Usage of polarization state to control ponderomotive phase plate

Abstract

To provide the usage of the polarization state to control a ponderomotive phase plate.SOLUTION: A ponderomotive phase plate, also called a laser phase plate or a standing wave optical phase plate, includes a first mirror and a second mirror that define an optical cavity. An electron beam passes through the focal point of the optical cavity. A laser with a variable polarization angle of the laser light is coupled into the optical cavity. A standing wave of polarized laser light with an antinode at the focal point of the optical cavity causes a variable modulation of the electron beam. The variable modulation of the electron beam is controllable by the variable polarization angle of the laser light. In a transmission electron microscope, an image plane accepts the electron beam modulated by the standing wave optical phase plate. The image formed at the image plane is based on the variable polarization angle of the polarized laser light.SELECTED DRAWING: Figure 13

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit under 35 USC § 119(e) of U.S. Patent Application No. 62 / 901,160, filed September 16, 2019, the entire contents of which are hereby incorporated by reference herein. This application is also related to U.S. Patent Application No. 15 / 939,028, entitled OPTICAL-CAVITY BASED PONDEROMOTIVE PHASE PLATE FOR TRANSMISSION ELECTRON MICROSCOPY, filed March 28, 2018, and granted as U.S. Patent No. 10,395,888 on August 27, 2019, which claims the benefit of priority from U.S. Provisional Patent Application No. 62 / 479,044, filed March 30, 2017, the entire contents of which are hereby incorporated by reference herein.

[0002] [Statement of Government Support] This invention was made with Government support under Contract No. DE-AC02-05CH11231 awarded by the Department of Energy and under Award No. R01GM126011 awarded by the National Institute of General Medical Sciences of the National Institutes of Health. The Government has certain rights in this invention.

[0003] The present disclosure relates generally to transmission electron microscopy (TEM), and more specifically to an optical cavity phase plate for a TEM. [Background technology]

[0004] Although modern transmission electron microscopes have become powerful imaging tools that achieve resolutions about 1000 times higher than optical microscopes, their imaging performance for thin biological samples has remained relatively low. Such samples are weakly scattering "phase objects", i.e., they show virtually no absorption of incident electrons. As a result, the intensity of the transmitted electron beam remains equal to that of the incident beam, and full images of such objects show no contrast. Historically, electron microscopes have therefore required specialized preparation of samples, where heavy metal "stains" provide contrast. These procedures are difficult and time consuming, and moreover, they are known to modify the structure, thus limiting the resolution at which meaningful information can be obtained.

[0005] Even for unstained phase objects, the object (sample) structure is transferred onto the phase of the matter wave representing the transmitted electrons. As Zernike found for optical microscopy, invisible phase modulation can be converted into visible amplitude contrast. Light passing through the sample is decomposed into undiffracted and diffracted components. The undiffracted light is focused by the objective lens into a high-brightness spot at the center of this plane. The diffracted light is arranged around this center. Diffraction by the fine structure of the object results in larger diffraction angles. Thus, the diffraction orders corresponding to the fine details of the image (i.e. having small dimensions) are located far away from the center, whereas large-scale structures give rise to diffracted light close to the center. Mathematically, the intensity distribution in this back focal plane is given by the spatial Fourier transform of the transmittance of the sample, which is called the Fourier transform plane.

[0006] If the sample is a pure phase object, there is a spatial phase relationship between these components. By offsetting these phase relationships, the phase modulation is converted to amplitude modulation. Maximum conversion and therefore maximum phase contrast is obtained with a phase shift of 90 degrees or π / 2. In optical systems, this is done by a phase plate, which is essentially a plate of glass coated so that light passing through a small area in the center receives an extra phase shift.

[0007] Unfortunately, there is no simple phase plate for the electron beam, making it difficult to observe unstained biological samples. A partial solution is provided by cryo-electron microscopy methods. These avoid the associated occurrence of staining and structural artifacts, and generate a certain amount of phase contrast by observing the sample in deliberately defocused conditions in combination with deliberate spherical aberration. The desired conversion of phase to amplitude contrast can be achieved by optimizing the compromise between the phase distortion caused by defocus and that caused by spherical aberration. However, the phase shift varies continuously across the spectrum of spatial frequencies. As a result, this "simple" method works well for small features in the image, but contrast for larger features is lost. Since it is necessary to have substantial contrast even for large features in order to see biological macromolecules, it is often necessary to use much larger amounts of defocus. Unfortunately, this results in a reduction in resolution. Also, the contrast transfer function oscillates multiple times in the region of higher spatial frequencies. Defocusing is thus an imperfect way to generate phase contrast in images of biological macromolecules.

[0008] One conventional technique utilizes a thin carbon film as a phase plate in transmission electron microscopy. The thickness of this film causes the scattered electrons to undergo a π / 2 phase shift while the axial electrons pass through a 1 μm diameter central hole. The main drawback of this technique is that these phase plates "degrade" on a time scale of days or weeks. Also, they are very difficult to fabricate reproducibly. In addition to this, a small part of the useful signal is lost when the scattered electrons pass through the thin carbon film.

[0009] More recently, microfabrication techniques have enabled the construction of electron microscope phase plates. A focused non-diffracting beam is passed through a small hole in an electrode that is biased by tens to hundreds of millivolts, depending on the particular electrode geometry in the device, resulting in the desired phase shift. Electrostatic shielding of the electrode prevents interaction with the scattered electrons, so that they do not receive any additional phase.

[0010] Another embodiment of a phase plate uses a long, very thin bar magnet placed across the electron diffraction pattern in close proximity to the non-diffracting electron beam. The phase shift is generated by the Aharonov-Bohm effect, which is caused by the difference in magnetic vector potentials on either side of the long bar magnet. [Prior art documents] [Patent documents]

[0011] [Patent Document 1] U.S. Pat. No. 10,395,888 [Patent Document 2] U.S. Patent Application Serial No. 15 / 939,028 [Patent Document 3] U.S. Patent Application Serial No. 13 / 487,831 Summary of the Invention [Problem to be solved by the invention]

[0012] All such efforts are currently limited by the short time it takes for the physical device to become charged, possibly due to contamination accumulation on the surface when the device is bombarded by the intense electron beam. This causes undesirable electric fields, which lead to uncontrolled phase shifts in the electron beam at various scattering angles. This makes the image virtually unreproducible and uninterpretable. Similarly, electrodes block the diffracted beam closest to the center, thus reducing the contrast for large structures in the image. A similar problem is faced with thin film phase plates. [Means for solving the problem]

[0013] In one embodiment, a system for transmission electron microscopy includes a transmission electron microscope (TEM). The TEM has a back focal plane. A mirror forms an optical cavity. The focal point of the optical cavity is positioned at the back focal plane of the TEM. The optical cavity is positioned to allow an electron beam provided by the TEM to pass through the focal point of the optical cavity. The optical cavity is operable to receive a laser beam. A laser having a variable polarization angle of the laser light is coupled to the optical cavity. The laser is operable to provide a laser beam of a specified wavelength and variable polarization angle to enter the optical cavity. The laser beam is reflected from the mirror and provides a standing wave optical phase plate focused at the back focal plane of the TEM. The standing wave optical phase plate causes modulation of the electron beam. An image plane of the TEM is positioned to receive the electron beam modulated by the standing wave optical phase plate. The image plane forms an image with variable image contrast enhancement according to the variable polarization angle of the laser light.

[0014] In one embodiment, a transmission electron microscope generates an electron beam. The TEM has a back focal plane. The electron beam is received along an axis passing through a center of an optical cavity. The optical cavity is positioned at the back focal plane. The optical cavity is defined by a first mirror and a second mirror. A laser beam having a variable polarization angle of the laser light is received into the optical cavity. The laser beam is reflected from the first mirror into the second mirror generating a standing wave optical phase plate that is focused into the back focal plane of the TEM. The standing wave optical phase plate causes modulation of the electron beam. The TEM images the electron beam onto an image plane. The image plane is positioned to receive the electron beam modulated by the standing wave optical phase plate. Varying the polarization angle of the laser light changes the contrast enhancement of the image.

[0015] In one embodiment, the ponderomotive phase plate includes a first concave mirror, a second concave mirror, and a laser. The first concave mirror and the second concave mirror are positioned to define an optical cavity. The optical cavity is positionable to pass the electron beam through a focal point of the optical cavity. The laser has a variable polarization angle of the laser light. The laser and the laser light are coupled into the optical cavity. The laser is operable to have a standing wave of the laser light having an antinode at the focal point of the optical cavity to cause a variable modulation of the electron beam. The variable modulation of the electron beam is controllable by the variable polarization angle of the laser light.

[0016] Other aspects and advantages of the embodiments will become apparent from the following detailed description, taken in conjunction with the accompanying drawings, which illustrate, by way of example, the principles of the described embodiments.

[0017] The described embodiments and their advantages can be best understood by referring to the following description in conjunction with the accompanying drawings, which in no way limit any changes in form and detail that may be made to the described embodiments by those skilled in the art without departing from the spirit and scope of the described embodiments. [Brief description of the drawings]

[0018] [Figure 1] FIG. 1 depicts a schematic geometry of an embodiment of a phase-contrast TEM in which a narrow-waist optical cavity mode is focused into the back focal plane of the TEM objective lens and retardation is applied to the transmitted wave but not to the scattered wave. [Diagram 2] FIG. 1 illustrates the experimentally measured dependence of intracavity circulating power on input laser power, with grey shading representing the measurement uncertainty. [Diagram 3] The intensity distribution of the fundamental mode of a near-concentric Fabry-Perot cavity is depicted, with the horizontally elongated inset showing the interference fringes of the standing wave. [Figure 4] FIG. 13 depicts the contrast transfer function (CTF) of a TEM with a cavity-based laser phase plate averaged over azimuth angle. [Diagram 5]FIG. 1 is a schematic diagram of an embodiment of a laser system including a cavity inside an electron microscope cylinder and coupling optics, a delivery laser system, and a cavity characterization tool. [Figure 6] FIG. 2 is a cross-sectional view of an optical cavity including a housing, a cavity mirror, and a coupling aspheric lens in one embodiment. [Figure 7] FIG. 1 depicts a cavity mount in one embodiment in which the wires shown connect to piezoelectric actuators used for precision alignment of the cavity mirrors. [Figure 8] FIG. 1 is a perspective view of a laser phase plate composed of three cavities with overlapping focal volumes in one embodiment. [Figure 9A] FIG. 1 depicts the interference pattern produced by a three-cavity arrangement featuring a hexagonal lattice of interference peaks. [Figure 9B] FIG. 13 depicts the rotationally averaged contrast transfer function of a three-cavity phase plate (upper curve) compared to a single-cavity (black) (lower curve) for f=2.5 mm. [Figure 10] FIG. 1 illustrates an embodiment of a transmission electron microscope including an optical cavity phase plate according to an embodiment of the present disclosure. [Figure 11] FIG. 1 illustrates the intensity pattern of a resonant optical cavity phase plate according to an embodiment of the present disclosure. [Figure 12] FIG. 1 illustrates a system for generating an optical phase plate for modulating the phase of an electron beam according to an embodiment of the present disclosure. [Figure 13] 1 is a flow diagram of a method for enhancing phase contrast in an electron beam image. [Figure 14] FIG. 1 depicts a schematic diagram of yet another embodiment of a phase-contrast TEM featuring laser light with variable polarization angle coupled into an optical cavity. [Figure 15A] FIG. 15 depicts a half-wave plate 1502 with a rotator for changing the polarization angle of laser light in a phase contrast TEM embodiment and a ponderomotive phase plate embodiment. [Figure 15B]FIG. 1 depicts an optical fiber coupler with a rotator for changing the polarization angle of laser light in a phase contrast TEM embodiment and a ponderomotive phase plate embodiment. [Figure 15C] FIG. 1 depicts a laser with polarized laser light and a rotator to change the polarization angle in a phase contrast TEM embodiment and a ponderomotive phase plate embodiment. [Figure 16] FIG. 15 is a schematic diagram with actions showing a sensor at the image plane of the phase contrast TEM of FIG. 14 or a variation thereof in a phase contrast TEM embodiment and a ponderomotive phase plate embodiment, and a controller that analyzes the Ronchigram for use in controlling the polarization angle of the laser light. [Figure 17] 1 is a flow diagram of a method for transmission electron microscopy that can be performed by various embodiments of a TEM with a ponderomotive phase plate. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0019] Described herein is a laser phase plate with an optical cavity and phase contrast transmission electron microscopy using same. Figures 1-13 relate to an optical cavity based ponderomotive phase plate for transmission electron microscopy. Figures 14-16 relate to the use of polarization states to control the ponderomotive phase plate. Examples of these specific embodiments are illustrated in the accompanying drawings. While certain variations are described with respect to these specific embodiments, this description is not intended to be limited to these described embodiments. Rather than being limited, it is intended to cover alternatives, modifications, and equivalents that may be included within the spirit and scope defined by the claims.

[0020] In the following description, numerous specific details are set forth in order to provide a thorough understanding of the embodiments of the present disclosure. Certain exemplary embodiments may be practiced without some or all of these specific details. In other instances, well-known process operations are not described in detail so as not to unnecessarily obscure the present disclosure.

[0021] Various techniques and features of embodiments of the present disclosure are described below, sometimes in the singular, for purposes of clarity, but it should be noted that some embodiments include multiple iterations of a technique or multiple embodiments of a feature unless otherwise noted.

[0022] The terms "about" or "approximately" and the like are used to be synonymous and to indicate that the value modified thereby has an understood range associated therewith, which may be ±20%, ±15%, ±10%, ±5%, or ±1%. The term "substantially" is used to indicate that a value is close to a target value, where close can mean, for example, that the value is within 80% of the target value, within 90% of the target value, within 95% of the target value, or within 99% of the target value.

[0023] Described herein are optical power amplification cavities suitable for use as phase masks for electron beams in electron-based imaging systems such as transmission electron microscopes (TEMs) or electron-based spectroscopy systems. Depending on the configuration, such phase masks can function as several electron-optical elements for the coherent manipulation of electron beams, including: Phase plates for Zernike phase contrast electron microscopy, A Bragg beam splitter for coherently splitting the electron beam into two with a controllable split ratio; A transmission grating for an electron beam that splits the input beam into several diffraction orders, A temporal phase modulator similar to the electro-optic modulators used in conventional optical microscopes; A local temporal phase modulator that can be used in conjunction with an energy filter as a beam stop for dark-field electron microscopy, A spatio-temporal phase modulator similar to the acousto-optical modulators (deflectors) used in conventional optical microscopes.

[0024] One embodiment design of the power amplification optical cavity includes multiple cavity mirrors (e.g., spherical dielectric mirrors) at least one of which is characterized by a highly curved front surface. The front (concave) surfaces of these mirrors have a reflective coating disposed thereon. The rear (convex) surfaces of these mirrors can have an anti-reflective coating disposed thereon. The intracavity laser field configuration generates an effective potential for electrons that transfers a spatially patterned phase shift, which can also be time modulated, onto the electron beam transmitted through the cavity.

[0025] Advantages of optical cavity electro-optic elements over conventional elements based on material structures (e.g., optical cavity phase plates over material phase plates) include: (1) the amount of phase shift is substantially constant and does not change with the length of time the phase plate is used, as was the case with all previous devices that direct a physical object near or into an electron beam; (2) the amount of phase shift can be adjusted as needed to "tune" the phase shift from approximately zero degrees to 360 degrees; and (3) negligible electron losses (undesirable electron scattering) in the optical cavity elements.

[0026] Advantages of optical cavity electron optical elements over elements based on pulsed laser systems include the ability to use optical cavity phase plates with continuous electron illumination rather than with a pulsed electron gun (as was the case with previous pulsed laser phase plates). This feature makes optical cavity phase plates uniquely suitable for use in standard continuous electron emission microscopes and for applications where very high electron exposures are used. Cavity-based systems are also characterized by precisely controllable phase shift patterns determined by the laser field configuration of the cavity modes. The absence of distortions and defects typically present in the laser beam profile is highly desirable to avoid unwanted electron scattering due to laser field anomalies.

[0027] Features of the optical cavity phase plate include: Near-concentric Fabry-Perot cavity configuration or another cavity configuration characterized by a high-intensity focus, Highly reflective dielectric coatings on curved surfaces, An arrangement of three or more piezoelectric actuators for aligning the cavity and for controlling the cavity mode shapes; Phase plates for TEMs that are characterized by a phase shift profile that needs not to be a single spot but an extended pattern with multiple maxima and minima inducing a contrast transfer function suitable for the particular purpose (e.g. a standing wave with a waist of several wavelengths can be used as a Zernike phase plate, while a larger waist is preferred to act as a Bragg beam splitter), Phase plates for TEMs based on electron-light interaction (one embodiment is an approximately spherical cavity with high-finesse high-numerical aperture (high NA) mirrors), A mirror with a front (concave) and a concentric rear (convex) surface for efficient coupling into the cavity modes; A mirror having a rear surface on which an anti-reflective coating is disposed for improved coupling efficiency; Simultaneous injection into the cavity of laser fields at several corresponding (resonant) frequencies to provide time modulation and control of the phase pattern applied to the electron beam.

[0028] The feedback and alignment mechanisms and methods for optical cavity phase plates can include: A feedback mechanism to keep the mode shape and size stable (e.g., a charge-coupled device (CCD) camera and / or a segmented detector monitoring the transmitted beam, the reflected beam, or both); A feedback topology in which the laser frequency remains locked to the cavity following both a change and a systematic change in the cavity length, or alternatively the cavity length is altered such that the resonant frequency of the cavity remains locked to the laser frequency; A method for aligning the cavity with respect to the electron beam by monitoring the TEM image, its Fourier transform, or other mathematical construct to determine the degree of misalignment and provide feedback for alignment; Feedback to the cavity modes can help keep the laser phase plate aligned with the beam; Injecting a master laser with a laser beam transmitted through the cavity (e.g. as a frequency locking mechanism).

[0029] Further features of the optical cavity phase plate can include: Heat pipes to remove heat from the cavity, Heat shield to protect the cavity structure, -Controlling cavity length by temperature, Controlling the mirror curvature by controlled preheating using an auxiliary laser, a heating circuit, or a thermoelectric device; Using multi-cavity configurations to reduce spot size and increase intensity; Bringing the laser to the cavity using an optical fiber; - Protecting the laser system from reflected light using optical isolators; The laser focal spot size is larger than the electron focal spot size to avoid scattering of the electron beam; Controlling the intracavity field configuration by deforming the mirror (e.g. by gradually squashing it).

[0030] Additional uses / features may include: · Varying the laser power to adjust the amount of phase shift (this feature can be used to invert or otherwise adjust the contrast); The ability to use optical cavity phase plates for high electron doses in a TEM (high electron doses generally do not allow the use of Volta phase plates), The ability to induce fast modulation of the phase shift by modulating the intracavity laser power (leading to modulated electronic wavefunctions that can be used to deliver oscillating or pulsed electromagnetic fields to a microscopic object (i.e. a form of spectroscopy)); The formation of sidebands at the focal spot can be used in combination with energy filters to substantially focus a transmitted (i.e., non-diffracted) electron beam for full-field dark-field TEM; Lossless controllable electronic beam splitter.

[0031] Various embodiments have some or all of the following features: At least one of the mirrors has a reflective surface with a radius of curvature of 5 cm or less to create a small focal spot inside the cavity; The reflectivity of each of the mirrors comprising the cavity is about 0.9 or greater; the mirror containing the cavity has a reflective surface coated with a reflective dielectric coating; the plurality of mirrors includes a first mirror and a second mirror; a cavity housing in which an optical cavity is disposed and one or more of the mirrors containing the cavity are mounted in an adjustable suspension, the adjustable suspension being operable for adjusting the angle and position of the mirror; the adjustable suspension includes a piezoelectric actuator; The cavity amplifies the power of the laser beam by a factor of about 10 or more.

[0032] In some embodiments, the shape of the rear surface of the mirror (i.e., the non-reflective surface) may not be important and may not need to be convex and concentric with the front surface of the mirror. For example, it is contemplated that the rear surface of one or both of the mirrors could be flat. In some embodiments, one feature that defines a cavity suitable for a phase plate is that (as opposed to a typical optical cavity) the cavity can focus the beam to a small focal spot and at the same time amplify the laser power by a large factor and can withstand the high power that can be obtained. This is not found in known optical cavities. Features present in the optical cavity for these embodiments include (1) a short radius of curvature of at least one of the mirrors and (2) low round trip loss resulting in high reflectivity / low absorptivity of all mirrors.

[0033] An advantage of optical cavity phase plates in cryo-electron microscopy (cryo-EM) is that the amount of phase contrast can be 100% of a specified value at the beginning of data collection. Furthermore, the amount of phase contrast can remain at the specified value throughout the data collection period, which may last 24 hours or longer per cryo-EM session. This means that all parts of the collected data can be considered to be of equal quality, as opposed to some data being significantly worse than others. Further advantages of optical cavity phase plates include higher versatility and reliability. Furthermore, it is theoretically expected that a phase shift of 270 degrees rather than 90 degrees will result in better performance.

[0034] Further details regarding the operation of an optical cavity phase plate according to one embodiment are described in U.S. patent application Ser. No. 13 / 487,831, filed Jun. 4, 2012, which is incorporated herein by reference.

[0035] Recently, transmission electron microscopy (TEM) of unstained flash-frozen biological samples (cryo-EM) has become the main source of structural information in molecular biology. The lower limit for the molecular mass of accessible particles has been reduced to 100-200 kDa, and in some cases even below 100 kDa. Theoretical considerations indicate that reconstruction of particles as small as about 40 kDa should be possible. Nevertheless, determining the structure of macromolecular complexes with sizes between 40 kDa and 100-200 kDa, as well as addressing larger but more flexible complexes, requires further improvements in the cryo-EM technique.

[0036] One route towards TEM reconstruction of smaller particles is the development of Zernike phase plates, which allow phase-contrast imaging without defocus. An important step in this direction was the development of the "Volta" phase plate.

[0037] However, significant opportunities remain to achieve further improvements. In the Volta design, the phase shift is induced by the exposure of the amorphous carbon foil to the electron beam and continues to increase during data collection. Only a moderate number of images can be acquired while the phase shift is in the usable range of 40-120°, and then the carbon film must be moved to expose a new spot. Many images are forced to be acquired with non-optimal phase shifts. Even within the same phase shift, the phase shift and its drift rate vary from spot to spot. These characteristics make it difficult to use carbon foil phase plates for cryo-EM structural studies that require high-yield image acquisition with consistent parameters.

[0038] Placing material structures in the path of a high-energy electron beam inevitably leads to charging and degradation. Time-varying behavior is therefore an unavoidable drawback of phase contrast devices based on material objects in the electron stream of a TEM. An additional drawback is the small but noticeable 18% electron scattering in the Volta phase plate.

[0039] Here we propose to circumvent this limitation by using a strong laser focus as an electron wave retarder. Such a laser-based phase plate, or "ponderomotive" phase plate, does not require a material object to be inserted into or positioned near the electron beam. This phase plate applies a constant phase shift indefinitely, has a predictable and reproducible contrast transfer function, and may not feature any substantial electron losses. The phase shift can be varied as desired by changing the laser power.

[0040] A central concept in various embodiments is that a laser beam focused into the back focal plane of a TEM objective lens 1018 (see embodiment in FIG. 10 described in more detail below) can be used to act as a Zernike phase plate to delay the phase of the transmitted wave relative to the scattered wave. The controllable and stable phase shift provided by a laser phase plate will enable the cryo-EM community to take full advantage of the improved image contrast in Zernike phase contrast TEM.

[0041] The physical mechanism of electron retardation is explained below. The goal for the embodiments described here is to construct better Zernike (quarter-wave) plates for phase-contrast TEM by controlling the phase of the electron wave with a laser. This concept is borrowed from the field of atomic physics, where lasers are used to manipulate both the motional and internal degrees of freedom of ensembles of cold atoms. These methods enable many experiments aimed at testing the fundamentals of quantum mechanics and general relativity, searching for the fields responsible for dark energy, and making precise measurements of fundamental constants.

[0042] The physical principle behind laser-based coherent control of electron waves can be explained in terms of classical physics as follows: Relativistic electrons traveling through an intense oscillating laser field undergo small-scale micromotion, resulting in an effective "ponderomotive" potential. This potential is expressed by the following equation: TIFF2025041925000002.tif8170 where I is the laser intensity, e and m are the charge and mass of the electron, ε0 is the dielectric constant of vacuum, c is the speed of light, and ω is the optical frequency. This repulsive potential imposes a negative phase shift (retardation) on the passing electrons.

[0043] A more rigorous description of the interaction of relativistic electrons with an intense laser beam can be given in terms of quantum electrodynamics (QED), where the cause of the phase shift is stimulated Compton scattering. In this process, an electron either absorbs a photon from a laser beam and then re-emits this photon by stimulated emission stimulated by this same beam, or undergoes these two events in the reverse direction. Electrons that undergo stimulated Compton scattering against a monochromatic laser beam emerge with the same energy as when the scattering began, without loss of coherence. In contrast, spontaneous Compton scattering causes electrons to lose coherence, but the fraction of electrons that undergo such an event can be as low as 10 under conditions appropriate for various embodiments. -7 The inventors have noted that higher order scattering processes involving three or more photons can cause electrons to scatter inelastically, thereby losing coherence. However, these processes are limited to laser intensities of I≈10 13 W / cm 2 (for a 1 μm laser wavelength), which far exceeds the intensities that these embodiments will handle.

[0044] The required laser intensity is explained below. The proportionality coefficient between the ponderomotive potential and the laser intensity in equation (1) is small, so high laser intensity is required. The power P and the focus waist w0 (beam radius is 1 / e of the peak intensity) 2For an electron beam passing orthogonally through a focused Gaussian beam with θ (measured at θ = 1), the phase shift is given by TIFF2025041925000003.tif8170In the above formula, α is the fine structure constant, and β=v e / c, and v e is the electron velocity, and γ=(1-β 2 ) -1 / 2 To estimate the required optical power, an electron energy U = 300 keV is substituted as is commonly used in cryo-EM studies. According to equation (2), for a laser wavelength λ = 1064 nm and a beam waist of w0 = 7 μm, the laser power required to produce a maximum 90° phase shift is P ≈ 300 kW.

[0045] Providing such a refractive power would be straightforward for a pulsed laser system, but the pulsed phase plate would additionally require a synchronous pulsed electron gun 1024 (see FIG. 10). The main goal of these embodiments is to develop a laser phase plate that is compatible with conventional cryo-EM instruments based on continuous wave (CW) laser systems.

[0046] In the following we describe the enhancement of resonant fields in optical cavities.

[0047] 1 depicts a schematic geometry of an embodiment of a phase-contrast TEM. A narrow-waist optical cavity mode is focused into the back focal plane of the TEM objective lens, e.g., the diffraction plane 104, and a delay is applied to the transmitted wave 106 but not to the scattered wave 108. Electrons from the scattered wave 108 are detected by an electron detector 112 and imaged as a result of the delay in the transmitted wave 106 in a high-contrast view of the sample in the sample plane 102.

[0048] In some embodiments, the laser intensity is enhanced by resonating in a high-finesse near-concentric Fabry-Perot cavity (see optical cavity 110 in FIG. 1). This type of cavity is characterized by an hourglass-shaped fundamental mode with a small waist and a large spot size on the face of the mirrors 114, 116. The small waist concentrates the power in a high-intensity focal point and confines the phase shift to a narrow region around the focus of the transmitted wave 106. The large spot size on the mirrors 114, 116 helps to prevent laser-induced breakdown of the mirror coating and spread the heat load over a larger area. Furthermore, the two opposing waves propagating in the Fabry-Perot cavity form a standing wave, and the intensity is quadrupled at the antinodes. For a wavelength λ of 1064 nm and a beam waist of w0=7 μm, an intracavity circulating power 204 of P=75 kW is sufficient to reach a maximum 90° phase shift.

[0049] Recent work towards the above mentioned goal has demonstrated a near-concentric cavity operating at a wavelength of λ = 1064 nm and with a finesse of F = 22,000. Delicate alignment of the near-concentric cavity was performed using a specially designed flexure-based mount with three piezoelectric actuators, which allowed reaching a waist (radius at 1 / e2 intensity) of 7 μm.

[0050] Figure 2 depicts the experimentally measured dependence of the intracavity circulating power 204 on the input laser power 206. The grey shading 206 represents the measurement uncertainty. At an input power 202 of P0=2.2 W, the cavity reached a circulating power 204 of P=7.5 kW, corresponding to a record CW optical intensity of 40 GW / cm2 (see Figure 2). The phase shift corresponding to these beam parameters is 9.2° for a 300 keV electron beam. Therefore, a realistic 10x power increase seems sufficient to reach the goal of giving a 90° phase shift.

[0051] In the following the laser field configuration and contrast transfer function are described.

[0052] FIG. 3 depicts the intensity distribution 302 of the fundamental mode of a near-concentric Fabry-Perot cavity. The horizontally stretched inset 304 illustrates the interference fringes of a standing wave. In an ideal Zernike phase plate, a 90° delay is applied only to the transmitted wave that is focused at the center of the electron diffraction plane 104, and there is no phase shift in the remainder of this plane. This embodiment of a cavity-based laser phase plate has a Gaussian standing wave intensity profile (shown in FIG. 3) and applies a harmonically modulated phase shift to a stripe-shaped region of the diffraction plane 104. Below we discuss the consequences of this phase profile and why this phase plate effectively functions as a Zernike phase plate in cryo-EM single particle analysis.

[0053] Phase contrast extends to spatial frequencies low enough to image small (≈5 nm or less) protein complexes. The “cut-on” frequency at which phase contrast is effective is determined by the contrast transfer function (CTF) of the imaging system. The correspondence between spatial frequency ν and radius r in the back focal plane of the TEM objective is given by TIFF2025041925000004.tif6170In the above formula, f is the focal length of the TEM objective lens, λ e is the electron wavelength. Typical parameters for a microscope used in cryo-EM are λ e = 1.97 pm (corresponding to an electron energy of U = 300 keV) and f = 2.5 mm. In the early stages of development of the embodiment, including the development of the first prototype and initial data collection, an FEI Titan TEM was used with additional relay optics to magnify the electron diffraction pattern in the plane of the phase plate. This additional magnification results in an effective focal length of f = 20 mm, which relaxes the requirements for the tightness of the laser focus.

[0054] FIG. 4 illustrates the focus CTF 402 of a cavity-based phase contrast TEM, calculated for an electron microscope and averaged over azimuthal angles. For clarity, the graph shows the CTF at zero defocus and does not consider spherical aberration. At very low spatial frequencies 404, the curve starts with zero contrast. The CTF 402 reaches 50% at low spatial frequencies 404 down to about 1 / (240 nm), a scale set by equation (3) where r=λ / 2 represents the size of the standing wave fringes (see FIG. 2). The oscillations are caused by the interference fringes of a standing laser wave. The CTF reaches nearly maximum (>80%) contrast at frequencies larger than 1 / (10 nm), a scale set by the radius of the laser beam. Thus, D≈0.5ν -1 For particles with a size of 5 nm or smaller, the information contained in the electronic phase is transmitted with nearly maximum contrast. Many macromolecules with molecular masses smaller than 100-200 kDa have diameters of about 5 nm or less. Cavity-based phase plates give nearly ideal contrast for these macromolecules. Larger particles (100 nm) are imaged with only moderate reduction in contrast.

[0055] Image artifacts due to standing wave phase profiles do not significantly distort the particle image. Multislice numerical modeling of cryo-EM imaging of hemoglobin, a 64 kDa particle embedded in 30 nm amorphous ice, confirms that the cavity-based phase plate works well for small particles. The structure of the hemoglobin molecule was downloaded from the Protein Data Bank. 20 e / Å 2 The acquired images show significant contrast enhancement by the laser phase plate compared to the defocused image produced with a defocus of 1 μm.

[0056] Numerical modeling allows for the evaluation of possible artifacts due to the anisotropic phase shift profile of the laser waveplate. Images including those of hemoglobin simulated with an ideal phase plate, which is considered to phase shift only the transmitted wave and the image using a cavity-based laser phase plate, were studied. The simulation highlights any differences by not showing any shot noise or noise from ice density variations. Even with the highlighting, only slight vertical streaking can be discerned. The simulation showed the difference between the two images in a grayscale that amplifies this difference signal by a factor of 10 until it reaches a visible level. This difference is well below the noise level and is therefore not expected to affect particle identification and classification.

[0057] Another side effect of the standing wave phase shift pattern is that it acts as a diffraction grating for the electron wave, generating additional weak "ghost" images. These ghost images are displaced from the first order image by a distance δx=2nfλe / λ, where n is the diffraction order, f is the focal length, and λe is the electron wavelength. Even for a maximum shift of 90°, the amplitude of such ghost images is less than 20e / Å. 2 is well below the shot noise for illumination of 100 nm. In dense samples, superpositions between ghost images of one particle and the primary image of another can be expected to occur. However, the ghost images are too faint to be visible in the individual micrographs and can be expected to cancel each other out and average out of the fused data, resulting in a negligible contribution to noise in the average image used for density map reconstruction.

[0058] A tighter laser focus is required to adapt the laser phase plate to a standard cryo-EM system. At a later stage, we developed an embodiment that includes an advanced phase plate prototype with a narrower focus suitable for use with a regular TEM system with a typical focal length f=2.5 mm. For this purpose, we increased the numerical aperture of the cavity modes and operated the cavities close to concentric. In one approach, it was unclear whether this operation degraded the cavity finesse. In another scheme, we used a multi-cavity configuration, which entailed less risk but required additional steps of cavity alignment and frequency locking. Both concepts are explained in the "Methods" section.

[0059] Essentially, we propose to develop a Zernike phase plate for a TEM operating at 300 kV based on the ponderomotive potential of a high-intensity CW laser circulating in an optical cavity. It is shown that sufficient laser intensity for a phase shift of ~9° and a factor of 10 increase (considered to give an optimal phase shift of 90°) are within the reach of modern mirror fabrication techniques. Numerical simulations show that the cavity-based phase plate can be effectively used as a Zernike phase plate for cryo-EM single particle reconstruction. Below we detail our approach, which includes a continuous-wave laser phase plate module with sufficient laser intensity to shift the phase of a 300 keV electron beam by 90°.

[0060] To construct a laser phase plate based on a near-concentric cavity, custom-made highly curved cavity mirrors with high reflectivity and low loss are required. Additionally, appropriate delivery lasers and opto-mechanical systems are employed to maintain cavity alignment. System embodiments must also be compact, vacuum compatible, and non-magnetic to operate within the TEM column. Below, we outline an approach to construct a system that meets these requirements.

[0061] In the following, embodiments of a laser system are described.

[0062] 5 is a schematic diagram of an embodiment of a laser system including a cavity 110 in an electron microscope column 526 and coupling optics, a delivery laser system, and a cavity characterization tool. In terms of acronyms of the system components, FA=fiber amplifier 502, FC=fiber coupler 504, PDH PD=Pound-Drever-Hall locked photodiode 506, FI=Faraday isolator 508, CL=coupling lens 510, CM=cavity mirror 512, BS=beam splitter 514, FPD=fast photodiode 516, DA=digital data acquisition 518, e.g., camera 524 and fast photodiode 516. In one embodiment, a Nd:YAG laser wavelength (1064 nm) was chosen, which allows the use of commercially available narrow linewidth lasers and highly optimized optical coatings. A low power (<100 mW) master laser 522 is amplified using an Ytterbium Fiber Amplifier (FA) 502 from IPG Photonics or Nufern, which provides single mode, single frequency, polarization maintaining (PM) amplification up to 50 W.

[0063] The laser beam is coupled into a high power single mode pm fiber from NKT Photonics capable of delivering at least 20 W of optical power at 1064 nm. This power is delivered to a small assembly attached to the microscope that includes a Faraday isolator (to prevent the laser radiation from reflecting back into the laser) and a beam steering mirror (to spatially adapt the fiber output to the cavity mode). A camera 524 and a photodetector, e.g., FPD 516, observe the mode shape transmitted by the cavity 110 for optimization purposes and to measure the cavity finesse using cavity ring-down spectroscopy.

[0064] As demonstrated in recent work, the master laser frequency is stabilized ("locked") to the resonance of the optical cavity 110 using the Pound-Drever-Hall method. To this end, a portion of the light reflected from the cavity 110 is detected by a photodetector (PDH PD) 506 and used to control the laser frequency by a PDH locking circuit 520.

[0065] Below we describe the cavity mirrors 512. For a Fabry-Perot cavity fabricated with two identical mirrors 512, the power amplification M is determined by the transmission and reflection coefficients T and R of the cavity mirrors, and the mode overlap integral Q: TIFF2025041925000005.tif7170

[0066] Since |Q|≦1, the power boost is limited by the quality of the cavity mirrors, where the reflectivity must be as close to unity as possible and the transmittance must be as high as possible. The two coefficients mentioned above are constrained by the law of conservation of energy to R+T+L=1, where the loss factor L=S+A includes the scattering factor S and the absorption factor A. In general, the absorption is a property of the dielectric material used to produce the mirror coating. In general, the absorption is less than 3 parts per million (ppm) in near-infrared reflective coatings produced by ion beam sputtering (IBS) by vendors such as Advanced Thin Films, Inc. and Research Electro-Optics (both located in Boulder, Colorado, USA).

[0067] Scattering, on the other hand, depends primarily on the smoothness of the mirror substrate and is therefore determined by the quality of the mirror polishing process. Scattering is given by S=(4πδh), where δh is the root-mean-square (RMS) surface roughness. 2 / λ 2 Methods for polishing flat fused silica substrates are well developed, so that surface roughnesses smaller than 0.1 nm and scattering on the order of 3 ppm are routinely obtained. However, polishing the high curvature concave mirrors required for near-concentric cavities remains challenging.

[0068] One embodiment utilizes a recently developed polishing process that allows reaching an RMS surface roughness of about 0.1-0.2 nm on substrates with radii of curvature as short as 10 mm. This should result in S well below 10 ppm. In one embodiment, the mirror 512 is coated with a state-of-the-art highly reflective IBS coating from either Advanced Thin Films or Five Nines Optics. Based on these figures, these mirrors support amplification factors in the range of M=8,000-13,000.

[0069] One plan, in case the IBS coating process used by the coating companies mentioned above does not work well on the new substrates produced by Perkins Precision, is to purchase mirrors 512 with a sufficiently small radius of curvature available from Layertec and LaserOptik. The reflectivity of these mirrors is expected to be sufficient to reach an amplification factor of M=4,000-6,000. In case neither supplier is likely to be able to reach the performance target specified in their proposal, there is a second alternative option to purchase mirrors 512 available from Advanced Thin Films, which have a larger radius of curvature and can support a mode waist of 8-10 μm with a power amplification factor of 10,000.

[0070] The mirror transmission coefficient will be measured immediately prior to installation in the cavity 110. The reflectivity of the mirror 512 will be measured using the ring-down spectroscopy technique utilized for this type of cavity.

[0071] For a cavity amplification factor of M=5,000, an input power of 15 W is needed to reach a maximum phase shift of 90°. Even with a circulating power of up to 75 kW, direct laser damage of the mirror coating is unlikely, and using a mirror 512 with a radius of curvature of Rcurv=20 mm, a spot diameter on the mirror surface of w1=(fλ) / (πw0)=1 mm is provided, which induces a maximum surface intensity lower than 0.5 mW / cm2, well below the laser damage threshold for high reflectivity mirror coatings in the near infrared range, providing an intensity of 100 mW / cm2.

[0072] The large mode size on the mirror surface necessitates special attention to surface quality, since any scratches in the center of the mirror 512 will increase the round-trip loss rate. This risk can be mitigated by specifying a scratch-dig surface quality of 10-5 or better, and by ordering mirrors in batches of 10 or 12 to select the best performing mirrors.

[0073] In the following the cavity optical setup is described.

[0074] 6 is a cross-sectional view of an optical cavity that, in one embodiment, includes a housing 606, multiple cavity mirrors 604, and a coupling aspheric lens 602. The optics on the right are placed on a flexure suspension that can be tilted or moved axially.

[0075] The alignment of the near-concentric resonator with a small mode waist is highly sensitive to the position and orientation of these mirrors 604 relative to each other, as the desired configuration is very close to the degeneracy point, where a set of modes have the same frequency and the resonator becomes unstable. In this configuration, small angular shifts of the mirrors 604 result in significant lateral displacements of the modes. In a near-concentric optical resonator, the mode waist size can be controlled by adjusting the distance between the mirrors 604, and this adjustment must be made with sub-micron precision.

[0076] 7 illustrates the cavity mount in one embodiment. Wires 702 are shown connecting to piezoelectric actuators (within housing 606) that are used for precision alignment of the cavity mirror 604.

[0077] For the various embodiments, it was planned to replicate the cavity mount (see Figs. 6 and 7) used in previous work on near-concentric resonators. It was shown that flexure suspensions carved from a single aluminum block could be used to align the near-concentric cavity with a mode waist w0 = 7 μm. The mount was small enough to allow installation in a TEM cylinder with a cylindrical access port of φ25 mm diameter. For the optical bench experiments, the cavity 110 was positioned in a small vacuum chamber with input and output anti-reflection coated laser quality optical ports. One of the mirrors 604 was left stationary in the mount, while the position of the second mirror 604 was controlled using a piezoelectric actuator in combination with three fine screws for coarse alignment. The entire mount assembly was made of non-magnetic materials (aluminum body, 316 series stainless steel screws with silicon carbide tips, piezoelectric ceramic).

[0078] Thermal control is described below. Operation under vacuum creates additional challenges in removing heat dissipated within the cavity. Absorption in the IBS coated mirrors is only 1-3 ppm of the circulating power, so each of the two cavity mirrors 604 must dissipate a maximum of 200 mW at resonance. Care must be taken to remove heat that accumulates within the cavity mirrors to avoid thermal deformation. Numerical simulations have shown that the thermal conductivity of the fused silica substrate limits the temperature increase at the center of the mirror to approximately 30 K, and communication with the manufacturer of this mirror has confirmed that a temperature increase of 30 K is highly unlikely to result in mirror damage.

[0079] The local temperature increase will also result in an increase in the local radius of curvature, on the order of δR≈300 nm. When the cavity 110 is induced to operating power, the distance between the mirrors 604 will be increased by 2δR to keep the cavity 110 at a selected distance to the concentricity (which determines the mode waist). By changing the cavity length, the resonant frequency of the cavity will be lowered by ≈7 GHz. The master laser used in one embodiment (NP Photonics Rock fiber laser module or equivalent) has a frequency tuning range of about 30 GHz, so that the shift in resonant frequency can be easily followed by the frequency-locked master laser.

[0080] However, additional heat load is introduced from light scattered on the mirror surface and absorbed in the cavity. Assuming that at least half of the input power is either transmitted or reflected, it is necessary to be able to remove up to 10 W dissipated in the laser module. This task is made more difficult by the need to suspend the cavity on a support structure of about 200 mm length in vacuum. The aluminum cavity mount body should provide sufficient thermal conductivity to limit the temperature increase to 10 K, but to avoid changes in the cavity alignment driven by temperature changes, it was planned to actively control the cavity temperature with a thermoelectric cooler. It was planned to use a thermoelectric set (cold plate and controller) from TE Technology, capable of absorbing up to 28 W and maintaining the temperature well within 0.1 K of the set point.

[0081] To facilitate external heat transfer, it was planned to use up to three copper / water heat pipes with a diameter of 4-6 mm, running in grooves along the support structure. Such heat pipes, commonly used in the electronics industry for cooling chips, have a thermal resistance well below 0.1 K / W, providing significantly higher heat removal capabilities compared to all-copper or aluminum rod suspensions without introducing vibrations associated with circulating coolant. At the same time, the copper / water heat pipes are vacuum compatible and non-magnetic, and therefore suitable for use in a TEM environment.

[0082] The following describes the tests that were planned to be performed in an optical bench configuration to ensure that the phase plate prototype met its target specifications.

[0083] The numerical aperture of the cavity modes will be measured by recording the far-field pattern of the beam transmitted through the cavity and fitting it to a Gaussian profile. The estimated mode waist will be w0 = 7 μm or smaller.

[0084] The optical power circulating within the cavity will be at least 75 kW, which will be calculated using the measured transmission coefficients of the output cavity mirrors and the transmitted power measured using a calibrated power meter.

[0085] The system is vacuum gauge measured at 2·10 -7 It will operate in a vacuum chamber with a pressure not exceeding mbar.

[0086] The system will be able to maintain cycling power and numerical aperture continuously for at least 2 hours without manual adjustment of any degree of freedom, and will be able to maintain these parameters for at least 10 hours with intermittent control adjustment.

[0087] In one embodiment, the electron microscope is a low-base FEI Titan TEM equipped with a lateral-insertion Gatan cryo-holder, allowing for the study of cryo-EM samples. Images will be recorded with a 16 megapixel Gatan K2 Summit direct detection camera. As mentioned above, the microscope has additional relay optics that magnify the electron diffraction pattern and extend the effective focal length to 20 mm. The microscope has four ports (three of which have an acceptance width of φ25 mm) that give advantageous access to the diffraction plane 104 at the height of the conjugate back focus plate. One of these ports will be used to insert the cavity module, and the transmitted laser beam will be directed into the port that is approximately diametrically opposed (offset by 170°) and has a prism attached to the cavity mount.

[0088] The insertable laser module will be made of non-magnetic materials to avoid disturbing the magnetic fields of the TEM electron optics, i.e. aluminum alloys (cavity suspension system), piezoelectric ceramics, fused silica (mirrors, lenses). The small micrometer screws we decided to use are made of 316 series stainless steel with silicon carbide ball tips. Titanium screws can be used as an alternative option if the extremely weak magnetic permeability of stainless steel is found to disturb the TEM alignment.

[0089] The laser beam source and frequency locking system will be positioned adjacent to the electron microscope. The light will be directed to the insertable module using a flexible high power polarization maintaining single mode fiber. One advantage of fiber coupling is that alignment of the optics to the electron beam can be performed by moving the entire insertable optical module without disturbing the internal alignment of this module.

[0090] The insertable module will be suspended in a mount that allows three-axis position adjustment of the module with respect to the TEM cylinder. The laser focus, and therefore the module, must be positioned vertically within 100 μm of the diffraction plane 104. This ensures that the transmitted electron wave will traverse a high intensity area when it is focused to a size smaller than the laser wave interference fringes. The horizontal position perpendicular to the cavity optical axis must be controlled to within 1 μm. This ensures that any misalignment is significantly smaller than the waist of the laser beam. Finally, the horizontal position along the cavity axis must be stable to better than 100 nm. Since the half-width of the standing wave fringes is only 266 nm, this stability ensures that the unscattered electron beam will pass through the highest point of the standing wave. Meanwhile, all maxima within ≈50 μm around the beam waist have approximately the same intensity (as shown in FIG. 5), and any of these maxima can be used to delay the transmitted wave. A piezoelectric actuator will be incorporated into the suspension system to keep the longitudinal position of the cavity stable.

[0091] To place the cavity focus at the center of the conjugate Fourier plane, a two-step procedure was planned. First, for a rough alignment, it was planned to use a φ1 mm circular aperture cut into the cavity mount to pass the electron beam so as to center the cavity focus under the electron beam without a sample inserted. This position can be found by moving the assembly while observing the TEM image, noting that the electron beam is blocked by the edge of the beam aperture. Then, for a finer alignment, an amorphous carbon film can be used as the sample. Observing the Fourier transform of the image with the appropriate defocus reveals the position of the laser focus relative to the center of the TEM Fourier plane. Using the image as a guide, the center of the cigar-shaped focus can be guided to the center of the Fourier plane.

[0092] For fine adjustment of the module position along the cavity axis, it was planned to observe the Thon rings, again using the defocused image of amorphous carbon. When the unscattered beam passes through a node of the standing wave, the Thon rings should have their regular pattern. However, when the unscattered electron beam is phase shifted by the standing wave, these rings will be shifted by the same phase, providing quantitative feedback for fine alignment.

[0093] With the system aligned, the power of the laser system can be calibrated by observing the shift of the Thon ring with various laser powers.

[0094] The delicate alignment of the near-concentric resonator is easily disturbed by vibrations and thermal expansion. On the optical bench, the alignment can be restored manually by a piezoelectric actuator. However, to be advantageous to the TEM user population, the phase plate needs to be able to operate without frequent human intervention.

[0095] One goal was to eliminate this difficulty by automating some of the adjustments required to keep the cavity aligned. As verified in previous studies, the main types of misalignment are easily observed with a CCD camera monitoring the laser beam transmitted through the cavity, and the necessary adjustments are easy. When the cavity 110 is nearly perfectly aligned, there are three main misalignment modes: (i) pitch tilt and (ii) yaw tilt of the mode with respect to the cavity axis, and (iii) change in numerical aperture of the mode. These three modes can be measured by observing the power laser beam with a camera and fitting its intensity profile with a Gaussian distribution for the two coordinates of the center of gravity and the beam width. The coordinate of the beam center will be used to adjust the mirror tilt to realign the mode with the cavity axis, and the size of the mode will be used to stabilize the cavity length (and therefore the mode waist). For these purposes, a piezoelectric actuator is integrated into the mirror mount. The bandwidth of this feedback system does not need to be high, since in an optical table adjustments are only required once every few operating hours. Provided that the vibration isolation of the TEM system is at least as good as the optical table, it was anticipated that adjustments would not need to be very frequent, and therefore standard computer interface tools would be more than sufficient to provide the necessary bandwidth.

[0096] It was envisaged that it may be necessary to periodically check the alignment of the unscattered electron beam with respect to the maxima of the standing laser wave by monitoring the Thon ring pattern in the observed image. One embodiment for implementing this feature is as a software connection that analyzes the TEM image and sends a feedback signal to an actuator that controls the alignment of the phase plate module with respect to the electron beam.

[0097] In principle, a laser phase plate can be used either in the back focal plane or in any suitable conjugate plane on the TEM cylinder.

[0098] Higher numerical apertures require highly curved mirrors that cannot be accommodated by the polishing processes currently used for high finesse mirrors such as those planned for use in the various embodiments. However, advanced processes such as ion beam milling are capable of doing this in principle. Numerical apertures as high as 0.36 (at λ=1064 nm) have been reported for near-concentric cavities, corresponding to a mode waist of w0=0.94 μm. A cavity-based ponderomotive phase plate with a focus as tight as that would enable phase-contrast imaging in a conventional TEM with a focal length of f=2.5 mm.

[0099] However, ion beam milling produces surfaces that are not sufficiently smooth for low loss mirrors, and must be superpolished before a reflective coating can be applied (some cavities have been reported to have a finesse of only about 600). In the past, it would not have been clear whether very high NA ion milled substrates could be polished to a surface roughness small enough for the fabrication of high finesse cavity mirrors. Furthermore, even if the substrate were sufficiently smooth, it was not certain in the planning stages whether a highly reflective dielectric coating could be applied uniformly enough to such a high numerical aperture mirror.

[0100] When the straightforward approach of increasing the numerical aperture was unsuccessful, another embodiment relied on the use of a miniature assembly containing two or more crossed cavities. One possible approach was to use a three-cavity configuration to generate an interference pattern characterized by a narrow intensity peak.

[0101] Figure 8 is a perspective view of a laser phase plate 800 consisting of three cavities 802, 804, 806 with overlapping focal volumes. The electron beam 808 is shown as a vertical line. As shown in Figure 8, the cavities 802, 804, 806 were planned to be placed in a horizontal plane with their axes at 60° to each other and their focal volumes intersecting. The numerical aperture of each cavity 802, 804, 806 can be moderate. Due to the constructive interference between these cavities, each cavity 802, 804, 806 needs to have only 1 / 9 of the laser power required to generate the same phase shift using a single cavity.

[0102] It was planned to frequency lock the three cavities 802, 804, 806 to a single feed laser using the Pound-Drever-Hall method, which ensures mutual coherence of the intracavity fields.

[0103] FIG. 9A illustrates an interference pattern 900 produced by a three-cavity arrangement featuring a hexagonal lattice of interference peaks. The three-cavity mode interference pattern forms a hexagonal lattice of narrow intensity peaks with a half-maximum diameter of 382 nm (see FIG. 9A). The transmitted electron wave will be aligned to pass through the central maximum intensity peak. The additional high intensity peaks generate "shading zones" in the diffraction plane 104 with a contrast transfer function close to zero, and such zones are small and isolated. The rotationally averaged CTF for this configuration assuming a focal length f=2.5 mm is shown in FIG. 9B.

[0104] FIG. 9B plots the rotationally averaged contrast transfer function of a three-cavity phase plate (upper curve 902) compared to a single cavity (black) (lower curve 904) for f=2.5 mm. Due to the narrow central peak, it already reaches 90% of its maximum value at a spatial frequency of about ν=(20 nm)−1 and remains close to unity at higher frequencies with a minor dip due to an additional intensity peak. Thus, the three-cavity configuration should allow maximum contrast observation of particles with diameters up to about 10 nm, which is sufficient for most particles with molecular masses of 200 kDa or less.

[0105] The cavities 802, 804, 806 can be positioned with respect to the electron beam (and thus with respect to each other) by deactivating all but one of the cavities, translating this one longitudinally until the phase shift of the transmitted wave (as observed by the Thon ring shift) is maximized, and repeating this procedure for all the cavities.

[0106] Below, we present the milestones for reaching the milestone of a prototype laser phase plate and achieving continuous power of 40 GW / cm in a near-concentric optical cavity. 2 The steps for achieving laser intensity are described.

[0107] By manipulating the free-space electron wave function with a laser field, new electron-optical elements for transmission electron microscopy can be achieved. In particular, Zernike phase plates enable high-contrast imaging of soft materials, opening new opportunities in structural biology and materials science. Zernike plates can be implemented with tight, intense continuous laser focusing, which shifts the phase of the electron wave by the ponderomotive potential. Here, a near-concentric cavity is used to focus 7.5 kW of circulating laser power at 1064 nm into a 7 μm waist, setting a record for continuous-wave laser intensity and establishing the path to ponderomotive phase-contrast TEM.

[0108] Transmission electron microscopy (TEM) has emerged as a very important source of structural information with atomic resolution in both molecular biology and materials science. One limitation of TEM is that specimens composed of light elements, such as biological macromolecules, are nearly transparent to the electron beam, leading to weak image contrast. In optical microscopy, the problem of observing thin transparent objects such as biological cells has been solved by the invention of Zernike phase contrast microscopy. Introducing Zernike-type phase contrast into electron microscopy has been the goal of an increasingly intense research effort. More recently, phase contrast in TEM has been dramatically revealed using carbon foil-based phase plates. Nevertheless, significant potential for improvement still exists, as exposure to the electron beam over time changes the properties of the carbon foil, altering its contrast transfer function and limiting the time during which the phase plate can be optimally used for imaging.

[0109] Controlling free-space electron propagation with lasers offers an alternative to electron optics. Charged particles traveling through a strong laser field undergo small-scale oscillatory motion, resulting in an effective "ponderomotive" potential. Experiments using electron scattering on a standing laser wave have shown that this ponderomotive potential can be used to create diffraction gratings and beam splitters for electron beams. It has recently been proposed that a laser beam focused on the back focal plane of a TEM objective can function as a Zernike phase plate. Unlike material phase plates, laser phase plates are essentially immune to charging and electron beam damage, and impart negligible electron losses. An additional advantage is the potential for development to rapidly vary the phase retardation by varying the laser power.

[0110] Free-space manipulation of the high-energy electrons used in TEM requires very high laser intensities. The phase delay induced by a focused Gaussian laser beam can be calculated as TIFF2025041925000006.tif8170In the above equation, α is the fine structure constant, c is the speed of light, m ​​is the electron mass, β and γ are relativistic factors of the electron, P is the beam power, ω is the laser angular frequency, and w is the beam waist (see also equation (2)). Another possible requirement for the ponderomotive phase plate is that the focal spot size should not exceed a few micrometers. By equation (5), giving the electron a phase shift of π / 2 over a distance of a few microns with typical TEM energies of 200-300 keV requires laser intensities in the range of hundreds of GW / cm2. As a result, most experiments using electron scattering on light are performed with pulsed laser systems. However, continuous operation is desirable for cryo-EM and other high-resolution TEM applications where the signal-to-noise ratio is a limiting factor.

[0111] The laser power of continuous wave (CW) systems can be increased using power storage cavities. Low-loss cavity mirrors have been shown to withstand intensities up to 0.1 GW / cm2. The much higher intensities required for ponderomotive phase plates can be achieved in focusing cavities such as near-concentric Fabry-Perot resonators. In this configuration, the fundamental mode has an hourglass shape, and the laser power concentrated in a small focal spot at its center is distributed over a large area on the mirror surface, thereby preventing mirror damage. Tight intracavity focusing at low power has been demonstrated in near-concentric cavities of moderate finesse. At the same time, average circulating powers up to 670 kW have been provided in focusing cavities configured to amplify ultrashort pulse trains for intracavity harmonic generation and optical comb spectroscopy in the extreme ultraviolet spectral range. However, the combination of high power and tight cavity focusing required for laser phase plates has not yet been realized.

[0112] Here we report reaching milestones for a prototype laser phase plate implemented as a high-finesse, high-numerical aperture near-concentric cavity. Its fundamental mode is characterized and a numerical model is used to analyze its TEM properties in the presence of an intracavity laser field. At 7.5 kW circulating CW laser power, a maximum intensity of 41 GW / cm2 is revealed, sufficient to delay a 300 keV electron beam by 0.16 rad, previously achieved only with pulsed laser systems.

[0113] In the following, experimental results are reported. The optical system shown in Fig. 5 includes a near-concentric cavity 110 and a CW feed laser 522 operating at a wavelength γ = 1064 nm. The feed laser 522 in this embodiment is an external cavity diode laser, frequency locked to the cavity using the Pound-Drever-Hall method and a fiber amplifier 502. The cavity 110 is designed for insertion in a plane conjugate to the back focal plane of the TEM objective, with the electron beam entering orthogonally to the optical axis. The cavity has a diameter of 12.7 mm, a radius of curvature of 12.7 mm, and a specified reflectivity R = 1-(10±5)·10 -5 The meniscus shape allows efficient coupling into high numerical aperture cavity modes with a single aspheric lens 510.

[0114] The cavity mount (see Figs. 6 and 7) allows adjustment of the tilt and axial position of one of the mirrors 604 housed in a flexure suspension. The entire cavity housing 606 is machined from a single aluminum block to ensure accurate centering of the mirror 604 and to provide effective thermal conductivity to cool the cavity. Alignment of the near-concentric cavity 110, which requires an angular accuracy better than 1 μrad, is achieved by three fine-pitch micrometer screws that press against three piezoelectric actuators located in pockets in the aluminum block to provide coarse alignment. The high-power optical module, including the cavity 110, coupling lens 602, and mirror alignment optics, is fabricated small enough to fit into a 25 mm diameter cylindrical space, facilitating subsequent integration into a TEM system.

[0115] The cavity 110 simulates the environment of a conventional TEM cylinder and prevents unwanted ionization of air molecules. -7 The cavity 110 is suspended in a vacuum chamber evacuated to 1000 Å (mbar). The tilt and axial motion degrees of freedom of one of the mirrors were used to steer the cavity 110 into a near-concentric configuration. To characterize the size of the focus within the cavity, the laser frequency was tuned to oscillate around the fundamental mode of the cavity 110. The transmitted beam was collimated by an aspheric lens (focal length 25 mm) and steered into a CMOS image sensor. A 2D Gaussian profile was fitted to the mode image to obtain the fundamental mode width in the far field, which is the inverse of the size of the focus. This image provides a small ellipticity determined by the negligible astigmatism of the cavity mirrors. The two major axes of the ellipse are aligned along the NA a =0.0469±0.0005 and NA b This corresponds to a numerical aperture of NA = 0.0524 ± 0.0005. b The mode waist corresponding to is s=λ(πNA) -1 =6.46 μm.

[0116] The reflectivity of the cavity mirrors 114, 116 was measured using the cavity ring-down (CRD) technique, in which light is briefly injected into the optical cavity 110 and the subsequent power increase and decay of the transmitted light is observed. To avoid the need for a pulsed laser source or optical modulator, the TEM of the cavity was 00 Injection of light into the optical cavity 110 was achieved by rapidly sweeping the laser frequency across the longitudinal mode resonance of the mode (fast swept cw-CRD).

[0117] Under these conditions, the transmitted electric field amplitude is well modeled by the inverse Fourier transform such that the transmitted power of the product of the cavity transfer function and the spectrum of the linearly chirped laser field is given by: TIFF2025041925000007.tif12170In the above equation, R is the cavity mirror reflection coefficient, L is the cavity length, and η is the frequency sweep rate. Using this model, we adapted R, which serves as a target fitting parameter, to the experimentally measured CRD profile.

[0118] The measured CRD profile is shown in Figure 4 along with a least-squares best fit of this profile to the model represented by equation (6). Expressing the cavity mirror reflectivity in terms of the cavity mirror transmittance T and loss factor L as R = 1 - (T + L), the fitted profile corresponds to a cavity mirror transmittance plus loss factor of 137.9 ± 0.4 ppm. This value corresponds to a cavity finesse given by: TIFF2025041925000008.tif9150

[0119] The seed laser was locked to the cavity using the Pound-Drever-Hall method, and sidebands were generated by direct RF modulation of the seed laser current (e.g., by RF generator 1216, see FIG. 12). The reflected beam was separated by a Faraday isolator 508 and directed into a photodiode 506. The RF signal from this diode was demodulated and used as the error signal.

[0120] In order to estimate the power circulating in the cavity, in addition to the cavity finesse, the coupling efficiency and the transmission-to-loss ratio of the mirrors are needed. Both parameters are related to the cavity transmission coefficient T cav and the reflection coefficient R cav Denoting the modal overlap between the input beam and the fundamental cavity mode as Q, we obtain TIFF2025041925000009.tif8170

[0121] With the laser frequency locked to the cavity resonance, R cav =0.34±0.03, T cav = 0.32 ± 0.03 was measured. The cavity parameters were extracted, and |Q| 2 = 0.75 ± 0.05 and T / (T+L) = 0.65 ± 0.05 were obtained. Together with the CRD data, these parameters allowed the mirror transmittance T = 90 ± 7 ppm and the amplification factor to be determined as TIFF2025041925000010.tif10150

[0122] Using the determined cavity parameters, the input power was increased. With the cavity chamber held at atmospheric pressure, the input power was increased beyond 300 mW, but only 5.5 GW / cm was obtained. 2 The onset of nonlinear optical effects in air at a circulating power of about 1 kW, corresponding to a maximum intensity of 1000 Hz, does not lead to any further increase in the transmitted power, likely due to the appearance of such effects. In an evacuated chamber, intracavity powers of up to 7.5±0.6 kW were reached. The intracavity circulating power 204 (inferred from the transmitted power) as a function of input power 202 is shown in FIG. 2. The graph is approximately linear at higher powers with small deviations that may result from thermally induced deformations of the cavity housing that modify the cavity alignment. The maximum power was limited due to concerns regarding the risk of thermal damage to mirrors that were not sufficiently thermally coupled to their mounts. For the measurement mode parameters mentioned above, the maximum measured power was (41±4) GW / cm. 2This corresponds to a maximum intensity of 0.16 rad, which would result in a phase delay of 0.16 rad for a 300 kV electron beam. Repeated CRD measurements at lower powers confirmed that no damage to the mirror occurred during high power operation.

[0123] In the following, numerical modeling is described. To evaluate the effect of laser phase plates on the TEM of biological macromolecules, numerical simulations were performed to perform TEM imaging of human hemoglobin embedded in amorphous ice. This tetrameric complex has a molecular mass of about 64 kDa, which is too small for conventional TEM reconstruction, but has been resolved to a resolution of 3.2 A using phase contrast TEM with a carbon foil phase plate.

[0124] Modeling of a TEM image (not shown) of a hemoglobin molecule using a cavity-based ponderomotive phase plate was tested, showing (a) a ribbon diagram of the molecule, (b) the atomic potential, and (c) the phase shift induced by the fundamental mode of the optical cavity. A magnified image shows the individual interference fringes of the standing wave. Panel (d) shows a simulated TEM image of the molecule in the same orientation as in (a,b), without shot noise and with a shifted "ghost" image corresponding to the first diffraction order. A dose of 20 e / A produced a simulated conventional TEM image defocused by 1 pm. A focused image formed using a cavity-based ponderomotive phase plate at the same electron dose is shown. The cavity numerical aperture in this model is NA=0.05, which is consistent with the experimentally revealed parameters. The model assumes that the intracavity power is intensity-tuned to provide a maximum of 2 retardation, with the maximum value requiring another increase in the refractive power by almost 10 times.

[0125] Multislice simulations were performed using an accelerating voltage of 300 kV, a pixel size of 0.2 A, and a spherical aberration of 1.3 mm. In these simulations, the spatial and temporal finite coherence of the electron beam was neglected, since the shot noise and thermal smearing of the potential nearly determined the information limit of the simulations. The hemoglobin structure used was downloaded from the Protein Data Bank. The thermal vibration of the protein atoms was assumed to be 0.1 A and applied as an envelope function. The continuum model of amorphous ice developed by Shan and Sigworth was used to model the embedding potential around the hemoglobin structure and was numerically integrated in 3D.

[0126] The modeling results were examined: a spatial map of the phase shift induced by a Gaussian standing wave cavity mode is shown in FIG. 3, where a zoomed-in plot (see inset 304) shows the individual minima and maxima of the standing wave.

[0127] A side effect of passing the electron beam through a standing laser wave is that the standing wave acts as a diffraction grating for the electrons generating an additional, weaker "ghost" image. where n is the diffraction order, f is the focal length of the TEM objective, and λ e When is the electron wavelength, these ghost images are separated from the primary image by δx=2nfλ e / λ. First-order ghost images (not shown) rendered without shot noise are visible, but would be invisible with shot noise. Such ghost images have amplitudes well below the shot noise, so they will not be visible in the individual images and will make an insignificant contribution to the noise in the average image used to reconstruct the density map.

[0128] A simulated TEM image (not shown) of a hemoglobin molecule was investigated. Conventionally, highly transparent biopolymers are made visible by defocusing the imaging system from the sample plane 102 (see FIG. 1) and generating a phase contrast image using an oscillatory contrast transfer function. Stronger defocusing leads to stronger contrast at low spatial frequencies, but also leads to a loss of contrast at higher spatial frequencies. The 1 μm defocus used in this case is a value that still allows the reconstruction of density maps with near atomic resolution. The shot noise is typically 20 e / Å, which is used in TEM protein structure studies as an optimum between radiation damage, which increases with dose, and shot noise, which decreases with dose. 2 The model assumes an effective dose of 1000 nm. Focused images of hemoglobin using a ponderomotive phase plate at the same electron dose (not shown) were investigated. A phase shift of up to π / 2 was assumed at the intensity maximum. The phase-contrast image reveals a strong signal at low spatial frequencies compared to the defocus-contrast image, which is expected to enable classification and alignment of particle projections for macromolecules at least as small as hemoglobin.

[0129] Numerical results, examined with reference to Fig. 3, reveal that standing waves established in the focus resonator give rise to a contrast transfer function suitable for phase contrast imaging. Importantly, the well-defined spatial structure of the cavity modes ensures that the contrast transfer function can be accurately taken into account when interpreting the EM images. The experimentally revealed intensities are close to an order of magnitude smaller than those required to impart a π / 2 phase shift to a 300 keV electron beam, but could be sufficient for a first demonstration of ponderomotive retardation. Moreover, the use of state-of-the-art mirrors allows the cavity finesse to be reduced to 2·10 5 You should be able to increase the focus strength to 10 12 W / cm 2To increase the input power to well above 10 W, it should be sufficient to increase the input power to 30 W, which can be achieved using commercially available NIR wavelength fiber amplifiers.

[0130] In this work, one embodiment developed is a laser-based Zernike phase plate. However, several other tools can be considered that use high-intensity intracavity CW laser fields. For example, quantum imaging methods based on interaction-free measurement concepts have been proposed. A significant obstacle to implementing these concepts lies in the absence of high-quality beam splitters for the electronic wave function. A CW standing wave in an optical cavity can act as a highly ordered and virtually lossless phase grating, coherently splitting the electron beam into two paths by Kapitza-Dirac scattering in the Bragg regime. It is conceivable that such a beam splitter could also enable various electronic interferometry schemes that mimic different families of optical interferometers used for measurement and sensing.

[0131] Finally, it is noted that the type of cavity constructed can be relevant for a wide range of experiments. The combination of the small mode volume and the open and accessible geometry of the high NA near-concentric resonator can be advantageous for cavity QED experiments. Furthermore, the ability to accumulate very high circulating power can be used to implement ultra-deep dipole traps as well as to trap and cool nanoparticles.

[0132] Essentially, we have developed a high-finesse optical cavity 110 with a tightly focused fundamental mode. Numerical simulations verify that such a field configuration can function as a ponderomotive phase plate for a TEM. We have demonstrated that a near-concentric Fabry-Perot cavity can be used to produce tens of GW / cm in a CW laser system. 2It has been shown that light intensities in the range of 1000 nm to 1000 nm can be reached. These results represent an important step towards ponderomotive phase-contrast TEM and, more generally, pave the way towards laser-based coherent control of free-space electron wavefunctions.

[0133] Below, research results and embodiments of a transmission electron microscope 1002 with an optical cavity phase plate are described.

[0134] 10 illustrates one embodiment of a transmission electron microscope 1002 including an optical cavity phase plate according to an embodiment of the present disclosure. An electron gun 1024 generates an electron beam 1012, which is collimated by a condenser 1020, focused by a sample objective lens 1018, passed to the sample plane 102 (see FIG. 1), passed to a Fourier transform plane 1004 in a laser cavity 1008, and focused by a projection lens 1022 to generate an image 1016.

[0135] In an embodiment, the strong electric field in a tightly focused laser beam forms a phase plate (see FIG. 10). A phase plate inserted in the Fourier transform plane 1004 of an electron microscope 1002 (i.e., the back focal plane or diffraction plane 104 in FIG. 1) is constructed from a strong focused laser beam 1014 from a laser 1006.

[0136] The laser cavity 1008, an approximately spherical resonant optical cavity, is used to shape the focus and further enhance the laser beam phase plate intensity. The electron beam 1012 travels through holes at opposite ends (e.g., top and bottom) of the spherical optical cavity housing 1010. The interaction of the strong electric field of the laser beam with the charge of the electrons produces a phase shift in the electron beam that corresponds to an extension of the electron's "path length," which can be visualized as perturbing the electron trajectory to extend the travel path distance. When this extension is 1 / 4 of the electron's de Broglie wavelength, it provides the desired π / 2 phase shift.

[0137] Figure 11 illustrates the intensity pattern 1102 of a resonant optical cavity phase plate according to an embodiment of the present disclosure. The resonant enhancement of the laser beam in the spherical optical cavity housing 1010 reduces the high power optical beam requirement. This gives a narrow intensity maximum 1104 at the center (approximately 0.5 wavelength radius as shown in Figure 11) that is a good match to the size of the non-diffracting electron beam in the Fourier transform plane. Electrons outside the center see a relatively low electric field and undergo a negligible phase shift.

[0138] In a simple conventional model, the function of a laser as a phase plate is due to the laser's alternating electric field perturbing the electrons' trajectories, thus lengthening their path length. A quantum mechanical treatment shows that the phase shift is given by

[0139] TIFF2025041925000011.tif7170

[0140] where h is the reduced Planck constant, α is the fine structure constant, ρ is the photon density, λ is the laser wavelength, τ is the time it takes for an electron to traverse the focus, and m is the electron mass. Away from the optical focus at the center of the cavity, the photon density ρ drops off rapidly, and therefore there is no significant phase shift apart from the optical focus. The photon density is scaled by the laser power P as ρ=4P / (π 2 c 2 The electric field can be expressed as 1 / e with w0 = λ / 2, which is a good approximation to the intensity distribution inside the cavity. 2 Assume that the laser has a Gaussian intensity profile with an intensity "waist" radius. This profile is the minimum focus that can be achieved for a laser of a given wavelength; larger focuses are possible, but result in higher required laser power. To estimate the transit time τ, v e Using the same model, τ=λ / v is the electron beam velocity. e A more accurate estimation, taking into account the actual intensity distribution in the cavity (Fig. 11), is τ = sqrt(π / 2)λ / v eSubstituting this into equation (1), the phase is given by TIFF2025041925000012.tif10170

[0141] A laser phase plate ensures that very few electrons are lost as they traverse it, with only about one electron in a million being lost due to Compton scattering by a photon.

[0142] Using equation (9), we can calculate the laser power P required to produce a phase shift of δ=π / 2 as a function of electron velocity and laser wavelength. For a 100 keV electron microscope, the electron velocity v e is about 10 8 m / s.

[0143] According to equation (9), the desired π / 2 phase shift depends on the product of the laser wavelength and the laser beam power. It is desirable to work with the lowest possible laser power P that can be compromised with a larger laser wavelength λ.

[0144] Conversely, the laser wavelength determines the size of the laser focus, which must match the size of the non-diffracting electron beam. In particular, if the focus is too large, a loss of contrast to larger structures in the sample results. For example, note the tobacco mosaic virus, which is rod-shaped with a diameter of d = 18 nm. To achieve the maximum amount of contrast over this diameter, a phase contrast of up to 1 / (2d) = 1 / (36 nm) is required for spatial frequency. For example, the electron optical focal length in an electron microscope (e.g., FEI Titan, available from FEI Company, 5350 NE Dawson Creek Drive, Hillsboro, Oregon 97124, USA) is f = 20 mm, and the electron wavelength λ e is 3.7 picometers at 100 keV. By resolving a spatial frequency of 1 / (36 nm), λ e This leads to a maximum phase plate radius of about / (2d)=20 mm×36 nm / (3.7 pm)≒2 μm, i.e., a maximum wavelength of about 4 μm.

[0145] High power lasers at this wavelength are difficult to achieve. The choice of laser wavelength is also constrained by the availability of reliable commercial high power lasers. For example, by equation (9), a laser wavelength of 1064 nm requires about 5 kW laser power to generate the required π / 2 phase shift for a 100 keV electron beam. At this wavelength, an optical beam focus radius of about 0.5 μm is obtained. This optical beam focus radius is believed to allow phase contrast for structures with sizes up to 80 nm and meet the above mentioned requirements.

[0146] Pulsed lasers can easily achieve kW-level powers over nanosecond (ns) durations, but the use of these pulsed lasers would also require pulsing the electron beam. However, such pulsing is not available in most commercial TEM devices. Most importantly, however, limitations on the cathode current density in the electron gun introduce new challenges to the operation of electron microscopes in a pulsed mode. Therefore, continuous wave high intensity optical fields are desirable.

[0147] The intensity of a laser beam can be increased by the use of an optical cavity to achieve resonant enhancement. In such a cavity, the laser radiation is reflected back and forth between the cavity mirrors. When the laser wavelength is a half-integer multiple of the mirror separation, the radiation on all round trips adds constructively, providing a resonant intensity enhancement. This enhancement occurs when the radiation has one of the resonant frequencies given by: TIFF2025041925000013.tif7170 where n=1,2,3,... is the number of modes (the above formula holds to sufficient accuracy for cavities when the length L is much larger than the wavelength). For resonant laser light, the intensity will be enhanced by a factor given by 1 / (1-R) ​​(11) R in the above equation is the reflectivity of the mirror. A perfect reflector is considered to have a value of R=1.

[0148] In addition to the intensity enhancement, the cavity must provide a very tight focus of the laser beam. A tightly focused beam diverges rapidly, i.e. requires an optical system with a large numerical aperture (NA). Thus, the smallest focus results from the strongly diverging electromagnetic mode TEMn01 in a spherical cavity that completely encapsulates the optical beam (see Figs. 11 and 12). The electromagnetic field in a tightly focused beam is not negligible. The use of this spherical cavity has the additional advantage that the electric field in such a spherical cavity is known from analytical calculations.

[0149] With reference to FIG. 10, an optical cavity 1008 can be arranged, for example, with an electron beam 1012 traveling vertically downward (e.g., along the z-axis), entering the cavity 1008 through a hole at the top and exiting through another hole at the bottom. A laser beam 1014 is shown oriented horizontally and can have a polarization perpendicular to the plane of the figure. The electric field far from the center of the optical cavity is zero on a circle in the xy plane that includes the top and bottom of the cavity. Thus, the holes for the electron beam do not incur any discernible loss of refractive power, and these holes are large enough not to inhibit the electron flow for a radius of, for example, about 1 mm, but which can be larger or smaller. For a cavity radius of 10 mm or more, these holes will dissipate no more than 0.01% of the circulating power of the cavity. Furthermore, this allows the cavity to be constructed from two hollow hemispheres that are joined to create a single sphere after fabrication. The hemispheres are joined in the vanishing field plane so that an imperfect join will not result in any appreciable loss of optical power.

[0150] The reflectivity of the mirror surface, i.e. the high internal reflection coefficient, sets the intensity enhancement factor. Reflectivities of R=99.99% and higher with dielectric coatings, corresponding to an intensity enhancement of 10,000 times and more, are state of the art. Unfortunately, however, dielectric coatings require precise thicknesses that are difficult to achieve on highly curved surfaces such as two hemispheres, but can eventually be achievable. Metallic coated mirrors are easier to fabricate as the coating thickness is not critical. Metallic reflectivities of 99% at the first micron are currently available, giving a power enhancement of 100 times. This reflectivity can be sufficient when using the most advanced high power lasers available.

[0151] In an embodiment, the required power can be reduced by increasing the wavelength, but this will increase the spot size. At a CO2 laser wavelength of 10 μm, the power circulating in the cavity would require only 500 W for a phase shift of π / 2, and the power enhancement factor when using metal mirrors is thought to be about 200. Thus, only 2.5 W of laser power would be required. At a focus size of ≈5 pm, some loss of contrast for large-scale features of the sample would occur, as discussed above. Nevertheless, such a configuration would be simpler in terms of the required laser power.

[0152] The laser cavity 1008 can be created by joining two hemispheres with radii of, for example, 8-10 mm to form a spherical optical cavity housing 1010. The size is determined by the space available in the electron microscope for the placement of the phase plate. The hemispheres can be ground to a quadratic shape in a block of beryllium-copper to achieve good thermal conductivity and mechanical stability, or alternatively be Invar® to achieve a low thermal expansion coefficient. Note that it is not as important to maintain a precisely spherical shape, since the cavity can be intentionally distorted anyway, as explained below. The hemispheres are polished to λ / 10 and gold coated on the inside. Better polishing is not necessary due to the relatively high loss factor of the gold mirror. The hemispheres can be attached to each other by one or more piezoelectric transducers (PZTs) to tune the optical cavity for frequency stabilization at one optical frequency while suppressing other competing modes that may result in phase gratings of different standing wave periodic strengths. On the order of 50 W of laser power will be dissipated on the cavity walls. Heat can be carried away by liquid cooling. For a 10°C temperature increase (easily compensated for by liquid cooling), a hemisphere of radius 10 mm expands by approximately 160 nm for BeCu or 16 nm for Invar®. This expansion will result in a shift in the resonance frequency that must be compensated for by active feedback (described below). Furthermore, to be able to achieve resonance at any laser frequency, the dynamic range of the PZT must be at least half the laser wavelength (see FIG. 10). A gold surface at λ=1 μm has R=0.99, resulting in a power enhancement factor of 100. From equations (9, 10) and assuming a 75% power transmission from the laser to the cavity, a 67 W laser would be required. A single-frequency fiber laser currently available from IPG photonics (50 Old Webster Road, Oxford, MA 01540, USA) provides a 50 W laser at this wavelength.Since the phase contrast varies as the sine of the phase shift and therefore as the sine of the laser power, >90% of the optimal phase contrast is available with a 50 W laser. A higher power laser 1006 would operate with a lower cavity coupling efficiency, allowing for lower costs of fabricating the optical cavity.

[0153] To efficiently couple the laser radiation into the cavity, it is desirable to obtain an approximately spherically symmetric mode from the guided laser beam. This is because, in addition to the desired resonant mode with the tightest focus (see FIG. 11), the cavity can resonate with a large number of other modes due to spherical symmetry. In a precise sphere, many of these modes have the same resonant frequency as the desired mode. Thus, some distortion of the cavity, which can be done simply by adjusting the distance between the hemispheres, breaks the cavity mode degeneracy and the desired mode now has a clearly different resonant frequency. The laser radiation will mainly excite this mode if it has this frequency. Other modes that would resonate at different frequencies are made non-resonant with the cavity and are therefore suppressed.

[0154] 12 illustrates a system for generating an optical phase plate for modulating the phase of an electron beam. in Coupling of the laser beam can be achieved through the hole 1204 of r. The transmission of power from the laser 1224 into the cavity mode is optimized when it compensates for losses in other parts of the cavity (i.e., losses due to the finite reflectivity of the metal surfaces). For the desired resonant mode shown in FIG. 11, this condition is met when cav 1=(2 / 3)(4r ) where R is the radius of the cavity 1202 and R is the reflectivity of the cavity mirror. cav 2 / r m 2 )(1-R) ​​or R = 0.99 for r in / r cav =[8(1-R) / 3] 1 / 2= 0.16.

[0155] Even if this condition is met, the coupling efficiency may still not be 100%. Consider a time-reversal situation where the cavity generates light, which exits through a coupler. The coupler is small compared to the size of the TEMn01 mode resonating in the cavity. Thus, the electric field exiting through the coupler has a nearly uniform intensity across the diameter of the coupler and is nearly zero outside (this is when ignoring diffraction at the edges, which is appropriate since the coupler radius is much larger than the wavelength, and therefore diffraction effects can be practically ignored). Time-reversal defines the required shape of the laser beam with optimal power transfer entering the cavity. However, the laser generates a Gaussian mode. The optimal power transfer between the Gaussian mode and the truncated ("top hat") cavity mode is 50.4% and occurs when the waist radius parameter of the Gaussian beam at the coupler plane is equal to the coupler radius. Also assume a coupling lens of appropriate focal length. This efficiency can theoretically be improved to 100% by transforming the Gaussian beam into a uniform intensity beam with a beam shaper, which is basically a phase grating. Such techniques with efficiencies of 84-90% have been reported.

[0156] For the cavity to apply maximum intensity enhancement, the frequency of the laser must match one of the cavity resonances. To achieve resonance despite thermal and other drifts in the laser and cavity, a feedback mechanism based on the Pound-Drever-Hall method is used (e.g., as shown in FIG. 12). For this purpose, an electro-optic modulator 1206 is used, which is tuned to a frequency ω m and modulation index β mThe laser beam is then coupled through a beam shaper 1220 and a coupling lens 1222 into the cavity, and a reflected signal due to radiation exiting the optical cavity is detected by a detector 1218. An isolator 1208 can be used to couple the reflected signal to the detector 1218. In another embodiment, a beam splitter and a mirror can be used to couple the reflected signal to the detector 1218. The resonant properties of the cavity convert the phase modulation into an amplitude modulation. When the laser frequency and the resonant frequency match, the detected reflected component of the amplitude modulation (AM) reaches a minimum and ideally disappears or else a non-zero AM occurs. This detected reflected component is detected by amplitude and phase using a double balanced mixer (DBM) 1210, whose output is converted to a signal with a modulation frequency ω m , which is low pass filtered to suppress . This leads to a signal with zero phase crossing at resonance. This signal is fed back by feedback 1212 through a suitable servo 1214 to the laser frequency actuator, which keeps the cavity resonance aligned with the laser 1224. Due to the high signal power in this system, a very weak modulation may be sufficient to achieve a high signal to noise ratio feedback signal.

[0157] Many uses and applications can be envisioned. In biochemistry, significant improvements can be achieved in determining the structure of multiprotein complexes and macromolecular machines. In cell biology, significant improvements can be achieved in determining the location and spatial relationships of such complexes within whole cells. In both cases, Zernike phase contrast is expected to improve the ability to image unstained samples embedded in amorphous ice, i.e. in a life-like state.

[0158] In an embodiment, the method of use includes recording images 1016 of biopolymer and supramolecular structures. Such studies require achieving the maximum image contrast physically possible. Compared to existing defocusing methods for imaging unstained samples in a TEM, the contrast of the focused Zernike optical cavity phase contrast microscope is thought to be stronger by a factor of 10 or more (see above), without degrading or destroying the signal at high resolution as in the case of the use of defocusing.

[0159] In addition to biological applications, embodiments may characterize the microstructure of soft materials.

[0160] In an embodiment, the optical cavity phase plate can be applied to generate extremely deep three-dimensional optical traps. The trap depth can be in the range of tens or even hundreds of Kelvin, for example to trap room temperature atoms even for difficult to cool chemical species and determine their location in space more accurately than 0.5 microns. Such traps are advantageous for a wide range of atoms or molecules, since the enormous intensity in the cavity would make it pointless to closely match the laser frequency to the atomic or molecular transitions. This allows for spectroscopic measurements of very weak transitions for such atoms. One example is the nuclear spectroscopy of thorium-229 atoms using a laser. Laser spectroscopic measurements of the ≈5-6 eV transitions in the thorium-229 nucleus could be a breakthrough in precision measurements, since it allows for the construction of a "nuclear" clock based on transitions between nuclear energy levels rather than in electron shells. However, this spectroscopic measurement has been hampered by the lack of a suitable method for positioning the atoms. Only in the case of a positioned atom can the probe laser be tightly focused as required to generate sufficient intensity. The dipole trap proposed here can solve this problem and allows direct laser spectroscopic measurements of this transition, which can lead to more precise clocks (since the nuclei are less affected by the environment) as well as the examination of the time-varying nature of the fundamental "contrast" with unprecedented precision.

[0161] Providing a dielectric coating technology capable of producing a uniform and controllable dielectric layer on high NA cavity interior surfaces allows for higher resonance enhancement, thus enabling the use of lower power lasers or longer laser wavelengths at lower cost (thereby potentially enabling the use of lower NA cavities).

[0162] It can be seen that, compared to the conventional microstructured phase plates mentioned above, the resonant cavity phase plate has several advantages: it does not use any mechanical electrodes in the electron beam; the optical elements that need to be brought into the laser beam can be far enough away (1 mm or more) thus avoiding the problems associated with blurring and distorting the image; it eliminates the problem of short device lifetime that currently limits the performance of thin-film type phase plates; it eliminates the problem of partial loss of scattered electrons that occurs with both thin-film phase plates and electrostatic or magnetic phase plates; it does not cause partial loss of signal and makes the electron microscope more productive and more efficient by eliminating the interruptions caused by the need to replace microstructured or thin-film phase plates that have deteriorated over time or become contaminated when hit by the electron beam.

[0163] By converting phase contrast to amplitude contrast, resonant optical cavity phase TEM can capture significant signal from very small amounts of unstained and therefore unaltered material. The method is therefore advantageous for all research programs that use electron microscopy to determine the structure of unstained biological materials or low atomic number materials in general, including organic polymers and other soft materials. These programs include biological and materials science research, medical schools, private laboratories, or chemical companies developing new polymeric materials. Use is also likely to expand to include research institutes in the fields of biotechnology and pharmaceuticals.

[0164] 13 is a flow diagram of a method for enhancing phase contrast in an electron beam image, which can be implemented using a transmission electron microscope, an optical cavity, and a laser.

[0165] In action 1302, an electron beam is generated in a transmission electron microscope. In action 1304, the electron beam is received along an axis defined by a first mirror and a second mirror and passing through a center of an optical cavity positioned at a back focal plane of the transmission electron microscope.

[0166] Action 1306 includes accepting a laser beam into the optical cavity, where the laser beam is reflected off a first mirror and a second mirror to generate a standing wave optical phase plate, which is focused to a back focal plane to cause modulation of the electron beam.

[0167] In action 1308, the angle or position of the first mirror or the second mirror is adjusted using a piezoelectric actuator in an adjustable suspension. In action 1310, the electron beam is imaged onto an image plane of a transmission electron microscope. The image plane is positioned to receive the electron beam modulated by the standing wave optical phase plate.

[0168] The embodiments described below with reference to Figures 14-16 extend the functionality of ponderomotive phase plates by allowing control of the spatial profile of the ponderomotive phase plate through control of the polarization state of the light used for the ponderomotive phase plate. In the context of transmission electron microscopy imaging, this control can be used to eliminate the presence of image artifacts due to electron diffraction in the phase plate. For the same reasons, in one embodiment, this control can be used as a switchable electron beam splitter. Various embodiments and variations thereof described herein provide similar control over the spatial profile of the phase plate.

[0169] The spatial profile of a ponderomotive phase plate results from the spatial profile of the ponderomotive potential that the light generates. However, the spatial profile of the ponderomotive potential not only depends on the light intensity, but also on the polarization state of the light when the incident charged particles (e.g. electrons) have a significant fraction of the speed of light. In particular, the depth of the standing wave in the phase plate can be changed by rotating the polarization angle. For particles with a speed greater than 1 / sqrt(2) times the speed of light, the depth of the standing wave can be made zero.

[0170] Since the standing wave structure of the phase plate diffracts passing particles, the embodiments described herein can be used to control the amount of diffraction occurring within the phase plate. When using phase plates for transmission electron microscopy imaging, eliminating the electron diffraction effect can be desirable as it eliminates the presence of image artifacts due to electron diffraction. Eliminating the electron diffraction effect can make the process of aligning the electron beam with respect to the phase plate easier and faster since the spatial profile of the phase plate changes less rapidly. Controlling the depth of the standing wave also provides a way to control the maximum phase shift imparted to the electron beam, which adjusts the amount of image contrast enhancement provided by the phase plate for phase contrast transmission electron microscopy. It can be advantageous to use various embodiments to switch between two phase plate profiles (with or without standing waves) during normal data collection in order to more accurately reconstruct data about the sample.

[0171] Another application is as a switchable electron beam splitter (or beam splitter) due to the fact that the standing wave structure can be enabled and disabled as quickly as the polarization state of the light can be changed. One embodiment uses an electro-optic modulator to enable and disable the diffraction effect acting on the electron beam to rapidly switch the polarization of the laser light, thereby enabling and disabling the beam splitting. It is believed that the electro-optic modulator can be used in various embodiments of the ponderomotive phase plate or laser phase plate, including in a transmission electron microscope and further embodiments described below.

[0172] Yet another application is as electronic pulse slicing. A sufficiently strong laser beam completely diffracts the incident electron beam. None of the exiting electron beams travel in the same direction as the incident electron beam. All of the incident electron beams are deflected at some specific angle. If the device is working at this stage and then the polarization is switched, this action slices the electron beam. Rapid switching of the polarization results in electronic pulse slicing.

[0173] A further application is the temporal phase modulation of an electron beam focused through a single antinode of a standing wave of laser light in a laser phase plate. When the electron beam is focused and passes through the antinode of this wave, where the electric field of the standing wave is highest, the electron beam undergoes a phase shift. The phase shift depends on the polarization state of the laser light. By rapidly changing the polarization state, the amount of phase shift that the electron beam experiences from the laser light changes, and this change occurs at the frequency of the change in the polarization state of the laser light. This effectively amplitude modulates the electron beam after it has propagated some distance. A sample placed next to the electron beam experiences repulsive or attractive pulses of this beam at the frequency of the change in the polarization state of the laser light. It is believed that an apparatus so designed and operated can control the properties of an electron beam for use in studying the chemical properties of a sample or other properties of a material.

[0174] Various embodiments are provided by modifying the ponderomotive phase plate configuration described in Figures 1-13 to allow the polarization state of light fed into the Fabry-Perot optical cavity to be rotated to any angle relative to the electron beam axis. In particular, in some embodiments, a half-wave plate, a common optical element, is inserted in the path of the laser beam input into the cavity and rotated. The rotation of the half-wave plate, or more generally the rotation of the polarization angle of the laser beam through various mechanisms, can be a discrete preset value or span a continuous range and can be manually or automatically controlled in various embodiments.

[0175] These embodiments are believed to be novel for several reasons. First, it is generally non-obvious to experts in the field that the ponderomotive potential should have a polarization dependence for fast particles. However, prior to the disclosure of the present invention, there were several papers from other academic research groups that confirmed the existence of this effect in computer simulations and described potential applications. Second, the embodiments described herein are believed to be the first experiments to reveal this effect. Third, the proposed application of this effect for use in a transmission electron microscope has not been reported before.

[0176] Various embodiments have potential applications. The most likely commercial application of the present invention is in the operation of ponderomotive phase plates for image contrast enhancement in transmission electron microscopes as a method to eliminate image artifacts due to electron diffraction from the phase plate. The present group is currently in the process of entering into a collaborative research and development agreement with Thermo Fisher Scientific to further develop the ponderomotive phase plate technology described herein.

[0177] There is no known competing technology that removes the standing wave structure from a ponderomotive phase plate. The advantages of ponderomotive phase plates over other types of phase plates are described herein.

[0178] FIG. 14 depicts in schematic form yet another embodiment of a phase contrast TEM 1434 featuring a laser light with a variable polarization angle coupled into an optical cavity 1424. Varying the polarization angle of the laser light changes the image contrast enhancement of the image of the sample 1404 formed at the image plane 1428 of the transmission electron microscope 1434. Below, a mechanism for varying the polarization angle of the laser light is described with reference to FIGS. 15A-15C, and further mechanical, electromechanical, and electro-optical embodiments may be readily devised following the teachings herein. In one embodiment, the laser with variable polarization angle 1412 has an electro-optical modulator used to vary the polarization.

[0179] In operation, an electron beam 1402 of a transmission electron microscope 1434 is directed at a sample 1404 at an object plane 1406. A portion of the electron beam 1402 is diffracted by the sample 1404 and travels as a diffracted electron beam 1436. The undiffracted portion of the electron beam 1402 is focused onto a focal point 1422 of an optical cavity 1424 by an objective lens 1408, typically made of an iron-core magnetic device or electromagnetic field coils. On the other hand, the diffracted electron beam 1436 that diverges from the sample 1404 cannot be focused by the objective lens 1408 onto the focal point 1422 of the optical cavity 1424.

[0180] Polarized laser light from a laser 1412 with a variable polarization angle is coupled into an optical cavity 1424 by a coupler 1416, which in various embodiments could be air, gas, vacuum, or optical fiber, and other types of couplers could be readily devised. The optical cavity 1424 is formed by two concave mirrors 1418, 1420, which in various embodiments focus the laser light into a focal point 1422 of the optical cavity 1424 as described above. An antinode of the polarized laser light at the focal point 1422 of the optical cavity 1424 causes modulation of the electron beam 1402. Most of the diffracted electron beam 1436 scattered from the sample 1404 does not pass through the focal point 1422 of the optical cavity 1424 and is therefore not significantly modulated by the polarized laser light.

[0181] The unscattered, undiffracted portion of the electron beam 1402 focused on the focal point 1422 undergoes a phase shift due to the focused polarized laser light, whereas the scattered beam portion that does not intersect the light wave does not undergo a phase shift. The electron beam 1402 and the diffracted electron beam 1436 are directed by the projection lens 1426 to an image plane 1428 where they form an image that can be captured by an electronic camera 1430 (or other sensor in yet another embodiment). Like the objective lens 1408, the projection lens 1426 is also typically made of iron-core magnetic devices or electromagnetic field coils. Moreover, the electronic camera 1430 works on a principle similar to that of an optical electronic camera that captures pixels using, for example, a charge-coupled device (CCD) or an exposure random access memory (RAM). The phase shift difference between the unscattered beam and a particular portion of the scattered diffracted electron beam 1436 determines how much contrast enhancement the ponderomotive phase plate 1410 (or laser phase plate) applies at the image plane 1428.

[0182] Varying the polarization angle of the laser light affects how much of the standing wave structure is present in the ponderomotive phase plate 1410, which can be adjusted from a maximum to zero. The absence of standing waves results in only weak image contrast enhancement, but eliminates the presence of ghost images from the scattered diffracted electron beam 1436. When the standing waves are at a maximum, the image contrast enhancement is at a maximum, and therefore the presence of ghost images is also at a maximum. Various compromises are available in intermediate settings. Since it can be difficult to achieve alignment of the electron beam to the laser beam antinodes, one potential side effect of setting the laser polarization angle such that "ghost" images are eliminated is that such alignment can be made easier. In one embodiment, such alignment is advantageous for generating phase contrast images. Note that such advantages may be independent of other benefits noted herein.

[0183] FIG. 15A depicts a half-wave plate 1502 with a rotator 1504 for changing the polarization angle of the laser light in a phase contrast TEM embodiment and a ponderomotive phase plate embodiment. Polarized laser light from a laser 1503 passes through a half-wave plate 1502 made of a birefringent material that affects the phase angle of the polarized light passing through. The rotator 1504 sets the rotation angle of the half-wave plate 1502 and therefore the phase angle of the polarized laser light. The rotator 1504 could be implemented as a dial that could have a friction mechanism with the half-wave plate, a gear tooth mechanism that engages with gear teeth on the half-wave plate, a belt drive mechanism, or even another mechanism and could be set manually, for example, with a shaft or knob, or actuated by a motor, for example, a stepper motor or other electric motor. The motor-driven rotator 1504 could be controlled manually, for example, by a switch or button, or could be controlled automatically, for example, by a feedback path and a controller, as shown in FIG. 16.

[0184] FIG. 15B depicts a fiber optic coupler with a rotator 1512 for changing the polarization angle of the laser light in a phase contrast TEM embodiment and a ponderomotive phase plate embodiment. A collar 1506 of the fiber optic coupler secures a fiber optic fiber 1508 or cable to a laser 1503 with polarized laser light. Another collar 1510 at the other end of the fiber optic fiber 1508 or cable is rotated by a rotator 1512, which rotates this end portion of the fiber optic fiber 1508 or cable relative to the end secured to the laser 1503, thereby rotating the polarization angle of the polarized laser light from the laser 1503. There are many possibilities for how the rotator 1512 can be implemented with respect to manual, motor-driven, or automatic control similar to the rotator 1504 in FIG. 15A.

[0185] Figure 15C depicts a laser 1503 with polarized laser light and a rotator 1516 to change its polarization angle in a phase contrast TEM embodiment and a ponderomotive phase plate embodiment. By directly rotating the body of the laser 1503, the polarized laser light generated by the laser 1503 will have the same rotation angle as the body. There are many possibilities for how the rotator 1516 can be implemented, similar to the rotator 1504 in Figure 15A.

[0186] FIG. 16 depicts a schematic with actions showing a sensor 1606 at an image plane 1428 of the phase contrast TEM 1434 embodiment of FIG. 14 and the phase contrast TEM 1434 embodiment of the ponderomotive phase plate 1410 or variations thereof, and a controller 1602 analyzing a Ronchigram for use in controlling the polarization angle of the laser light. The Ronchigram is formed at the image plane 1428 and has standing wave interference fringes that vary with the polarization angle of the laser light. At a particular polarization angle for a particular electron speed, the standing wave interference fringes disappear. The sensor 1606, which in one embodiment may be an electronic camera 1430, detects the standing wave interference fringes at the image plane 1428 and sends a signal to the controller 1602. The controller 1602 performs various actions including analyzing the Ronchigram as action 1604 and controlling the polarization angle as action 1608. For example, software, firmware, or hardware in the controller 1602 could look for the strength and configuration of the standing wave fringes, direct a motor-driven rotator to change the polarization angle of the laser light, analyze the change in the standing wave fringes as the polarization angle is changed, and correlate the rotator position and associated polarization angle with the appearance of the standing wave fringes. For example, the controller 1602 could look for a polarization angle setting of the laser light that results in the disappearance of the standing wave fringes, and this setting and additional settings developed by the controller (or selected by the user) could be the phase plate profile. In one embodiment, the controller 1602 could analyze the contrast in the image to determine the tradeoff between contrast and ghost images, and then the optimal setting for the polarization angle.

[0187] FIG. 17 is a flow diagram of a method for transmission electron microscopy that can be performed by various embodiments of a TEM with a ponderomotive phase plate.

[0188] In action 1702, the TEM generates an electron beam. The electron beam can pass through the sample.

[0189] In action 1704, the electron beam is received into an optical cavity of the ponderomotive phase plate, and an undiffracted, unscattered portion of the electron beam is focused to a focal point of the optical cavity.

[0190] In action 1706, a laser beam having polarized laser light is injected into the optical cavity to form a standing wave optical phase plate and cause modulation of the electron beam. Suitable mechanisms and techniques for doing so are described above.

[0191] Action 1708 images the electron beam into an image plane of the TEM. When a sample is used, the image shows the sample through a phase contrast transmission electron microscope. In addition, the image also shows a Ronchigram with standing wave interference fringes (which may be zero amplitude for certain settings of tilted polarization of the laser light as a degenerate case).

[0192] In action 1710, the polarization angle of the laser light is changed to change the contrast enhancement of the image. In various embodiments, it is contemplated that this change can be accomplished by manual adjustment or automatic adjustment by the system.

[0193] Although the disclosure of the present invention and its advantages have been described in detail, it should be understood that various changes, substitutions, and alterations can be made therein without departing from the spirit and scope of the disclosure of the present invention as defined by the appended claims. Moreover, the scope of this application is not intended to be limited to the particular embodiments of the processes, machines, manufacture, compositions of matter, means, methods, and steps described herein. Those skilled in the art will readily appreciate from the embodiments of the present disclosure that existing or later developed processes, machines, manufacture, compositions of matter, means, methods, or steps that perform substantially the same function or produce substantially the same result as the corresponding embodiments described herein can be utilized in accordance with the present disclosure. Accordingly, the claims are intended to include within their scope such processes, machines, manufacture, compositions of matter, means, methods, or steps.

[0194] In the foregoing specification, the invention has been described with reference to specific embodiments. However, those skilled in the art will recognize that various modifications and changes can be made without departing from the scope of the invention as set forth in the following claims. The specification and figures are therefore to be regarded in an illustrative rather than a restrictive sense, and all such modifications are intended to be included within the present invention. [Explanation of symbols]

[0195] 1302 The action of generating an electron beam in a transmission electron microscope 1306 The action of accepting a laser beam into an optical cavity 1404 Sample 1424 Optical Cavity 1434 Phase contrast TEM

Claims

1. a transmission electron microscope (TEM) having a back focal plane; a plurality of mirrors forming an optical cavity, the focal point of the optical cavity being positioned at the back focal plane of the TEM, the optical cavity being positioned to allow an electron beam provided by the TEM to pass through the focal point of the optical cavity, the optical cavity being operable to accept a laser beam; a laser coupled to the optical cavity and operable to provide a laser beam of a specified wavelength and variable polarization angle incident on the optical cavity, the laser beam being reflected off the plurality of mirrors to provide a standing wave optical phase plate focused at the back focal plane of the TEM, thereby causing modulation of the electron beam; an image plane of the TEM positioned to receive the electron beam modulated by the standing wave optical phase plate to form an image responsive to the variable polarization angle; A system comprising:

2. A half-wave plate; a rotator arranged to hold and rotate the half-wave plate to provide the variable polarization angle of the laser light; 2. The system of claim 1, further comprising:

3. a fiber optic member coupling the laser to the optical cavity and being bendable or rotatable to provide the variable polarization angle of the laser light; 2. The system of claim 1, further comprising:

4. an electronic camera or one or more sensors positioned at the image plane and operable to analyze a Ronchigram to provide feedback for automatic control of the variable polarization angle of the laser light; 2. The system of claim 1, further comprising:

5. 2. The system of claim 1, wherein the polarization angle of the laser light is variable between at least a first phase plate profile having a standing wave in a Ronchigram formed at the image plane and a second phase plate profile having no standing waves in the Ronchigram.

6. 10. The system of claim 1, wherein the polarization angle of the laser light has two or more preset values.

7. 10. The system of claim 1, wherein the polarization angle of the laser light has one or more of manual adjustment or automatic adjustment.

8. generating an electron beam in a transmission electron microscope (TEM) having a back focal plane; receiving the electron beam along an axis passing through a center of an optical cavity defined by a first mirror and a second mirror positioned at the back focal plane; accepting a laser beam having a variable polarization angle of laser light into the optical cavity, the laser beam generating a standing wave optical phase plate that is reflected off the first mirror and the second mirror and focused onto the back focal plane of the TEM, thereby causing modulation of the electron beam; imaging the electron beam onto an image plane of the TEM positioned to receive the electron beam modulated by the standing wave optical phase plate to form an image; varying the polarization angle of the laser light; The method according to claim 1, further comprising:

9. rotating a half-wave plate to provide the variable polarization angle of the laser light; 9. The method of claim 8, further comprising:

10. rotating or bending a fiber optic member coupling a laser to the optical cavity to provide the variable polarization angle of the laser light; 9. The method of claim 8, further comprising:

11. analyzing the Ronchigram formed in the image plane based on the output of a sensor or electronic camera; controlling the variable polarization angle of the laser light based on the analyzing step; 9. The method of claim 8, further comprising:

12. said varying the polarization angle of said laser light to vary the contrast enhancement of said image; The step of varying the polarization angle of the laser light includes: changing between a first phase plate profile having a standing wave in a Ronchigram formed at the image plane and a second phase plate profile having no standing waves in the Ronchigram; Including, 9. The method of claim 8.

13. The step of changing the polarization angle of the laser light includes: determining the polarization angle of the laser light based on two or more preset values; Including, 9. The method of claim 8.

14. The step of changing the polarization angle of the laser light includes: determining the polarization angle of the laser light based on manual or automatic adjustment; Including, 9. The method of claim 8.

15. a first concave mirror and a second concave mirror positioned to define an optical cavity positionable to pass the electron beam to a focal point of the optical cavity; a laser having a variable polarization angle of laser light coupled into said optical cavity and having a standing wave with an antinode at said focal point, thereby operable to cause a variable modulation of said electron beam that is controllable by said variable polarization angle of said laser light; A ponderomotive phase plate comprising:

16. a transmission electron microscope (TEM) having the focal point of the optical cavity positioned at a back focal plane; an imaging device positioned at an image plane of the TEM and operable to form an image having variable image contrast enhancement controllable by the variable polarization angle of the laser light; 16. The ponderomotive phase plate of claim 15 further comprising:

17. a half-wave plate positioned to couple laser light into the optical cavity; a rotator operable to rotate the half-wave plate such that the laser light has the variable polarization angle responsive to a rotation of the half-wave plate when incident on the half-wave plate and when coupled into the optical cavity to provide the standing wave with the antinode at the focal point.

16. The ponderomotive phase plate of claim 15 further comprising:

18. a fiber optic coupler positioned to couple the laser to the optical cavity; a rotator operable to rotate one end of the fiber optic coupler relative to an opposite end of the fiber optic coupler to change the polarization angle of the laser light relative to the optical cavity; 16. The ponderomotive phase plate of claim 15 further comprising:

19. a rotator coupled to the laser and operable to rotate the laser to change the polarization angle of the laser light relative to the optical cavity; 16. The ponderomotive phase plate of claim 15 further comprising:

20. further comprising an electron beam source; The laser having a variable polarization angle of laser light is operable as a switchable electronic beam splitter or an electronic pulse slicing device using the first concave mirror and the second concave mirror.

16. The ponderomotive phase plate of claim 15.

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