Soft x-rays have been shown to be useful for imaging thick biological sections or even whole cells at better than 50 nm resolution with useful native contrast and without the need for staining. 1 Soft x-ray radiation within the so-called "water-window" energy regime (between the absorption edge of C (285 eV) and O (540 eV)) produces strong contrast from intercellular components but is ~10X less attenuated by surrounding water. 2 The brightest and most intense sources for soft-x-ray radiation are by far synchrotrons which can produce sub-second images at wavelengths tunable through the soft-x-ray regime. In comparison, the few laboratory sources available currently are typically limited to one or a few wavelengths and exposure times are source-brightness limited to many seconds or minutes. 3 A laboratory-based (aka "compact") soft-x-ray microscope with sufficient imaging performance has distinct advantages in cost, size, and ability to be located close to the biological laboratory. This paper compares the laboratory-based soft-x-ray sources we have investigated for both 2D and 3D imaging of hydrated biological material in the 27-100 nm resolution range.The most straightforward method of soft-x-ray generation is ionization matter by a beam of electrons. We have used an SEM probe at 5 keV to create a focused spot of 50-100 nm on a target to produce a soft x-ray point source (Fig. 1). Using probe energies below 5 keV effectively shut off the V/Ti/Sc Kα lines leaving a sharp Lα line (V: 511 eV, Ti: 452 eV, Sc: 395 eV) in the soft-x-ray range. The spot is then used as a pointprojection x-ray source with magnifications of 10-1000+X on to a soft-x-ray sensitive CCD detector ((20µm) 2 1.7Mpixel) 100-300 mm away. An advantage of this technique is the lack of x-ray optics which are costly, complex, delicate, and inefficient. As a result, living cells imaged with the lens-less technique can survive 10-50X longer compared to systems using Fresnel zone plate (FZP) optics. Other aspects of the point projection method include essentially infinite depth-of-field, an inherent phase contrast condition (that must be phase retrieved at these energies to preserve resolution), and cone-beam (half-angle 50 mrad typical) imaging parameters. 4 A 1µA beam current produces reasonable images (2X binning) of dehydrated samples in ~1 minute with a resolution of ~100 nm. Images with 300 photons/pixel through 5µm and 10µm of vitreous ice are expected to take ~5 min and ~20 min when the system is fully functional in spring 2008. A resolution of ~70 nm should be achievable using the V target with a 5X increase in imaging time (although demonstration pending). Ultimate resolution from Ti is expected to be ~80nm and Sc to be ~100nm due to lower density.Laser-pulsed plasma (LPP) sources permit significantly greater source brightness, deliver higher radiance to the sample and permit shorter exposure times. 5,6 In our second generation construction (full functionality expected summer 2008) an ultrafast laser (1064 nm, 800 ps, 180 mJ, 300 Hz) is focused...

Electron tomography is an important tool for electron microscopists trying to understand the 3-dimensional structure of their specimens. Many TEM specimens, particularly biological ones, are sensitive to the total electron dose used during imaging. Several groups have demonstrated procedures for minimizing the dose during electron tomography [1,2,3,4]. Here we describe an approach to minimizing the electron dose for use with the Gatan TEM Tomography Software [5]. By offsetting the tracking areas from the data acquisition area the electron dose on the specimen is reduced by a factor of 4. In addition to the low-dose capability, this solution is supported on a broad range of microscopes from different vendors, is fully integrated into the Gatan Microscopy Suite, and benefits from the image processing capabilities and other tools that this provides.The acquisition of a tomographic tilt series would be straightforward were it not for the mechanical limitations of a typical microscope stage. As a specimen is tilted from one angle to the next the goniometer imposes both random and systematic shifts in X, Y, and Z on the specimen. These shifts can cause a loss of focus or lose the field of interest after a small number of tilts, depending on the magnification and the properties of the given stage.Gatan's tomography software allows automated and pre-calibration based methods to correct for stage errors [1]. In the automated method, drift in the X-Y plane is measured by comparing an image from the current tilt angle to a reference image. To measure the drift in the Z direction the beam-tilt induced image-shift method is used [6].Here we describe an extension to the Gatan TEM Tomography Software that allows the user to define regions for automated X/Y and Z tracking that are displaced along the tilt axis from the data acquisition area. Each of the tracking regions and the data acquisition area can have different illumination conditions (spot size, brightness). The electron dose in the tracking regions can be increased over that in the data acquisition area in order to improve the signal-to-noise ratio of the images used in determining the drift and hence improve the reliability of these measurements.A graphical user interface is provided to help set up the automated tracking regions. The user adjusts the relative offsets of the two tracking regions from the data acquisition region by means of sliders ( Figure 1) and can record the illumination conditions to be used at each of the 3 regions. The software assists the user by constraining the region's offset to lie on the tilt axis and allows the user to preview a sample image from any of the 3 regions (Figure 2).For users with electron-dose sensitive specimens the ability to acquire tracking data from regions other than the area of interest should enable acquisition of tilt series from specimens that would otherwise suffer too much damage to be useful or track with a higher signal-to-noise ratio than before while still reducing the overall electron dose on the area of inter...

Soft x-rays have been shown to be useful for imaging whole cells or microns-thick sections at better than 50-nm resolution with useful native contrast and without the need for staining.1 Soft x-ray radiation within the so-called "water-window" energy regime (between the absorption edge of C (285 eV) and O (540 eV)) produces good absorption contrast from intercellular components but ~10x less contrast from surrounding water. 2 The brightest and most intense sources for soft-x-ray radiation are by far synchrotrons which can produce sub-second images at wavelengths tunable through the soft-x-ray regime. In comparison, the few laboratory sources available currently are limited to one or a few wavelengths and exposure times are source-brightness limited to many seconds or minutes. 3 However laboratory-based (aka "compact") soft-x-ray microscopes have distinct advantages in cost, size, and ability to be located close to the biological laboratory. This paper demonstrates results from the compact soft-x-ray microscope (Gatan Model 550 SXM) we have built for both 2D and 3D imaging of hydrated biological material with 50-60 nm full-period resolution.The SXM has a laser-pulsed plasma (LPP) source 4 based on an ultrafast laser that is focused on a target stream of methanol. The superheated target produces an intense source of soft-x-ray radiation emanating from a region tens of microns across and dominated by the C-VI line (368 eV) with an estimated brightness of >10 10 photons/sr/μm 2 /s. Other transitions are present but are filtered by the condenser system which produces hollow cone illumination at the sample and is subsequently imaged onto a 2Kx2K CCD using an objective zone-plate. The SXM can host multiple objective zone plates (to trade off resolution against depth-of-field) -the current system has a 30-nm zone plate (resolution image shown in Fig. 1a) for high-resolution 2D, and a 48-nm zone plate for tomography.Specimens for the SXM can either be placed on whole TEM grids, 600 μm-wide TEM grids for full-tilt tomography, or inside pipettes. Specimens are then plunge-frozen in liquid ethane to cryo-protect the cells in vitreous ice. Once transferred into the SXM, samples are typically viewed with a built-in fluorescence light-microscope which is extremely useful for locating areas of interest. Switching between light objectives and x-ray objectives takes seconds and positions are precisely correlated. Fast soft x-ray imaging with quality sufficient for navigation can be several images per second and high-quality images take 10-100 seconds.In the experiment shown in Figures 1-3, we sought to measure the thylakoid membrane volume in cyanobacteria -these membranes are essentially invisible in 2D whole cell imaging except where membranes break spherical symmetry. In Figures 1bc, as-acquired projections show the bacteria embedded in ice supported by a lacey carbon film. These films are useful in supporting "droplets" of water containing bacteria and the lacey filaments also serve as a built-in resolution standard throughou...

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Extended abstract of a paper presented at Microscopy and Microanalysis 2008 in Albuquerque, New Mexico, USA, August 3 – August 7, 2008