What is cryo-imaging?
Imaging cryo-preserved biological samples allows us to probe the inner workings of cells preserved in a near-native stage in unprecedented detail, radically changing how we understand the inner workings of cells and pathogens. Accurate, high-quality 3-dimensional subcellular positioning data for regions, structures, or proteins of interest is vital for efficient ultrastructural and near-atomic resolution cryo-tomography of biological samples, using, for example, electron cryo-tomography (cryoET) or soft X-ray cryo-tomography (cryoSXT).
The Pathogen Cryo-Imaging Facility in Cambridge (PaCIFiC) allows the preparation and high-resolution confocal cryo-microscopy analysis of vitrified samples. PaCIFiC is a containment level 2 (CL2) facility that prepares and imagines non-hazardous and infectious samples. Specifically, PaCIFiC enables:
- Vitrification of cells grown on EM grids
- Confocal cryo-microscopy, including sample quality analysis and subcellular 3D targeting for downstream electron and X-ray cryo-tomography
- Vitrification of aqueous samples for tomography or single-particle analysis
- Preparation and imaging of samples containing hazard group 2 pathogens at CL2
PaCIFiC is currently under commissioning but will soon be available to users. If you want to use PaCIFiC, please get in touch with us (see below).
Equipment
PaCIFiC houses a Leica EM GP2 for vitrifying (plunge-freezing) samples and a Leica STELLARIS 5 confocal cryo-microscope, in addition to facilities for biological sample preparation (cell culture and infection with hazard group 2 pathogens).
EM GP2
The EM GP2 can prepare grids for single particle analysis and vitrifying cells grown on electron microscopy grids for cryo-imaging. Please see image above.
- Automatic single- or multi-sided blotting
- Environmental control (temperature and humidity)
- Viewing system (Leica S9E) for accurate alignment
STELLARIS 5 Cryo-Confocal
-
White light laser (485-790 nm) plus solid-state lasers (405 and 448 nm)
- 50× 0.9 NA cryo CLEM objective (HC PL APO 50x/0.90)
- Cryo-CLEM stage with liquid nitrogen reservoir
- 5 Power HyD detection channels
- Tau sense modality
- Lightning Confocal Super Resolution modality
- Transfer shuttle with cartridges for bare grids or autogrids (clipped grids)
- ‘Cover box’ for avoiding contamination during transfer shuttle grid loading/unloading
- K5 sCMOS camera for brightfield/reflection/epifluorescence grid mapping
- LAS X software (including Coral Cryo)
Biological sample preparation
- Access controlled facility (CL2) for the use of hazard group 2 pathogens
- Class 2 microbiological safety cabinet
- PHCBI multi-gas incubator (MCO-50M-PE IncuSafe)
- Temporary storage space (4°C and liquid nitrogen)
- Recirculating fume hood
Biological safety
Work involving biological containment (up to CL2) will require submitting a risk assessment, which will need to be discussed with the facility and approved by the Department of Pathology biological safety committee. We can support you in this process—please contact us (see below).
Booking
Once trained, University of Cambridge members (i.e. internal users) can book access via PPMS. For training, please get in touch with us (see below).
External academic/industrial users can only book by contacting us (see below).
Usage charges
Please note, there are access charges related to instrument use. For the University of Cambridge members, they are as follows (from April 1, 2024):
- Class 2 microbiological safety cabinet, £10 per hour
- Leica EM GP2, 2-hour session - £57
- Leica STELLARIS 5 Cryo-Confocal, £50 per hour (minimum booking: 3 hrs)
The rates are reviewed regularly. During the PaCIFiC commissioning phase, we will offer introductory discounts for using the cryo-confocal. For information regarding these and the rates for external academic groups and industrial users, please contact us (see below).
Consumables
Access charges for the EM GP2 and STELLARIS 5 cryo-confocal include gas usage (ethane and liquid nitrogen) but do not include consumable items (e.g., electron microscopy grids, C-clips, storage boxes, etc.). However, we stock some of them that may be available for purchase. Please ask for details.
Access charges for the class 2 microbiological safety cabinet include reasonably using essential consumables (gloves, filter tips, plastic serological pipettes). They do not include tissue culture dishes/flasks or medium—please bring your own or speak to us for details.
Contact details
PaCIFiC is currently under commissioning. If you are interested in using PaCIFiC please get in touch with the directors, Professor Colin Crump cmc56@cam.ac.uk and Professor Stephen Graham scg34@cam.ac.uk
*Cover image features EM GP2