Xplore3d

Manufactured by Thermo Fisher Scientific

The Xplore3D is a compact, high-resolution 3D microscope designed for detailed imaging and analysis of samples. It provides advanced imaging capabilities with its versatile optical system and high-performance camera.

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8 protocols using xplore3d

Tilt Series Tomography of Skin Biopsies

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Two hundred nm sections of embedded skin biopsy specimens were collected on glow discharged carbon grids, and 10 nm colloidal gold fiducial markers were applied. Using a linear tilt scheme, single axis tilt series were collected on a Tecnai BioTwin Spirit TEM (FEI) operated at 120 kV. Images captured over a tilt range of +/- 68° (1° increments) at 1 μm defocus were recorded on a 2048 x 2048 pixel UltraScan 1000 Gatan CCD camera using the automated tomography acquisition software (Xplore 3D, FEI). Resulting images had a binning factor of 1 and pixel size of 2.1 nm. The tilt series were aligned using either Inspect 3D (FEI) or IMOD software package (version 4.2.5) and SIRT reconstructions of 35 iterations were performed. Movies were generated through the volume ortho-slices and volren models created from unfiltered tomograms with inverted contrast by thresholding colors based on pixel intensity within the volumes using the Amira Visualization Package (version 5.3.0, Visage Imaging, Carlsbad, CA).

Kirshenbaum A.S., Cruse G., Desai A., Bandara G., Leerkes M., Lee C.C., Fischer E.R., O’Brien K.J., Gochuico B.R., Stone K., Gahl W.A, & Metcalfe D.D. (2016). Immunophenotypic and Ultrastructural Analysis of Mast Cells in Hermansky-Pudlak Syndrome Type-1: A Possible Connection to Pulmonary Fibrosis. PLoS ONE, 11(7), e0159177.

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Cryo-TEM Imaging and 3D Tomography

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Grids were transferred to a Gatan 626 cryo holder (Gatan, Pleasanton, USA) and inserted into a Tecnai T12 transmission electron microscope (FEI Company, USA) operating at 120 kV. Images were recorded on a OneView CMOS detector (Gatan Inc., Pleasanton, USA) using low-dose conditions. EM images used for Fig. 5 were collected on a Tecnai F20 transmission electron microscope (FEI Company, USA), operating at 200 kV. Images were recorded on a US 4000 (Gatan Inc, Pleasanton). A tilt series were acquired using the software package Xplore3D (FEI Company, Eindhoven, NL), using a discontinuous tilt scheme from −54 ° to +60 °, using 3 ° increments, under low dose conditions. The tomogram was reconstructed using IMOD and 6 iterations of the simultaneous iterative reconstruction technique.

Tuijtel M.W., Koster A.J., Jakobs S., Faas F.G, & Sharp T.H. (2019). Correlative cryo super-resolution light and electron microscopy on mammalian cells using fluorescent proteins. Scientific Reports, 9, 1369.

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Ultrastructural Analysis via STEM Tomography

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Plastic-embedded samples were sectioned to 1.5 μm. Sections were stained with 0.2% uranyl acetate in ethanol for 2 h and lead citrate for 10 min. Grids were carbon coated and 20 nm gold applied to both sides of the grid. Imaging was performed at 300 kV on a FEI Tecnai TF30, 300 keV field-emission gun. The microscope was equipped with a Gatan STEM bright-field detector and a Model 3000 in-column high angle annular dark-field detector (Fischione). Xplore3D (FEI Company) was used to obtain tomographic image tilt series at two different magnifications, pixel sizes of 3.31 nm and 2.47 nm. STEM tomograms were calculated from double-axis tilt series ranging from 68° to 68°, 2° increment.

Pokrovskaya I.D., Aronova M.A., Kamykowski J.A., Prince A.A., Hoyne J.D., Calco G.N., Kuo B.C., He Q., Leapman R.D, & Storrie B. (2016). STEM Tomography Reveals that the Canalicular System and α-Granules Remain Separate Compartments During Early Secretion Stages in Blood Platelets. Journal of thrombosis and haemostasis : JTH, 14(3), 572-584.

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TEM Imaging and Tomographic Reconstruction

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For TEM, grids were loaded in a room temperature single-tilt holder (FEI Company, USA), and inserted into a Tecnai T12 transmission electron microscope (FEI Company, USA) operating at 120 kV, equipped with either an Eagle CCD camera (FEI Company, Eindhoven, NL) or a OneView CMOS detector (Gatan Inc., Pleasanton, USA). Large tile-scans were acquired as described by Faas et al.^{63 (link)} using a defocus of −1 μm and a nominal magnification of 6,500× . Images were binned 2× , corresponding to a final pixel size of 3.3 nm. Tomographic tilt series were acquired using the software package Xplore3D (FEI Company, Eindhoven, NL), at a nominal magnification of 21,000× , and were binned 2× for a final pixel size of 1 nm. Automatic focussing to −2 µm was performed prior to each image acquisition. A linear tilt scheme was used from −60° to + 60° in 2° increments. Tomograms were reconstructed with the software package IMOD^{64 (link)} using patch-tracking alignment and weighted back-projection.

Tuijtel M.W., Mulder A.A., Posthuma C.C., van der Hoeven B., Koster A.J., Bárcena M., Faas F.G, & Sharp T.H. (2017). Inducing fluorescence of uranyl acetate as a dual-purpose contrast agent for correlative light-electron microscopy with nanometre precision. Scientific Reports, 7, 10442.

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Transmission Electron Microscopy Tomography

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Samples were prepared as described in TEM section. Sections (200 nm) were collected on glow discharged carbon grids, and 10 colloidal gold fiducial markers were applied. Using a linear tilt scheme, single axis tilt series were collected on a Tecnai BioTwin Spirit TEM (FEI) operated at 120 kV. Images captured over a tilt range of +/− 68° (1° increments) at 1µm defocus were recorded on a 2048 × 2048 pixel UltraScan 1000 Gatan CCD camera using the automated tomography acquisition software (Xplore 3D, FEI). The tilt series were aligned using the IMOD software package (version 4.8.40). All measurements were done using the Amira Visualization Package (version 6.0.0, FEI).

Justis A.V., Hansen B., Beare P.A., King K.B., Heinzen R.A, & Gilk S.D. (2016). Interactions between the Coxiella burnetii parasitophorous vacuole and the endoplasmic reticulum involve the host protein ORP1L. Cellular microbiology, 19(1), 10.1111/cmi.12637.

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Electron Tomography Imaging Workflow

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For electron tomography, tilt series acquisitions were performed on uranyl acetate and lead citrate-stained 250–300-nm thick resin-embedded sections. Data were obtained between −60° and +60° by using increments of one to two degrees and collected using Xplore3D (FEI, Eindhoven, The Netherlands) on a FEI Tecnai20 200 kV TEM or using Tomography 4.0 (FEI) on a Tecnai G2 Spirit BioTWIN TEM at 120 kV. Images were recorded using an Eagle 4 K CCD (FEI) or a Gatan^® US1000 2 K CCD. Tilt series alignments and back projection reconstructions were performed using eTomo (IMOD) software (University of Colorado, Boulder). Manual contours of features of interest within the tomography reconstructions were drawn using 3dmod (IMOD) software and images were exported to NIH Image J (v1.48r) for the generation of model animations.

Davis L.J., Bright N.A., Edgar J.R., Parkinson M.D., Wartosch L., Mantell J., Peden A.A, & Luzio J.P. (2021). Organelle tethering, pore formation and SNARE compensation in the late endocytic pathway. Journal of Cell Science, 134(10), jcs255463.

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Cryo-TEM Characterization of Nanocapsules

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The shape, morphology and size distribution of the nanocapsules was evaluated using low-dose cryo-transmission electron microscopy (cryo-TEM) and cryo electron tomography. Cryo-TEM enables the investigation of colloidal solutions close to their native state. A drop of solution (3µL) was applied on hydrophilic TEM Quantifoil grids. The grids were then blotted for 2 sec and plunged into the liquid ethane pool using a FEI Vitrobot mark IV, in order to produce a thin vitreous ice layer with embedded assemblies in the holes of the grid. Digital cryo-TEM images were acquired using a FEI Tecnai Spirit operated at 120kV using a Gatan 626 cryo-transfer tomography holder. The tomographic series was acquired using automated acquisition software Xplore3D from FEI. Next, the series was aligned and reconstructed using the simultaneous iterative reconstruction technique (SIRT) based on 30 iterations carried out using the FEI inspect 3D tomography package. The measurements of size distribution were carried out in Amira (3D visualisation software).

Bai J., Wang J.T., Rubio N., Protti A., Heidari H., Elgogary R., Southern P., Al-Jamal W.T., Sosabowski J., Shah A.M., Bals S., Pankhurst Q.A, & Al-Jamal K.T. (2016). Triple-Modal Imaging of Magnetically-Targeted Nanocapsules in Solid Tumours In Vivo. Theranostics, 6(3), 342-356.

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Electron Tomography of Doped Molecules

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Tilt series for electron tomography were acquired with the software Xplore 3D (FEI Company). The tilt series used a Saxton scheme spanning angles of ±70°with a tilt step varying from 0.9°to 2.5°, for a total of 79 images per tilt series. 30 Reconstructions were done using the software IMOD (University of Colorado). 31 The series were aligned using crosscorrelation, and the simultaneous iterative reconstruction tomography (SIRT) algorithm (run for 25 iterations) was used to reconstruct the volumes. The final reconstructions were visualised using the software Visualizer-evo (System In Frontier Inc.). 3D meshes representing the dopant clusters were created by applying intensity thresholds on the reconstructions. The threshold values for each sample were optimised relative to each other by keeping a ratio of 5 : 20 : 40 dopant voxels for the samples with concentrations of 5 mol%, 20 mol% and 40 mol% Mo(tfd-COCF 3 ) 3 . A red colour in the volumes was chosen to represent the position of the dopant molecule and a slightly lower intensity threshold was added as a semi-transparent yellow colour to the figures to represent potential wobble of dopant fluorinated pendant groups during acquisition of the tilt series. The positions of individual dopant species were fit to data by adding correctly sized markers to the reconstructions.

Persson G., Järsvall E., Röding M., Kroon R., Zhang Y., Barlow S., Marder S.R., Müller C, & Olsson E. (2022). Visualisation of individual dopants in a conjugated polymer: sub-nanometre 3D spatial distribution and correlation with electrical properties. Nanoscale, 14(41).

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