GB2235061A - Stereo microscopy with single shot x-ray exposure - Google Patents

Abstract

A soft x-ray microscope simultaneously takes two or more exposures to allow three dimensional imaging by stereo viewing. The source of illumination 10 may be a soft x-ray laser. The condenser optics 16 may consist of a multilayer coated mirrors and the imaging optics 20 may be zone plates. The specimen chamber 18 may have a moveable, snout like arrangement (36, Fig. 4) with silicon nitride windows (34 and 38, Fig. 4) to allow transmission viewing of hydrated specimens with a minimum of material in the x-ray path, but adequate supplies of nutrient material in the close vicinity of the specimen. A magnified image 22 may be produced on with high resolution film or a CCD array. <IMAGE>

Description

STEREO MICROSCOPY WITH SINGLE SHOT X-RAY EXPOSURE This invention relates to x-ray microscopy and stereo imaging x-ray microscopy, especially that made with flash x-ray sources, or sources of very high brightness, when the two stereo views need to be made simultaneously.

A soft x-ray laser imaging microscope is potentially a laboratory and clinical tool of great importance.

The primary incentive to investigate soft x-ray imaging is the possibility of looking at hydrated biological specimens at greater resolution than is possible with the light microscope without the potentially damaging specimen preparation used in electron microscopy, and eventually in an hydrated state.

This is important both for research - a living cell has not been seen at a resolution greater than that possible with a light microscope - and in medical diagnostics, where the increased resolution of hydrated specimens could improve diagnostics of many diseases.

One of the potential uses of such an x-ray stereo microscope is in cancer diagnosis. The advantage of looking at biological specimens at high resolution (i.e. 10 times better than obtainable with a light microscope) in clinical pathology is that the shape of the cell nucleus has been shown to be of considerable importance in diagnosing both the existence of cancer and in determining its level of malignancy - i.e. whether it has progressed from proliferation to invasion of surrounding tissue, or it is starting to generate secondary deposits elsewhere in the body. A high resolution three dimensional view of the tissue section will be of considerable use to the clinical pathologist in making a diagnosis.

In principle, stereo imaging is straight forward. Two views are taken of the same object from slightly different angles, and parallax is used to provide the depth information. If the images are arranged so that the parallax variation matches that normally perceived by the human brain, the 3 D variation is seen directly, with no need for computer analysis.

The angle difference is normally introduced by tilting the specimen. A complication with the laser based x-ray microscopy is that the very intense burst of x-rays used to create the image is likely to result it some damage to the specimen. Because the exposure time is so short - of the order of nanoseconds - the damage only manifests itself after the initial picture is taken. However, by the time the specimen has been tilted ready for the next image, the damage will have become apparent. For stereo imaging with the soft x-ray laser microscope, it is necessary to take both view at the same time.

A problem with soft x-ray microscopy is that even at the optimal 4.3 nm in the water window, the total thickness of water that can be tolerated is only 10 microns. If the whole chamber is limited to this thickness, it makes the supp;y of n'jtrients to the specimen difficult. The novel feature of the proposed environmental chamber is the movable snout, which should allow large volumes of nutrients, whilst enabling very small optical path through the specimen in the region of interest By having the snout like arrangement that we propose, there can be copious supplies of nutrient to the cell, and by moving the snout to the point of interest under a light microscope, and then lowering using interference positioning, the region to be imaged can have the required small total thickness.When using the 18.2 nm laser, where the total allowed water and specimen thickness is only 0.5 microns, the vail of a living cell can be viewed, without impacting on the rest of the cell. Moreover, if the snout is of the order of ten microns in diameter, the window on it can be very much thinner than the 100 nm silicon nitride window required on the exit side of the chamber, allowing more flux through the system.

Optimum stereo results - i.e. those most easily viewed - require the correct tilt between the two views of the specimen. This depends on both the final magnification at which the image is going to be viewed and on the specimen thickness. As discussed by Hudson and Makin ("The optimum tilt angle for electron stereo microscopy', Jl. Phy. E: Sci. Instru., 3: 311 (1970)) the parallax Y in the final image is Y=2hMsin(2) --(1) where the parallax is the difference in distance between a corresponding point in the two images, M is the final magnification, 0 is the half angle of titt and h is the vertical separation of points in the object.According to Cheng et al ["Recent Advances in contact imaging of biological materials", X-ray Microscopy, ed Cheng and Jan, Springer-Verlag, 1987.], Y is usually fixed at between 3 and 5 mm for optimal viewing. For instance, a half micron thick specimen at total magnification of 15 000, has e equal to 17.5 degrees as the best stereo angle.

As the single shot optics system will have a fixed tilt angle, it is vital that this initial decision, which impacts on all the optics, is made after careful consideration of the intended use of the microscope.

The reason that the condenser is a two reflection multilayer coated mirror system, is that this gives the maximum flux throughput. Two reflection systems, using silicon-molybdenum multilayer coatings offer normal incidence reflectivity of up to 60% per surface at 18.2 nm [N. Ceglio, "The revolution in x-ray optics", To be published in Proc. of Berkeley workshop "X-ray imaging for the life sciences", May 24-26, 989 and would seem to be the best choice. The imaging is done hy zone plates, as these are the only elements with proven diffraction limited resolution at these wavelengths.The resolution of zone plates is determined by the width of the outermost zones, and is a problem of micro fabrication. Several groups have demonstrated zone plates which should be capable of 20 nm resolution, with 5% efficiency [ C.Buckley et al " Zone Plate Replication by Contact X-ray Lithography", X-ray Microscopy, Springer-Verlag, 247-253, (1 987)i.

A specific embodiment of the invention will now be described with reference to the attached drawings in which: Figure 1 is a schematic view of the stereo x-ray microscope; Figure 2a is a schematic view of the condenser optics system; Figure 2b is a schematic view of an alternative arrangement of the condenser system; Figure 3a is a schematic view of the imaging optics of the microscope; Figure 3b is a schematic view of an alternative arrangement of the imaging optics; Figure 4a is a cross-sectional view of the specimen holder; Figure 4b is a plan view of the specimen holder; Figure 5a is a cross-sectional view of a specimen holder for contact microscopy; Figure 5b is an isometric view of a specimen holder for contact microscopy; and Figure 6 is a schematic layout of an imaging x-ray microscope showing the arrangement for specimen alignment.

The illuminating source for the microscope is an x-ray laser 10, as described in US patents 4,704,718 ("Apparatus and method for generating soft x-ray lasing action in a confined plasma column through the use of a picosecond laser") and 4,771,430 ("Enhancement of soft x-ray lasing action with thin blade radiators").

Such a laser provides a suitable bright, monochromatic light with low divergence. Other sources of soft ray illumination, such as synchrotron ligtit, undulators, laser produced plasmas or z-pinch devices could be used. The light is then focussed onto the specimen 18 by a condenser system consisting of well known x-ray reflecting multilayer mirrors. A standard Schwarzchild system has two spherical surfaces. In one embodiment of the stereo microscope, this Swarzchild system is adapted by having a first spherical mirror 12, and two concentric spherical mirrors 14 and 16, each of which defines an optical axis off set from each other by the appropriate angle necessary for good stereo imaging. An alternative arrangement of the condenser system is shown in figure 2b in which the first multilayer mirror 12 is now two flat mirrors, splitting the incoming light into two beams.The second elements 14 and 16 could be conic sections, or other aspherical surfaces capable of good focus. However, because of the practical difficulty of machining such aspherics to the tolerances required for soft x-ray imaging, they would most likely be spherical mirrors. The fact that a single spherical mirror will not give aberration free illumination of the specimen is not important, as the imperfections in the illumination do not necessarily affect the final image resolution.

The imaging of the specimen 18 is performed by a pair of matching zone plates 20 which produce a magnified image 22 on either high resolution film or a CCD array or other x-ray detecting device of appropriate spatial resolution.

The imaging zone plates 20 can either be of conventional circular design, but mounted at an angle to each other so that they are perpendicular to each other, as shown in figure 3a, or they can be of elliptical design and mounted flat as shown in figure 3b. The reason for doing this is that the small diameters (about 80 microns) and small spacing apart (about 100 microns) may make the physical realization of the design in figure 3a difficult, and that it may be easier to draw the elliptical patterns to the required accuracy using the established electron beam techniques.

The holder for the specimen 18 is shown in detail in figures 4 a and b.

The specimen is isolated from the vacuum by being sandwiched between two thin windows 34 and 38. These could be made from silicon nitride, which at a thickness of 100 nm , and dimension as large as 0.25 mm square, can with stand vacuum. Using the movable snout shaped upper window holder 36, the window 38 can be very much smaller - maybe 10 microns in diameter - allowing the use of even thinner windows, and materials other than silicon nitride, such a carbon films. The other reason for having the snout shape is that it allows a reasonably large reservoir of cell nurturing medium to surround the specimen, whilst maintaining a ve.y small transit thickness for the x-rays in the region of interest. (This can be compared with a design for a specimen holder for contact x-ray imaging shown in figure 5.In this the image is recorded on a photosresist 58, which is spun on a silicon substrate 62. The gold undercoating 60 is placed under the resist to assist in viewing the final developed contact pattern in the scanning electron microscope, by providing enhanced contrast for the backscattered electrons (essentially allowing the SEM to measure resist thickness). The copper or similar metal coating 54 is evaporated on to prevent cells on that part of the resist. In that way the prepared substrate can have specimens grown directly onto the resist, giving close contact between the specimen 18 and the resist 58, which is necessary for good contact imaging, without affecting the separation. The front window of silicon nitride 34 is on an etched silicon substrate 32, held the appropriate distance away by spacer 56.

By loading the entire device under liquid, bowing of the window 34 is obviated.

However, because of the need to keep spacer 56 of the order of 10 microns or less, the cell is surrounded by only a very small amount of nutrient medium enough to allow it to survive for minutes but not hours). In the snout version for the imaging microscope, the snout is moved laterally by rotatable off set disks 48, rotating on axis 48. The snout containing disk 36 is held against the positioning disks 48 by the springs 44. Vertical positioning is achieved by thumbscrews 42. The o-ring 40 doubles as springs against which the vertical screws load and act to seal the contents of the environmental chamber against vacuum.

One further problem of doing high resolution soft x-ray microscopy with a flash source such as the laser 10, is the need to have the specimen in position to within a fraction of a micron, as the depth of resolution of such imaging systems is about 0.1 micron. One means of achieving this is shown in figure 6, which is a schematic of an imaging microscope, which can also be a stereo microscope. In this version, the light from the laser 10 is deflected through 90 degrees by a flat, multilayer coated mirror 11, useful to obtain a vertical microscope axis where the source axis is horizontal. The x-rays are then focussed onto the specimen 18 by the condenser mirrors 27 before being imaged onto the detector 22 by the zone plates 20. Once the system has been aligned over a number of shots, using a test object, the movable beam splitter 21 and the mirror 13 are brought into place. One now has a conventional Michelson type interferometer, in which one arm has the condenser mirror 27 and the specimen 18 and the other arm is a matching optic 17 with a reflecting mirror 19. Illumination is provided by the source 25, suitably collimated by the optic 23. The interference is observed with the eyepiece 15. By adjusting the mirror 19, fringes ate obtained on the specimen using a long coherence source, such as a HeNe laser, or Mercury lamp. Once these are obtained, the system is further adjusted, with a mercury lamp, until white light fringes are seem. This means that the optical path lengths are equal to within a fraction of a micron. The test specimen is removed, the object of interest is inserted, and positioned so that white light fringes appear, indicating that it has been positioned at the correct point.

Claims (19)

1. STEREO MICROSCOPE WITH SINGLE SHOT X-RAY

   EXPOSURE CLAIMS 1. A soft x-ray microscope adapted so as to take two or more exposures simuitaneousiy.
2. 2. A soft x-ray microscope adapted so as to take two or more exposures simultaneously, as in claim 1, in which the condenser system is a muftilayer coated mirror system.
3. 3. A soft x-ray microscope adapted so as to take two or more exposures simultaneously, as in claim 1, in which the condenser system is a Schwarzchild mirror system, coated with an appropriate multilayer coating.
4. 4. A soft x-ray microscope adapted so as to taKe two or more exposures simultaneourly, as in claim 1, in which the condenser system is two Schwarzchild mirror systems, in which cnh ~e first mirror is common.
5. 5. A soft x-ray microscope adapted so as to take two or more exposures simultaneously, as in claim 1, in which tne condenser system is a combinatior of a flat first mirror and off axis conic sections as the secondary mirrors.
6. 6 A soft x-ray microscope adapted so as to take two or more exposures simuitaneously, as in claim 1, in which fix condenser system is a constructed of diffraction optics.
7. 7. A soft x-ray microscope adapted so as to take two cr more exposures simultaneously, as in claim 1, in which the condenser system is constructed of zone plates.
8. 8. A soft x-ray microscope adapted so as to take two or more exposures simultaneously, as in claim 1, in which the imaging optics are two or more zone plates.
9. 9. A soft x-ray microscope adapted so as to take two or more exposures simultaneously, as in claim 1, in which the imaging optics are two or more elliptical zone plates.
10. 10. A soft x-ray microscope adapted so as to take two or more exposures simultaneously, as in claim 1, in which the imaging optics are multlayer coated mirror systems.
11. 11. A soft x-ray microscope adapted so as to take two or more exposures simultaneously, as in claim 1, in which the imaging optics are multilayer coated mirrors of the Schwarzchild type.
12. 12. A soft x-ray microscope adapted as in any of claims 2 to t1, in which the specimen is observed in a holder having a snout arrangement to minimize total thickness in the region of imaging, but allow a large volume of nutrient medium to be in close proximity to the specimen.
13. 13 A soft x-ray microscope adapted as in claim 12 in which the snout is moveable.
14. 1 A xrry microscope adapted in claim 12 or 13 in which the x-ray @ansmitting widows ar: constructed from silicon nitride.
15. 15. A soft x-ray microscope adapted as in any of claims 2 to 14 in wr.r; te source of x-rays is a soft x-ray laser.
16. 16. A soft x-ray microscope adapted as in any of claims 2 to 14 in which the source of x-rays is a laser produced plasma.
17. 17. A scft x-ray microscope adapted as in any of claims 2 to 14 in which the source of x-rays is such as to allow exposures in less than a second.
18. 18. A rPt x-ray microscope adapted as in any of claims 2 to 17 in which the specimen postitioning is done by optical interferometry.
19. 19. A soft x-ray microscope adapted as in any of claims 2 to 17 in which the specimen postitioning is done by white light optical interferometry.

    20 A soft x-ray microscope substantially as described herein with reference to figures 1 to 6.