Patent Application
20130140459
This invention relates to sample preparation and imaging in electron microscopy. In particular the invention relates to three-dimensional imaging of cathodoluminescence emitted by a sample.
Morphometry is an important and growing discipline within many spheres of biological science. Structural biologists require 3D information over extensive volumes. For example, in neuroscience, current models are based on real data obtained from serial sectioning brain tissue and subsequent reconstruction. Realistic and meaningful analysis requires morphometric analysis at the ultrastructural level over large sample volumes. Large volumes are required in order to be statistically relevant and usable for model building. Electron microscopy is key to providing information at the ultrastructural level.
Using electron microscopy, the classical method to obtain such data was serial sections collected on grids and observed in a transmission electron microscope (TEM). This is a long and difficult process requiring much skill. Sections are obtained as ribbons using an ultra-microtome. Ribbons must be divided manually. Multiple sections are then collected on grids.
Sections on grids are processed in various chemicals. All these steps are risky as the grids or sections can be broken. http://en.wikipedia.org/wiki/Ultramicrotomy.
Serial block-face scanning electron microscopy (SBFSEM) is a relatively new technique compared to the classical method mentioned above, whereby 3D information is gathered by sequentially scanning a freshly microtomed sample block face and the resultant multiple 2D images provide structural detail in 3D. With SBFSEM, the microtome operates in-situ in the electron microscope.
This method automates the cutting and subsequent imaging of the specimen. Grids are no longer used because sections are not cut for collection. It is the block of tissue itself that is directly introduced inside the scanning electron microscope and its surface is repeatedly shaved and scanned to obtain a stack of aligned images. The images collected through scanning depend on the contrast mechanism and therefore detector or detectors employed. A back scattered electron signal is commonly employed with the SBFSEM technique as this provides ultrastructure as revealed through staining techniques. The raw images obtained can be directly exported to software for reconstruction and quantification.
This technique called Scanning Block Face Scanning Electron Microscopy (SBFSEM) has been commercialized by Gatan® under the trade name 3View® . In this technique, structural information about the specimen is typically gathered by imaging with a back scattered electron signal. A relevant paper is this field is by W. Denk and H. Horstmann entitled “Serial block-face scanning electron microscopy to reconstruct three-dimensional tissue nanostructure”. Plos. Biology, 2004.2(11): pp. 1900-1909. Additional references describing the use of SBFSEM include: H. E. J. Armer et al., “Imaging Transient Blood Vessel Fusion Events in Zebrafish by Correlative Volume Electron Microscopy.” Plos. One 4(11), November 2009: p. 4.; John B. West et al., “Structure-Function Studies in Blood and Air Capillaries in Chicken Lung Using 3D Electron Microscopy.” Respir Physiol Neurobiol, 2010 Feb. 28 170 (2): p. 204; Xiaokun Shu et al., “A Genetically Encoded Tag for Correlated Light and Electron Microscopy of Intact Cells, Tissues, and Organisms.” Plos. Biology, 2011 9(4): pp. 5, 6; Thomas Müller-Reichert et al., “Three-Dimensional Reconstruction Methods for Caenorhabditis elegans Ultrastructure.” Methods in Cell Biology Vol. 96, 2010: pp. 348-49; Armin Zankel et al., “3D Elemental Mapping in the ESEM.” G.I.T. Imaging & Microscopy February 2011: pp. 2-4; Jacques Rouquette et al., “Revealing the High-resolution Three-dimensional Network of Chromatin and Interchromatin Space: A Novel Electron-microscopic Approach to Reconstructing Nuclear Architecture.” Chromasome Research (2009) 17:801-810.; and Debarshi Mustafi et al., “Defective Photoreceptor Phagocytosis In a Mouse Model of Enhanced S-Cone Syndrome Causes Progressive Retinal Degeneration.” FAESB J. 25: pp. 3157-3176 (2011). All references cited herein are incorporated by reference.
It is possible to implement this technique in a range of scanning particle instruments since the core principle is based on the automated sequential removal of a surface layer of a block face followed by imaging. In a related scanning technique, the specimen is sequentially milled by an energetic charged focused ion beam (FIB) to reveal a fresh plane of a block face for subsequent imaging by scanning a focused electron beam. This process is described in the Müller-Reichert and Armer publications cited above. This sequential approach is used to build a 3D data set of the imaged volume of the specimen. Again, with this technique, structural information is gathered by imaging with the back scattered electron signal. This is a competing approach to the 3View® (SBFSEM technique) for performing 3D structural microscopy in a scanning microscope, since with both approaches a thin layer is removed in-situ and the freshly revealed block face is imaged. With the 3View® approach, the volume of material that can be cut in an automated fashion is much greater than using this FIB approach, which is typically slower per unit volume of material removed.
Kristina D Micheva and Stephen Smith, (US Patent application 20080152207, Jun. 26 2008). have described a technique for 3D imaging from a bulk specimen whereby the specimen is first cut into thin sections. Each section is imaged away from and independent of the cutting equipment, and then recombined to form a 3D data. This patent describes the collection of various signals as a result of the energized stimulation including cathodoluminescence. In this array technique, the sections which are cut away from the bulk specimen are imaged in an array. In this approach, there is more emphasis on software to re-align images in a sequence to form the 3D image. In this approach, the imaging takes place after all the cutting has been performed, not after each cut.
Related to the Micheva/Smith technique is serial sectioning, imaging and subsequent reconstruction in the TEM. This technique is similar to that described above in that it is the cut sections which are prepared and then imaged in order to provide the ability recombine them to form 3D information.
Optical microscropy techniques can provide fluorescence imaging in 3D by using a confocal approach. The depth in the specimen which provides the 2D image is controlled by the localized focusing of the light stimulation. This technique is different in that the stimulation is by photons, and the specimen is not cut to provide information in the third dimension.
It is possible to obtain some depth and therefore 3D information using Cathodoluminescence from bulk specimens from comparing the 2D images recorded at different accelerating voltages of the stimulating beam. F. K. Toth et al, “Depth Profiling of GaN by Cathodoluminescence Microanalysis”, in 74 Applied Physics Letters 8 pp. 1114-16 (February 1999).
At low energies, the signal emerges from material closer to the irradiated surface than it does when using a higher energy beam wherein the signal emerges from a range of depths. This technique is different from that described herein since the depth of penetration is limited, the deconvolution of information leads to losses in 2D resolution, and because the same surface area needs to be irradiated multiple times. This makes the technique unsuitable for beam sensitive specimens such as resin-embedded material which is the primary subject contended for the 3D CL invention.
Cathodoluminescence is the light created by stimulation using energetic electrons. The definition has also been relaxed to apply to the creation of light stimulated by other energetic particles. This definition does not extend to stimulation by photons, even though the term fluorescence is sometimes used to describe the act of light emission without reference to the act of stimulation.
With cathodoluminescence, the electrons or charged particles can be focused to a beam or remain unfocused to cover an area of interest. The stimulated light is measured as the beam is located at a defined position, rastered over an area, or illuminates an area of interest in an unfocused manner. The wavelength of photons associated with Cathodoluminescence typically ranges from 180 nm to 3000 nm. The local intensity, spectroscopic make-up and transient behavior of the light with regard to the period of stimulation can be analyzed, for example the onset of luminescence, the decay of luminescence, or the growth or quenching behavior associated with changes stimulated by the electrons/charged particles. Cathodoluminescence can be associated with components which are intrinsic or extrinsic to the specimen. Cathodoluminescence can also be studied as a function of the specimen temperature. An intrinsic luminescence could be a background luminescence, whilst an extrinsic feature can be associated with an intentional marker or label.
Cathodoluminescence from a bulk specimen can take place in a high vacuum or low vacuum conditions associated with the scanning electron microscope, or FIB instrument. The bulk specimen contains intrinsic or extrinsic features which luminesce in response to the stimulation. These features extend into the depth of the specimen, but only those in proximity to the surface compared to the penetration depth of the stimulating energetic beam are caused to luminesce. The collection of luminescence forms the basis of a 2D image. This technique is standard and has been practiced for many years. Cathodoluminescence can only be collected from underneath the specimen where the bulk of the specimen is transparent to the wavelength of the light, and if the specimen holder which supports the specimen is also transparent. For example in the paper below, simultaneous CL and BSE are collected (
An important aspect of collecting a 3D data set in this manner is that unlike confocal or transmissive techniques, the spatial resolution obtained in each 2D image does not diminish as a function of depth since each acquisition is from a fresh block face.
The luminescence usually has no favored directionality. However, the light emission from the specimen will have strongly favored directionality because of the effect of total internal reflection in the specimen. For a specimen with a flat surface, the light emission distribution always follows Lambertian distribution, which means that the intensity peaks normal to the surface.
Most specimens suitable for cutting with a microtome or milling will be sensitive to the dose by the stimulant. Therefore the design of the equipment to optimize the light collection efficiency is important to the success of the technique. It is not sufficient to have poor collection efficiency using optics which present a poor solid angle to the luminescence source. This is because the energy and dose of the stimulation needs to be kept low in order to achieve the desired resolution in X,Y and Z and also in order to maintain the structural integrity of the medium to be cut. For this reason the design of the collection optics is an important consideration.
There is a need, therefore, for a system for obtaining layered cathodoluminescence images of a sample wherein the light collecting equipment is highly efficient and wherein the microtoming or Focused Ion Beam equipment does not interfere with the efficiency of the light collecting equipment and wherein the position of the sample with respect to the light collecting equipment is not disturbed in the microtoming or ion beam milling process.
The invention is directed to an apparatus for imaging cathodoluminescence from a specimen in 3D using an electron microscope.
In an embodiment, the microtoming apparatus is adapted to reflect the cathodoluminescence to a light detector after making a cutting pass across the specimen. The microtome is further adapted to allow the cathodoluminescence imaging to take place in synchrony with collection of the back scattered signal. In a further embodiment an alternative adaption is to allow the cathodoluminescence imaging to take place in synchrony with the collection of secondary electrons.
In a further embodiment, the microtome is transparent and the cathodoluminescence is reflected to the light detector total internal reflectance within said microtome. In a further embodiment, the microtome is mounted on a support containing integral light detectors and the cathodoluminescence is detected while the microtome is passing across the specimen. In a further embodiment, the light detectors comprise a plurality of filters for simultaneous measurement of different wavelengths of light.
In a further embodiment, the light detector is mounted in a support for the microtome and in the path of an electron beam of the electron microscope.
In a further embodiment, the specimen and a specimen support are at least partially transparent and the light detector detects light internally reflected by the specimen support. In a further embodiment of this configuration, there is a highly reflective surface positioned above the specimen to enhance the amount of light reflected to the light detector.
In a further embodiment, the apparatus includes a mirror and a mirror actuator, and the mirror actuator positions the mirror to reflect the cathodoluminescence to the light detector after the microtome has passed across the specimen. In a further embodiment of this configuration, the mirror actuator is attached to the pole piece of the electron microscope or to the microscope chamber.
In a further embodiment, the specimen is optically transparent and has a milled face cut by the microtome and an opposite face parallel to the milled face and the light detector is positioned to receive light emitted at the opposite face. In a further embodiment of this configuration, there is included a reflector positioned to reflect light emitted by the milled surface back into the specimen.
In a further embodiment, there is a microtome support and a fiber optic mounted to the microtome support, wherein the fiber optic directs light to the light detector while the microtome is passing across the specimen.
In a further embodiment there is a remotely actuated fiber optic holder, wherein the holder is actuated to place a fiber optic to collect light from the specimen after the microtome has passed across the specimen.
In a further embodiment the specimen includes reflective surfaces to reflect light to the photo detector.
In a further embodiment, the apparatus includes a linear carriage, a dead stop and a mirror, wherein the microtome and mirror are mounted in and moved along the linear carriage and wherein the mirror is positioned against the dead stop when the microtome has completed a pass across the specimen.
In a further embodiment of the invention, there is an apparatus for simultaneous imaging of backscattered electrons and cathodoluminesence from a specimen in an electron microscope and the apparatus includes: a focused ion beam (FIB) generator, a light director; a light detector; a backscattered electron detector. The FIB generator directs a beam to mill a surface of the specimen and the light director is positioned to direct light from the specimen surface to the light detector after the beam has milled the surface. In a further embodiment of this configuration, the beam also mills structures for reflecting light to the light director and a reflective surface is deposited onto surfaces of the milled structures by introduction of localized gas in combination with the FIB.
Several embodiments of the invention are directed to obtaining a 3D CL data set through sequentially imaging and microtoming a block face. When performed in an automated fashion, voxels of dimension X, Y, Z are acquired, whereby the Z dimension is influenced by the thickness of each sequential cut to the block. With multiple detectors and analysis techniques, each voxel can be associated with structural, chemical, elemental and luminescence information.
Embodiments of the invention disclosed here relate to the equipment which allows the cutting, the imaging, and the collection of the emitted CL light signal in synchrony, or with the option of being in the same sequential acquisition as other signals providing matching structural information. In various embodiments, different types of CL signals can be collected with high efficiency, and simultaneously with other signals associated with the stimulating beam in the confined space of the instrument. High efficiency is required since photons are in short supply when the injection conditions are optimized for maximizing spatial resolution in either 2D or 3D. Moreover, using novel light collection optics prevents the need for a macroscopic movement of the specimen in the X, Y or Z direction in order to switch between imaging to collect luminescence, imaging to collect other structural signals, or cutting. If such movement were to be incurred, it would introduce potential misalignments in the 3D data set and require post processing re-alignment. Novel light collection techniques also enhance the resolution of the technique since this allows use of shorter working distances, and lower accelerating voltages, (which are directly linked to spatial resolution in a scanning electron microscope). In a low vacuum electron microscope, they are also associated with lower losses associated with scattering by gas molecules.
The following embodiments disclose optical devices for in-situ collection of light emitted from the freshly revealed block face, that do not interfere with the fundamental operation of the 3D acquisition techniques, whether this is achieved through in-situ microtomy: Scanning Block Face Scanning Electron Microscopy (SBFSEM) or FIB milling.
Certain embodiments disclose ways to collect light with high efficiency without moving the specimen a macroscopic distance, such as using the SBFSEM technique based on in-situ microtomy while using the mechanical movement of a knife between successive images. CL light is either collected and redirected towards light detection equipment which is remote from the specimen, or else detection sensors are positioned in suitable locations in close proximity to the specimen either permanently, or temporarily through a controlled manner. The presence or introduction of light collection, light coupling or light detection equipment does not interfere with the general performance of the instruments as a tool for sequentially cutting or milling specimens in-situ. When light is redirected, e.g. using a mirror or light pipe, the light detection equipment can be inside the microscope chamber, or attached to the outside, or else relatively remote to the chamber.
In this embodiment, the knife holder/reflector has a hole 11 through the reflecting portion. This allows a clear path for the primary electrons 60 to the specimen. The knife/reflector is moved across the specimen 30 to expose a new layer and then momentarily stopped at the end of the cut to allow the freshly revealed surface to be scanned by the particle beam, with the resultant specimen luminescence to be reflected and imaged by the photon detector 20. This process is repeated to collect a 3D CL image.
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In a further embodiment a lens is used to enhance the solid angle of light collected into a more remote light detecting equipment. The lens can be fixed in an optimum position, attached to the knife holder in an optimum position, or else attached to an arm which is repositioned mechanically and automatically between imaging and cutting.
Note that in all cases employing a mirror, additional cutouts can be provided in the mirror surface which sacrifice some of the potential solid angle and light collecting efficiency, but which provide simultaneous collection using other imaging signals such as back scattered or secondary electron signals.
While the invention has been described in detail and with reference to specific examples thereof, it will be apparent to one skilled in the art that various changes and modifications can be made therein without departing from the spirit and scope thereof.
