The invention relates to the field of microscopy, more particularly electron microscopy. More particularly, the present invention relates to methods and systems for extracting wave function information using phase plates in electron microscopy, such as for example in transmission electron microscopy.

Background of the invention

The importance of phase plates for phase contrast imaging has been amply demonstrated in optics. Already in the sixties, attempts have been made to introduce phase plates in a TEM. However the problem in electron microscopy is that a phase plate typically consists of a very thin layer that has to be traversed by the electron, and, because of the strong interaction of electrons with matter, the effect of the plate on the phase and amplitude of the outgoing electrons is very difficult to control. Earlier 2000, a carbon film was used as a phase plate. The thickness of carbon film was around 30nm. A hole in the center of the carbon film allowed that the scattered beam has shifted π /2. Besides problems with absorption contrast, caused by attenuation of scattered beam through the carbon film, also problems with charging occurred.

Recently however, using advanced methods of nanotechnology, it becomes possible to control the dimensions and quality of the phase plates to such an extent that the amplitude of the electron wave is less affected and the phase can be controlled. Reported examples of phase plates are electrostatic phase plates. In one example a phase plate comprising a multilayer structure is suggested.

Several groups have reported regarding phase plates for phase contrast enhancement. Alloyeau et al. in their abstract "Contrast Transfer Function Design by an Electrostatic Phase Plate for contrast enhancement in transmission electron microscopy" and Hsieh et al. in their abstract "Contrast Transfer Function Design by an Electrostatic Phase Plate" in Microsc. Microanal 13 (2007) describe the fabrication of an electrostatic phase plate.

Nagayama et al have also reported the use of phase plates for electron microscopy in Biophys. Rev. (2009) p37-42.

The availability of phase plates for electron microscopes opens possibilities which have not been completely explored yet.

Summary of the invention

It is an object of embodiments of the present invention to provide good methods and systems for obtaining wave function information, based on microscopy such as electron microscopy using a variable phase inducing means. It is an advantage of embodiments according to the present invention that methods and systems are provided that allow retrieving the whole wave function, even for thicker objects. It is an advantage of embodiments according to the present invention that methods and systems are provided for obtaining wave function data, the methods and systems being robust and easy to implement. It is an advantage of embodiments according to the present invention that efficient and accurate methods and systems for obtaining wave function information are provided. It is an advantage of embodiments according to the present invention that wave function information can be obtained by relatively simple processing of image intensities obtained via microscopy, more particularly electron microscopy, using a variable phase inducing means, e.g. a set of phase plates or a variable phase plate such as for example a tuneable variable phase plate. The above objective is accomplished by a method and device according to the present invention.

The present invention relates to a method for obtaining wave function data from microscope measurements, the method comprising receiving electron microscope image intensity data acquired using a variable phase inducing means inducing a phase variation over time, the electron microscope image intensity data taking into account image intensity data recorded for a plurality of different induced phases over time, and extracting wave function data from the electron microscope image intensity data. The different phases may be induced subsequently during electron microscopy image intensity data recording. The wave function data may comprise at least phase information. It is an advantage of embodiments according to the present invention that wave function data can be derived from electron microscope measurements by the use of a variable phase inducing means. Such data may be used e.g. in the processing of electron microscope data for deriving predetermined information. The received electron microscope image intensity data may comprise image intensity data recorded for a plurality of different induced phases, wherein said extracting comprises first combining the plurality of image intensity data recorded for different induced phases.

Extracting wave function data may comprise deriving the wave function data from the electron microscope image intensity data by distinguishing a component related to the variable phase inducing means from a component comprising the wave function data.

The component related to the variable phase inducing means may be a component being function of an aperture function of the phase inducing means.

Extracting wave function data can be obtained by applying a high pass filter on the acquired image intensity data. It is an advantage of embodiments according to the present invention that extraction of the information can be easily performed using conventional techniques such as applying a high pass filter.

Receiving microscope image intensity data acquired using a variable phase inducing means may comprise receiving, for each phase, image intensity data weighted by applying a phase dependent image intensity recording time. It is an advantage of embodiments according to the present invention that electron microscope image data can be recorded in a single recording step and that the step of combining the data is inherently performed, resulting in an efficient manner for obtaining wave function data, advantageously the full wave function. It is an advantage of embodiments of the present invention that good results can be obtained, even for strong phase objects. The image recording times may be goniometric functions of the phase shift angle Θ induced by the variable phase inducing means. It is an advantage of embodiments according to the present invention that the image recording times required for obtaining the correct microscope image intensity data can be easily determined. Receiving microscope image intensity data may comprise acquiring transmission microscope image intensity data using a variable phase inducing means inducing different phases over time.

The phase inducing means may be a variable electrostatic phase plate.

The present invention relates to an image processing system for extracting wave function data, the image processing system comprising an input port adapted for receiving electron microscope image intensity data acquired using a variable phase inducing means inducing different phases over time, the microscope image intensity data taking into account image intensity data for a plurality of different phases induced over time, and a processor for extracting wave function data from the electron microscope image intensity data acquired.

The present invention furthermore relates to a transmission electron microscope for obtaining electron microscope images, the transmission electron microscope comprising a variable phase inducing means for inducing a variably settable phase shift in an aperture of the variable phase inducing means over a phase shift angle, a controller for controlling acquisition of electron microscope image intensity data using a variable phase inducing means inducing different phases over time, the electron microscope image intensity data taking into account image intensity data for a plurality of different induced phases, and an image processing system as described above. In addition thereto the present invention also may relate to an optical microscope comprising a variable phase inducing means for inducing a variably settable phase shift over time in an aperture of the variable phase inducing means over a phase shift angle, a controller for controlling acquisition of microscope image intensity data using a variable phase inducing means, the microscope image intensity data taking into account image intensity data for a plurality of different induced phases, and an image processing system as described above. The present invention also relates to a computer program product for performing, when implemented on a computing device, a method as described above.

The present invention also relates to a machine readable data storage device storing such a computer program product or to the transmission of such a computer program product over a local or wide area telecommunications network.

Particular and preferred aspects of the invention are set out in the accompanying independent and dependent claims. Features from the dependent claims may be combined with features of the independent claims and with features of other dependent claims as appropriate and not merely as explicitly set out in the claims. These and other aspects of the invention will be apparent from and elucidated with reference to the embodiment(s) described hereinafter.

Brief description of the drawings

FIG. 1 is a flow chart of an exemplary method for obtaining wave function information according to an embodiment of the present invention.

FIG. 2 is a schematic representation of the aperture function for a phase plate as can be used in an embodiment of the present invention.

FIG. 3 is a schematic representation of an image processor for obtaining wave function information according to an embodiment of the present invention.

FIG. 4 is a schematic representation of a transmission electron microscope according to an embodiment of the present invention.

The drawings are only schematic and are non-limiting. In the drawings, the size of some of the elements may be exaggerated and not drawn on scale for illustrative purposes.

Any reference signs in the claims shall not be construed as limiting the scope.

In the different drawings, the same reference signs refer to the same or analogous elements. Detailed description of illustrative embodiments

Where in embodiments of the present invention reference is made to wave function data, reference is made to data regarding the wave function. The wave function data may be wave function data comprising a phase-related component or phase-related information. The wave function data may be data comprising at least a part of the wave function. The wave function data advantageously may comprise the full wave function.

In a first aspect, the present invention relates to a method for obtaining wave function data from electron microscope measurements. The method is especially suitable for use with electron microscope measurements. The obtained wave function data may be used for processing measurements, more particularly electron microscope measurements, thus allowing to extract particular information, such as for example to reconstruct an exit wave for an object which then may be used for reconstructing the object upon imaging. Nevertheless, the invention is not limited thereto but only limited by the claims. The method according to embodiments of the present invention comprises receiving electron microscope image intensity data, e.g. electron microscope image intensity data, acquired using a variable phase inducing means. Such a variable phase inducing means may be a set of phase plates or a variable phase plate, like for example a continuous variable phase plate. The acquired image intensity data thereby takes into account a plurality of image intensity data recorded for different induced phases over time by the variable phase inducing means. It may be the result of combining such image intensity data recorded for a plurality of different induced phases over time or it may be received as a set of separate image intensity data, each part of the set of image intensity data recorded for a different induced phase. In the latter case an additional combining step may be performed. As will be shown, advantageously for each phase the corresponding image intensity data may be given a particular weight. In some embodiments, this weight can be provided by recording the image intensity data for a predetermined time, being function of the phase Θ . The method furthermore comprises extracting wave function data from the received image intensity data. The obtained wave function data advantageously is the complete wave function. It is an advantage of embodiments according to the present invention that wave function data, advantageously the complete wave function, can be efficiently and rather easily derived from microscopy intensity measurements, more particularly electron microscopy intensity measurements. By way of illustration, embodiments of the present invention not being limited thereto, standard and optional steps of a method for obtaining wave function data are illustrated in FIG. 1.

The method 100 for obtaining wave function data comprises receiving 110 microscope image intensity data, more particularly electron microscope image intensity data, acquired using a variable phase inducing means, e.g. a set of phase plates or a variable phase plate. The electron microscope image intensity data, according to embodiments of the present invention, takes into account electron image intensity data recorded for a plurality of different phases induced over time, induced by the variable phase inducing means. In other words, different phases are subsequently induced such that different phases apply over time. Such receiving 110 may be receiving of previously recorded image intensity data, for each phase separately or combined, via an input port. Alternatively, the electron microscope image intensity data also may be received by acquiring of the data using an electron microscope comprising a variable phase inducing means. Examples of obtaining such data may include obtaining separate electron image intensity data for different induced phases by recording image intensity data for a plurality of different phase plates, each received separately. In another, more advantageous, embodiment the image intensity data for different induced phases may be received in a combined, e.g. after integration, and obtaining then may comprise recording the data in a single experiment whereby the different phases are introduced by tuning a variable phase plate, such that the received image intensity data is received as integrated data.

In advantageous embodiments, the image intensity data for a given phase may be given a certain weight dependent on the phase. Such a weight may be introduced during processing steps for extracting the wave function by providing weight factors to a set of separate image intensity data. Nevertheless, in one advantageous embodiment, the weight may be inherently provided in the recorded image intensity data, e.g. by adjusting the recording time for image intensity data corresponding to each of the phases at which data is recorded. The image intensity data at one phase requiring a higher weight then typically may be recorded over a longer recording time than image intensity at another phase requiring less weight. As will be shown for one advantageous example, predetermined recording times as function of the phase shift angle Θ may be used. In one example, the image intensity data that is used for a given phase thus may be electron image intensity recorded over a recording time being proportional to cos θ and electron image intensity recorded over a recording time being proportional to sin θ, resulting in a fixed weight factor for a fixed phase shift angle, i.e. for image intensity data corresponding with a fixed phase shift angle.

In some examples, the range of phase angles of interest, preferably the range [0, 2π], may be samples in different angular increments, In some examples angular increments may be in the order of a few degrees resulting in between 10 to 100 intervals.

The method also comprises extracting 130 wave function data, advantageously the whole wave function, from the electron image intensity data. Where the received data comprises receiving separate image intensity data for different phases, the method 100 furthermore advantageously may comprise combining 120 the different electron image intensity data for different phases, advantageously each with their own weight factor. Combining the electron image intensity data in preferred embodiments may be integrating the electron image intensity data, optionally multiplied with their weight factor, over all possible phases. Combining may be a discrete summation of the different contributions at different phases, semi-discrete summation or, if continuous data would be available, continuous integration. The more numerous the measurements at different phases, the more accurate the result can be. In some advantageous embodiments, the combination of the different electron image intensity data may have already been performed during the imaging process by subsequent measurement of the electron image intensity data for the different phases. Also in this case, the weighting factors may have been taken into account experimentally as described above. In this way combined weighted electron image intensity data can be directly measured, e.g. by varying the variable phase inducing means over time over a plurality of phases representative for a phase range of interest, typically [0,2π[. In some examples, the required voltage for covering this range via for example an electrostatic phase plate may vary depending on the design of such an electrostatic phase plate. The total recording time for recording the image intensities if CCD's are used typically is of the order of 1 second, although embodiments of the present invention are not limited thereby. The total recording time may depend on the source used and on the number of imaging particles obtained. The latter may be selected so as to have a sufficient signal to noise ratio. Extracting wave function data may comprise distinguishing a component related to the phase inducing means from a component comprising the wave function. The component related to the phase inducing means may be a component being function of an aperture function of the phase inducing means. Separation between the components can for example be performed by applying a high pass filter on the acquired image intensity data, such as for example a Laplacean filter. In one example the processed image intensity data may be a function of the wave function ψ(τ) and an expression dependent on the aperture function of the phase inducing means, i.e.

whereby is the Fourier transform of the aperture function of the phase inducing

means.

In one particular embodiment, the combined contributions of electron image intensity data integrated over the phase angle, are function of the wave function and the aperture function as follows

whereby is the Fourier transform of the aperture function of the phase inducing

means and is the wave function. Based on the relation between the integrated

electron image intensity and the function of the wave function and aperture function, wave function data can be extracted.

I n em bodiments according to the present invention, advantageously, the whole wave function in the image plane can be derived.

I n an optional step, when the whole wave function in the image plane is derived, the method also comprises determining 140 an exit wave for the imaged object. Such information may for example be used for reconstruction of objects, such as for obtaining an atomic structure of an object.

The method 100 furthermore optionally may comprise outputting 150 the obtained wave function data or exit wave data and/or using such data for other electron microscopy image processing such as for example image analysis or tomography.

It is an advantage of embodiments according to the present invention that methods and systems are provided that filter out incoherent backgrounds due to inelastic scattering in the object and in the phase inducing means since they are independent from θ and cancel in the integration. Where necessary the sensitivity to noise may be taken into account. It is also clear that, when the central beam would be partly absorbed in the phase inducing means so that only a fraction of (ψ) is transmitted, the final result is only affected by a constant factor. I n this way the reconstructed wave functions will not suffer from artefacts such as the Stobbs factor.

I n some embodiments according to the present invention, the variable phase inducing means, e.g. a variable phase plate or set of phase plates, may be selected to have a central active part that is sufficiently small in order not to influence the small spatial frequencies that are relevant for large structures such as macromolecules. Furthermore in some embodiments according to the present invention, diffraction effects of the central beam on the edges of the central area should be avoided by arranging or manufacturing the variable phase inducing means such that no edges of the interacting part of the phase inducing means cross the central beam. The interaction region thus must also be sufficiently large. Typically the size of the interacting portion thus may be chosen intermediate.

Without wishing to be bound by theory, features and advantages of at least some embodiments of the present invention could be explained based on the following theoretical principles and algorithm, embodiments of the present invention not being limited thereto. Whereas a particular shape of the phase inducing means, e.g. a phase plate, is used in the following description, embodiments of the invention are not limited thereby. Whereas a particular mathematical formulation is used, embodiments of the present invention are not limited thereto.

I n the present example, a disk-like phase plate is considered being a foil with a central, active area, that is put in the focal plane of the objective lens to shift the phase of the central beam (g = o) in diffraction space over a phase angle Θ with respect to the other beams (g≠θ). The aperture function of this central area can then be defined as

for g in the central area

[4]

for g outside the central area

Consider now an electron wave that crosses the phase plate. After transmission,

the electron wave then becomes ereby g is being indicative of where the wave crosses the phase plate. It is to be noticed that the same result can be obtained with the central area of the phase plate inactive and the outer region shifting the phase of the electron wave over - Θ . Fourier transforming in real space then yields

where is the Fourier transform of and * is a convolution product.

If the central area of the phase plate is very small it acts as a low pass filter so that the convolution product with in real space acts as an averaging over an area, the size

of which is inversely related to the size of

This averaging can be denoted as

If one defines

or

with

the Fourier transforming becomes

For the intensity one can obtain

yielding

If the intensity is integrated with a weighted cosine and sine function of θ , we obtain respectively

In a real experiment, this can be done by, for each phase, keeping the phase θ constant while recording image intensity data during a time proportional to cos θ respectively sin # . In practice, since one cannot record with negative weighting functions, integration in two steps will be required in combination with subtracting of the results afterwards.

Now one can reconstruct the wavefunction based on and and (ψ) and

(δ) can be separated using a high pass filter. Such a high pass filter may be implemented for example by Fourier transforming the (wave) function and multiplying the Fourier transform with a function that puts more weight to the large spatial frequencies. Selection of such a high pass filter, and e.g. of a weighting function can be performed as common in image processing, whereby for example caution is taken to avoid artifacts that can occur with sharp edges. The latter illustrates how wave function data, in the present example being the whole wave function, can be extracted from electron microscopy using a variable phase inducing means, further indicating optional features and advantages of embodiments of the present invention. Note that the method does not make any assumptions of the relative values of (ψ) and (δ) and can thus be used even for strongly scattering objects.

In a second aspect, the present invention also relates to an electron microscopy image processing system for obtaining wave function data. The electron microscopy image processing system according to embodiments of the present invention comprises an input port adapted for receiving microscope image intensity data, more particularly electron microscope image intensity data, acquired using a variable phase inducing means, e.g. a phase plate, whereby the data takes into account electron microscopy image intensity data recorded for a plurality of different phases induced over time by the variable phase inducing means. The image processing system also comprises a processor for extracting wave function data from the electron microscopy image intensity data. The received data may be obtained in weighted form, i.e. for each phase a weighting factor may be applied to the corresponding electron microscopy image intensity data, or the processor may be adapted for applying a weighting factor to the different image intensity data. The image processor furthermore may comprises components performing the functionality as expressed by one or more features of the method for obtaining wave function data as described above. The image processing system may be adapted for performing a method as described in the first aspect in an automated and/or automatic way. The image processing system may be implemented in a software manner or as dedicated hardware. The processor may operate according to a predetermined algorithm. It may make use of predetermined instructions, look up tables, etc. By way of illustration, embodiments of the present invention not being limited thereto, an example of an image processing system 200 comprising an input port 210 and a processor 220 as described above is shown in FIG. 3. The processor 220 may be adapted for extracting the wave function data and optionally also for combining data. The system furthermore typically may comprise a memory 230 for temporarily storing data and an output means 240 for outputting the obtained results. The latter may be by displaying the results e.g. on a display, plotter, printer, etc. or it may be by outputting data signals, e.g. through an output port. In one aspect, the present invention also relates to a transmission electron microscope for obtaining electron microscopy images. The transmission electron microscope according to embodiments of the present invention comprises a variable phase inducing means for inducing a phase shift over time in an aperture of the variable phase inducing means over a phase shift angle and a controller for controlling acquisition of electron microscope image intensity data recorded taking into account image intensity data recorded for the plurality of different phases induced over time by the variable phase inducing means. The electron microscope furthermore comprises an image processing system as described in the second aspect. By way of illustration, the present invention not being limited thereto, an example of a transmission electron microscope is shown in FIG. 4. A high-resolution electron microscope 300 is shown comprising an electron source 303 which is fed by a high-voltage generator 305, and also comprises a number of Ienses307 which are fed by a lens power supply source 309. The electron microscope 301 also comprises a detection system 311, the detected information being applied to the image processing system 313. The electron beam 315 is incident on an object 317. High- resolution images of the object 317 can be recorded. According to embodiments of the present invention, the electron microscope 300 also comprises a variable phase inducing means 319, such as for example a variable electrostatic phase plate, a set of phase plates that can be altered depending on the phase required, etc. The electron microscope 300 also comprises a controller 321 for controlling the imaging. Further features and advantages may be as expressed in other aspects of the present invention. Embodiments according to the present invention also encompass optical coherent microscope having corresponding components as described above. In one aspect, embodiments of the present invention also relate to computer- implemented methods for obtaining wave function data based on an image obtained in an electron microscope using a variable phase inducing means inducing different phases over time during image recording. Embodiments of the present invention also relate to corresponding computer program products. The methods may be implemented in a computing system. They may be implemented as software, as hardware or as a combination thereof. Such methods may be adapted for being performed on computer in an automated and/or automatic way. In case of implementation or partly implementation as software, such software may be adapted to run on suitable computer or computer platform, based on one or more processors. The software may be adapted for use with any suitable operating system such as for example a Windows operating system or Linux operating system. The computing means may comprise a processing means or processor for processing data. According to some embodiments, the processing means or processor may be adapted for processing image data obtained in an electron microscope using a variable phase inducing means according to any of the methods as described above. Besides a processor, the computing system furthermore may comprise a memory system including for example ROM or RAM, an output system such as for example a CD-rom or DVD drive or means for outputting information over a network. Conventional computer components such as for example a keybord, display, pointing device, input and output ports, etc also may be included. Data transport may be provided based on data busses. The memory of the computing system may comprise a set of instructions, which, when implemented on the computing system, result in implementation of part or all of the standard steps of the methods as set out above and optionally of the optional steps as set out above. The obtained results may be outputted through an output means such as for example a plotter, printer, display or as output data in electronic format.

Further aspect of embodiments of the present invention encompass computer program products embodied in a carrier medium carrying machine readable code for execution on a computing device, the computer program products as such as well as the data carrier such as dvd or cd-rom or memory device. Aspects of embodiments furthermore encompass the transmitting of a computer program product over a network, such as for example a local network or a wide area network, as well as the transmission signals corresponding therewith. While the invention has been illustrated and described in detail in the drawings and foregoing description, such illustration and description are to be considered illustrative or exemplary and not restrictive. It will be appreciated, however, that no matter how detailed the foregoing appears in text, the invention may be practiced in many ways, and is therefore not limited to the embodiments disclosed. Other variations to the disclosed embodiments can be understood and effected by those skilled in the art in practicing the claimed invention, from a study of the drawings, the disclosure and the appended claims.

In the claims, the word "comprising" does not exclude other elements or steps, and the indefinite article "a" or "an" does not exclude a plurality. A single processor or other unit may fulfill the functions of several items recited in the claims. The mere fact that certain measures are recited in mutually different dependent claims does not indicate that a combination of these measures cannot be used to advantage. Any reference signs in the claims should not be construed as limiting the scope.

It should be noted that the use of particular terminology when describing certain features or aspects of the invention should not be taken to imply that the terminology is being re-defined herein to be restricted to include any specific characteristics of the features or aspects of the invention with which that terminology is associated.