Vitrification of thick samples for soft X-ray cryo-tomography by high pressure freezing

Weiner, Allon; Kapishnikov, Sergey; Shimoni, Eyal; Cordes, Sandra; Schneider, Gerd; Elbaum, Michael

doi:10.1016/j.jsb.2012.10.005

Cited by 30 publications

(23 citation statements)

References 36 publications

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“…Infected red blood cells derived from a culture are cryo-freezed to preserve their hydrated native structure 8, 9 , so the sample to be studied is like an insect in amber. The freezing is done so fast that the ice is vitrified into an amorphous structure rather than into ice crystals.…”

Section: Introductionmentioning

confidence: 99%

Biochemistry of malaria parasite infected red blood cells by X-ray microscopy

Kapishnikov

Leiserowitz

Yang

et al. 2017

Sci Rep

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Red blood cells infected by the malaria parasite Plasmodium falciparum are correlatively imaged by tomography using soft X-rays as well as by scanning hard nano-X-ray beam to obtain fluorescence maps of various elements such as S and Fe. In this way one can deduce the amount of Fe bound either in hemoglobin or in hemozoin crystals in the digestive vacuole of the malaria parasite as well as determine the hemoglobin concentrations in the cytosols of the red blood cell and of the parasite. Fluorescence map of K shows that in the parasite’s schizont stage the K concentration in the red blood cell cytosol is diminished by a factor of seven relative to a pristine red blood cell but the total amount of K in the infected red blood cell is the same as in the pristine red blood cell.

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Section: Introductionmentioning

confidence: 99%

Biochemistry of malaria parasite infected red blood cells by X-ray microscopy

Kapishnikov

Leiserowitz

Yang

et al. 2017

Sci Rep

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“…Cells may be loaded into capillaries [18] or grown on carbon-coated grids [19,20]. Samples are then prepared by plunge-freezing, or more recently by HPF [21], both of which are highly effective at preserving dynamic and volatile cellular structures. Samples are then imaged in a cryo-SXT microscope at one of three synchrotron beamlines currently operational worldwide (the Advanced Light Source in the USA, BESSY II in Germany, ALBA in Spain).…”

Section: Introductionmentioning

confidence: 99%

Imaging endosomes and autophagosomes in whole mammalian cells using correlative cryo-fluorescence and cryo-soft X-ray microscopy (cryo-CLXM)

et al. 2014

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Cryo-soft X-ray tomography (cryo-SXT) is a powerful imaging technique that can extract ultrastructural information from whole, unstained mammalian cells as close to the living state as possible. Subcellular organelles including the nucleus, the Golgi apparatus and mitochondria have been identified by morphology alone, due to the similarity in contrast to transmission electron micrographs. In this study, we used cryo-SXT to image endosomes and autophagosomes, organelles that are particularly susceptible to chemical fixation artefacts during sample preparation for electron microscopy. We used two approaches to identify these compartments. For early and recycling endosomes, which are accessible to externally-loaded markers, we used an anti-transferrin receptor antibody conjugated to 10 nm gold particles. For autophagosomes, which are not accessible to externally-applied markers, we developed a correlative cryo-fluorescence and cryo-SXT workflow (cryo-CLXM) to localise GFP-LC3 and RFP-Atg9. We used a stand-alone cryo-fluorescence stage in the home laboratory to localise the cloned fluorophores, followed by cryo-soft X-ray tomography at the synchrotron to analyse cellular ultrastructure. We mapped the 3D ultrastructure of the endocytic and autophagic structures, and discovered clusters of omegasomes arising from ‘hotspots’ on the ER. Thus, immunogold markers and cryo-CLXM can be used to analyse cellular processes that are inaccessible using other imaging modalities.

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“…Improved cryo soft X-ray microscopy was established recently to image large native frozen samples by tomography (Schneider et al, 2010;Weiner et al, 2013). This method is applicable to samples of up to 10 µm thickness at a resolution in the range of 36 to 70 nm.…”

Section: Introductionmentioning

confidence: 99%

Cryo FIB-SEM: Volume imaging of cellular ultrastructure in native frozen specimens

Schertel

Snaidero

Hong-mei

et al. 2013

Journal of Structural Biology

174

113

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Volume microscopy at high resolution is increasingly required to better understand cellular functions in the context of three-dimensional assemblies. Focused ion beam (FIB) milling for serial block face imaging in the scanning electron microscope (SEM)is an efficient and fast method to generate such volume data for 3D analysis. Here, we apply this technique at cryo-conditions to image fully hydrated frozen specimen of mouse optic nerves and Bacillus subtilis spores obtained by high-pressure freezing (HPF). We established imaging conditions to directly visualize the ultrastructure in the block face at −150°C by using an in-lens secondary electron (SE) detector. By serial sectioning with a focused ion beam and block face imaging of the optic nerve we obtained a volume as large as X=7.72 µm, Y=5.79 µm and Z=3.81 µm with a lateral pixel size of 7.5 nm and a slice thickness of 30 nm in Z. The intrinsic contrast of membranes was sufficient to distinguish structures like Golgi cisternae, vesicles, endoplasmic reticulum and cristae within mitochondria and allowed for a threedimensional reconstruction of different types of mitochondria within an oligodendrocyte and an astrocytic process. Applying this technique to dormant Bacillus subtilis spores we obtained volumes containing numerous spores and discovered a bright signal in the core, which can not be related to any known structure so far. In summary, we describe the use of cryo FIB-SEM as a tool for direct and fast 3D cryo-imaging of large native frozen samples including tissues.

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Vitrification of thick samples for soft X-ray cryo-tomography by high pressure freezing

Cited by 30 publications

References 36 publications

Biochemistry of malaria parasite infected red blood cells by X-ray microscopy

Biochemistry of malaria parasite infected red blood cells by X-ray microscopy

Imaging endosomes and autophagosomes in whole mammalian cells using correlative cryo-fluorescence and cryo-soft X-ray microscopy (cryo-CLXM)

Cryo FIB-SEM: Volume imaging of cellular ultrastructure in native frozen specimens

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