WO2010136319A1

WO2010136319A1 - Correlative optical and particle beam microscopy

Info

Publication number: WO2010136319A1
Application number: PCT/EP2010/056229
Authority: WO
Inventors: Torsten Sievers; Lutz Hoering
Original assignee: Carl Zeiss Ag
Priority date: 2009-05-27
Filing date: 2010-05-07
Publication date: 2010-12-02
Also published as: DE102009022912A1; DE102009022912B4

Abstract

The invention relates to a system for the correlative optical microscopy and particle beam microscopy of a sample (3, 17), comprising: an SPI microscope (1) for the optical representation of the sample (3), which represents the sample (3) along a z-direction and illuminates the sample substantially perpendicular thereto, wherein the SPI microscope comprises a sample moving device (12) that moves the sample (3) such that different sample regions, which have different positions at least along the z-direction before the movement starts, are represented, a control device (13) that captures coordinate data associated with a position defined at least in the z-direction of structures detected in the sample (3), a sample cutting apparatus (10, 11; 19, 20) that cuts the sample (3) on the basis of the coordinate data, and a particle beam microscope (15) for the particle beam microscopy of the sample (17) remaining after cutting.

Description

Correlative optical and particle beam microscopy

The invention relates to the microscopy of an object with a combination of optical microscopy and particle beam microscopy.

For the purposes of this description, the term optical microscopy is understood to mean a microscopy method which uses radiation which obeys the optical laws, in particular in the visible range, ie light. Particle beam microscopy in the sense of this description is given when imaging takes place by means of a beam of charged particles, for example in the form of electron beam microscopy. As far as in this description of light microscopy or electron beam microscopy is mentioned, this is only an example of optical microscopy or particle beam to understand microscopy.

For biological and material science samples in particular, it is often desirable to study both with optical microscopy, eg with light microscopy, and with particle beam microscopy, eg electron microscopy. The prior art uses complicated microscopes, which can perform both microscopy methods. Such a microscope is known for example from EP 0849765 A2 or US 6683316 B2. Such combination microscopes are particularly expensive because the optical microscope must be completely installed in the vacuum chamber, which is required for particle beam microscopy, and a sample table must be provided, which shifts the sample between two microscopes in a vacuum. The result is a relatively large vacuum volume and also a high cost in the production of the optical microscope, which must then be created in vacuum-compatible design. One waives the particle beam microscopy on the arrangement of the object in a vacuum, such. B. in the combination microscope according to US 20080308731 A1 suffers the imaging quality, since the electrons are scattered on a membrane and in air. An alternative for the use of such combination microscopes is the sequential use of individual devices. This is known, for example, from the publication: MS Lucas, P. Gasser, M. Günthert, J. Mercer, A. Helenius and R. Wepf: Correlative 3D microscopy: LSM and FIB / SEM tomography used to study cellular entry of vaccinia virus, A. Aretz, B. Hermanns-Sachweh, J. Mayer (Eds.): EMC 2008, Vol. 3: Life Science, p. 361 -362, Springer-Verlag Berlin Heidelberg 2008. There, the optical image of the sample is first taken, for example with confocal laser scanning microscopy. Subsequently, an attempt is made to record the sample region already optically imaged in this way (also referred to as region of interest - ROI) with an electron microscope. The image of the most voluminous sample takes place in that the sample is removed in layers by means of a focused ion beam and imaged with the electron microscope.

The sequential use of the confocal laser scanning microscope and the electron microscope has the disadvantage that the position, in particular the axial position of the examined in each microscope sample area can not be assigned exactly to the corresponding position of the examination in the other microscope. The correlation of the data obtained in each case is therefore difficult or at least very much subject to errors. A correlative optical 3D microscopy and particle beam microscopy is thus not or only partially possible. In another known approach, a sample is embedded. The investigation of living biological samples is therefore no longer possible. Then, as described in KD Micheva and Stephen J. Smith: Array Tomography: A New Tool for Imaging the Molecular Architecture and Ultrastructure of Neural Circuits, Neuron 55, 25-36, June 5, 2007, the specimen with an ultramicrotome in Thin sections disassembled. Assuming a typical sample size of 5 to 10 μm, with a slice thickness of 100 nm, approximately 50 to 100 slices are produced, which must first be analyzed first by light microscope and then by electron microscope. The resulting 2D image series are then fused to form a 3D image data set. Subsequently, the 3 D image data sets from optical microscopy and particle beam microscopy are superimposed. The disadvantage of this approach is that serial cuts must be generated, which has hitherto not been automated, but requires time-consuming, error-prone and tedious manual work. Due to this manual work, the assembly of the 2D image series into a 3D image data set is also very complicated because individual sections can be twisted relative to one another and in any case are shifted relative to each other.

The only hitherto known solution for correlating the image data from the two microscopes uses an additional mechanism which presses a pointed object, for example a needle, into the sample so as to form a narrowing impression in the sample produce. On the one hand, a corresponding mechanism must be integrated into the optical microscope, which in itself is disadvantageous. On the other hand, the impression must be made outside the image field in order not to destroy the actual sample information. However, the transmission of the information to the sample area is automatically faulty. Another disadvantage is the additional expenditure of time associated with the assignment of the impression to the actually interested sample structure. Finally, this approach is limited to resin or plastic embedded samples.

The invention is therefore based on the object to provide a correlative optical and particle beam microscopy, with which the image information from the two microscopy methods can be optimally correlated to each other.

This object is achieved according to the invention by a system for correlative optical microscopy and particle beam microscopy of a sample, comprising: an SPI microscope for optically imaging the sample which images the sample along a z-direction and illuminates it substantially perpendicular thereto, the SPI microscope Microscope a sample movement device, which moves the sample so that different sample areas, which are at least along the z-direction before movement start, come to the illustration, and a control device which detects coordinate data, which a defined at least in the z-direction position of sample structures detected in the sample, a sample cutter which cuts the sample based on the coordinate data, and a particle beam microscope for particle beam microscopy of the sample left after cutting.

The object is further achieved by a method for correlative optical microscopy and particle beam microscopy of a sample comprising: the sample is first optically imaged by SPI microscopy, the sample being imaged along a z-direction and illuminated substantially perpendicular thereto, the sample is moved so that different sample areas, which are at least along the z-direction before movement start in different positions, mapped and coordinate data are detected, which are assigned to a defined at least in the z direction layer of structures detected in the sample, based on the coordinate data which is cut and the sample remaining after cutting is imaged by particle beam microscopy. According to the invention, therefore, a plurality of devices are provided, which are combined to form a system. The term "system" can also be understood to include other process features in the sense of usage or usage instructions for the devices. Thus, the invention combines a SPI microscope with a particle beam microscope and further provides a sample cutting device or the (loading) cutting the sample between the two microscopy operations or in at least one of the microscopy operations.

In a sample, especially in a biological sample, the refractive index is usually not locally constant, but obeys a distribution that is related to the sample structure. Because of this distribution, the axial position can not be determined via the focus position of the optical microscope, since the variations of the refractive index result in a corresponding variation of the z-position of the focus with otherwise identical optical arrangement. Since the refractive index in the sample can not be detected three-dimensionally, the variation of the focal point can not be corrected. Thus, the position of structures to be detected or detected in the sample in the z-direction can not be determined with sufficient accuracy.

The use of a per se known SPI microscope, as described for example in WO 2004/053558, allows an exact determination of the axial position of a sample area to be examined. The imaging of the sample with the SPI microscope avoids these disadvantages of the varying refractive index in that the illumination of the sample takes place transversely to the imaging direction. As in the cited WO 2004/053558, the disclosure content of which is fully incorporated herein, the SPI microscope illuminates the sample by a light sheet which penetrates the focal plane of the transversely projecting lens. The illumination is achieved by optics whose optical axis is substantially perpendicular to the optical axis of the observation lens, and the example, a cylindrical lens o.a. which produces the light sheet. The light sheet is opposite the microscope objective, i. fixed the focus plane, so that the z-coordinate of the imaged sample plane is known with high precision - and this completely independent of the refractive index variation of the sample. If you move the sample along the z-direction, you can record the deep structure of the sample and thus obtain a three-dimensional image. By an additional rotational movement about an axis substantially perpendicular to the axes of the illumination and imaging beam axis, an isotropic sample image can be achieved.

The image data on the sample structure thus generated are, as already mentioned, highly accurate and independent of the variation of the refractive index of the sample.

After the optical imaging thus achieved, the sample is cut. This can take place in one embodiment of the invention in that a microtome is integrated with the sample-cutting device in a sample chamber of the SPI microscope. Of course, other sample cutting devices in the sample chamber are possible. The integration of the sample cutting device in the sample chamber has the advantage that the previously determined Coordinates of the sample structures can be considered when cutting the sample. This cutting of the sample causes the sample to be reduced to sample areas, which are subsequently imaged in particle beam microscopy. Because of the coordinates previously determined in optical microscopy, it is therefore possible to cut out a specific sample area in the sample chamber of the SPI microscope in a defined manner.

Alternatively or additionally, the portion of the sample may also be cut off by ablation in the particle beam microscope if it has a suitable double particle beam source emitting a corresponding particle beam for sample ablation. This offers itself as a sole cutting when the sample to be examined is already so small that it can be examined in the particle beam microscope, or reach the achievable with Teilchenstrahlablation Abtragsdicken.

Thus, in one embodiment of the invention, the following sequence is carried out:

a) taking a 3D image by means of an SPI microscope (either by three-dimensional sample displacement or by two-dimensional imaging and displacement of the sample in the z-direction), b) optionally determining the image position relative to the sample holder, c) optionally chemical fixation of the sample, d) determination of a sample region of interest from the image data obtained, e) excision of the region of interest by means of the microtome, f) removal of the remaining or by cutting sample, g) optionally embedding the sample, h) transfer to the particle beam microscope, i) transfer the coordinates of the sample area of interest to the

Particle beam microscope, j) taking a 3D image by imaging the sample with particle beam microscopy, possibly with ablation of the sample with particle beam.

The invention achieves a reduction of biological samples to a volume which can be easily imaged by particle beam microscopy without losing the coordinates of sample regions of interest or could no longer be corrected. Manual serial cuts are no longer required. In this way, living biological samples can also be imaged using correlative optical and particle beam microscopy. In particular, it is of course possible to split the sample in the SPI microscope into several sections and to examine them by electron microscopy.

An isotropic imaging in the SPI microscope can be achieved in particular by a rotation of the sample about an axis lying substantially perpendicular to the z-direction.

It is essential for the correlation of the image data from SPI microscopy and particle beam microscopy that the sample is no longer changed in its structure after SPI microscopy, at least in the sample region of interest. There is only a reduction to dimensions useful in particle beam microscopy, possibly several times.

The correlation of the image data obtained in the two microscopes is particularly simple when a data communication channel is provided between SPI microscope and particle beam microscope, from the SPI microscope to the particle beam microscope coordinates of areas and / or structures in the sample or one or more dimensional images be transferred to the sample.

It is understood that the features mentioned above and those yet to be explained below can be used not only in the specified combinations but also in other combinations or alone, without departing from the scope of the present invention.

The invention will be explained in more detail for example with reference to the accompanying drawings, which also disclose characteristics essential to the invention. Show it:

1 is a schematic representation of an SPI microscope in the system according to the invention,

2 shows a particle beam microscope of the system according to the invention,

3 is a perspective view of some elements of the microscope of Fig. 1,

4 shows a schematic view of the sample chamber, sample holder and microtome of the SPI microscope of FIG. 1 in a view along the optical imaging axis and FIG

Fig. 5 is an illustration of the elements shown in Fig. 4 in a respect to the Fig. 4 taken from above view. The following describes devices that are combined into a system. Figs. 1 and 2 schematically show two devices which can form the system. In Fig. 1, an SPI microscope 1 is shown, which uses a selective plane illumination (hence the abbreviation SPI) to image a sample, preferably three-dimensional. The microscope 1 has for this purpose a sample chamber 2, in which a biological sample 3 to be examined is arranged. The sample chamber 2 can be partially seen again in a perspective view in FIG. 3 and FIGS. 4 and 5. In all figures, the same reference numerals have been used for structurally and / or functionally identical elements.

As described in WO 2004/053558, the SPIM microscope 1 effects a sample imaging by illuminating the sample 3 with illumination radiation transversely to the imaging direction. For this purpose, the microscope 1 has a light source 4, which illuminates the sample 3 with illumination light formed in the form of a light sheet 6 through an illumination window 5 in the sample chamber 2. This results in the sample 3, a light section 7. Orthogonal thereto, the sample with an imaging device 8, which has a protruding into the sample chamber 2 lens 9, the sample imaged. The optical axis of this figure is referred to below as the z-direction or z-axis and is substantially perpendicular to the light sheet 6. Essential for the microscope 1 is that the distance between the focus of the lens 9 and light section 7, which generated by the light sheet 6 is, is spatially fixed. It is therefore always known for the SPI microscope in which spatial position, i. E. z- coordinate, a captured image is located. This assignment is independent of the refractive index in the sample.

A three-dimensional image of the sample is obtained by displacing the sample 2 along the optical axis of the objective 9. For this purpose, a suitably adjustable sample stage 12 is provided in the sample chamber 2, which like the entire microscope 1 is connected via unspecified or illustrated data connections to a control unit 13. Due to the displacement of the sample 3, the observation plane, i. the light section

7 through the sample. If the imaging device 8 generates a two-dimensional image with the objective 9, a displacement of the light section 7 is sufficient for 3D imaging

Sample movement along the z-axis, i. the observation axis. Uses the

Imaging device 8, however, a confocal detection, is for a three-dimensional

Figure additionally provide a displacement of the sample 3 along the plane of the light section 7. Additional possible sample movements will be discussed in more detail later with reference to FIG. 3.

The sample 3 is to be examined not only in the SPI optical microscope 1, but also in a particle beam microscope 15, as shown for example in FIG. For this is provided that from the sample 3, a sample region 17 of interest is cut off or cut out. In order that this can be achieved with reference to the generated three-dimensional sample image, a microtome 10 is provided in the sample chamber 2, which has a knife 1 1 suitable for cutting the sample 3. This microtome is also controlled by the control unit 13. With him, for example, by driving the sample table 12, a certain, interesting sample area out of the sample 3 or cut off from this. In this cutting, reference is made to the previously generated (ideally three-dimensional) sample image so that the sample table and microtome are relatively brought into a specific position and then a high-precision excision of the sample region of interest takes place by possibly automatic microtome actuation.

This sample area 17 (e.g., sample cut) is then placed in a vacuum chamber 18 of the electron beam microscope 15 (see Fig. 2) and placed thereon on a corresponding sample holder or sample table 18. The electron beam microscope is formed in this embodiment as a double-beam microscope. It has two beam sources, namely an ion beam source 19, which directs an ion beam 20 onto the sample area 17 now serving as a sample, and an electron-optical system in the form of an electron beam device 21, which directs an electron beam 22 onto the sample, and a detector 14 for detection of electrons backscattered from the sample. Due to its high energy, the ion beam 20 causes the removal of sample material, as known to those skilled in the art. The arrangement is now used to image the sample by changing the image with the electron beam 22 and ablation with the ion beam 20 in the volume. The sample 17 is naturally destroyed.

Due to the three-dimensional imaging in the microscope 1 with in particular high-precision information about z-coordinates of sample areas in the system, a sample area of interest 17, for example by the microtome 10, are isolated so that the image data from the microscope 1 and the electron microscope 15 are highly precisely correlated can be obtained, which are obtained from both microscopes maximum well associated with each other image information on the sample 3. Due to the previously effected (3D) imaging in the microscope 1, one knows exactly the position which the sample area 17 shown in the particle beam microscope had in the original sample 3, and can thus correlate the image data obtained by electron microscopy to the optical image data.

As already mentioned, FIG. 3 schematically shows the area of the sample chamber 2 of the microscope 1. In this embodiment, the sample table (not shown) is located below the sample 3, which is located in a cylinder perpendicular to the optical axes of FIG Illumination and imaging optics is rotatable. A three-dimensional shot Sample 3 is now obtained by displacing the sample along the z-axis by a z-displacement, shown schematically in FIG. In addition, the sample is possibly rotated again after such a shift and the image is repeated. This provides several sets of data that are appropriately merged into an isotropic and high-resolution image data set.

The sample 3 is a biological material which is embedded in an agarose gel 26 by way of example. Furthermore, the sample in the sample chamber 2 is lapped with water, which achieves an adaptation to the refractive index of the sample and improves the optical resolution.

The process of cutting the sample is shown schematically in Figs. 4 and 5. The representation of FIG. 4 corresponds substantially to a view of FIG. 1 from below (based on the illustration of FIG. 1). Fig. 5, however, shows a representation which substantially corresponds to Fig. 1. The sample chamber 2 has an access for the sample 3, which is embedded in the aforementioned agarose gel 26. It is located further in a cylinder 29, which consists for example of a glass tube. Guided in the cylinder, the sample can be moved along the cylinder axis, whereby the agarose gel cylinder can be transferred into and out of the sample chamber 2.

In the embodiment illustrated here, a biological sample 3 is examined. For high-resolution investigations, it is necessary to fix the state of the sample. This is usually done by adding a chemical fixative. For this purpose, 29 supply lines 30, which may be formed for example as capillaries, are provided in the cylinder. The fixer can be added over it and after fixation the sample does not change.

After fixation, sample 3 should be further examined by electron microscopy. Due to the much higher resolution of an electron microscope and the regularly low removal rate of an ion beam, it is advantageous or necessary for most samples 3 to bring only one sample region 17 of interest to the sample 3 for electron microscopy, ie to transfer it to the electron microscope. Consequently, in most cases, the sample, if appropriate after fixation, must be tailored to the sample area 17 of interest. In order to be able to perfectly correlate the image data of the electron microscope with the image data of the optical microscope, the cutting must of course take place in an exact and known position. For this purpose, an access for the already mentioned microtome 10 is provided in the sample chamber 2. It is preferably supplied to the illumination window 5 opposite side and includes next to the knife 11, a collecting plate 10, come to lie on the cut slices of the sample. For each slice, at least the z-coordinate of the cut boundaries is known due to the previous imaging. By removing the collecting plate 32, the cut can then be removed from the sample chamber 2. To produce a cut while the knife 11 is advanced in the direction of arrow 31.

As far as has previously been made reference to a cylindrical sample holder, this may in particular be designed so that it can not only be used in the optical microscope 1, but also in the electron microscope 15 can be used.

The mentioned fixation of the sample is advantageous for certain sample types. For other sample types, however, a direct, timely fixation of the sample during observation in the microscope 1 is not required.

Of course, the imaging of the sample by particle beam microscopy can be carried out particularly preferably with an electron beam microscope. Alternatively, however, an ion source, e.g. working with helium ions. The same applies to the removal of the sample area 17 in the particle beam microscope 15. For this purpose, a variety of ion sources come into question.

The optical microscopy of the sample can be carried out both in fluorescence microscopy and in the bright field method.

Claims

claims

A system for correlative optical microscopy and particle beam microscopy of a sample (3, 17), comprising: an SPI microscope (1) for optically imaging the sample (3) which images the sample (3) along a z-direction and in Illuminated substantially perpendicular thereto, wherein the SPI microscope, a sample movement means (12), which moves the sample (3) so that different sample areas, which are at different locations at least along the z-direction prior to movement, come to the illustration, and a control device (13), which records coordinate data associated with a layer defined at least in the z-direction of structures detected in the sample (3), a sample-cutting device (10, 11, 19, 20) which determines the sample on the basis of the coordinate data ( 3), and - a particle beam microscope (15) for particle beam microscopy of the sample (17) remaining after trimming.

The system of claim 1, wherein the particle beam microscope (15) comprises a dual particle beam source (19, 21), wherein one of the particle beams (20) ablates the sample (17), thus realizing the sample cutting apparatus.

3. System according to any one of the above claims, wherein in a sample chamber (2) of the SPI microscope (1), a microtome (10, 11) is integrated as a sample cutting device.

4. System according to any one of the above claims, wherein the SPI microscope (1) comprises a fixing device (29, 30) for chemical fixation of the sample (3), wherein preferably the fixing means (29, 30) in a sample chamber (2) of the SPI microscope (1) is integrated.

A system according to any one of the preceding claims, wherein the sample moving means (12) is adapted to displace the sample (3) substantially along the z-direction and / or rotating the sample about an axis substantially perpendicular to the z-direction.

6. System according to any one of the above claims, wherein a data communication channel 5 between SPI microscope (1) and particle beam microscope (15) is provided over the from

SPI microscope (1) to be transferred to the particle beam microscope (15) coordinates of areas in the sample.

7. A method for correlative optical microscopy and particle beam microscopy of a sample (3, 17) comprising: the sample (3) is first optically imaged by SPI microscopy, wherein the sample (3) is imaged along a z-direction and imaged Illuminated substantially perpendicular to the sample (3) is moved so that different sample areas, which are at least along the z-direction before movement in different positions, mapped and5 coordinate data are detected which a defined at least in the z-direction position of in the Sample are detected on the basis of the coordinate data, the sample (3) is trimmed, and the remaining after trimming the sample (17) is imaged by means of particle beam microscopy. 0

8. The method of claim 7, wherein a portion of the sample (3) is cut off by means of particle beam ablation in the particle beam microscope (15).

9. Method according to one of the above method claims, wherein a part of the sample (3) 5 is cut off by means of a microtome (10, 11) integrated into the SPI microscope (1).

10. The method according to one of the above method claims, wherein the sample after imaging with the SPI microscope (1) and prior to trimming the sample (3) is chemically fixed, preferably still in a sample chamber (2) of the SPI microscope (1 ). 0

11. The method according to any one of the above method claims, wherein the sample (3) is displaced substantially along the z-direction and / or is rotated about an axis lying substantially perpendicular to the z-direction. 5