PIE-scope, integrated cryo-correlative light and FIB/SEM microscopy.

The PIE-Scope setup The illumination source is a four wavelength combiner with a single multimode fibre output (Toptica iCLE-50 405, 488, 561, 640 nm). The choice between a multi-band-pass dichroic (Chroma 89402 m) and a Thorlabs 10/90 (R/T) beam splitter allows for fluorescence or reflected light imaging. The maximum laser power used during imaging corresponded to 0.5 mW out of the fibre, which was further reduced to 0.2 mW at the back-focal plane of the objective, although there might be occasions where more laser power is required for imaging, we noted that these conditions have provided sufficient illumination on all tested samples. Currently, the camera used is a Basler acA1920-155um with USB3.0 interface. This camera is a heated CMOS detector with a quantum efficiency in the visible range of ~70%, it represents a cost-effective solution which comes with a dark noise of 6.6 e - . The major advantage of this component, beside its price, is that it does not require active cooling, making the system less susceptible to vibrations. If single-molecule imaging is required, or if the fluorescence signal is extremely dim, then cooled CMOS cameras such as the Hamamatsu Flash4.0 V3 should be used. In this case, in order to ensure that no vibration is introduced in the system, the camera cooling lines should be connected to the FIB/SEM water chiller. The feedthrough from the atmosphere to the high-vacuum is realised through a glass window (Thorlabs VPCH42-A). The optical path is steered using three axes mirror holders embedded in the custom build microscope body/flange visible in Figure 2a . The focus drive is a high-vacuum-compatible piezoelectric linear positioner (Smaract SLC-2445HV). The objectives tested were Olympus MPLFNL 10x/0.3, MPLFNL 20x/0.45, UPFLN 20x/0.8 and MPLAPON 50x/0.95. The microscope body and the adapters were machined at the Monash University Mechanical and Aerospace Engineering Workshop. All atmosphere parts were made of Aluminium alloy 7075. All vacuum parts were machined from Stainless Steel 316 l. Mounting of the microscope body on the vacuum chamber of the FIB/SEM occurs directly on the middle front GIS port (GIS2 on ThermoFisher instruments). The only strict requirement of this design is that neither a gas injection system (GIS) nor detector can be mounted on the front GIS ports at the FIB side of the chamber, we resolved this issue by repositioning our GISs from the flanges GIS1 and GIS2 to the rear flanges (GIS4 and GIS5). Although this can be a limitation for highly equipped material science microscopes, on systems where biological imaging and lamella cryo-preparation is the major focus this will not result in a problem. Also, with the advent of multi-line-GISs offered by most instrument manufacturers, it is extremely unlikely that all GIS flanges will be required. All custom components were designed using Autodesk Inventor Pro 2016 and Siemens NX. Parts were fabricated in the Mechanical and Aerospace engineering mechanical workshop at Monash University. Ray tracing was performed using Zemax OpticsStudio. The control software has been developed using National Instruments LabVIEW 2015, Python three with Numpy 1.15, Scipy 1.1.0 and ThermoFisher AutoScript 4.0. All drawings, latest Bill of Material (matched to the stable software released), a stable software release, list of dependencies and instructions are available at https://www.demarco-lab.com/resources . Sample preparation All Caenorhabditis elegans strain RJP3088 (Pets-5::mCherry +Pelt-2::GFP) ( Juozaityte et al., 2017 ) was maintained at 20°C on NGM plates seeded with Escherichia coli OP50 bacteria (Porta-de-la-Riva, Fontrodona et al.). Isolation of L1 larvae was performed as previously described ( Porta-de-la-Riva et al., 2012 ). Larvae were deposited on a holey carbon EM-grid and plunge-frozen in liquid ethane. C. elegans samples were kept in M9 medium, concentrated to a density of 3–4 worms / µL. 5 µL of solution were deposited on a Cu-300-mesh Quantifoil R-2/2 grid which was glow discharged for 30 s using a Pelco EasyGlow. The excess of medium was manually blotted using Whatman filter paper grade 595 (Merck) and vitrified by plunge freezing in liquid ethane. Drosophila melanogaster used were a cross between Engrailed-Gal4 (BL30564) and UAS-eGFP (BL6874) from the Bloomington Drosophila Stock Center (Indiana). Flies were raised on standard media at 25°C. Drosophila brains at the third instar larval stage were dissected in cold phosphate buffered saline before being mounted in 20% (w/v) dextran solution prior vitrification. Samples were high-pressure frozen using a Leica EM-PACT2 on 200 µm deep planchettes. In order to expose the sample to the surface, therefore reducing the milling time required, we performed manual freeze-fracture by scraping the surface with a scalpel under liquid nitrogen. The difference in density between the dextran solution and the sample itself ensures an easy separation. Saccharomyces cerevisiae cells were cultured at overnight 30°C in 2 YPD+. Cell growth was monitored through the measurement of the optical density. Cells were harvested when the optical density reached 0.6, washed in PBS and stained using Nuclear-ID (Enzo Life Sciences). 5 µL were deposited on a Cu-300-mesh Quantifoil R-2/2 grid which was previously glow discharged for 30 s in a Pelco EasyGlow. Grids were manually blotted for 5 s using Whatman 595 blotting pads and plunge-frozen in liquid ethane.

Additional files 10.7554/eLife.45919.011 Transparent reporting form