Cryo-soft-X-ray tomography (cryoSXT) and cryo fluorescence microscopy imaging
of uninfected and herpes simplex virus (HSV)-1 infected cultured cells.
November 2021
Kamal Nahas, Viv Connor, Maria Harkiolaki, Colin Crump and Stephen Graham

Files
===
tomographic_collection_parameters.csv - Experiment and CryoSXT data 
                                        collection parameters
*.st - X-ray data (tilt series)
*.rec - reconstructed tomograms
*.tif - fluorescence imaging of EM grids used for CryoSXT data collection

All .st and .rec files have been compressed using xz (https://tukaani.org/xz/)
and can be uncompressed using "unxz <filename.xz>".

The file F48_tomogram.rec.xz was split into two chunks using the Unix "split"
command to reduce the file size below 2 Gb. The file can be reconstituted using
the Unix "cat" command, e.g.
"cat F48_tomogram.rec.xz.1 F48_tomogram.rec.xz.2 > F48_tomogram.rec.xz"

The .st and .rec files can be opened using image viewing programs such as
Fiji (https://imagej.net/software/fiji/). The .rec files can also be opened 
with ChimeraX (https://www.rbvi.ucsf.edu/chimerax/). 

Cryo-soft-X-ray tomography collection parameters
===
An UltraXRM-S/L220c X-ray microscope (Carl Zeiss Xray microscopy) was used to
collect the datasets at Beamline B24 at Diamond Light Source. The raw data,
known as a tilt series, consists of a collation of images collected from the
same field of view at different angles. These were collected within a maximum
range of -70° and +70° degrees and at increments of 0.2° or 0.5° and with an
exposure time of 0.5 seconds or 1 second. 500 eV X rays were focused with a
zone plate objective capable of a nominal resolution of 25 nm or 40 nm. Full
details on parameters used for individual tilt series can be found in the
attached tomographic_collection_parameters.csv file. Tomograms were
reconstructed in Imod version 4.9.2. For each field of view, a tilt series and
a reconstructed tomogram are provided.

Detection of HSV-1 by cryo-soft-X-ray tomography
===
Tomograms were collected from human foreskin fibroblast cells that had been
immortalised with hTERT (HFF-hTERT). The ultrastructure of uninfected cells was
compared with that of HSV-1-infected cells to determine if HSV-1 particles
could be detected with cryoSXT. In this dataset, we note capsids in the nuclei,
viral particles in the nuclear envelope and cytoplasm, and virions at the
exposed cell surface and at cell junctions.

Changes to cytoplasmic vesicles and mitochondria during HSV-1 infection
===
We used human osteosarcoma cells (U2OS) to study changes to cytoplasmic
vesicles and mitochondria. A population of synchronously infected cells
progress through infection at different rates and this could affect the state
of these cellular compartments. To account for this, we used a recombinant of
HSV-1, known as the timestamp virus, which contains two fusion proteins with
different temporal expression. This allowed us to distinguish between early
stages of infection (using the immediate early protein eYFP-ICP0) and late
stages (using the late protein gC-mCherry). First we collected fluorescence
data from infected cells to identify early-stage and late-stage cells for
subsequent imaging by cryoSXT. To collect fluorescence data, we used a Zeiss
AxioImager2 microscope with an achromatic 50× air objective (Zeiss LD EC
Epiplan-Neofluar 50x/0.55 DIC M27; NA=0.55; free working distance=9.1 mm) with
the following filters: Zeiss 46 HE YFP filter (Excitation 500±25 nm, Emission
535±30 nm) and the Zeiss 64 HE mPlum filter (Excitation 587±25 nm, Emission
647±70 nm). This fluorescence data is supplied here in the form of a map of the
whole sample grid. Second, we imaged these early-stage and late-stage cells in
addition to uninfected cells on the X-ray microscope as described above. Data
were collected from three independent replicates.