Journal: Journal of Structural Biology

Article Title: Correlative VIS-fluorescence and soft X-ray cryo-microscopy/tomography of adherent cells

doi: 10.1016/j.jsb.2011.12.012

Figure Lengend Snippet: Sample grid preparation for soft X-ray cryo-microscopy/tomography at the BESSY II U41-XM beamline. (A) Due to the confined space for sample tilting in the microscope, a special grid design (IFR-1, left) is needed. For comparison, a standard 400 × 100 mesh grid for electron microscopy is shown (diameter: 3.05 mm, right). (B) Retaining the outer dimensions, an improved mesh design (HZB-2) with smaller slots helps to stabilize standard perforated carbon film (tested: Quantifoil R2/2). Furthermore, it alleviated targeting for correlative microscopy. (C) In order to prevent liquid to spread over the whole grid, liquid blocker (PAP pen) was applied as a thin line near the observation area of the grid. (D) Alignment marker bead suspension was blotted and dried on the carbon film of the glow-discharged grid. Note the green liquid blocker line on the carbon-coated side of the right grid. (E) On-grid cell incubation was performed in commercially available microscope slide growth chambers. (F) Grid handling with tweezers, and finally vitrification by plunge freezing of the sample, has to spare the clamping end of the grid for proper mounting in the Gatan model 630 specimen cryo-holder.

Article Snippet: After 5 min of drying, the grids were submerged in 1 ml of fresh complete medium in plastic microscope slide growth chambers (μ-slide 2×9 well, Ibidi GmbH, Munich, Germany).

Techniques: Microscopy, Tomography, Comparison, Electron Microscopy, Marker, Suspension, Incubation

Journal: Journal of Structural Biology

Article Title: Correlative VIS-fluorescence and soft X-ray cryo-microscopy/tomography of adherent cells

doi: 10.1016/j.jsb.2011.12.012

Figure Lengend Snippet: Correlative live-cell pre-scanning and in-column fluorescence cryo-microscopy of PrV pUL31/pUL34-GFP coexpressing EFN-R cells grown on a HZB-2 grid. For most images, two or three detection channels were combined: gray, phase-contrast image at the focal plane of the cell nuclei; green, GFP fluorescence from tagged PrV pUL34; red (only in light cryo-microscopic images), red fluorescence of FluoSpheres beads (diameter 200 nm, 580/605 nm excitation/emission). (A) Montage of live-cell light microscopic images taken at 20×-magnification after 36 h of incubation at 37 °C and 5% CO 2 , exhibiting optimal cell density for our experiments. Higher resolved imaging data were acquired from grid slots marked below with Roman numerals. (B) Enlarged view of grid slot number I illustrating the confinement of the target protein, GFP-tagged PrV pUL34, to the nuclei. Note the shading/absorption of epi-fluorescence by the carbon support foil. (C) Montage of fluorescence cryo-microscopic images taken with a 100× long distance objective (N.A. 0.75) of the same grid slot number I shown in (B), after further 3.5 h of incubation and plunge freezing. The images were gathered with the light cryo-microscope installed inside of the HZB TXM. The Arabic numerals mark the nuclei (and the order) where subsequently soft X-ray tilt series had been taken (tilt series 3 was acquired from grid slot II, cf . E). Red fluorescing FluoSpheres were applied, in excess as correlation and alignment markers, before cell seeding and incubation, as depicted in D, and additionally before plunge freezing. (D) Montage of in-column fluorescence cryo-microscopic images taken of the same grid slot number I shown in (B) and (C), after X-ray exposure for the four tilt series marked in (C) and in (E). As camera settings were kept exactly the same as in (C), it demonstrates the almost complete bleaching of both fluorophores. Another two grid slots of the sample grid depicted in (A) (E, F – II; G, H – III) were imaged by in-column fluorescence cryo-microscopy before (E, G) and after (F, H) X-ray exposure for four tilt series taken close to the spots marked by Arabic numerals (E – for 3, camera frame/X-ray irradiated area at 0° tilt angle marked by yellow rectangle; cf . C for 1, 2 and 4). Note the fluorescence intensity gradient in (F) where highest bleaching was observed near the exposure spot 3. More distant areas were also affected, due to horizontal tilting during tilt series acquisition, stray light and/or inefficiency of the condenser beam stop. As a control (G, H), grid slot number III was not exposed to direct X-rays, neither for searching nor for tilt series acquisition. Scale bar is 200 μm (A) or 20 μm (B–H), respectively.

Article Snippet: After 5 min of drying, the grids were submerged in 1 ml of fresh complete medium in plastic microscope slide growth chambers (μ-slide 2×9 well, Ibidi GmbH, Munich, Germany).

Techniques: Fluorescence, Microscopy, Incubation, Imaging, Irradiation, Control