Use of polarization states to control the 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 U.S.C. § 119(e) of U.S. Patent Application No. 62 / 901,160, filed September 16, 2019, which is thus incorporated herein by reference in its entirety. This application also claims the benefit of priority from U.S. Provisional Patent Application No. 62 / 479,044, filed March 30, 2017, which is also incorporated herein by reference in its entirety, and relates to the specification of U.S. Patent Application No. 15 / 939,028, filed March 28, 2018, and granted U.S. Patent No. 10,395,888, granted August 27, 2019.

[0002] [Statement of government support] This invention was made with government support under contract number DE-AC02-05CH11231, given by the U.S. Department of Energy, and under award number R01GM126011, given by the National Institute of General Medical Sciences of the National Institutes of Health. The government has certain rights to this invention.

[0003] The disclosure of this invention relates in general to transmission electron microscopy (TEM), and more specifically to optical cavity phase plates for TEM. [Background technology]

[0004] While modern transmission electron microscopes have become powerful imaging tools, achieving resolutions approximately 1000 times higher than optical microscopes, their imaging performance for thin biological samples remains relatively low. Such samples are weakly scattering "phase objects," meaning they exhibit virtually no absorption of incident electrons. Consequently, the intensity of the transmitted electron beam remains equal to that of the incident beam, and a complete image of such objects lacks contrast. Historically, electron microscopy has therefore required special preparation of samples, such as heavy metal "staining," to provide contrast. These procedures are difficult and time-consuming, and furthermore, they are known to alter the structure, thus limiting the resolution from 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 transmitted electrons. As Zernike discovered with respect to optical microscopy, invisible phase modulation can be converted into visible amplitude contrast. Light passing through the sample is decomposed into non-diffracted and diffracted components. The non-diffracted light is focused by the objective lens to a high-intensity spot at the center of this plane. The diffracted light is arranged around this center. Diffraction due to the fine structure of the object results in a larger diffraction angle. Therefore, diffraction orders corresponding to fine details in the image (i.e., having small dimensions) are located far from the center, while large structures cause diffracted light closer to the center. Mathematically, the intensity distribution in this back-focus plane is given by the spatial Fourier transform of the sample's transmittance, which is called the Fourier transform plane.

[0006] When the sample is a pure phase object, a spatial phase relationship exists between these components. By offsetting these phase relationships, phase modulation is converted to amplitude modulation. The maximum conversion and therefore the maximum phase contrast is obtained using 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 covered so that light passing through a small area in the center undergoes an extra phase shift.

[0007] Unfortunately, there is no simple phase plate for an electron beam, which makes it difficult to observe unstained biological samples. A partial solution is provided by cryo-electron microscopy. These avoid the occurrence of artifacts associated with staining and generate a certain amount of phase contrast by observing the sample under intentionally defocused conditions in combination with intentional spherical aberration. By optimizing the compromise between the phase distortion due to defocus and that due to spherical aberration, a desirable conversion of phase to amplitude contrast can be achieved. However, the phase shift varies continuously across the spectrum of spatial frequencies. As a result, this "simple" method works well for small features within the image, but contrast is lost for larger features. Since it is necessary to have substantial contrast even for large features in order to view biological macromolecules, it is often necessary to use a much larger amount 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. Defocus is thus an imperfect way to generate phase contrast within the image of biological macromolecules.

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

[0009] More recently, microfabrication techniques have made it possible to construct electron microscope phase plates. The focused non-diffracted beam is passed through small holes in electrodes that are biased by tens to hundreds of millivolts depending on the specific electrode shape within the device, thereby resulting in the desired phase shift. The electrostatic shielding of the electrodes prevents interaction with scattered electrons, so that they do not receive an additional phase.

[0010] Another embodiment of the phase plate uses long, very thin rod magnets placed across the electron diffraction pattern in the immediate vicinity of the non-diffracting electron beam. The phase shift is generated by the Aharonov - Bohm effect due to the difference in the magnetic vector potential on either side of the long rod magnet.

Prior Art Documents

Patent Documents

[0011]

Patent Document 1

Patent Document 2

Patent Document 3

Summary of the Invention

Problems 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, which is likely due to the accumulation of contamination on the surface when the device is bombarded by a powerful electron beam. This causes an unwanted electric field, which leads to uncontrolled phase shifts in the electron beam at various scattering angles. This makes the image substantially irreproducible and uninterpretable. Also, the electrodes block the diffraction beam closest to the center, i.e., reduce the contrast for large structures in the image. Thin - film phase plates face similar problems.

Means for Solving the Problems

[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 in the back focal plane of the TEM. The optical cavity is positioned to allow the electron beam provided by the TEM to pass through the focal point of the optical cavity. The optical cavity is operable to accept a laser beam. A laser having a variable polarization angle of 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 for entering the optical cavity. The laser beam is reflected from the mirror and provides a standing-wave optical phase plate focused in the back focal plane of the TEM. The standing-wave optical phase plate causes modulation of the electron beam. The image plane of the TEM is positioned to accept 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 (TEM) generates an electron beam. The TEM has a back focal plane. The electron beam is received along an axis passing through the center of an optical cavity. The optical cavity is positioned in the back focal plane. The optical cavity is defined by a first mirror and a second mirror. A laser beam with a variable polarization angle of 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 focused in 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 the image plane. The image plane is positioned to receive the electron beam modulated by the standing-wave optical phase plate. Changing 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 and second concave mirrors are positioned to define an optical cavity. The optical cavity is positionable to allow an electron beam to pass through its focal point. The laser has a variable polarization angle of laser light. The laser and laser light are coupled to the optical cavity. The laser is operable to have a standing wave of laser light with a wave antinode at the focal point of the optical cavity to cause 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, along with the accompanying drawings illustrating the principles of the embodiments described.

[0017] The embodiments described and their advantages can be best understood by referring to the following description together with the accompanying drawings. These drawings do not limit any modifications of form and detail that can be made to the embodiments described by those skilled in the art without departing from the spirit and scope of the embodiments described. [Brief explanation of the drawing]

[0018] [Figure 1] This figure schematically depicts the shape of an embodiment of a phase-contrast TEM in which a constricted waist optical cavity mode is focused on the back focal plane of the TEM objective lens, and the delay is applied to the transmitted wave rather than the scattered wave. [Figure 2] This figure illustrates the experimental measurement dependence of the circulating power within the cavity on the input laser power, with gray shading indicating measurement uncertainty. [Figure 3] The inset shows the interference fringes of a standing wave, with the intensity distribution of the fundamental modes of a near-concentric Fabry-Perot cavity plotted and extended horizontally. [Figure 4] This figure shows the contrast transfer function (CTF) of a TEM with a cavity-based laser phase plate averaged over the azimuthal angle. [Figure 5]This is a schematic diagram of an embodiment of a laser system including an internal cavity and coupling optical system of an electron microscope cylinder, a feeding laser system, and a cavity characterization tool. [Figure 6] In one embodiment, this is a cross-sectional view of an optical cavity including a housing, a cavity mirror, and a coupled aspherical lens. [Figure 7] This diagram illustrates a cavity mount in one embodiment, in which the wire shown is connected to a piezoelectric actuator used for precise alignment of a cavity mirror. [Figure 8] This is a perspective view of a laser phase plate composed of three cavities having overlapping focal volumes in one embodiment. [Figure 9A] This diagram illustrates the interference pattern generated by a three-cavity configuration characterized by a hexagonal grid of interference peaks. [Figure 9B] This figure shows the rotation-averaged contrast transfer function (upper curve) of a 3-cavity phase plate compared to a single-cavity (black) (lower curve) for f=2.5mm. [Figure 10] This figure shows one embodiment of a transmission electron microscope including an optical cavity phase plate according to an embodiment of the disclosure of the present invention. [Figure 11] This figure shows the intensity pattern of a resonant optical cavity phase plate according to an embodiment of the disclosure of the present invention. [Figure 12] This figure shows a system for generating an optical phase plate for modulating the phase of an electron beam according to an embodiment of the disclosure of the present invention. [Figure 13] This is a flowchart illustrating a method for enhancing phase contrast in electron beam images. [Figure 14] This figure shows a schematic shape of yet another embodiment of a phase-contrast TEM, characterized by a laser beam coupled to an optical cavity and having a variable polarization angle. [Figure 15A] This figure shows a half-wave plate 1502 having a rotator for changing the polarization angle of laser light in an embodiment of a phase contrast TEM and an embodiment of a ponderomotive phase plate. [Figure 15B]This figure depicts an optical fiber coupler having a rotator for changing the polarization angle of laser light in an embodiment of a phase contrast TEM and an embodiment of a ponderomotive phase plate. [Figure 15C] This diagram illustrates a laser having polarized laser light and a rotating device for changing the polarization angle in an embodiment of a phase contrast TEM and an embodiment of a ponderomotive phase plate. [Figure 16] Figure 14 is a schematic diagram showing an action involving a sensor in the image plane of a phase contrast TEM or a variation thereof, and a controller for analyzing a ronchigram used to control the polarization angle of the laser light, in an embodiment of a phase contrast TEM and an embodiment of a ponderomotive phase plate. [Figure 17] This is a flowchart of a method for transmission electron microscopy that can be performed by various embodiments of a TEM having a ponderomotive phase plate. [Modes for carrying out the invention]

[0019] This specification describes laser phase plates having optical cavities and phase-contrast transmission electron microscopy using the same. Figures 1 to 13 relate to optical cavity-based ponderomotive phase plates for transmission electron microscopy. Figures 14 to 16 relate to the use of polarization states for controlling the ponderomotive phase plate. Examples of these specific embodiments are illustrated in the accompanying drawings. Certain modifications are described with respect to these specific embodiments, but this description is not intended to limit to these embodiments described. Rather than limiting, it is intended to cover alternatives, modifications, and equivalents that may be included in the spirit and scope defined by the claims.

[0020] The following description provides numerous specific details to offer a complete understanding of the embodiments of the disclosure of the present invention. Certain exemplary embodiments can be implemented without using some or all of these specific details. In other instances, known processing operations are not described in detail so as not to unnecessarily obscure the disclosure of the present invention.

[0021] Various technologies and mechanisms of the embodiments disclosed in this invention will be described below, sometimes in singular form, for clarity. However, it should be noted that some embodiments include multiple iterations of the technology or multiple embodiments of the mechanism, unless otherwise specified.

[0022] The terms “approximately” or “nearly” are synonyms and are used to indicate that the value they modify has an understood range that can be ±20%, ±15%, ±10%, ±5%, or ±1%. The term “substantially” is used to indicate that a value is close to a target value, which can mean, for example, that the value is within 80%, 90%, 95%, or 99% of the target value.

[0023] This specification describes refractive power amplification cavities suitable for use as phase masks for electron beams in electron-based imaging systems or electron-based spectroscopic measurement systems, such as transmission electron microscopes (TEMs). Depending on the configuration, such phase masks can function as several electron-optical elements for coherent manipulation of the electron beam, including: • Phase plate for Zernike-type phase-contrast electron microscopy, • Bragg beam splitter for coherently splitting an electron beam into two with a controllable splitting ratio. • A transmission diffraction grating for an electron beam that splits the input beam into several diffraction orders. • A time-phase modulator similar to the electro-optic modulator used in conventional optical microscopes. A local time-phase modulator that can be used as a beam diaphragm for dark-field electron microscopy when used in conjunction with an energy filter. A spatiotemporal phase modulator similar to the acousto-optic modulators (deflectors) used in conventional optical microscopes.

[0024] The design of one embodiment of a power-amplifying optical cavity includes a plurality of 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 placed thereon. The rear (convex) surfaces of these mirrors may have an anti-reflective coating placed thereon. The in-cavity laser field configuration generates an effective potential for electrons, which transfers a spatially patterned phase shift, which can also be time-modulated, onto the electron beam passing through the cavity.

[0025] The advantages of optical cavity electron-optical elements over conventional elements based on material structure (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 in the case of all previous devices that guide 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 to 360 degrees; and (3) negligible electron loss (undesirable electron scattering) in the optical cavity element.

[0026] The 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 irradiation rather than with pulsed electron guns (as in the case of earlier pulsed laser phase plates). This feature makes optical cavity phase plates particularly suitable for use in standard continuous electron emission microscopes and applications where very high electron exposures are used. Cavity-based systems are also characterized by a precisely controllable phase shift pattern determined by the laser field configuration of the cavity modes. The absence of distortions and defects commonly present in the laser beam profile is highly desirable in avoiding undesirable electron scattering caused by anomalies in the laser field.

[0027] The features of an optical cavity phase plate may include the following: • Near-concentric Fabry-Perot cavity configuration or other cavity configuration featuring a high-intensity focus. • Highly reflective dielectric coating on curved surfaces, Arrangement of three or more piezoelectric actuators for aligning the cavity and controlling the cavity mode shape, • Phase plates for TEM characterized by a phase-shift profile, which should be an extended pattern having multiple maxima and minima that induce a contrast transfer function suitable for a particular purpose, rather than a single spot (for example, a standing wave with several wavelength waists can be used as a Zernike phase plate, while larger waists are preferred for functioning as a Bragg beam splitter). • Phase plate for TEM based on electron-optical interaction (one embodiment is a nearly spherical cavity with a high-finesse high numerical aperture (high NA) mirror), • A mirror having a front (concave) surface and a rear (convex) surface concentric with the front (concave) surface for efficient coupling to the cavity mode. • A mirror having a rear surface on which an anti-reflective coating is placed to improve coupling efficiency. • Simultaneous input of the laser field into the cavity at several corresponding (resonant) frequencies to provide time modulation and time control of the phase pattern applied to the electron beam.

[0028] Feedback and alignment mechanisms and methods for optical cavity phase plates may include the following: • A feedback mechanism to maintain the shape and size of the mode (e.g., a charge-coupled device (CCD) camera and / or segmentation detector that monitors the transmitted beam, reflected beam, or both), A feedback topology in which the laser frequency remains locked to the cavity following both a change in cavity length and a systematic change, or alternatively, the cavity length is changed so that the cavity's resonant frequency remains locked to the laser frequency. A method for aligning a cavity with an electron beam by monitoring a TEM image, its Fourier transform, or other mathematical constructs to determine the degree of mismatch and provide feedback for alignment. • Feedback to the cavity mode can help keep the laser phase plate aligned with the beam. • The step of injecting the master laser using a laser beam that penetrates the cavity (for example, as a frequency locking mechanism).

[0029] Further features of the optical cavity phase plate may include: • Heat pipes for removing heat from the cavity. • Heat shield to protect the cavity structure, • Control of cavity length by temperature, • Controlling the mirror curvature by controlled preheating using an auxiliary laser, heating circuit, or thermoelectric device. • By using a multi-cavity configuration, the spot size can be reduced and the intensity increased. • Using optical fibers to bring the laser to the cavity, • Protecting the laser system from reflected light using an optical isolator. • To avoid scattering of the electron beam, the laser's focal size must be larger than the electron's focal size. • Controlling the configuration of the cavity field by deforming the mirror (for example, by gradually crushing it).

[0030] Additional uses / features may include: • Changing the laser power to adjust the amount of phase shift (this feature can be used to invert the contrast or adjust it otherwise). • The ability to use an optical cavity phase plate for high electron irradiation doses within the TEM (generally, a Voltaic phase plate cannot be used for high electron irradiation doses). • By modulating the laser power within the cavity, high-speed modulation of the phase shift can be induced (this can lead to a modulated electron wave function that can be used to send an oscillating or pulsed electromagnetic field to a microscope object (i.e., a form of spectroscopy)), • By using sideband formation at the focal point in combination with an energy filter, the transmitted (i.e., non-diffractive) electron beam can be substantially focused toward a full-field dark-field TEM. • Lossless, controllable electron beam splitter.

[0031] Various embodiments have some or all of the following features: • To generate a small focal point inside the cavity, at least one of the mirrors has a reflective surface with a radius of curvature of 5 cm or less. • The reflectivity of each mirror, including the cavity, is approximately 0.9 or greater. The mirror, including the cavity, has a reflective surface covered with a reflective dielectric coating. • Multiple mirrors include a first mirror and a second mirror. A cavity housing in which an optical cavity is located, and one or more of the mirrors, including the cavity, are mounted within an adjustable suspension, the adjustable suspension being operable to adjust the angle and position of the mirrors. • The adjustable suspension includes a piezoelectric actuator. The power of the laser beam is amplified by approximately 10 times or more by the cavity.

[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 a concentric convex surface with respect to the front surface of the mirror. For example, one or both rear surfaces of the mirrors may be flat. In some embodiments, one feature that defines a cavity suitable for a phase plate is that (contrary to typical optical cavities) the cavity can focus the beam to a small focal point, while simultaneously amplifying the laser power by a large factor and being able to withstand the high power obtained. This is not seen in known optical cavities. Features present in the optical cavity with respect to these embodiments include (1) a short radius of curvature of at least one of the mirrors, and (2) low round-trip losses resulting in high reflectivity / low absorptivity of all mirrors.

[0033] An advantage of optical cavity phase plates in cryo-electron microscopy (CryoEM) is that the phase contrast can be set to 100% of the specified value at the start of data acquisition. Furthermore, the phase contrast can remain at the specified value throughout the data acquisition period, which may last 24 hours or longer for each CryoEM session. This means that all parts of the acquired data can be considered to be of equal quality, in contrast to some data being considerably worse than others. Yet another advantage of optical cavity phase plates includes greater diversity and reliability. In addition, it is theoretically expected that a 270-degree phase shift instead of 90 degrees will result in better performance.

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

[0035] Recently, transmission electron microscopy (TEM) of unstained, flash-frozen biological samples (low-temperature EM) has become a primary source of structural information in molecular biology. The lower limit of the molecular mass of accessible particles has been reduced to 100–200 kDa, and in some cases, below 100 kDa. Theoretical considerations suggest that it should be possible to reconstruct particles as small as approximately 40 kDa. Nevertheless, determining the structures of polymeric complexes with sizes between 40 kDa and 100–200 kDa, as well as addressing larger and more flexible complexes, requires further improvements in low-temperature EM technology.

[0036] One avenue for TEM reconstruction of smaller particles is the development of Zernike phase plates, which enable phase-contrast imaging without defocusing. A key step in this direction was the development of the "Volta" phase plate.

[0037] However, there remains a significant opportunity 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 acquisition. Only a moderate number of images can be acquired while the phase shift is within the usable range of 40–120°, after which the carbon film must be moved to expose new spots. At suboptimal phase shifts, many images must be acquired. Even within the same phase shift, the phase shift and its drift rate differ from spot to spot. These characteristics make it difficult to use carbon foil phase plates in low-temperature EM structural studies that require high-yield image acquisition with consistent parameters.

[0038] Placing material structures in the path of high-energy electron beams inevitably leads to charging and degradation. Therefore, time-varying behavior is an unavoidable drawback of phase-contrast devices based on material objects within the electron stream of a TEM. An additional drawback is the small but discernible 18% electron scattering within the Voltaic phase plate.

[0039] Here, we propose circumventing this limitation by using a strong laser focus as an electron wave delay device. Such a laser-based phase plate or "ponderomotive" phase plate does not require a material object 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 feature virtually no electron loss. The phase shift can be changed as desired by changing the laser power.

[0040] A central concept in various embodiments is that a laser beam focused on the back focal plane of the TEM objective lens 1018 (see the embodiment in Figure 10, described in more detail below) can be used 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 the laser phase plate allows the Cryogenic EM Society to take full advantage of the improved image contrast achieved with Zernike phase contrast TEM.

[0041] The physical mechanism of electron delay is described below. The goal of the embodiments described here is to construct a better Zernike (quarter-wavelength) plate for phase-contrast TEM by controlling the phase of the electron wave using a laser. This concept is drawn from the field of atomic physics, where lasers are used to manipulate both the motion and internal degrees of freedom of a collective of cold atoms. These methods enable many experiments aimed at verifying the foundations of quantum mechanics and general relativity, exploring the fields that give rise to dark energy, and precisely measuring fundamental constants.

[0042] The physical principle behind laser-based coherent control of electron waves can be explained from the standpoint of classical physics as follows: Relativistic electrons traveling through a strong oscillating laser field undergo small-scale tremors, resulting in an effective "ponderomotive" potential. This potential is expressed by the following equation: In the above equation TIFF0007837591000001.tif8170, I is the laser intensity, e and m are the charge and mass of the electron, ε0 is the permittivity of vacuum, c is the speed of light, and ω is the optical frequency. This repulsive potential imposes a negative phase shift (delay) on the passing electrons.

[0043] A more rigorous explanation of the interaction between relativistic electrons and strong laser beams can be given from the standpoint of quantum electrodynamics (QED), where stimulated Compton scattering is the cause of the phase shift. In this process, electrons either absorb a photon from the laser beam and then re-emit this photon through stimulated emission induced by the same beam, or they undergo these two events in opposite directions. Electrons subjected to stimulated Compton scattering from a monochromatic laser beam emit with the same energy at which the scattering began, without losing coherence. In contrast, spontaneous Compton scattering causes electrons to lose coherence, but the fraction of electrons undergoing such an event is 10 under conditions appropriate to various embodiments. -7 To that extent. The inventors focused on the fact that electrons may be inelastically scattered by higher-order scattering processes involving 3 or 4 or more photons, thereby potentially losing coherence. However, these processes are performed when the laser intensity is I≒10 13 W / cm 2 It is not expected to become significant until it reaches (for a laser wavelength of 1 μm) and far exceeds the intensity processed by these embodiments.

[0044] The required laser intensity is explained below. The proportionality constant between the ponderomotive potential and laser intensity in equation (1) is small, requiring high laser intensity. Power P and focus waist w0 (beam radius) are 1 / e of the peak intensity. 2For an electron beam passing orthogonally through a focused Gaussian beam (which is measured in this case), the phase shift is given by the following equation: In the above equation TIFF0007837591000002.tif8170, α is the fine structure constant, and β = v e / c and v e γ = (1-β) 2 ) -1 / 2 Therefore, to estimate the required refractive power, the electron energy U = 300 keV is substituted, as is commonly used in low-temperature EM studies. According to equation (2), at a beam waist with laser wavelength λ = 1064 nm and w0 = 7 μm, the laser power required to produce a maximum phase shift of 90° is P ≈ 300 kW.

[0045] While such refractive power is easily achieved in pulsed laser systems, the pulsed phase plate is thought to require an additional synchronous pulsed electron gun 1024 (see Figure 10). The main objective of these embodiments is to develop a laser phase plate that is compatible with conventional low-temperature EM equipment based on continuous-wave (CW) laser systems.

[0046] The following explains how to enhance the resonant field in an optical cavity.

[0047] Figure 1 shows a schematic shape of an embodiment of a phase-contrast TEM. A constricted waist optical cavity mode is focused within the back focal plane of the TEM objective lens, for example, within the diffraction plane 104, and a delay is applied to the transmitted wave 106 rather than the scattered wave 108. Electrons from the scattered wave 108 are detected by the electron detector 112 and imaged in a high-contrast view of the sample in the sample plane 102 as a result of the delay in the transmitted wave 106.

[0048] In some embodiments, the laser intensity is increased by resonance in a high-finesse near-concentric Fabry-Perot cavity (see optical cavity 110 in Figure 1). This type of resonator features an hourglass-shaped fundamental mode with a small waist and a large spot size on the surfaces of mirrors 114, 116. The small waist concentrates the power within the high-intensity focus and confines the phase shift to a narrow region around the focus of the transmitted wave 106. The large spot size on mirrors 114, 116 prevents laser-induced destruction of the mirror coatings and helps diffuse the thermal load over a wider area. Furthermore, two opposing waves propagating within the Fabry-Perot cavity form standing waves, with the intensity quadrupled at the wave antinodes. With a wavelength λ of 1064 nm and a beam waist of w0 = 7 μm, a cavity circulating power of P = 75 kW 204 is sufficient to achieve a maximum phase shift of 90°.

[0049] Recent research toward the aforementioned goals has revealed a near-concentric cavity operating at a wavelength of λ=1064nm with a finesse of F=22,000. Precise alignment of the near-concentric cavity was achieved using a specially designed flexible base mount with three piezoelectric actuators, thereby enabling a waist of 7μm (radius at 1 / e2 intensity).

[0050] Figure 2 illustrates the experimental measurement dependence of the cavity circulating power 204 on the input laser power 206. The gray shaded area 206 represents measurement uncertainty. With an input power of P0 = 2.2 W 202, the cavity reached a circulating power 204 of P = 7.5 kW, corresponding to a recorded CW light 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 tenfold power increase is considered sufficient to achieve the goal of giving a 90° phase shift.

[0051] The laser field configuration and contrast transfer function are described below.

[0052] Figure 3 depicts the intensity distribution 302 of the fundamental mode of a nearly concentric Fabry - Perot cavity. The horizontally extended inset 304 illustrates the interference fringes of the standing wave. In an ideal Zernike phase plate, a 90° delay is applied only to the transmitted wave focused at the center of the electron diffraction plane 104, and there is no phase shift within the remainder of this plane. This embodiment of the cavity - based laser phase plate has a Gaussian standing - wave intensity profile (shown in Figure 3) and applies a harmonic - modulation phase shift to the striped region of the diffraction plane 104. Below, the results of this phase profile and why this phase plate substantially provides the function as a Zernike phase plate in cryo - EM single - particle analysis will be discussed.

[0053] Phase contrast extends to spatial frequencies low enough (≒5 nm or less) to image small protein complexes. The "cut - off" frequency at which phase contrast becomes effective is determined by the contrast transfer function (CTF) of the imaging system. The correspondence between the spatial frequency ν and the radius r within the back - focal plane of the TEM objective system is given by the following equation. TIFF0007837591000003.tif6170In the above equation, f is the focal length of the TEM objective lens, and 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 U0 = 300 keV) and f = 2.5 mm. In the initial stage of the development of the embodiment, including the development of the first prototype and initial data collection, a FEI Titan TEM with a relay optical system added to magnify the electron diffraction pattern on the surface of the phase plate was used. This additional magnification results in an effective focal length of f = 20 mm, which relaxes the requirements for the precision of laser focusing.

[0054] Figure 4 illustrates the cavity-based phase contrast (CTF) of a TEM, calculated and averaged over azimuthal angles for an electron microscope. For clarity, this graph shows the CTF at zero defocus and does not account for spherical aberration. At very low spatial frequencies of 404, this curve begins with zero contrast. CTF402 reaches 50% at low spatial frequencies of 404 up to approximately 1 / (240 nm), a scale set by equation (3), where r = λ / 2, representing the size of the standing wave interference fringes (see Figure 2). The oscillations are caused by the interference fringes of the standing laser wave. CTF reaches nearly maximum contrast (>80%) at frequencies greater than 1 / (10 nm), a scale set by the radius of the laser beam. Thus, D ≈ 0.5ν -1 For particles of 5 nm or smaller, the information contained within the electronic phase is transmitted with nearly maximum contrast. Many polymers with molecular masses smaller than 100-200 kDa have a diameter of approximately 5 nm or less. Cavity-based phase plates provide nearly ideal contrast for these polymers. Larger particles (100 nm) are imaged with only a very moderate reduction in contrast.

[0055] Image artifacts resulting from standing wave phase profiles do not significantly distort particle images. Multislice numerical modeling of low-temperature EM imaging of hemoglobin, a 64 kDa particle embedded in 30 nm amorphous ice, has confirmed that cavity-based phase plates function well for small particles. The structure of the hemoglobin molecule was downloaded from the Protein Databank. 20 e / Å 2 The irradiation dose was used. The acquired image shows significant contrast enhancement by the laser phase plate compared to the defocused image generated with a 1 μm defocus.

[0056] Numerical modeling enables the evaluation of artifacts that may arise from the anisotropic phase shift profile of the laser waveplate. A study was conducted on images, including hemoglobin, simulated using an ideal phase plate that is thought to phase-shift only transmitted waves and images using a cavity-based laser phase plate. This simulation highlights any difference by not showing shot noise or noise from changes in ice density. Even with this highlighting, only slight vertical streaking is discernible. The simulation displayed the difference between the two images on a grayscale, amplified tenfold until this difference signal reached a visible level. This difference is far below the noise level and therefore is 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 electron waves, 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 with a shift of up to 90°, the amplitude of such ghost images is 20e / Å. 2 The irradiation is well below shot noise. In high-density samples, it can be expected that superposition occurs between the ghost image of one particle and the primary image of another particle. However, the ghost images are too weak to be visible in individual micrographs and are expected to cancel each other out and average out from the fused data, resulting in only a negligible contribution to noise in the average image used for density map reconstruction.

[0058] Adapting laser phase plates to standard low-temperature EM systems requires more precise laser focusing. Later, embodiments were developed, including a prototype of an advanced phase plate with a narrower focus suitable for use with conventional TEM systems having a typical focal length f=2.5mm. To achieve this, the numerical aperture of the cavity modes was increased to operate the cavities near concentricity. In one approach, it was unclear whether this operation degraded cavity finesse. Another approach, less risky, employed a multi-cavity configuration requiring additional steps of cavity alignment and frequency locking. Both concepts are described in the "Methods" section.

[0059] Essentially, we proposed developing a Zernike phase plate for a TEM operating at 300kV based on the ponderomotive potential of a high-intensity CW laser circulating within an optical cavity. Sufficient laser intensity and a tenfold increase (considered to provide an optimal 90° phase shift) for a ~9° phase shift have been shown to be within the reach of current mirror fabrication techniques. Numerical simulations demonstrate that a cavity-based phase plate can be effectively used as a Zernike phase plate for low-temperature EM single-particle reconstruction. Below, we detail a method involving a continuous-wave laser phase plate module with sufficient laser intensity to shift the phase of a 300keV electron beam by 90°.

[0060] To construct a laser phase plate based on a near-concentric cavity, a specially designed, highly curved cavity mirror with high reflectivity and low loss is required. Furthermore, an appropriate feed laser and optics-mechanical system are employed to maintain cavity alignment. The system embodiment must also be compact, vacuum-compatible, and non-magnetic to operate within a TEM cylinder. The following outlines a method for constructing a system that meets these requirements.

[0061] The following describes an embodiment of the laser system.

[0062] Figure 5 is a schematic diagram of an embodiment of a laser system including a cavity 110 in an electron microscope cylinder 526, a coupling optical system, a feeding laser system, and a cavity characterization tool. Regarding the acronyms of the system components, FA = fiber amplifier 502, FC = fiber coupler 504, PDH PD = pound-Dreber-Hall-lock photodiode 506, FI = Faraday isolator 508, CL = coupling lens 510, CM = cavity mirror 512, BS = beam splitter 514, FPD = high-speed photodiode 516, and DA = for example, a camera 524 and a digital data acquisition unit 518 for the high-speed photodiode 516. In one embodiment, an Nd:YAG laser wavelength (1064 nm) was selected to allow the use of a commercially available narrow-linewidth laser and a highly optimized optical coating. The low-power (<100mW) master laser 522 is amplified using a ytterbium fiber amplifier (FA) 502 from IPG Photonics or Nufern. This provides single-mode, single-frequency, constant-polarization (PM) amplification up to 50W.

[0063] The laser beam is coupled into a high-power single-mode pm fiber from NKT Photonics, which has the capability to conduct a refractive power of at least 20 W at 1064 nm. This power is delivered to a small assembly mounted on a microscope, which 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 modes). A camera 524 and a photodetector, e.g., FPD 516, observe the mode shape transmitted through cavity 110 for optimization purposes and to measure cavity finesse using cavity ring-down spectroscopy.

[0064] As recent studies have revealed, the master laser frequency is stabilized ("locked") to the resonance of the optical cavity 110 using the Pound-Dreber-Hall method. For this purpose, the portion of light reflected from the cavity 110 is detected by a photodetector (PDH PD) 506 and used by a PDH lock circuit 520 to control the laser frequency.

[0065] The cavity mirror 512 is described below. For a Fabry-Perot cavity manufactured with two identical mirrors 512, the power amplification M is determined by the transmission coefficient T and reflection coefficient R of the cavity mirror, as well as the mode overlap integral Q, given by the following equation. TIFF0007837591000004.tif7170

[0066] Since |Q|≦1, power enhancement is limited by the quality of the cavity mirror, with reflectivity having to be as close to 1 as possible and transmittance having to be as high as possible. The two coefficients mentioned above are constrained to R+T+L=1 by the law of conservation of energy, in which case the loss rate L=S+A includes the scattering rate S and the absorptivity A. Absorption is generally a property of the dielectric used to produce the mirror coating. Generally, absorption is less than parts per million (ppm) in near-infrared reflective coatings produced by ion beam sputtering (IBS) by companies such as Advanced Thin Films, Inc. and Research Electro-Optics (both located in Boulder, Colorado, USA).

[0067] On the other hand, scattering primarily depends 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) when δh is the root mean square (RMS) surface roughness. 2 / λ 2 It can be approximated as follows. Methods for polishing flat fused silica substrates are well developed, so surface roughness of less than 0.1 nm and scattering of about 3 ppm are always obtained. However, polishing high-curvature concave mirrors required for near-concentric cavities remains difficult.

[0068] One embodiment utilizes a recently developed polishing process that enables achieving an RMS surface roughness of approximately 0.1–0.2 nm on a substrate having a short radius of curvature of about 10 mm. This should result in an S well below 10 ppm. In one embodiment, the mirror 512 is coated with a state-of-the-art high-reflectivity IBS coating from either Advanced Thin Films or Five Nines Optics. Based on these figures, these mirrors support amplification coefficients in the range of M = 8,000–13,000.

[0069] If the IBS coating process used by the aforementioned coating companies does not work well on new substrates produced by Perkins Precision, one plan is to purchase Mirror 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 amplification factors of M = 4,000–6,000. If neither supplier appears likely to be able to meet the performance targets specified in their proposal, a second alternative option exists: purchasing Mirror 512 available from Advanced Thin Films, which has a larger radius of curvature and can support a mode waist of 8–10 μm along with a power amplification factor of 10,000.

[0070] The mirror's transmittance coefficient will be measured immediately before installation into the cavity 110. The reflectance of the mirror 512 will be measured using the ring-down spectroscopy technique used for this type of cavity.

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

[0072] Any scratch in the central part of the Mirror 512 will increase the round-trip loss rate, so larger mode sizes on the mirror surface necessitate special attention to surface quality. 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] The cavity optical mechanism is described below.

[0074] Figure 6 is a cross-sectional view of an optical cavity in one embodiment, which includes a housing 606, a plurality of cavity mirrors 604, and a coupled aspherical lens 602. The optical system on the right is housed in a flexible suspension that can be tilted or moved in the axial direction.

[0075] The alignment of a near-concentric resonator and a small mode waist is highly susceptible to the relative positions and orientations of these mirrors 604, as the desired configuration is very close to the degeneracy point when a series of modes have the same frequency and the resonator becomes unstable. In this configuration, a small angular shift of the mirrors 604 results in significant lateral displacement 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 performed with submicron precision.

[0076] Figure 7 shows a cavity mount in one embodiment. The wire 702 shown connects to a piezoelectric actuator (located in the housing 606) used for precise alignment of the cavity mirror 604.

[0077] For various embodiments, it was planned to replicate the cavity mount used in previous studies on near-concentric resonators (see Figures 6 and 7). It was shown that a flexible suspension machined 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 inside a TEM cylinder using a cylindrical access port with a diameter of φ25 mm. For optical bench experiments, cavity 110 was positioned inside a small vacuum chamber with input and output anti-reflective coated laser-quality optical ports. One of the mirrors 604 was fixed within the mount, while the position of the second mirror 604 was controlled using a piezoelectric actuator in combination with three fine-pitch screws for coarse alignment. The entire mount assembly was made of non-magnetic materials (aluminum body, 316 stainless steel screws with silicon carbide tips, and piezoelectric ceramic).

[0078] The following describes thermal control. Operation under vacuum presents additional challenges in removing heat dissipated within the cavity. Absorption into the IBS coated mirror is only 1-3 ppm of the circulating power; therefore, each of the two cavity mirrors 604 must dissipate up to 200 mW in the resonant state. Care must be taken to remove the heat accumulated within the cavity mirror to avoid thermal deformation. Numerical simulations show that the thermal conductivity of the fused silica substrate limits the temperature increase at the center of the mirror to approximately 30 K, and contact with the mirror manufacturer confirms that a 30 K temperature increase is unlikely to cause mirror damage.

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

[0080] However, the light scattered on the mirror surface introduces an additional thermal load, which is absorbed within the cavity. Assuming that at least half of the input power is either transmitted or reflected, it is necessary to remove up to 10W of power radiated within the laser module. This challenge is further complicated by the need to suspend the cavity on a support structure approximately 200mm long in a vacuum. While the aluminum cavity mount body should provide sufficient thermal conductivity to limit the temperature increase to 10K, it was planned to actively control the cavity temperature using a thermoelectric cooler to avoid changes in cavity alignment driven by temperature changes. It was planned to use a TE Technology thermoelectric set (cooling plate and controller) capable of absorbing up to 28W and maintaining the temperature well within the setpoint of 0.1K.

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

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

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

[0084] The refractive force circulating within the cavity will be at least 75 kW. This power will be calculated using the measured transmission coefficient of the output cavity mirror and the transmitted power measured using a calibrated power meter.

[0085] The system measures 2.10 using a vacuum gauge. -7 It will operate in a vacuum chamber with a pressure not exceeding mbar.

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

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

[0088] The insertable laser module will be made of non-magnetic materials, i.e., aluminum alloy (cavity suspension system), piezoelectric ceramic, or fused silica (mirrors, lenses), to avoid disturbing the magnetic field of the TEM electron optics. The small micrometer screws chosen for use are made of 316 series stainless steel with silicon carbide ball tips. If the extremely low magnetic permeability of stainless steel is found to disturb the TEM alignment, titanium screws can be used as an alternative option.

[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 the optical system alignment with respect to the electron beam can be achieved by moving the entire insertable optical module without disturbing its internal alignment.

[0090] The insertable module will be suspended within a mount that allows for triaxial positioning of the module relative to the TEM cylinder. For laser focusing, the module must be positioned vertically within 100 μm of the diffraction plane 10⁴. This ensures that the transmitted electron wave traverses the high-intensity region when focused to a size smaller than the interference fringes of the laser wave. The horizontal position in the direction perpendicular to the cavity optical axis must be controlled within 1 μm. This ensures that any mismatch is considerably smaller than the waist of the laser beam. Finally, the horizontal position along the cavity axis must be more stable than 100 nm. Since the full width at half maximum of the standing wave interference fringes is only 266 nm, this stability ensures that the non-scattered electron beam passes through the highest point of the standing wave. Meanwhile, all maxima within approximately 50 μm around the beam waist have roughly the same intensity (as shown in Figure 5), and any of these maxima can be used to delay the transmitted wave. The suspension system will incorporate piezoelectric actuators to maintain a stable longitudinal position of the cavity.

[0091] A two-step procedure was planned to position the cavity focus at the center of the conjugate Fourier plane. First, for rough alignment, a φ1 mm circular opening cut out within the cavity mount was planned to be used to pass the electron beam through, so that the center of the cavity focus was positioned below the electron beam when no sample was inserted. This position could be found by moving the assembly and observing the TEM image, taking care that the electron beam was shielded by the edge of the beam opening during this process. Next, for finer alignment, an amorphous carbon film could be used as the sample. By observing the Fourier transform of the properly defocused image, the position of the laser focus relative to the center of the TEM Fourier plane was revealed. Using the image as a guide, the center of the cigar-shaped focus could be guided to the center of the Fourier plane.

[0092] To fine-tune the module position along the cavity axis, it was planned to observe the Thon rings again using defocused images of amorphous carbon. When a non-scattered beam passes through the nodes of a standing wave, the Thon rings should have their usual pattern. However, when the non-scattered electron beam is phase-shifted by the standing wave, these rings will be shifted by the same phase, providing quantitative feedback for fine-tuning.

[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 precise alignment of near-concentric resonators is easily disturbed by vibration and thermal expansion. Alignment can be manually restored on the optical bench using a piezoelectric actuator. However, to benefit the TEM user base, the phase plate needs to be operable without frequent human intervention.

[0095] One goal was to overcome 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 using a CCD camera that monitors the laser beam passing through the cavity, and the necessary adjustments are straightforward. When cavity 110 is nearly perfectly aligned, there are three main misalignment modes: (i) pitch tilt and (ii) yaw tilt of the mode relative to the cavity axis, and (iii) change in the numerical aperture of the mode. These three modes can be measured by observing the power laser beam using a camera and fitting a Gaussian distribution relating two coordinates of the centroid and beam width to its intensity profile. The coordinates 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 thus the mode waist). For these purposes, piezoelectric actuators are incorporated within the mirror mount. Within the optical table, adjustments are only required once every few operating hours, so the bandwidth of this feedback system does not need to be high. Assuming that the vibration isolation of the TEM system is at least as good as that of the optical table, it was thought that adjustments would not need to be made very frequently, and therefore, it was expected that standard computer interface tools would be more than sufficient to provide the necessary bandwidth.

[0096] We considered that it may be necessary to periodically check the alignment of the non-scattered electron beam with respect to the highest point of the steady-state laser wave by monitoring the Thon ring pattern of the observed image. One embodiment of this feature is 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, the laser phase plate can be used either in the back focal plane or in any suitable conjugate plane on the cylinder of the TEM.

[0098] Higher numerical apertures require highly curved mirrors that cannot be addressed by the polishing processes currently used for high-finesse mirrors, such as those planned for use in various embodiments. However, the advanced process of ion beam milling has the capability to do this in principle. For near-concentric cavities, high numerical apertures of around 0.36 (at λ=1064 nm) have been reported, corresponding to a mode waist of w0=0.94 μm. Cavity-based pondelomotive phase plates with similarly precise focus are thought to enable phase-contrast imaging with a standard TEM having a focal length of f=2.5 mm.

[0099] However, ion beam milling produces surfaces that are not sufficiently smooth for low-loss mirrors, requiring superpolishing before reflective coatings can be applied (some cavities have been reported to have a finesse of only about 600). In the past, it was unclear whether ion-milled substrates with very high numerical apertures could be polished to a surface roughness small enough to be suitable for the manufacture of high-finesse cavity mirrors. Furthermore, even if the substrate was sufficiently smooth, it was not certain at the planning stage whether a highly reflective dielectric coating could be applied sufficiently uniformly to such high numerical aperture mirrors.

[0100] If the direct approach of increasing the numerical aperture was unsuccessful, another embodiment decided to use a small assembly containing two or more crossing cavities. One possible approach was to use a three-cavity configuration to generate an interference pattern characterized by narrow intensity peaks.

[0101] Figure 8 is a perspective view of a laser phase plate 800, which consists of three cavities 802, 804, and 806 having overlapping focal volumes. The electron beam 808 is shown as a vertical line. As shown in Figure 8, the cavities 802, 804, and 806 were planned to be arranged 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, and 806 can be of a moderate magnitude. Due to constructive interference between these cavities, each cavity 802, 804, and 806 only needs to have 1 / 9 of the laser power required to produce the same phase shift using a single cavity.

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

[0103] Figure 9A illustrates the interference pattern 900 generated by a three-cavity configuration featuring a hexagonal grid of interference peaks. The interference pattern of the three-cavity modes forms a hexagonal grid of narrow intensity peaks with a half-maximum diameter of 382 nm (see Figure 9A). The transmitted electron wave is aligned to pass through the central maximum intensity peak. Additional high-intensity peaks create "shielding zones" within the diffraction plane 104 where the contrast transfer function is close to zero; such zones are small and isolated. The rotation-averaged CTF for this configuration, assuming a focal length f = 2.5 mm, is shown in Figure 9B.

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

[0105] Cavities 802, 804, and 806 can be positioned relative to the electron beam (and therefore relative to each other) by shutting down all but one cavity, translating this one longitudinally until the phase shift of the transmitted wave (observed by the Thon ring shift) is maximized, and repeating this procedure for all cavities.

[0106] The following describes the steps to reach the achievement target for the prototype laser phase plate, and the continuous 40 GW / cm² output within the near-concentric optical cavity. 2 This section explains the steps involved in achieving laser intensity.

[0107] By manipulating the free-space electron wave function using a laser field, novel electron-optical elements for transmission electron microscopy can be achieved. In particular, Zernike phase plates enable high-contrast imaging of soft materials and are expected to lead to new opportunities in structural biology and materials science. Zernike plates can be implemented using precise and strong continuous laser focusing, which shifts the phase of the electron wave by a ponderomotive potential. Using a near-concentric cavity, a circulating laser power of 7.5 kW at 1064 nm was focused into a 7 μm waist, establishing a record for continuous wave laser intensity and paving the way for ponderomotive phase-contrast TEM.

[0108] Transmission electron microscopy (TEM) has emerged as a crucial source of structural information with atomic resolution in both molecular biology and materials science. One limitation of TEM is that samples composed of light elements, such as biomacromolecules, are nearly transparent to the electron beam, resulting in weak image contrast. In optical microscopy, the problem of observing thin, transparent objects such as living cells was solved by the invention of phase-contrast microscopy by Zernike. Introducing Zernike-type phase contrast into electron microscopy has been the goal of increasingly intense research efforts. 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 alters the properties of the carbon foil, changing the contrast transfer function and limiting the time during which the phase plate can be optimally used for imaging.

[0109] Controlling free-space electron propagation using lasers provides an alternative to electron optics. Charged particles traveling through a strong laser field undergo small-scale vibrational motion, resulting in an effective "pondelomotive" potential. Experiments using electron scattering with stationary laser waves have shown that this ponderomotive potential can be used to generate 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 lens can function as a Zernike phase plate. Unlike material phase plates, laser phase plates are essentially unaffected by charging and electron beam damage and impose only negligible electron losses. An additional advantage is the potential for rapid variation of the phase delay by changing the laser power.

[0110] Free-space manipulation of high-energy electrons used in TEM requires extremely high laser intensity. The phase delay induced by a focused Gaussian laser beam can be calculated as follows: In the above equation, α is the fine-structure constant, c is the speed of light, m ​​is the electron mass, β and γ are the 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 size must not exceed a few micrometers. Equation (5) shows that giving electrons a π / 2 phase shift over a distance of a few microns at a typical TEM energy of 200-300 keV requires laser intensities in the range of several hundred GW / cm2. Consequently, most experiments using electron scattering for light are performed using pulsed laser systems. However, continuous operation is desirable for low-temperature EM and other high-resolution TEM applications where the signal-to-noise ratio is a limiting factor.

[0111] The laser power of a continuous-wave (CW) system can be amplified using a power storage cavity. Low-loss cavity mirrors have been shown to withstand intensities up to 0.1 GW / cm². The much higher intensities required for a ponderomotive phase plate can be achieved within a focus cavity, such as a near-concentric Fabry-Perot resonator. In this configuration, the fundamental mode has an hourglass shape, and the laser power, concentrated within a small focal point at its center, is dispersed over a large area on the mirror surface, thereby preventing mirror damage. Strict in-cavity focusing at low power has been demonstrated with near-concentric cavities of moderate finesse. Simultaneously, average circulating power up to 670 kW has been provided within a focus cavity configured to generate in-cavity harmonics and amplify ultrashort pulse trains for optical comb spectroscopy in the extreme ultraviolet spectral range. However, the combination of the high power required for a laser phase plate and tight cavity focusing remains unrealized.

[0112] Here, we report reaching the achievement benchmark for a prototype laser phase plate implemented as a high-finesse, high-aperture, near-concentric cavity. Its fundamental modes were characterized, and the TEM characteristics were analyzed using a numerical model in the presence of the laser field within the cavity. At a 7.5 kW circulating CW laser output, a maximum intensity of 41 GW / cm² was revealed, sufficient to delay a 300 keV electron beam by 0.16 rad, a feat previously only achievable with pulsed laser systems.

[0113] The experimental results are reported below. The optical system shown in Figure 5 includes a near-concentric cavity 110 and a CW-fed laser 522 operating at a wavelength γ = 1064 nm. In this embodiment, the fed laser 522 is an external cavity diode laser and is frequency-locked to the cavity using a pound-Dreber-Hall method and a fiber amplifier 502. The cavity 110 is designed for insertion into a plane conjugate to the back focal plane of the TEM objective lens, and the electron beam enters in a direction perpendicular 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 It is formed by two concave mirrors 512 (Layertec) having a convex rear surface and concentric with the front surface. This meniscus shape allows for efficient coupling to a high numerical aperture cavity mode using a single aspherical lens 510.

[0114] The cavity mount (see Figures 6 and 7) allows for adjustment of the tilt and axial position of one of the mirrors 604 housed in a flexible suspension. To ensure precise centering of the mirrors 604 and to provide effective thermal conductivity for cooling the cavity, the entire cavity housing 606 is machined from a single aluminum block. Alignment of the near-concentric cavity 110, which requires 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 rough alignment. The high-power optical module, including the cavity 110, the coupling lens 602, and the mirror alignment optical mechanism, is manufactured small enough to fit into a 25 mm diameter cylindrical space, thereby facilitating subsequent integration into a TEM system.

[0115] Cavity 110 simulates the environment of existing TEM cylinders and prevents undesirable ionization of air molecules 2.10 -7 It is suspended in a vacuum chamber reduced to mbar. The cavity 110 was guided to a near-concentric configuration using the tilt and axial motion degrees of freedom of one of the mirrors. To characterize the size of the focus within the cavity, the laser frequency was adjusted to oscillate around the fundamental mode of cavity 110. The transmitted beam was parallelized by an aspherical lens (focal length 25 mm) and guided into a CMOS image sensor. A two-dimensional Gaussian profile was applied to the mode image to obtain the fundamental mode width at the far field, which is the reciprocal of the size of the focus. This image provides a small ellipticity determined by the slight astigmatism of the cavity mirror. The two principal axes of the ellipse are NA a =0.0469±0.0005 and NA b This corresponds to a numerical aperture of 0.0524 ± 0.0005. b The corresponding mode waist is s=λ(πNA) -1 = 6.46 μm.

[0116] The reflectivity of cavity mirrors 114 and 116 was measured using the cavity ring-down (CRD) method, which involves briefly injecting light into the optical cavity 110 and observing the subsequent increase and attenuation of the transmitted light's power. To avoid the need for a pulsed laser source or optical modulator, the cavity was subjected to TEM. 00 The injection of light into the optical cavity 110 was achieved by rapidly sweeping the laser frequency across the longitudinal mode resonance of the mode (high-speed swept cw-CRD).

[0117] Under these conditions, the transmitted electric field amplitude can be well modeled by an inverse Fourier transform such that the transmitted power, which is the product of the cavity transfer function and the spectrum of the linear chirped laser field, is given by the following equation. In the above equation (TIFF0007837591000006.tif12170), R is the cavity mirror reflection coefficient, L is the cavity length, and η is the frequency sweep rate. Using this model, R, which functions as a target fitting parameter, was adapted to the experimentally measured CRD profile.

[0118] The measured CRD profile is shown in Figure 4 along with its least-squares optimal fit to the model represented by equation (6). If the cavity mirror reflectance is expressed as R = 1 - (T + L) with respect to the cavity mirror transmittance T and loss rate L, the fitted profile corresponds to a cavity mirror transmittance plus loss rate of 137.9 ± 0.4 ppm. This value corresponds to the cavity finesse given by the following equation. TIFF0007837591000007.tif9150

[0119] A seed laser was locked into the cavity using the Pound-Dreber-Hall method, and sidebands were generated by direct RF modulation of the seed laser current (e.g., by RF generator 1216; see Figure 12). The reflected beam was isolated by Faraday isolator 508 and guided into photodiode 506. The RF signal from this diode was demodulated and used as an error signal.

[0120] To estimate the power circulating within the cavity, in addition to cavity finesse, coupling efficiency and the permeability-to-loss ratio of the mirror are required. Both parameters are related to the permeability coefficient T of the cavity. cav and the reflection coefficient R cav This can be inferred from the measured values. If we denote the mode overlap between the input beam and the fundamental cavity mode as Q, the following equation can be derived. TIFF0007837591000008.tif8170

[0121] With the laser frequency locked to the cavity resonance, R cav =0.34±0.03, T cav A measurement of =0.32±0.03 was performed. The cavity parameter was extracted, and the result was |Q| 2 The values ​​obtained were =0.75±0.05 and T / (T+L)=0.65±0.05. By combining these with the CRD data, it became possible to determine the mirror transmittance T=90±7ppm and the amplification coefficient using the following equations. TIFF0007837591000009.tif10150

[0122] Using the determined cavity parameters, the input power was increased. With the cavity chamber held at atmospheric pressure, even when the input power was increased beyond 300 mW, the output power remained at 5.5 GW / cm². 2 At a circulating power of approximately 1 kW, corresponding to the maximum intensity, the transmission power does not lead to any further increase in transmitted power, likely due to the manifestation of a nonlinear optical effect in the air. In the exhausted chamber, the cavity power reached up to 7.5 ± 0.6 kW. The circulating power in the cavity 204 (estimated from the transmitted power) as a function of the input power 202 is shown in Figure 2. This graph is nearly linear at higher powers, with small deviations that may be caused by thermally induced deformation of the cavity housing correcting the cavity alignment. The maximum power was limited due to concerns about the risk of thermal damage to the mirror that was not sufficiently thermally coupled to the mount. For the measurement mode parameters described above, the maximum measured power was (41 ± 4) GW / cm². 2This corresponds to the maximum intensity, which is thought to result in a phase delay of 0.16 rad for a 300 kV electron beam. Repeated CRD measurements at low power have confirmed that no damage occurred to the mirror during high-power operation.

[0123] The numerical modeling is described below. To evaluate the effect of laser phase plates on TEM of biomolecules, numerical simulations of TEM imaging of human hemoglobin embedded in amorphous ice were performed. This tetrameric complex has a molecular mass of approximately 64 kDa, which is excessively small for conventional TEM reconstruction, but it is resolved to a resolution of 3.2 Å using a phase-contrast TEM with a carbon foil phase plate.

[0124] A model of the TEM image (not shown) of a hemoglobin molecule using a cavity-based ponderomotive phase plate was tested, showing (a) the ribbon diagram of the molecule, (b) the atomic potential, and (c) the phase shift caused by the fundamental mode of the optical cavity. The 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), with no shot noise and 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 experimentally determined parameters. This model assumes that the cavity power is intensity-tuned to produce a delay of up to 2 at its maximum value, which requires another increase of approximately 10 times the refractive power.

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

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

[0127] A side effect of passing an electron beam through a stationary laser wave is that the stationary wave acts as a diffraction grating for the electrons, generating an additional weak "ghost" image. n is the diffraction order, f is the focal length of the TEM objective system, and λ e When the electron wavelength is such that these ghost images are obtained from the first image by δx = 2nfλ e The displacement is / λ. A first-order ghost image rendered without shot noise (not shown) is visible, but is considered invisible if shot noise is present. Since the amplitude of such ghost images is well below the shot noise, these ghost images are not visible within the individual images and make an insignificant contribution to the noise in the average image used to reconstruct the density map.

[0128] We examined a simulated TEM image (not shown) of a hemoglobin molecule. Conventionally, very transparent biomolecules are made visible by defocusing the imaging system from the sample plane 102 (see Figure 1) and generating a phase-contrast image using an oscillation-contrast transfer function. Stronger defocusing results in stronger contrast at low spatial frequencies but leads to contrast loss at high spatial frequencies. The 1 μm defocus used in this case is a value that still allows for the reconstruction of density maps at near-atomic resolution. This shot noise is generally used in TEM protein structure studies as an optimal point between radiation damage that increases with dose and shot noise that decreases with dose, at a value of 20 e / Å. 2 This model assumes an effective dose. A focused image of hemoglobin (not shown) using a ponderomotive phase plate was examined at the same electron dose. A maximum phase shift of π / 2 was assumed at the maximum intensity. This phase-contrast image reveals a stronger signal compared to the defocus-contrast image at low spatial frequencies, and this strong signal is expected to enable the classification and alignment of particle projection images for macromolecules small at least as small as hemoglobin.

[0129] Numerical results, as examined with reference to Figure 3, revealed that the standing waves constructed within the focus resonator generate a contrast transfer function suitable for phase-contrast imaging. Importantly, the clearly defined spatial structure of the cavity modes ensures that the contrast transfer function can be accurately considered when interpreting the EM image. The intensity revealed experimentally is by an order of magnitude smaller than the intensity required to impart a π / 2 phase shift to a 300 keV electron beam, but it may be sufficient for the first demonstration of the ponderomotive delay. Furthermore, using state-of-the-art mirrors, the cavity finesse can be reduced to 2.10 5 It should be possible to increase the focus intensity to 10. 12 W / cm 2To increase the signal strength well beyond the limit, increasing the input power to 30W should suffice, and this increase can be achieved using a commercially available NIR wavelength fiber amplifier.

[0130] In this study, one embodiment developed is a laser-based Zernike phase plate. However, several other tools using a CW laser field in a high-intensity cavity can be considered. For example, quantum imaging methods based on the interaction-free measurement concept have been proposed. A major obstacle to implementing these concepts lies in the absence of a high-quality beam splitter with respect to the electron wave function. A CW standing wave in an optical cavity can function as a highly regular and virtually lossless phase diffraction grating, coherently splitting the electron beam into two paths by Kapitza-Dirac scattering in the Bragg region. Such a beam splitter could also enable various electron interferometry schemes that simulate various sequences of optical interferometers used for measurement and sensing.

[0131] Finally, it should be noted that the type of cavity constructed may be relevant to a wide range of experiments. The combination of a small mode volume and an open, accessible shape of a high-NA near-concentric resonator can be advantageous for cavity QED experiments. Furthermore, its ability to store very high circulating power can be used to implement ultradeep dipole trapping as well as to trap and cool nanoparticles.

[0132] Essentially, this involves the development of a high-finesse optical cavity 110 with precisely focused fundamental modes. Numerical simulations have verified that such a field configuration can function as a ponderomotive phase plate for TEM. Tens of GW / cm² in a CW laser system using a near-concentric Fabry-Perot resonator. 2It has been shown that light intensities in this range can be reached. These results represent an important step towards ponderomotive phase-contrast TEM and, more generally, pave the way for laser-based coherent control of free-space electron wave functions.

[0133] The following describes research results and embodiments of a transmission electron microscope 1002 having an optical cavity phase plate.

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

[0135] In this embodiment, a strong electric field within a precisely focused laser beam forms a phase plate (see Figure 10). The phase plate, inserted into the Fourier transform plane 1004 (i.e., the back focal plane or diffraction plane 104 in Figure 1) of the electron microscope 1002, is composed of a strongly focused laser beam 1014 from the laser 1006.

[0136] The laser cavity 1008, a nearly 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 located at opposite ends (e.g., top and bottom) of the spherical optical cavity housing 1010. The interaction between the strong electric field of the laser beam and the electron charge generates a phase shift within the electron beam corresponding to an extension of the electron's "optical path length," which can be visualized as causing the electron trajectory to fluctuate to lengthen the propagation path distance. A desirable π / 2 phase shift is provided when this extension is 1 / 4 of the electron's de Broglie wavelength.

[0137] Figure 11 illustrates the intensity pattern 1102 of a resonant optical cavity phase plate according to an embodiment of the disclosure of the present invention. Resonant enhancement of the laser beam within the spherical optical cavity housing 1010 reduces the requirements for a high-power optical beam. This gives a narrow intensity maximum value 1104 (radius of about 0.5 wavelengths as shown in Figure 11) at the center, which is a good match to the size of the non-diffracted electron beam in the Fourier transform plane. Electrons outside the center encounter a relatively low electric field and undergo only a negligible phase shift.

[0138] In a simple conventional model, the function of the laser as a phase plate stems from the alternating electric field of the laser causing fluctuations in the electron orbits, and thus extending the electron path length. Quantum mechanical treatments have shown that the phase shift is given by the following equation.

[0139] TIFF0007837591000010.tif7170

[0140] In the above equation, 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 required for an electron to cross the focus, and m is the electron mass. As we move away from the optical focus at the center of the cavity, the photon density ρ decreases sharply, and therefore, no significant phase shift exists outside the optical focus. The photon density is given by ρ = 4P / (π) when c is the speed of light and the laser power P is given by ρ = 4P / (π) 2 c 2 It can be expressed as λh). The electric field is a good approximation of the intensity distribution in the cavity, w0 = λ / 2 = 1 / e 2 Assume we have a Gaussian intensity profile with an intensity "waist" radius. This profile is the smallest focus achievable 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 When is the electron beam velocity, the same model can be used to determine τ = λ / v e Obtain the following. A more accurate estimation considering the actual intensity distribution of the cavity (Figure 11) is given by τ = sqrt(π / 2)λ / v eSubstituting this into equation (1), the phase is given by the following equation. TIFF0007837591000011.tif10170

[0141] When electrons cross a laser phase plate, most are not lost, and only about 1 in 1 million electrons are lost due to Compton scattering by photons.

[0142] Using equation (9), the laser power P required to generate a phase shift of δ = π / 2 can be calculated as a function of electron velocity and laser wavelength. For a 100 keV electron microscope, the electron velocity v e It is about 10 8 It is 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 operate at the lowest possible laser power P that allows a compromise with larger laser wavelengths λ.

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

[0145] High-power lasers at this wavelength are difficult to achieve. The selection of laser wavelengths is also constrained by the availability of reliable, commercially available high-power lasers. For example, according to equation (9), a laser wavelength of 1064 nm requires approximately 5 kW of laser power to produce the required π / 2 phase shift for a 100 keV electron beam. At this wavelength, an optical beam focus radius of approximately 0.5 μm is obtained. This optical beam focus radius is considered to enable phase contrast for structures up to 80 nm in size and thus satisfy the requirements mentioned above.

[0146] While pulsed lasers can easily achieve kW-level power over nanosecond (ns) durations, the use of these pulsed lasers also requires pulsed oscillation of the electron beam. However, such pulsed oscillation is not available in most commercially available TEM devices. Most importantly, the limitation of cathode current density within the electron gun introduces a new challenge to the operation of electron microscopes in pulsed oscillation mode. Therefore, a continuous-wave high-intensity optical field is desirable.

[0147] The intensity of a laser beam can be increased by using an optical cavity to achieve resonant enhancement. Within such a cavity, the laser radiation is reflected and travels back and forth between cavity mirrors. When the laser wavelength is a half-integer multiple of the mirror separation width, the radiation from all round trips is added together constructively, resulting in resonant intensity enhancement. This enhancement occurs when the radiation has one of the resonant frequencies given by the following equation: In the above equation, n=1,2,3,... represents the number of modes (the above equation adheres to sufficient precision of the cavity when the length L is considerably larger than the wavelength). For resonant laser light, the intensity is increased by the multiple given by the following equation. 1 / (1-R) ​​(11) In the above equation, R is the reflectivity of the mirror. A perfect reflector is considered to have a value of R=1.

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

[0149] Referring to Figure 10, the optical cavity 1008 can be configured such that, for example, an electron beam 1012 travels vertically downward (e.g., along the z-axis), enters the cavity 1008 through a hole at its top, and exits through another hole at its bottom. The laser beam 1014 is shown oriented horizontally and may have 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 result in a discernible loss of refractive power, and these holes are large enough not to suppress electron flow with radii of, for example, about 1 mm, but which can be larger or smaller. When the cavity radius is 10 mm or more, these holes will dissipate less than 0.01% of the circulating power of the cavity. Furthermore, this makes it possible to construct the cavity from two hollow hemispheres that are joined together to produce a single sphere after manufacturing. These hemispheres are joined within a vanishing field plane in such a way that even an imperfect junction does not result in a discernible loss of refractive power.

[0150] The intensity enhancement coefficient is determined by the reflectivity of the mirror surface, i.e., a high internal reflection coefficient. Reflectivity of R=99.99% and higher with dielectric coatings, corresponding to 10,000 times and greater intensity enhancements, is state-of-the-art. Unfortunately, dielectric coatings require precise thicknesses that are difficult to achieve on highly curved surfaces such as two hemispheres, although this can ultimately be achievable. Metallic coated mirrors are easier to fabricate because the coating thickness is not critical. A metallic reflectivity of 99% at the first micron, providing a 100-fold power enhancement, is conventionally available. This reflectivity can be sufficient when using the most advanced high-power lasers available.

[0151] In this embodiment, the required power can be reduced by increasing the wavelength, but this will increase the spot size. With a CO2 laser wavelength of 10 μm, the power circulating in the cavity is only 500 W for a phase shift of π / 2, and the power enhancement factor when using a metal mirror is considered to be approximately 200. Therefore, it is thought that only 2.5 W of laser power is required. With a focus size of approximately 5 pm, as discussed above, some loss of contrast for large feature areas of the sample is expected. Nevertheless, such a configuration is considered simpler from the standpoint of required laser power.

[0152] The laser cavity 1008 can be produced, for example, by joining two hemispheres having radii of 8-10 mm to form a spherical optical cavity housing 1010. The size is determined by the available space in the electron microscope for the placement of the phase plate. The hemispheres can be ground into a quadratic shape within a beryllium-copper block to achieve good thermal conductivity and mechanical stability, or alternatively, made of Invar® to achieve a low coefficient of thermal expansion. Note that maintaining a perfectly spherical shape is not critical, as the cavity can be intentionally distorted in any case, as described below. The hemispheres are polished to λ / 10 and the interior is coated with gold. Further polishing is unnecessary due to the relatively high loss rate of the gold mirror. The hemispheres can be mounted together by tuning the optical cavity toward frequency stabilization at one optical frequency while suppressing other competing modes that may result in phase diffraction gratings of different standing wave period intensities, using one or more piezoelectric transducers (PZTs). Approximately 50W of laser power will be dissipated on the cavity wall. The heat can be carried away by liquid cooling. For a temperature increase of 10C (easily compensated for by liquid cooling), a hemisphere with a radius of 10mm will expand by approximately 160nm in BeCu, or 16nm in Invar®. This expansion will result in a shift in the resonant frequency that must be compensated for by active feedback (explained below). Furthermore, in order to achieve resonance at any laser frequency, the dynamic range of the PZT must be at least half the laser wavelength (see Figure 10). A metal surface at λ=1μm 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 67W laser is considered necessary. A single-frequency fiber laser currently available from IPG photonics (50 Old Webster Road, Oxford 01540, Massachusetts, USA) provides a 50W laser at this wavelength.Since the phase contrast changes as a sine of the phase shift and therefore as a sine of the laser power, >90% of the optimal phase contrast is available with a 50W laser. Higher power lasers 1006 are thought to work with lower cavity coupling efficiencies, which would reduce the cost of fabricating the optical cavity.

[0153] To efficiently couple laser radiation into a cavity, it is desirable to obtain nearly spherically symmetric modes from the stimulated laser beam. This is because, in addition to the desired resonant mode with the most precise focus (see Figure 11), the cavity can resonate with numerous other modes due to its spherical symmetry. In a perfect sphere, many of these modes have the same resonant frequency as the desired mode. Therefore, some distortion of the cavity, which can be achieved simply by adjusting the distance between the hemispheres, breaks the cavity mode degeneracy, and at this stage the desired mode has a distinctly different resonant frequency. The laser radiation will primarily excite this mode if it has this frequency. Other modes that are thought to resonate at different frequencies are made cavity non-resonant and therefore suppressed.

[0154] Figure 12 illustrates a system for generating an optical phase plate to modulate the phase of an electron beam. It simply involves a radius r within cavity 1202. in The laser beam can be coupled through hole 1204. The transmission of power from 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 surface). In the desired resonant mode shown in Figure 11, this condition is met by r cav When is the radius of cavity 1202 and R is the reflectivity of the mirror in the cavity, then 1 = (2 / 3)(4r cav 2 / r m 2 )(1-R) ​​or R=0.99 for r in / r cav =[8(1-R) / 3] 1 / 2This condition is met when = 0.16.

[0155] Even when this condition is met, the coupling efficiency may still not be 100%. Consider a time-reverse scenario where the cavity generates light and this light is emitted through the coupler. The coupler is small compared to the size of the TEMn01 mode resonating within the cavity. Therefore, the electric field emitted through the coupler has a nearly uniform intensity across the coupler's diameter and is nearly zero outside (this ignores diffraction at the edges, which is appropriate because the coupler's radius is considerably larger than the wavelength, and therefore diffraction effects can be substantially ignored). The time reversal determines the required shape of the laser beam having optimal power transfer entering the cavity. However, the laser generates Gaussian modes. Optimal power transfer between the Gaussian modes and the truncated ("top-hat") cavity modes occurs at 50.4%, which occurs when the waist radius parameter of the Gaussian beam at the coupler surface is equal to the coupler's radius. We also assume a coupling lens with an appropriate focal length. This efficiency can theoretically be improved to 100% by using a beam shaping device, which is essentially a phase diffraction grating, to transform a Gaussian beam into a uniform intensity beam. Such techniques with efficiencies of 84-90% have been reported.

[0156] For the cavity to apply maximum intensity enhancement, the laser frequency must match one of the cavity resonances. To achieve resonance despite heat and other drifts within the laser and cavity, a feedback mechanism based on the Pound-Dreber-Hall method is used (for example, as shown in Figure 12). For this purpose, the electro-optic modulator 1206 is set to frequency ω m and modulation index β mPhase modulation is applied to the laser beam by the following: The beam is coupled to the cavity through the beam shaping unit 1220 and the coupling lens 1222, and the reflected signal resulting from the radiation emitted from the optical cavity is detected by the detector 1218. The reflected signal can be coupled to the detector 1218 using an isolator 1208. In another embodiment, the reflected signal can be coupled to the detector 1218 using a beam splitter and mirrors. The resonance characteristics of the cavity convert the phase adjustment into amplitude adjustment. When the laser frequency and the resonance frequency match, the detected reflected component of amplitude modulation (AM) reaches a minimum value and ideally disappears, or other non-zero AM occurs. This detected reflected component is detected by amplitude and phase using a double-balance mixer (DBM) 1210, and its output is the modulation frequency ω m Low-pass filtering is applied to suppress this, resulting in a signal with zero phase crossover at resonance. This signal is fed back to the laser frequency actuator via the appropriate servo 1214 by feedback 1212, which keeps the cavity resonance aligned with the laser 1224. Due to the high signal power in this system, very weak modulation is sufficient to achieve a feedback signal with a high signal-to-noise ratio.

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

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

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

[0160] In embodiments, optical cavity phase plates can be applied to generate extremely deep three-dimensional optical traps. Trap depths can range from tens of Kelvins to even hundreds of Kelvins, trapping room-temperature atoms, for example, even for chemical species that are difficult to cool, and determining their location in space with greater precision than 0.5 microns. Such traps are advantageous for a wide range of atoms or molecules, as the enormous intensity within the cavity renders it pointless to tightly match the laser frequency to the transitions of the atoms or molecules. This enables spectroscopic measurements of very weak transitions concerning such atoms. One example is nuclear spectroscopy of thorium-229 atoms using a laser. Laser spectroscopy of transitions of approximately 5-6 eV in the nucleus of thorium-229 can be a breakthrough in precision measurements, as it allows for the construction of a “nucleus” clock based on transitions between nuclear energy levels rather than in the electron shell. However, this spectroscopic measurement has been hampered by the lack of a suitable method for positioning the atom. Only in the case of a positioned atom can the probe laser be precisely focused as needed to generate sufficient intensity. The dipole trap proposed herein can solve this problem and enable direct laser spectroscopic measurements of this transition. This can lead to a more precise clock (since atomic nuclei are less affected by the environment), as well as an examination of the time variation of fundamental "contrasts" with unprecedented precision.

[0161] By providing dielectric coating technology that enables the fabrication of a uniformly controllable dielectric layer on the inner surface of high NA cavities, higher resonant enhancement is possible, thus enabling the use of lower-power lasers or longer laser wavelengths at lower costs (which is expected to enable the use of cavities with lower NA).

[0162] Compared to the conventional microstructured phase plates described above, resonant cavity phase plates offer several advantages, including: not using any mechanical electrodes in the electron beam; allowing optical elements that need to be brought into the laser beam to be sufficiently far away (1 mm or more), thus avoiding problems associated with image blurring and distortion; resolving the short device lifespan that currently limits the performance of thin-film phase plates; eliminating the problem of partial loss of scattered electrons that occurs in both thin-film phase plates and electrostatic or magnetic phase plates; and making electron microscopes more productive and efficient by eliminating partial signal loss and the need to replace microstructured or thin-film phase plates that have deteriorated over time or become contaminated when struck by the electron beam.

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

[0164] Figure 13 is a flowchart of a method for enhancing phase contrast in an electron beam image. This method can be performed using a transmission electron microscope, an optical cavity, and a laser.

[0165] Action 1302 generates an electron beam within the transmission electron microscope. Action 1304 receives the electron beam along an axis passing through the center of an optical cavity positioned at the back focal plane of the transmission electron microscope, determined by the first and second mirrors.

[0166] In action 1306, the laser beam is received into the optical cavity. The laser beam is reflected by the first and second mirrors to generate a standing wave optical phase plate. The standing wave optical phase plate is focused at the back focal plane, causing modulation of the electron beam.

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

[0168] The embodiments described below with reference to Figures 14-16 extend the functionality of a ponderomotive phase plate by enabling control of the spatial profile of the ponderomotive phase plate by controlling the polarization state of the light used in the ponderomotive phase plate. In transmission electron microscopy imaging, this control can be used to eliminate the presence of image artifacts caused by electron diffraction within the phase plate. For the same reason, 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 is derived from the spatial profile of the ponderomotive potential generated by light. However, the spatial profile of the ponderomotive potential depends not only on the intensity of the light, but also on the polarization state of the light when the incident charged particle (e.g., an electron) has a significant proportion 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 velocities greater than 1 / sqrt(2) times the speed of light, the depth of the standing wave can be made zero.

[0170] The standing wave structure of the phase plate diffracts passing particles, and the amount of diffraction occurring within the phase plate can be controlled using the embodiments described herein. When using a phase plate for transmission electron microscopy imaging, eliminating the electron diffraction effect is desirable because it eliminates the presence of image artifacts caused by electron diffraction. Eliminating the electron diffraction effect makes the process of aligning the electron beam to the phase plate easier and faster because the spatial profile of the phase plate does not change so abruptly. A method is also provided for controlling the maximum phase shift applied to the electron beam, which adjusts the amount of image contrast enhancement provided by the phase plate for phase contrast transmission electron microscopy by controlling the depth of the standing wave. To more accurately reconstruct data about a sample, it can be advantageous to switch between two phase plate profiles (with or without a standing wave) during normal data acquisition using various embodiments.

[0171] Another application is as a switchable electron beam splitter (or beam splitter), due to its ability to enable and disable standing wave structures as quickly as it can change the polarization state of light. One embodiment uses an electro-optic modulator that enables and disables diffraction effects acting on the electron beam, thereby enabling and disabling beam splitting, in order to rapidly switch the polarization of the laser light. The electro-optic modulator can be used in transmission electron microscopes and various embodiments of a ponderomotive phase plate or laser phase plate, including yet another embodiment described below.

[0172] Another application is electron pulse slicing. A sufficiently strong laser beam completely diffracts the incident electron beam. None of the emitted electrons travel in the same direction as the incident electron beam. All of the incident electron beam is deflected at some specific angle. If the device is operating at this stage and the polarization is then switched, this action slices the electron beam. Electron pulse slicing results from rapidly switching the polarization.

[0173] Another application is to temporally phase-modulate an electron beam focused through a single wave antinode of a standing wave of laser light within a laser phase plate. The electron beam undergoes a phase shift as it focuses and passes through this wave antinode, where the electric field of the standing wave is highest. This phase shift depends on the polarization state of the laser light. Rapidly changing the polarization state alters the amount of phase shift the electron beam receives from the laser light, and this change occurs at the frequency of the change in the laser light's polarization state. This effectively amplitude-modulates the electron beam after it has propagated over a certain distance. A sample placed next to the electron beam receives repulsive or induced pulses from this beam at the frequency of the change in the laser light's polarization state. A device designed and operating in this manner is thought to be able to control the characteristics of the 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 to 13, thereby enabling the polarization state of the light delivered into the Fabry-Perot optical cavity to any angle with respect to the electron beam axis. In particular, in some embodiments, a half-wave plate, a common optical element, is inserted into 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 discretely preset or over a continuous range, and in various embodiments, can be manually controlled or automatically controlled.

[0175] These embodiments are considered novel for several reasons. Firstly, the fact that the ponderomotive potential should have polarization dependence with respect to high-speed particles is generally non-obvious to experts in this field. However, prior to the disclosure of this invention, the existence of this effect had been confirmed by computer simulations, and there were several papers by other academic research groups describing its potential applications. Secondly, the embodiments described herein are considered to be the first experiments to reveal this effect. Thirdly, the application of this effect to the proposed use in transmission electron microscopy 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 a ponderomotive phase plate for image contrast enhancement in transmission electron microscopy as a method for eliminating image artifacts caused by electron diffraction from the phase plate. The group of the present invention 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 are no known competing techniques for removing standing wave structures from a ponderomotive phase plate. The advantages of the ponderomotive phase plate over other types of phase plates are described herein.

[0178] Figure 14 shows a schematic shape of yet another embodiment of a phase contrast TEM 1434, featuring a laser beam with a variable polarization angle coupled to an optical cavity 1424. By changing the polarization angle of the laser beam, the image contrast enhancement of the image of the sample 1404 formed on the image plane 1428 of the transmission electron microscope 1434 is changed. Below, with reference to Figures 15A to 15C, a mechanism for changing the polarization angle of the laser beam will be described, and it is expected that further mechanical, electromechanical, and electro-optical embodiments will be readily devised by following the teachings herein. In one embodiment, the laser 1412 with a variable polarization angle has an electro-optic modulator used to change the polarization.

[0179] During operation, the electron beam 1402 of the transmission electron microscope 1434 is directed towards the sample 1404 located on the object plane 1406. A portion of the electron beam 1402 is diffracted by the sample 1404 and propagates as a diffracted electron beam 1436. The non-diffracted portion of the electron beam 1402 is typically focused onto the focal point 1422 of the optical cavity 1424 by an objective lens 1408, which is usually made of an iron core magnetic device or an electromagnetic field coil. On the other hand, the diffracted electron beam 1436 diverging from the sample 1404 cannot be focused onto the focal point 1422 of the optical cavity 1424 by the objective lens 1408.

[0180] Polarized laser light from laser 1412 having a variable polarization angle is coupled to optical cavity 1424 by coupler 1416, which in various embodiments can be air, gas, vacuum, or optical fiber, and other types of couplers can be easily devised. Optical cavity 1424 is formed by two concave mirrors 1418, 1420 that focus the laser light to the focal point 1422 of optical cavity 1424 as described above in various embodiments. Wave antinodes of the polarized laser light at the focal point 1422 of optical cavity 1424 cause modulation of electron beam 1402. Most of the diffracted electron beam 1436 scattered from sample 1404 does not pass through the focal point 1422 of optical cavity 1424 and is therefore not as modulated by the polarized laser light.

[0181] The non-scattering, non-diffracting portion of the electron beam 1402, focused on the focal point 1422, undergoes a phase shift due to the focused polarized laser light, while the scattering beam portion that does not intersect with the light wave does not undergo a phase shift. The electron beam 1402 and the diffracting electron beam 1436 are guided to the image plane 1428 by the projection lens 1426, where an image is formed and can be captured by the electron camera 1430 (or other sensors in yet another embodiment). Like the objective lens 1408, the projection lens 1426 is typically manufactured from an iron core magnetic device or an electromagnetic field coil. Furthermore, the electron camera 1430 operates on a principle similar to an optic-electron camera that captures pixels using, for example, a charge-coupled device (CCD) or exposure random-access memory (RAM). The phase shift difference between the non-scattering beam and a specific portion of the scattering, diffracting electron beam 1436 determines how much contrast enhancement the ponderomotive phase plate 1410 (or laser phase plate) applies in the image plane 1428.

[0182] The degree to which standing wave structures are present in the ponderomotive phase plate 1410 is affected by changing the polarization angle of the laser beam, which can be adjusted from 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 maximum, the image contrast enhancement is maximum, and therefore the presence of ghost images is also maximum. Various compromises are available in intermediate settings. Since it can be difficult to align the electron beam with the laser beam wave antinodes, one potential side effect of setting the laser polarization angle to eliminate "ghost" images is that such alignment can be facilitated. In one embodiment, such alignment is advantageous for producing a phase contrast image. It should be noted that such advantages may be independent of other benefits highlighted herein.

[0183] Figure 15A depicts a half-wave plate 1502 having a rotor 1504 for changing the polarization angle of laser light in an embodiment of a phase contrast TEM and an embodiment of a ponderomotive phase plate. Polarized laser light from 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 it. The rotor 1504 sets the rotation angle of the half-wave plate 1502 and, therefore, sets the phase angle of the polarized laser light. The rotor 1504 has 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 yet another mechanism, and can be implemented as a dial which can be set manually using a shaft or knob or actuated by a motor, such as a stepper motor or other electric motor. The motor-driven rotor 1504 can be controlled manually, for example by a switch or button, or can be controlled automatically, for example by a feedback path and controller, as shown in Figure 16.

[0184] Figure 15B depicts an optical fiber coupler having a rotator 1512 for changing the polarization angle of laser light in an embodiment of a phase contrast TEM and an embodiment of a ponderomotive phase plate. A collar 1506 of the optical fiber coupler fixes the optical fiber fiber 1508 or cable to a laser 1503 having polarized laser light. Another collar 1510 at the other end of the optical fiber fiber 1508 or cable is rotated by the rotator 1512, and this rotation rotates this end portion of the optical fiber fiber 1508 or cable relative to the end fixed 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 regard to manual, motor-driven, or automatic control, similar to the rotator 1504 in Figure 15A.

[0185] Figure 15C depicts a laser 1503 having polarized laser light and a rotor 1516 for changing its polarization angle in an embodiment of a phase contrast TEM and an embodiment of a ponderomotive phase plate. 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. Similar to the rotor 1504 in Figure 15A, there are many possibilities for how the rotor 1516 can be implemented.

[0186] Figure 16 schematic shows an action involving a sensor 1606 on the image plane 1428 of the phase contrast TEM 1434 or a variation thereof in the embodiment of the phase contrast TEM 1434 and the Pondelomotive phase plate 1410 in Figure 14, and a controller 1602 that analyzes a Ronchigram for use in controlling the polarization angle of the laser light. The Ronchigram has standing wave interference fringes formed on the image plane 1428 that change with the polarization angle of the laser light. At a certain polarization angle for a certain electron velocity, the standing wave interference fringes disappear. In one embodiment, the sensor 1606, which may be an electron camera 1430, detects the standing wave interference fringes on the image plane 1428 and sends a signal to the controller 1602. The controller 1602 performs a variety of actions, including a step of analyzing the Ronchigram as action 1604 and a step of controlling the polarization angle as action 1608. For example, the software, firmware, or hardware within the controller 1602 can search for the intensity and arrangement of standing wave interference fringes, instruct a motor-driven rotor to change the polarization angle of the laser beam, analyze the changes in the standing wave interference fringes as the polarization angle changes, and correlate the rotor's position and associated polarization angle with the characteristics of the standing wave interference fringes. For example, the controller 1602 can search for a laser beam polarization angle setting that results in the disappearance of standing wave interference fringes, and this setting, along with any other setting developed by the controller (or selected by the user), can be used as the phase plate profile. In one embodiment, the controller 1602 can analyze the contrast in the image to determine the optimal setting for the polarization angle, and then for the point of compromise between contrast and ghost images.

[0187] Figure 17 is a flowchart of a method for transmission electron microscopy that can be performed by various embodiments of a TEM having 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 the optical cavity of the ponderomotive phase plate. The non-diffraction, non-scattering portion of the electron beam is focused to the focal point of the optical cavity.

[0190] Action 1706 involves injecting a laser beam with polarized laser light into an optical cavity to form a standing wave optical phase plate, thereby inducing modulation of the electron beam. Appropriate mechanisms and techniques for doing so are described above.

[0191] Action 1708 images the electron beam onto the TEM image plane. When the sample is used, the image shows the sample through a phase-contrast transmission electron microscope. Furthermore, the image also shows a ronchigram with standing wave interference fringes (which may have zero amplitude in certain settings of the polarization tilt of the laser light as in the case of degeneracy).

[0192] Action 1710 alters the contrast enhancement of the image by changing the polarization angle of the laser beam. In various embodiments, this alteration can be achieved by manual adjustment or automatic adjustment by the system.

[0193] While the disclosure and advantages of the present invention have been described in detail, it should be understood that various changes, substitutions, and modifications can be made herein without departing from the spirit and scope of the disclosure of the present invention as defined by the claims. Furthermore, the scope of this application is not intended to be limited to specific embodiments of the processes, machines, manufactures, material compositions, means, methods, and steps described herein. Those skilled in the art will immediately recognize from the embodiments of the disclosure of the present invention that existing or subsequently developed processes, machines, manufactures, material compositions, means, methods, or steps that perform substantially the same function or produce substantially the same results as the corresponding embodiments described herein can be utilized in accordance with the disclosure of the present invention. Accordingly, the claims are intended to include within their scope such processes, machines, manufactures, material compositions, means, methods, or steps.

[0194] The present invention has been described in this specification with reference to specific embodiments. However, those skilled in the art will recognize that various modifications and variations can be made without departing from the scope of the invention as set forth in the following claims. Accordingly, this specification and drawings should be understood as illustrative rather than restrictive, and all such modifications are intended to be included within the scope of the invention. [Explanation of Symbols]

[0195] 1302 Action to generate an electron beam in a transmission electron microscope 1306 Action to receive the laser beam into the optical cavity 1404 samples 1424 Light Cavity 1434 Phase contrast TEM

Claims

1. Transmission electron microscope (TEM) and, A plurality of mirrors forming an optical cavity, the optical cavity being positioned such that an electron beam provided by the TEM passes through the focal point of the optical cavity, A laser coupled to the optical cavity and having a variable polarization angle of laser light, wherein the laser provides a laser beam of a specified wavelength and the variable polarization angle to the optical cavity, and the plurality of mirrors are configured to reflect the laser beam to provide a standing wave optical phase plate, thereby causing modulation of the electron beam, In order to form an image corresponding to the variable polarization angle, the image plane of the TEM is positioned to receive the electron beam modulated by the standing wave optical phase plate, A system characterized by including

2. Half-wave plate and A rotating device is provided to hold and rotate the half-wave plate in order to provide the variable polarization angle of the laser light, The system according to claim 1, further comprising:

3. The system according to claim 1, further comprising an optical fiber member that couples the laser to the optical cavity, wherein the optical fiber member is bendable or rotatable to provide the variable polarization angle of the laser light.

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

5. The system according to claim 1, characterized in that the variable polarization angle of the laser light is variable between at least a first phase plate profile having standing waves in the ronchigram formed in the image plane and a second phase plate profile without standing waves in the ronchigram.

6. The system according to claim 1, characterized in that the variable polarization angle of the laser light has two or more preset values.

7. The system according to claim 1, characterized in that the variable polarization angle of the laser light is manually adjustable or automatically adjustable (one or more).

8. The steps include generating an electron beam inside a transmission electron microscope (TEM) and A step of receiving the electron beam along an axis passing through the center of an optical cavity defined by a first mirror and a second mirror, The receiving step of receiving a laser beam having a variable polarization angle of laser light into the optical cavity, wherein the laser beam is reflected from the first mirror and the second mirror to generate a standing wave optical phase plate, thereby causing modulation of the electron beam, The steps include: imaging the electron beam onto the image plane of the TEM, which is positioned to receive the electron beam modulated by the standing wave optical phase plate to form an image; The step of changing the variable polarization angle of the laser light, A method characterized by including the following.

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

10. A step of rotating or bending an optical fiber member that couples the laser to the optical cavity in order to provide the variable polarization angle of the laser light, The method according to 8, further comprising:

11. The steps include: analyzing the ronchigram formed on the image plane based on the output of a sensor or electronic camera; A step of controlling the variable polarization angle of the laser light based on the analysis step, The method according to 8, further comprising:

12. The step of changing the variable polarization angle of the laser light is for the purpose of changing the contrast enhancement of the image, The step of changing the variable polarization angle of the laser light is, A step of changing between a first phase plate profile having standing waves in the ronchigram formed on the image plane and a second phase plate profile without standing waves in the ronchigram, including, The method according to feature 8.

13. The step of changing the variable polarization angle of the laser light is: A step of determining the current angle of the variable polarization angle of the laser light based on two or more pre-set values, including, The method according to feature 8.

14. The step of changing the variable polarization angle of the laser light is: A step of determining the current angle of the variable polarization angle of the laser light based on manual or automatic adjustment, including, The method according to feature 8.

15. A first concave mirror and a second concave mirror positioned to define an optical cavity, A laser having a variable polarization angle of laser light coupled to the optical cavity, wherein the optical cavity and the wavelength of the laser light are configured to generate a standing wave having a wave antinode at the focal point of the optical cavity, the standing wave causing variable modulation of an electron beam passing through the optical cavity, and the variable modulation depends on the variable polarization angle of the laser light, and the laser, A poleromotive phase plate characterized by including the following:

16. A half-wave plate positioned to couple laser light to the optical cavity, A rotator that can be operated to rotate the half-wave plate such that the laser light has a variable polarization angle corresponding to the rotation of the half-wave plate when incident on the half-wave plate and when coupled to the optical cavity to provide the standing wave having the wave antinode at the focal point, The ponderomotive phase plate according to claim 15, further comprising the following:

17. An optical fiber coupler is arranged to couple the laser to the optical cavity, A rotary device that can be operated to rotate one end of the optical fiber coupler relative to the opposite end of the optical fiber coupler in order to change the variable polarization angle of the laser light with respect to the optical cavity, The ponderomotive phase plate according to claim 15, further comprising the following:

18. A rotor coupled to the laser and operable to rotate the laser in order to change the variable polarization angle of the laser light with respect to the optical cavity, The ponderomotive phase plate according to claim 15, further comprising the following:

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