Developing cryo on-grid lamella workflows for the JEOL JIB 4700F

Session

FIB Applications & EM Sample Prep Techniques in Biological Sciences

Authors

M Kobylynska (1), A Carbajal (1), D McGrouther (2), P.C Hawes (3), R.A Fleck (1)

Affiliations

1. Centre for Ultrastructural Imaging, King’s College London
2. JEOL (UK) Ltd.
3. The Pirbright Institute

Abstract text

The stability and emitter life of a field emission (FEG) source, coupled with the low kV sensitivity of a scanning electron microscope (SEM) equipped with in-lens or semi in-lens detectors, is a powerful tool. Modern FEGSEM systems (with improved detector sensitivity and signal-to-noise ratios) are now achieving sub-nm resolution at 1kV, making them highly capable tools for life science research, particularly when combined with high-resolution coating and fracture techniques.  

The improved performance of the FEGSEM has allowed the development of several instruments/techniques of direct benefit to life sciences: serial block-face scanning electron microscopy (SBFSEM), focused ion beam scanning electron microscopy (FIBSEM), and array tomography (AT).  Each aims to generate 3D ultrastructural information over extended x-y-z ranges with the intention of informing the life scientist about the interrelationship between multiple cells and their individual compartments. Thus, these developments help to overcome the principal limitation of EM: the loss of 3D understanding of ultrastructure and the possibility of “missing” rare ultrastructural events through the limited volume of tissue examined. By collecting ultrastructural data from a large volume, to be interrogated in silico; novel understandings of the interconnectivity between many individual cell types can be deduced.

These volume EM (vEM) approaches have principally been applied to chemically processed tissue, with their associated artefacts.  Cryo fixation and cryo electron microscopy (cryoEM) allows cells and tissues to be studied close to their native biological state.  This has commonly been applied to cryo electron tomography (cryoET) of thin sections of cells and tissues. Since most cell regions are too thick to allow penetration by the transmission electron beam, cryo sections or lamellae are produced from vitrified material. Vitrified cells and tissues are sectioned by focussed ion beam milling in a dual beam scanning electron microscope at cryogenic temperatures (cryoFIB). This supports the preparation of cryo samples 100-300 nanometers in thickness without the compression of the tissue, scoring and crevassing artefacts of CEMOVIS sections.  This controlled milling of vitrified material also allows the cryoFIB to be used to generate 3D volumes by a sequence of sectioning, then imaging of the exposed face of the tissue with the scanning electron beam followed by further sectioning and imaging.  However, high beam sensitivity of the vitrified sample, lack of heavy metal contrast and significant challenges to the generation of sufficient signal to noise makes the approach challenging.    

Here we report on adapting a JEOL JIB 4700F FIB for cryoFIB milling of tissues for cryo 3D volume imaging.  Adaptations to the FIB now support 24/7 operation of the instrument at an operating temperature below -169°C and ice contamination rates below 6nm.h-1.  Characterisation of instrument stability, optimisation of imaging conditions and the impact of different strategies for tracking a region of interest during sequential sectioning have been determined.  We have also optimised the vitrification of a reference sample (Euglena gracilis, CCAP 1224/5Z) for cryo 3D volume production and are developing cryo correlative workflows.