Ultramicroscopy. 2014 Aug;143:33-40. doi: 10.1016/j.ultramic.2013.10.013. Epub 2013 Nov 7.
Correlative cryogenic tomography of cells using light and soft x-rays.
Smith EA1, Cinquin BP1, Do M1, McDermott G1, Le Gros MA2, Larabell CA3.
Department of Anatomy, School of Medicine, University of California San Francisco, San Francisco, CA, United States; National Center for X-ray Tomography, Advanced Light Source, Berkeley, CA, United States.
Department of Anatomy, School of Medicine, University of California San Francisco, San Francisco, CA, United States; Physical BioSciences Division, Lawrence Berkeley National Laboratory, Berkeley, CA, United States; National Center for X-ray Tomography, Advanced Light Source, Berkeley, CA, United States. Electronic address: MALegros@lbl.gov.
Department of Anatomy, School of Medicine, University of California San Francisco, San Francisco, CA, United States; Physical BioSciences Division, Lawrence Berkeley National Laboratory, Berkeley, CA, United States; National Center for X-ray Tomography, Advanced Light Source, Berkeley, CA, United States. Electronic address: carolyn.larabell@ucsf.edu.
Correlated imaging is the process of imaging a specimen with two complementary modalities, and then combining the two data sets to create a highly informative, composite view. A recent implementation of this concept has been the combination of soft x-ray tomography (SXT) with fluorescence cryogenic microscopy (FCM). SXT-FCM is used to visualize cells that are held in a near-native, cryopreserved. The resultant images are, therefore, highly representative of both the cellular architecture and molecular organization in vivo. SXT quantitatively visualizes the cell and sub-cellular structures; FCM images the spatial distribution of fluorescently labeled molecules. Here, we review the characteristics of SXT-FCM, and briefly discuss how this method compares with existing correlative imaging techniques. We also describe how the incorporation of a cryo-rotation stage into a cryogenic fluorescence microscope allows acquisition of fluorescence cryogenic tomography (FCT) data. FCT is optimally suited for correlation with SXT, since both techniques image the specimen in 3-D, potentially with similar, isotropic spatial resolution.
© 2013 Elsevier B.V. All rights reserved.
Cell structure; Imaging; Molecular localization
10.1016/j.ultramic.2013.10.013
High Numerical Aperture Fluorescence Cryo Microscope (FCM)
Computer-aided design (CAD) of the FCM together with the associated instruments that combine to create a cryogenic imaging workbench. The FCM has two objective lenses and three cameras for high and low-magnification imaging. The high-magnification side images onto Andor EMCCD cameras (red/green channels) using a commercial spinning disk confocal microscope unit. A specimen mount/goniometer mounts thin glass capillaries and permits 360° rotation of the specimen. A 9L liquid Nitrogen bath keeps the microscope stable at ∼90°K for several hours without the need for refilling. Specimens are rapidly cryopreserved by rapid plunging into liquid propane using the device mounted to the right hand side of the microscope in this view. The inset shows a magnified view of the high NA (main) objective lens, a specimen capillary tube in an imaging cell filled with liquid propane immersion fluid, and the objective lens for the low-magnification microscope.
Correlative cryogenic tomography of cells using light and soft x-rays
Ultramicroscopy. ;143:33-40.
Soft X-ray Microscope: Optical layout together with an example of SXT data
Schematic representation of XM-2, a soft x-ray microscope located at the Advanced Light Source, Berkeley. A bend magnet (a) in the synchrotron lattice generates an intense swath of soft x-rays. A flat, nickel-coated mirror (b) delivers soft x-rays onto the condenser zone plate (c), which, in combination with the pinhole (d) act as both a monochromator and as a condenser lens to focus the beam onto the specimen mounted in the cryogenic specimen rotation stage. Soft x-rays transmitted by the specimen are focused on to a CCD camera by the objective zone plate (e) onto a CCD camera (f). The optical path taken by the soft x-ray beam is shown in blue.
SXT-FCM workflow
Outline of the SXT-FCM workflow. Shown here are Schizosaccharomyces pombe cells stained with the vital dye BODIPY-TR-C5-ceramide, which accumulates in Golgi. Step 1. Cells in suspension are pelleted by centrifugation, re-suspended in a small volume of media, and pipetted into thin-walled glass capillaries, the exterior of which have been decorated with fiducial markers (a). Step 2. The specimen is vitrified by rapidly plunging the capillary into liquid propane, and then imaged in the FCM (b). Step 3. The specimen is then cryo-transferred to the soft x-ray microscope where the specimen is imaged tomographically. The measured contrast is a function of the material's linear absorption coefficient (LAC). Step 4. Once the FCM and SXT data sets have been processed they are aligned using the fiducial markers to yield a correlated dataset (d).
Correlated CFT-SXT imaging of LysoTracker-labeled B Cells
Shown here are mouse B cells that are labeled with the vital dye LysoTracker. Left: A slice from a through-focus FCM dataset, the cell outline is indicated by orange dotted line. Middle: An orthoslice through the SXT reconstruction of the same cell. Right: Overlay of the fluorescence and SXT reconstructions; punctate structures visible in the SXT reconstruction are identified by FCM data as being acidic lysosomes. Scale bar = 3μm
Cryoelectron Microscopy/methods
Imaging, Three-Dimensional/methods
Microscopy, Confocal/methods*
Microscopy, Fluorescence/methods*
Tomography, X-Ray Computed/methods*
P41 GM103445/GM/NIGMS NIH HHS/United States
P41RR019664/RR/NCRR NIH HHS/United States
P41 RR019664/RR/NCRR NIH HHS/United States
R01 GM063948/GM/NIGMS NIH HHS/United States
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