoplasma contamination by PCR.

Preparation of cells for serial block-face scanning EM (SBEM)
Chlamydia-infected monolayers were fixed in a solution of 2% paraformaldehyde
and 2.5% glutaraldehyde in 0.1 M cacodylate buffer, pH 7.4 for 1 hour. Cells were stained for SBEM. Briefly, cells were washed 5X in cold 0.1 M cacodylate buffer then incubated in solution containing 1.5% potassium ferrocyanide and 2% osmium tetroxide supplemented with 2 mM calcium chloride in 0.1 M cacodylate buffer for 30 minutes on ice. After 5X 2-minute washes in doubled distilled water, cells were incubated in 1% thiocarbohydrazide for 10 minutes at room temperature. Following 5X 2-minute washes in double distilled water at room temperature, cells were placed in 2% osmium tetroxide in double distilled water for 10 minutes at room temperature. Cells were rinsed 5X 2 minutes with double distilled water at room temperature and subsequently incubated in 2% uranyl acetate at 4°C overnight. The next day, cells were washed 5X 2 minutes in double distilled water at room temperature and en bloc Walton's lead aspartate staining was performed for 10 minutes at 60°C. Following 5X 2-minute washes in double distilled water at room temperature, cells were dehydrated using a series of ice-cold graded ethanol solutions and then embedded in Durcupan ACM resin (Electron Microscopy Sciences). The resin was allowed to polymerize in a vacuum oven at 60°C for 48 hours. SBEM imaging was completed using a Gatan automated 3View system (Gatan Inc.) incorporated into a Zeiss Sigma or Merlin Compact Scanning Electron Microscope (Zeiss), and images were recorded at 60 nm cutting intervals.
3D EM Segmentation and Analysis
Complete three-dimensional reconstructions of Chlamydia inclusions were constructed and analyzed using the IMOD image processing software (University of Colorado, Boulder).