Patent ID: 12205789

DETAILED DESCRIPTION

The techniques herein typically are implemented in association with a cryo-EM system.FIG.1depicts a representative cryo-EM system100in which the techniques of this disclosure are practiced. The system comprises a transmission electron microscope102, and an associated control system103configured to control the position of the microscope102relative to a target being examined. Control system103includes microscope control software105, such as Leginon (Simons Electron Microscopy Center, New York Structural Biology Center), SerialEM (University of Colorado Boulder), Thermo-Fisher® EPU, and the like. For imaging, microscope102comprises an electron source104that emits a beam of electrons, an electromagnetic lens system106that focuses the beam of electrons down to a nanometer scale, a sample holder108that holds a sample to be observed (e.g., within a stage), a series of electromagnetic lenses110that transmit the electrons through the sample, a series of detectors112that are configured to detect the electrons as they are transmitted through the sample, a display114configured to display an image of the sample based on the detected electrons, and a computing system116(including microscope control software105) configured to control the operation of the electron source, the electromagnetic lenses, the detectors, and the display. The system may also include a software-based pipeline118that uses neural network models and computer vision algorithms to navigate cryo-EM grids at low- and medium-magnification to determine high-quality targeting locations for the microscope, preferably without human input. The models may comprise both pre-trained neural network models, together with models that provide learning on-the-fly during an active data collection. In this example, the software pipeline118is supported in a data store or memory of the computing system116and interoperates with the microscope control software105over an Application Programming Interface (API)120. In an alternative, the software pipeline118is integrated directly into the microscope control software105in the computing system.

FIG.2depicts a portion of the optics of a representative TEM from the gun lens to the objective lens, such as for the microscope shown inFIG.1. As depicted, the microscope200has six (6) lenses in its illuminating system, namely, a gun lens202, a first condenser lens (C1)204, a second condenser lens (C2)206, a third condenser lens (C3) 208, a minicondenser lens (MC) (not shown), and an upper and lower objective lens210and212. The C2 aperture is the beam shaping aperture. The drawing also depicts a ray diagram of the electrons passing through the column of the TEM. This is the TEM mode of illumination. In this mode, theoretically the beam on the specimen214is parallel and the C3 lens images the source on the front focal plane of the upper objective lens. In a typical optical alignment of this type, it is known that the C2 aperture of the microscope induces Fresnel fringes at the edge of the beam that illuminates the specimen. These fringes can cause artifacts and unusable areas for data collection. To address this, it is also known to provide so-called Fringe-Free Imaging (FFI) to configure the objective lens and the mechanical height of the sample stage such that the C2 aperture is imaged in focus at the specimen plane and the fringes are almost completely eliminated.

In the prior art, the optical system depicted inFIG.2uses a circular beam. According to this disclosure, and in contrast to this known approach, the hole in the C2 aperture216is square (as opposed to circular), which produces a square beam. This beam configuration provides significant advantages, e.g., as depicted in the imaging examples with different beam setups as shown inFIG.3. The first drawing (A) depicts the conventional approach (circular beam, without FFI) showing the specimen300, the circular areas302illuminated by the electron beam, and the areas304captured by the microscope camera (which typically are squares due to the physical characteristics of the camera). The second drawing (B) is also a conventional approach (the circular beam), but in this case using FFI, which allows for better tiling of the beam on the sample as compared to the first use case (A). The third drawing (C) depicts the imaging of the specimen using the approach of this disclosure, wherein a square beam is used, preferably with FFI. The square beam is formed by configuring the C2 aperture as square. As can be seen, by placing the square aperture at the C2 aperture position and using FFI, the beam can be tiled and virtually all of the specimen is accounted for without excess damage and without excluding any portions of the specimen from the imaging.

FIG.4depicts images of a biological specimen captured by the camera illuminated with either a circular beam (the prior art) or a square beam (according to this disclosure). Preferably, and as reflected in the right side drawing, the square beam has the exact size of the shortest dimension of the camera, and it is rotated such that it is square onto the camera. The size of the square beam is adjusted by changing the beam intensity. As noted above, to accomplish the rotation of the beam (to be square onto the camera) may be accomplished by adjusting a current in the projection lens (P2)218in the microscope. Adjusting the current in the P2 lens has the side effect of changing the magnification, rotation, and defocus of the image (which in turn requires re-calibrating pixel size, image shift matrices, and eucentric focus). In an alternative embodiment, a physical rotation of the square aperture itself is done. To this end, a worm wheel gear is installed through the (hollow) aperture stick of the microscope to rotate the square aperture while it is in the liner tube under vacuum (during illumination). This allows alignment of the square beam to the camera during normal cryo-EM operation.

A square beam has significant advantages in that it can be tiled to maximize the area imaged by the TEM. This is highly advantageous in many applications but especially in montage cryo-electron tomography for exhaustively imaging large biological specimens. The system includes control software such as PACEtomo (University of Tokyo, Japan) and SerialEM (available from University of Colorado Boulder) that are configured to tile (preferably, in a perfect manner) the square beam during imaging. In particular, and in one example embodiment (with PACEtomo and SerialEM together), the microscope is controlled such that the electron beam is shifted by beam-image shift in a manner that tiles the beam onto the sample with minimal overlap, such that a large area of sample is imaged or montaged. The stage can then be tilted with PACEtomo and SerialEM to collect tomograms of the sample for montage tomography.

In single particle analysis, a key application of biological cryo-TEM, using a square beam as described herein dramatically increases throughput. This is depicted inFIG.5. In particular, the left image shows the specimen targeted using a circular beam (as in the prior art) as compared to the right image, which shows the same specimen targeted using the square beam (as provided in this disclosure). The white squares depicted in the image represent the area of the specimen that is imaged by the camera. In this example, using a fringe-free circular beam (on the left) allows for up to 34 targets (i.e., 17×2) per stage movement with a round beam, but up to 85 (i.e., 17×5) targets with the square beam). This method of data collection for single particle analysis can be achieved with microscope control software such as SerialEM, Leginon (New York Structural Biology Center), EPU (ThermoFisher Scientific), or the like. There is no significant loss in imaging quality compared to the standard round beam method of imaging. Single particle analysis reconstructions of apoferritin (a biological test sample) images taken with the square beam achieve almost the same resolution as images taken with the round beam, with a marginal loss of resolution of only ˜0.1 Å. Experimental results also confirmed that correction of coma aberrations with the square beam can achieve the same degree of coma (<150 nm) as a circular beam.

Variants

The technique herein may be expanded to include other non-circular-shaped beams, such as hexagons, octagons, and the like. Further, other microscopy techniques that suffer from radiation damage to the sample during imaging can benefit by applying the techniques herein to improve their imaging setup.

What we claim is as follows.