73
Information should ideally be captured in 3D, to the highest resolution possible and give us clear views into the architecture and composition of cells and tissue without losing fi delity of information through artefacts induced because of harsh sample preparation procedures or microscopy requirements. At beamline B24, this is achieved via cryo-preservation where cells grown adherent or deposited on grid sample holders (3mm gold wafers coated with a perforated carbon support fi lm) are plunge frozen in liquid nitrogen and then kept cold throughout transfer and exposure to light. This ensures that the sample not only remains perfectly static during imaging but also sustains little or no damage during exposure to the harsh illumination conditions required for high resolution imaging. Moreover, as chemical fi xation or embedding are not required, no chemical alterations have been induced within the sample and therefore the risk of misinterpretations is minimised.
The beamline team
B24 currently has a core team of fi ve scientists all working towards development and automation of the methods offered. They are two biologists, one chemist and two physicists and their expertise range from molecular and cell biology to cryogenic sample handling, beamline instrumentation and software development.
Mapped 3mm sample grid carrying human HFF cells (the outline of cells can be seen in the square inset). The panel on the left shows tweezers used for transferring grids and two gold grids on a fi lm-covered surface for size.
Other necessary pre-requisites for successful biological imaging in general are reproducibility, accessibility and ease of use. New technology should deliver reliably, at all times, made available as soon as possible, and have clear access routes. At beamline B24, the correlative cryo-imaging platform has been put through it’s paces for the past year and research groups working on a number of areas from cancer biology, infection and pathogen clearance to bacteria, algae and archaea biology have benefi tted from the technology in this time while the beamline team has focused on streamlining user support, accessibility, sample preparation and mapping as well as data collection and processing. Data collection at B24 typically takes 3-5 days per project (time on both cryoSIM and cryoSXT) and results in many Gigabytes of 3D data (10-30 SIM data sets, 1-3 X-ray tomograms per SIM data set) of areas of 100s of microns in and across cells.
Nucleus Lipid droplet
Endoplasmic reticulum
Nucleus
The current B24 core team. Next to the cryoSXT microscope is Dr M Harkiolaki (outer left), Dr C Okolo (middle left), Mr T Fish (middle right), Dr I Kounatidis (outer right). A new member Dr M Koronfel has recently joined B24.
Nuclear envelope
Mitochondria 10 microns Cytoplasm Cytoplasm
Slice of 3D cryoSXT data from human U2OS cells showing the level of detail that can be visualised with soft X-ray tomography and also the organelles and features prominent in a cryo-preserved mammalian cell at near-physiological conditions.
The B24 group collecting and evaluating X-ray.
They are supported by beamline technicians, engineers and designers, as well as a host of other support teams within Diamond Light Source from building and facility maintenance to IT and software support and development. All company departments are to a great degree interconnected and provide rapid and relevant support to the beamlines though out the year. An enthusiastic and professional User Offi ce team provides a handy interface between the technology and innovation teams and the user community that engages with the beamlines and use the equipment available.
Development of new techniques transcends the remit of a single organisation and greatly benefi ts from cross-institutional collaborations. At B24, a productive collaboration with Micron at the Biochemistry department of the University of Oxford in the UK has resulted in the development and delivery of the cryoSIM. This endeavour has in turn seen the support of microscopy and scientifi c software experts from around the world and continues to develop as a collaborative project.
Slice of 3D cryoSXT data (left) showing an area of a cell infected with reovirus (the nucleus and cytoplasm are indicated by a blue and green arrow respectively; the interface of this cell with its neighbour is outlined with orange arrows). CryoSIM data (also 3D) is superimposed (right) to delineate organelles carrying viral load in the perinuclear area two hours post infection. This fi gure clearly shows the benefi ts of combining a high-resolution imaging techniques such as SXT (nanometer resolution of cell ultrastructure; organelles and other substructures clearly delineated) with 3D super resolution fl uorescence SIM (localisation of particles of interest, reovirus in this case, in 3D). A subset of multivesicular bodies identifi ed near the nucleus in SXT can now be identifi ed with SIM as carriers of viral load.
Software development at B24 is driven by efforts of the core team in close contact with the scientifi c software, motion and controls group at Diamond Light source but extends beyond the confi nes of the home institute and involves collaborations with software scientist across the globe. Such a collaboration with Dr P. Paul-Gilloteaux at the University of Nantes in France (the developer of 3D correlation software eC-CLEM) involves the tailored development of bespoke 3D image registration software specifi cally for SIM and SXT which is currently underway.
Endosomes
WWW.LABMATE-ONLINE.COM
Page 1 |
Page 2 |
Page 3 |
Page 4 |
Page 5 |
Page 6 |
Page 7 |
Page 8 |
Page 9 |
Page 10 |
Page 11 |
Page 12 |
Page 13 |
Page 14 |
Page 15 |
Page 16 |
Page 17 |
Page 18 |
Page 19 |
Page 20 |
Page 21 |
Page 22 |
Page 23 |
Page 24 |
Page 25 |
Page 26 |
Page 27 |
Page 28 |
Page 29 |
Page 30 |
Page 31 |
Page 32 |
Page 33 |
Page 34 |
Page 35 |
Page 36 |
Page 37 |
Page 38 |
Page 39 |
Page 40 |
Page 41 |
Page 42 |
Page 43 |
Page 44 |
Page 45 |
Page 46 |
Page 47 |
Page 48 |
Page 49 |
Page 50 |
Page 51 |
Page 52 |
Page 53 |
Page 54 |
Page 55 |
Page 56 |
Page 57 |
Page 58 |
Page 59 |
Page 60 |
Page 61 |
Page 62 |
Page 63 |
Page 64 |
Page 65 |
Page 66 |
Page 67 |
Page 68 |
Page 69 |
Page 70 |
Page 71 |
Page 72 |
Page 73 |
Page 74 |
Page 75 |
Page 76 |
Page 77 |
Page 78 |
Page 79 |
Page 80 |
Page 81 |
Page 82 |
Page 83 |
Page 84
