WO2007065711A1

WO2007065711A1 - Miscroscope specimen holder

Info

Publication number: WO2007065711A1
Application number: PCT/EP2006/011863
Authority: WO
Inventors: Ernst H. K. Stelzer; Francesco Pampaloni; Emmanuel Reynaud; Philipp KELLER; James Swoger
Original assignee: Europäisches Laboratorium für Molekularbiologie (EMBL)
Priority date: 2005-12-09
Filing date: 2006-12-08
Publication date: 2007-06-14

Abstract

Specimen holding device for a microscope comprising: a sealable chamber(30) that is mountable to an objective of the microscope and a specimen support for a microscope, the specimen support (10) comprising at least one window (12) formed in a wall of the specimen support (10), wherein in use a specimen (2) is viewable with the microscope through the at least one window (12) and wherein in use the specimen (2) is embedded in a matrix (4), the matrix (4) being through the at least one window in contact with a liquid surrounding at least partially said specimen support (10).

Description

Description Field of the Invention

The present invention is related to a method and apparatus for viewing specimens in light microscopy. Background to the Invention

Advances in the life sciences are strongly related to the ability to observe dynamic processes in live systems and to mimic relevant in vivo conditions.

For instance, biological cells usually grow and differentiate in soft, jelly-like, three- dimensional growth environments provided by, for example, the extracellular matrix (ECM). Consequently, the relevance of any measurement system or method that reduces the number of dimensions or constrains the temporal resolution in which the biological cells grow should be carefully evaluated. In particular, introduction of hard surfaces (e.g. cover slips) adds elements to the environment of the biological cell that are usually not present in living systems. hi the resulting and essentially two-dimensional growth environment, the dramatic change in the surface-over- volume ratio and the hard surface of the cover slip induces the biological cell to adapt by changing its metabolic function and, in general, its gene expression. An alternative interpretation is that the growth environment with the cover slip favours biological cells that can adapt to such an environment. This most likely pushes any biological system's response into a realm that is at least less physiologically relevant.

On a different level, in biophysical studies microtubules are often observed close to a hard surface which could account for why the behaviour of the microtubules in this setting differs from the behaviour that is observed in a more physiological situation. For instance, the micro- tubule growth rates and catastrophe frequencies are force-dependent (see, for example, Dog- terom M, Yurke B: Measurement of the force-velocity relation for growing microtubules. Science 1997, 278:856-860 and Janson ME, de Dood ME₅ Dogterom M: Dynamic instability of microtubules is regulated by force. JCe// Biol 2003,161:1029-1034) and in S. pombe microtubule bundles seem to bend (Brunner D, Nurse P: CLIP170-like tiplp spatially organ- izes microtubular dynamics in fission yeast. Cell 2000, 102:695-704.) rather than depolymer- ise spontaneously when they touch the yeast's cell surface.

The importance of "introducing the third dimension" in biology, as discussed in Abbott A: Biology's new dimension. Nature 2003, 424:870-872, is now being realised. The main drive does not stem from basic research in cell biology but rather from clinical scientists (Webb DJ, Horowitz AF: New dimensions in cell migration. Nat Cell Biol 2003, 5:690-692) who would like to take advantage of the results of modern molecular biology (Jacks T, Weinberg RA: Taking the study of cancer cell survival to a new dimension. Cell 2002, 111 :923-925). A quantitative analysis of live three-dimensional structures requires fast optical sectioning. Confocal fluorescence microscopy works well in relatively thin specimens. For large objects, however, a signal is scattered and rejected by a pinhole. An excellent resolution is retained by physically sectioning the specimen (Weninger WJ, Mohun T: Phenotyping transgenic embryos: a rapid 3-D screening method based on episcopic fluorescence image capturing. Nat Genet 2002, 30:59-65), but this destroys the specimen irretrievably and is not applicable to live preparations. Most methods that have been developed to enhance the resolution— e.g. 4Pi-confocal microcopy (Hell S, Stelzer EHK: Properties of a 4Pi confocal fluorescence microscope. JOpt SocAm 1992, A9:2159-2166 ), I5M microscopy (Gustafsson MGL, Agard DA, Sedat JW: I5M: 3D widefield light microscopy with better than 100 nm axial resolution. J Microsc 1999, 195 : 10- 16) and STED microscopy (Klar TA, Jakobs S, Dyba M, Egner A, Hell SW: Fluorescence microscopy with diffraction resolution limit broken by stimulated emission. Proc Natl Acad Sci USA 2000, 97:8206-8210). These methods require an excellent control over the phase of the wavefront of the light and hence cannot address challenges encountered in multicellular objects.

Nevertheless, some of the recent developments, for example with I⁵M by Gustafsson

(Gustafsson M: Nonlinear structured-illumination microscopy: Wide-field fluorescence imaging with theoretically unlimited resolution. Proc Natl Acad Sci USA 2005, 102:13081-13086) indicate an impressive potential for improvement in resolution. Other imaging techniques for large specimens, such as optical projection tomography (OPT) (see Sharpe J, Ahlgren U, Perry P, Hill B, Ross A, Hecksher-Sørensen J, Baldock R, Davidson D: Optical projection tomography as a tool for 3Dmicroscopy and gene expression studies. Science 2002, 296:541- 5451 and micro magnetic resonance imaging (μMRI) (Louie AY, Huber MM, Ahrens ET, Rothbacher U, Moats R, Jacobs RE, Fraser SE, Meade TJ: In- vivo visualization of gene expression using magnetic resonance imaging. Nat Biotechnol 2000, 18:321-325) cannot take advantage of fluorescent proteins and hence lack specificity. Two-photon microscopy regarded by many regard as the best technique for use with sensitive biological material (Feijo JA, Moreno N: Imaging plant cells by two-photon excitation. Protoplasma 2004, 223 : 1 -32) suffers from a moderate resolution (Stelzer EHK, Hell SW, Lindek S, Strieker R, Pick R, Storz C, Ritter G, Salmon N: Nonlinear absorption extends confocal fluorescence microscopy into the ultra-violet regime and confines the observation volume. Opt Commun 1994, 104:223-228). It is currently not clear how much damage two-photon microscopy actually creates with its relatively high average intensities (several mW).

Our suggestion for an optically sectioning instrument is based on light-sheet technology and is termed single plane illumination microscopy (SPEvI) (Huisken J₅ Swoger J, Del Bene F, Wittbrodt J, Stelzer EHK: Optical sectioning deep inside live embryos by selective plane il- lumination microscopy. Science 2004, 305:1007-1009) also described in international patent application WO 2004/053558. The disclosure of this patent application is incorporated herewith by reference. The SPIM is a new type of fluorescence microscope allowing optical sectioning of biological specimens by scanning the specimen through a tiny focused laser sheet. The SPEVI operates on four principles: illumination with a light sheet, observation along at least one direction perpendicular to the illumination plane, rotation of the specimen about an axis parallel to gravity, and a stationary chamber with the immersion medium. The SPEVI owes much to the 'Ultramikroskop', an orthogonal, darkfield illuminator invented by Sieden- topf and Zsigmondy in 1903 (Siedentopf H, Zsigmondy R: Uber Sichtbarmachung und Grδssenbestimmung ultramikroskopischer Teilchen. Ann Phys 1903, 10:1) to visualize nm- sized gold particles. The concept has been used in ophthalmic instruments (Campbell CJ, Koester CJ, Rittler MC, Tackberry RB (Eds): Physiological Optics. Harper and Rowe; 1974) and in a macroscope used by Voie to observe the cochlea (Voie AH, Burns DH, Spelman FA: Orthogonal-plane fluorescence optical sectioning: three-dimensional imaging of macroscopic biological specimens. JMicrosc 1993, 170:229-236). Fuchs described such a device (Fuchs E, Jaffe JS, Long RA, Azam F: Thin laser light sheet microscope for microbial oceanography. Opt Express 2002, 10:145-154) to observe microbes while Huber (Huber D, Keller M, Robert D: 3D light scanning macrography. JMicrosc 2001, 203:208-213) reconstructed mm-sized specimens using scattered light. The orthogonal arrangement of point illumination and point detection was also used in confocal theta fluorescence microscopy (Stelzer EHK, Lindek S: Fundamental reduction of the observation volume in far-field light microscopy by detection orthogonal to the illumination axis: confocal theta microscopy. Opt Commun 1994, 111:536-547) with lenses of high numerical aperture.

In the SPIM a three-dimensional data set is recorded by scanning the specimen through the stationary light sheet while recording the fluorescence light with a camera. The specimen can be as small as a few micrometers (e.g. microtubule asters or yeast cells), in the hundreds of micrometers range (e.g. Madin-Darby canine kidney [MDCK] cysts or endothelial spheroids) or even as large as several millimetres (e.g. zebrafish or medaka embryos). The properties of the detection lens depend on the necessary working distance and on the material required for the embedding procedure (agar, liquid, gas). Since the specimen is attached to a stage it can be rotated as well as translated, meaning that three-dimensional image stacks can be recorded along different directions (Swoger J, Huisken J, Stelzer EHK: Multiple imaging axis microscopy improves resolution for thick-specimen applications. Opt Lett 2003, 28:1654-1656). These independently recorded data sets can be combined into a single three-dimensional data set with a spatial resolution that is dominated by the lateral resolution of the detection system. However, from a practical point of view, the most important advantage of the SPIM is that only those parts in the specimen that are observed are in fact illuminated. Out-of-focus light is not generated. With single-photon excitation and laser powers in the μW range, the SPIM ensures dramatically reduced photobleaching, is less phototoxic and is particularly well suited to the observation of live and dynamic processes. The SPIM is a new type of fluorescence microscope allowing optical sectioning of biological specimens by scanning the specimen through a tiny focused laser sheet. The specimen has to be immersed in aqueous medium for imaging, as required by the use of water-dipping objective lenses. Thus, there is a need for a specimen holder that enable moving the specimen with respect to a microscope objective. In the prior art, the specimens are embedded into an agarose full cylinder or injected into a hollow cavity inside an agarose cylinder. The agarose cylinder is subsequently fixed to a plastic/metallic holder, which is then connected to the xyz stage. The main drawbacks of the current system are the intrinsic low mechanical and chemical stability of agarose, and the difficulty of setting a mechanically stable connection with the xyz stage.

Summary of the invention

The present invention provides a specimen support for a microscope, the specimen support comprising at least one window formed in a wall of the specimen support, wherein in use a specimen is viewable with the microscope through the at least one window and wherein in use the specimen is embedded in a matrix, the matrix being through the at least one window in contact with a liquid surrounding at least partially said specimen support

The present invention also provides a specimen support for a microscope, wherein in use a specimen is viewable with the microscope, the specimen being embedded in a droplet matrix, the droplet matrix being attached to the specimen support and being in contact with a liquid surrounding at least partially said specimen support.

The matrix may comprise a polymer gel, comprising bio-polymers like agarose, collagen Ma- trigel or similar or a combination thereof.

The specimen support may be of plastics material or any other material suitable for use in the liquid the chamber is filled with in use. The specimens support may be a rigid structure with an inner volume for taking up the specimen in the volume.

The specimen supports may be advantageously used with a holding device for a microscope comprising: a sealable chamber that is mountable to an objective of the microscope. The specimen support may be mounted movably within the sealable chamber wherein in use the specimen is viewable with the microscope.

The sealable chamber may be mounted removably to the objective. In use, during viewing the specimen, the objective is fixed with respect to the chamber. The objective may be sealed to the chamber, for example by an 0-ring in such a way that the working side of the objective, (the side or lens facing towards the specimen to be observed) is arranged inside the chamber.

In use the sealable chamber may be filled with liquid. In particular, the sealable chamber may be filled in use with an aqueous solution, cell culture media or similar, comprising compounds that shall come into contact with the specimen to be viewed. The sealable chamber may be connected to or be part of a perfusion system for filling the sealable chamber or replacing the solution inside the sealable chamber. The sealable chamber may also comprise sensors for measuring or controlling physical, chemical and/or biological conditions inside the sealable chamber.

The invention also provides a method for viewing a specimen with a microscope, the method comprising: dipping a specimen support into a solution comprising the specimen; pulling the specimen support out of the solution, wherein the specimen support is adapted to form at least one droplet of a liquid containing the specimen on the specimen support; arranging the at least one droplet attached to the specimen support in front of an objective of the microscope; viewing the specimen in the droplet with the microscope. The method may comprise adding a matrix forming agent, such as a gel-forming polymer as for example agarose, to the droplet thereby forming a droplet matrix.

The method is particularly useful for viewing a specimen inside a sealable chamber. The specimen supports, the specimen holding device comprising the sealable chamber and the method are each or in any combination particularly useful for viewing three-dimensional specimen, for example with a single plane illumination microscope. However, any other microscope may be used with the invention. Description of the figures

The features of the present invention may be better understood when reading the detailed description and the figures, wherein identical numbers identify identical or similar objects.

Figure 1 shows schematically a SPIM measurement principle with a specimen holder according to the invention.

Fig. 2 shows a cut through a measurement chamber for a light microscope that can be used with a SPIM.

Figs. 3a and 3b show the measurement chamber of Figs 2a and 2b in an assembled view (Fig. 3a) and an exploded view (Fig. 3b). Fig. 4a shows a specimen support according to the invention in greater detail.

Fig. 4b shows how the specimen is mounted into the specimen support of Fig. 4a

Fig. 5 shows how the specimen support of Fig.4a is mounted to the measurement chamber of Figs. 2a, 2b, 3a and 3b.

Fig. 6 shows how the specimen holding device comprising the measurement chamber and the specimen support can be used with a perfusion system. Fig. 7 shows a second type of a specimen support

Fig. 8 shows the droplet specimen support of Fig. 7 being used for high throughput screening.

Detailed Description of an illustrative example

Figure 1 shows schematically a SPIM measurement principle with a specimen holder 10 according to the invention. The specimen holder 10 comprises a specimen 2 that is attached to the specimen holder 10 such that in use with a microscope, a movement of the specimen holder 10 is equally moving the specimen 2. Only an objective 20 of the microscope is shown in Fig.l. The microscope may be any microscope known in the art. The invention may be used with, but is not limited to, a SPEvI described for example in international patent application WO 2004//053558.

An illumination light beam 22 may be used to illuminate the specimen or a portion of the specimen 2 attached to the specimen support 10. The illumination light beam 22 may be oriented along an axis Y that is substantially perpendicular to the observation direction defined by the objective 20 (indicated as Z-direction in Fig. 1). hi the SPIM, an incoming light beam is focussed to an illumination plane, for example by the use of cylindrical lenses. However, other illumination shapes and/or directions of the incoming light beam may be applied with the invention. For example bright field illumination may used as indicated by arrow 24 in the in Fig.l The specimen support 10 may be attached to a xyz stage (not shown in Fig 1) to move or rotate the specimen support 10 with the specimen 2 with respect to the objective 20 and the illumination light beam 22, 24.

The objective 20 may be a standard objective commercially available or any other form of an objective known in the art.

In many cases, in particular when biological specimens or specimens are viewed, the specimen 2 has to be in a controlled environment. Depending on whether water immersion or oil immersion or air objectives are used, care has to be taken to observe the specimen 2 through the corresponding medium.

Therefore the invention provides a sealable chamber 30. The sealable chamber 30 is illustrated in cut-through view Fig. 2. The sealable chamber 30 has an objective opening 31. The objective 20 is shown in Fig. 2 inserted into the objective opening 31 of the sealable chamber 30. The objective 20 is removably inserted into the objective opening 31 of the sealable chamber 30 in such a way that the side of the objective 20 is facing the specimen inside the sealable chamber 30, while the section of the objective 20 facing the microscope is arranged outside the sealable chamber 30. The objective opening 31 of the sealable chamber 30 is in use closed by the objective 20 and sealed by an O-ring 28.

The sealable chamber 30 has further windows or openings 32, 33 and 34. The openings 32, 33 and 34 may be closed and sealed by transparent or semi transparent, for example filtering, covers 42, 43, 44. The covers 42, 43, 44 may be made from glass or any other suitable material known in the art. The openings 32 and 33 are arranged to allow illumination perpendicular to the observation direction defined by the objective 20, for example by the illumination light beam 22. The transparent covers 42 and 43, closing the openings 32 and 33, are trans- parent to the illumination light. The transparent covers 42 and 43 may be used to filter out other wavelengths not used for illumination of the specimen 2. A third cover 44 covers the opening 34. The opening 34 may be used for bright field illumination and the cover 44 may chosen to be transparent for the desired wavelength range. It is to be understood that the number and arrangements of the openings 32, 33 and 34 and the covers 42, 43 and 44 are purely exemplary and that fewer or more ones of the openings may be used with the invention. A person skilled in the art will easily adapt the number, an arrangement of the openings and the material and the degree of transparency used for the covers to the specimen, and the observation conditions.

The covers 42, 43, 44 are attached removably or non-removably to the openings 32, 33, 34 to sufficiently seal the chamber 30 by means known in the art.

Fig. 3a shows a side view of the sealable chamber 30 of Figs 2a and 2. Fig. 3b shows an ex- ploded view of the sealable chamber 30 of Fig. 3a. The sealable chamber 30 further has a specimen support opening 36 through which the specimen support 10 and the specimen 2 can be inserted into the sealable chamber 30. The sealable chamber 30 further comprises a ground plate 37. A temperature control 38 may be mounted on the ground plate 37 that may be used for controlling and adjusting the temperature inside the sealable chamber 30. For example, electric heating elements may be inserted in the temperature control 38 that are contacted via contacts 39. Thus the temperature of the specimen can be kept at, for example, 37°C if biological specimens are viewed. The sealable chamber 30 further comprises a shutter 35 that can be used to shut and thereby seal the sealable chamber 30 when the sealable chamber 30 is removed from the objective 20 and the microscope. The sealable chamber 30 further comprises an inlet 48 and an outlet 49 for perfusing the seal- able chamber 30 during use. The inlet 48 and the outlet 49 may be in the form of standardized fittings that allow the attachment of tubing, valves or other liquid guiding means. Perfusion of the sealable chamber will be described below with respect to Fig. 6. Fig. 4a shows the specimen support 10 in greater detail. The specimen support 10 has a holder 11 having a window 12 formed in a wall. The holder 11 may be of plastics material. The holder 11 may have a tube-like structure and the window 12 may be formed by cutting a portion out of the wall of the tube like structured holder 11. The form of the holder 11 and the window 12 may be adapted by a person skilled in the art according to the type of the specimen 2 or the type of observation to be performed.

The holder 11 has an inner volume. In case of the tube-like structured holder 11 illustrated in Figs 1 and 4, the inner volume may be cylindrical. The specimen 2, for example biological specimens such as cell cultures, is provided in a three-dimensional matrix 4. The matrix 4 may be an agarose gel, collagen gel, Matrigel, Pu- ramatrix or another polymer gel or biopolymer gel or similar. Other ones of the matrices 4 known to a person skilled in the art may also be used with the present invention. The matrix 4 may have substantially the same cross section or smaller as the inner volume of the holder 11. The matrix 4 comprising the specimen 2 can thus be inserted into the inner volume of the holder 11. According to the invention, the matrix 4 and, in particular, the specimen 2 are arranged inside the holder 11 such that they can be viewed through window 12 of holder 11.

Fig. 4b shows how the specimen is mounted into the specimen support of Fig. 4a. The matrix 4 may comprise a container, having substantially the form of a beaker formed of agarose-gel. The container may have an outer dimension of substantially the same cross section or smaller thanthe inner volume of the holder 11. The container may be filled with a specimen-gel mix (ii). The specimen-gel mix may comprise a gel different than agarose, such as but not limited to collagen . The beaker may be closed by an agarose cover to form a closed agarose container (iii). The container containing the specimen may thus form the matrix 4. The matrix forming container comprising the specimen 2 may then be inserted into specimen support 10 (iv).

It is to be understood that the gels described are purely exemplary and that any other gel suited and known to a person skilled in the art can be used.

The window 12 may provide direct access to the matrix 4 comprising the specimen 2 such that the matrix 4 is, in use, in contact with the atmosphere surrounding the holder 11. The window or a portion of the window 12 may also be closed by a first layer separating the matrix 4 or the specimen 2 from the atmosphere surrounding the holder 11. The first layer may be a foil for example from a plastics material. The first layer may also be permeable for certain components of the atmosphere surrounding the holder 11.

The first layer may also replace the matrix and the specimen 2 may be stored inside inner volume and prevented form leaking out by the first layer.

Returning nor to Fig. 4, the holder 11 may comprise a base 15 forming the bottom of the inner volume and keeping the matrix 4 in a desired position inside holder 11. The base 15 may be made from a plastics material or any other material usable as a first closing of the inner volume of holder 11. The base 15 may also be integrated into holder 11.

The holder may also comprise a second closing 16 for fixing the matrix 4 in the inner volume of the holder 12. The second closing 16 may be movable in the inner volume of the holder or may serve as a cork or stopper for the matrix 4. The matrix 4 may thus be fixed between the base 15 and the second closing 16 in the inner volume of the holder 11.

The second closing 16 may also be an anti-bacterial filter providing a sterile sealing of the matrix 4 comprising the specimen 2.

The specimen support 10 comprises an attachment member 18 for mounting the specimen support 10 to a specimen support moving device (not shown). The specimen support moving device may enable the movement of the specimen support 10 comprising the specimen 2 in one, two or three dimensions. The specimen support moving device may for example be a xyz stage known in the art, for example driven manually, by piezo elements, stepper motors or any other actuators known. The specimen support moving device may also enable rotation of the specimen support 10 comprising the specimen 2.

The attachment member 18 may be a magnet or any other mounting system that allows mounting of the specimen support 10 on the specimen support moving device. A movement of the specimen support moving device is thus directly transmitted to the specimen support 10 and the specimen 2.

A sealing member 60 is further attached to the specimen support 10. The sealing member 60 may be a flexible protective foil, for example made from plastics material. The sealing member 60 may be attached with a connector 61 to the specimen support 10.

Fig. 5 illustrates the insertion of the specimen support 10 into the sealable chamber 30. The specimen support 10 is inserted through a specimen support opening 36 without contact with the walls of the sealable chamber 30. When inserted in the sealable chamber 30, the specimen support 10 is essentially supported by the specimen support moving device. Thus the speci- men support 10 comprising the specimen 2 can be moved by the specimen support moving device with respect to the sealable chamber 30 and the objective 20. Movement of the specimen 2 inside the sealable chamber 30 is, in principle, only limited by the size of specimen support opening 36 that is larger, than the cross-section of the specimen support 10. The movement of the specimen 2 may also be limited by the range of the specimen moving device or other means.

The sealing member 60 covers the specimen support opening 36, when the specimen support 10 is inserted into the sealable chamber 30. The sealing member 60 may be attached to the sealable chamber 30, for example, by a sealing O-ring 63 that fixes the sealing member 60 in the form of the flexible foil to a rim defining the specimen support opening 36.

The sealing member 60 may also be attached otherwise to the sealable chamber. For example the sealing member may be attached, for example glued, to the sealing member 60 prior to insertion of the specimen support 10. The specimen support 10 may, after insertion, be sealed to the sealing member 60 by the connector 61. The connector 61 may have the form of an O- ring or a stopper. The sealable chamber 30 may in use be filled with liquid or any other atmosphere that is provided to the specimen 2 during observation. For example, the specimen 2 may be a cell culture and the sealable chamber 30 may be filled with culture media, know in the art for cell culturing. Once the specimen support 10 is inserted in the sealable chamber 30 and thus at least partially into the liquid inside the sealable chamber 30, the specimen 2 is via the matrix 4 and the window 12 in contact with the liquid.

The specimen 2 and the liquid inside the sealable chamber 30 can be kept separate from the outside environment because the sealable chamber 30 allows complete sealing. The term "sealing" is in the context of the invention to be understood as sterile sealing or otherwise clean. Thus the specimen 2, in particular in case of a biological specimen can be kept sterile inside the sealable chamber 30, at least during observation. In addition, the specimens 2 can be observed that require a clean, possible dust-free or contamination free environment.

The term "sealing" may also be understood as toxic or potentially hazardous sealing. Thus the specimen 2 inside the sealable chamber 30 can comprise toxic or other potentially dangerous components that are prevented from exiting the sealable chamber 30.

Fig. 6 illustrates how the sealable chamber 30 can be connected to a perfusion system. The perfusion system allows liquid to be filled into, emptied from and changed inside the sealable chamber 30 and thereby controlling the environment or the atmosphere at the specimen 2 attached to the specimen support 10.

The inlet 48, for example shown in Figs. 3a and 3b, may be connected with tubing to a liquid reservoir 72. A pump 73, for example a peristaltic pump, may be employed to pump liquid from the reservoir 72 into the sealable chamber 30. When cell cultures are used as the specimens 2 the reservoir 72 may contain cell culture medium, PBS or similar. A CO₂ supply 74 may be provided to the cell culture medium to control the CO₂ concentration of the medium. The inlet 48 may also be connected via valves to several reservoirs for changing the liquid composition inside the chamber and thus altering the conditions for the specimen 2 inside the sealable chamber 30, for example prior or during observation. Any perfusion system known in the art may use with the present invention.

The outlet 49, for example shown in Figs. 3a and 3b, may be connected with tubing to a liquid waste 76 taking up the material exiting the sealable chamber. A pump 77 may be provided for pumping liquid out of the sealable chamber and into the waste 76. Fig. 7 shows a second type of a specimen support 100 which is termed a droplet specimen support 100. The droplet specimen support 100 of Fig. 7 may be used in parallel or alternatively to the specimen support 10 described above with respect to Figs. 4 and 5. The droplet specimen support 100 can comprise an O-shaped portion 110 for taking-up and supporting a droplet.

For example, the O-shaped portion 110 of the droplet specimen support 100 may be dipped into a solution containing the specimen 2. By pulling out the O-shaped portion 110 of the droplet specimen support 100 out of the solution, a droplet 200 of the solution comprising the specimen remains attached to the O-shaped portion. Thus the droplet 200 comprising the specimen can be moved or otherwise handled.

The droplet 200 attached to the droplet specimen support 100 and comprising the specimen is then arranged in front of the objective 20 and illuminated by an illumination light beam 22, 24. The droplet may be arranged such that the focus of the objective is inside the droplet thus focussing on the specimen.

The droplet attached to the droplet specimen support 100 and comprising the specimen may be arranged inside the sealable chamber 30 as described with respect to Fig. 5 for the specimen support 10.

A matrix-forming agent may be injected or otherwise inserted into the droplet 200 comprising the specimen. For example, agarose or another polymer or a bio-polymer may be inserted into the droplet 200 for forming a gel-like droplet matrix 400 comprising the specimen. The droplet matrix 400 may be formed by collagen gel, Matrigel, Puramatrix or another polymer gel or biopolymer gel or similar. Other ones of the droplet matrices 400 known to a person skilled in the art may also be used with the present invention. The droplet matrix 400 may stay at the O-shaped portion 110. The droplet specimen support 100 and the matrix droplet comprising the specimen may be arranged in front of the objective 20 as described above.

The droplet matrix 400 allows the use of water or oil immersion objectives because matrix droplet 400 can be brought in contact with water, oil or any other liquid without changing the matrix droplet 400 considerably.

Thus the droplet matrix 400 may also be surrounded by a liquid, for example when the droplet matrix 400 is arranged inside the sealable chamber 30. The matrix droplet 400 may thus be perfused. Fig. 8 shows how the droplet specimen support 100 of Fig. 7 may be used for high throughput screening. The specimens may be stored or cultured in arrays in the form of multi-well plates or similar apparatus as known in the art. A plurality of the droplet specimen support 100 may be used to form a droplet 200 comprising the specimen or a droplet matrix 400 at each O- shaped portion 110 of the specimen support 100. Thus many identical or different specimen can be investigated in parallel or sequentially.

By dipping and pulling out of the O-shaped portion 110 a droplet 200 comprising the specimen and possibly a droplet matrix 400 can be obtained in a fast and simple manner. Thus, the system and method can be easily automated, for example by using robotics known in the art and a high throughput may be obtained.

It is obvious to a person skilled in the art that the same solution comprising the specimen can be used sequentially by repeating the dipping, preferably with different droplet specimen supports 100.

Examples:

Case study: microtubule asters In the studies of microtubule asters, we transferred the two-dimensional experiments of microtubule dynamic instability (Mitchison T, Kirschner M: Dynamic instability of microtubule growth. Nature 1984, 312:237-242) performed between two closely spaced glass flats to a three-dimensional environment and used the SPIM for imaging (Figure 1, 7). Experi- ments were performed in vitro using Xenopus laevis egg extracts, providing a physiological yet biochemically easily modifiable system. Three-dimensional specimen preparation ensured a minimal area of artificial surfaces and unconstrained development of the asters in three dimensions. Apart from addressing the fundamental questions of microtubule dynamics, this approach allowed the phrasing of questions that specifically focussed on three-dimensional aspects of microtubule structural dynamics.

Among these issues are the centrosome's three-dimensional movement and rotation during aster polymerization and spindle formation. Analysis of the structural homogeneity of the aster allowed the relation of the angular microtubule distributions to the centrosome's internal structure. Structural configurations (e.g. for the centrosomes' centrioles) are well-known from electron microscopy (see, for example, Chretien D, Buendia B, Fuller SD, Karsenti E: Reconstruction of the centrosome cycle from cryoelectron micrographs. J Struct Biol 1997,

120:117-133 and Keryer G, di Fiore B, Celati C, Lechtreck KF, Mogensen M, Delouvee A, Lavia P, Bornens M, Tassin AM: Part of Ran is associated with AKAP450 at the centrosome: involvement in microtubule-organizing activity. MoI Biol Cell 2003, 14:4260-4271). This raises the question of whether these configurations have effects on the spatial dependency of microtubule nucleation that are observable in live systems. Surfaces obviously impair microtubule growth (as discussed in Dogterom M, Yurke B: Measurement of the force-velocity relation for growing microtubules. Science 1997, 278:856-860 and Janson ME, de Dood ME, Dogterom M: Dynamic instability of microtubules is regulated by force. J Cell Biol

2003,161:1029-1034) and significantly influence aster structure. Hence, it is crucial to either avoid or precisely characterize the asters' surface contact in these studies. In our SPIM data sets, the evaluation of three-dimensional microtubule length distributions over time effectively takes all of the asters' microtubules into account. This provides a very good statistical basis to test and improve theoretical models of dynamic instability (Verde F, Dogterom M, Stelzer E, Karsenti E, Leibler S: Control of microtubule dynamics and length by cyclin A- and cyclin B-dependent kinases in Xenopus egg extracts. JCe// Biol 1992, 118:1097-1108; Dogterom M, Leibler S: Physical aspects of the growth and regulation of microtubule struc- tures. Phys Rev Lett 1993, 70:1347-1350; Dogterom M, Maggs AC, Leibler S: Diffusion and formation of microtubule asters: physical versus biochemical regulation. Proc Natl Acad Sci USA 1995, 92:6683-6688). Finally, the elastic properties of the microtubules could be determined from the thermal fluctuations of the filaments' three-dimensional position and geome- try.

It is believed that fast three-dimensional aster dynamics has not been successfully investigated with confocal fluorescence microscopes, mainly because of fluorophore photo bleaching. With single-view SPIM, however, we achieved a time resolution of three seconds for the en- tire three-dimensional volume of a typical interphasic aster without a significant effect of photo bleaching even after 15 minutes of continuous observation. Multi-view SPIM

(mvSPIM) recorded the entire three-dimensional volume of a stabilized microtubule aster with an isotropic resolution. The aster data sets are recorded along several directions and fused by image processing. The resulting isotropy is a crucial feature in the quantitative inves- tigation of three dimensional structures.

The three-dimensional imaging and specimen preparation in SPIM provide several advantages for the investigation of cytoskeletal filament dynamics. The imaging yields three-dimensional structural information instead of two-dimensional projections of fluorescent structures. Un- constrained filament growth along all dimensions eliminates uncharacterized interactions of the specimen with artificial surfaces. Additionally, the strongly reduced surface-over-volume ratio in SPIM specimen preparation minimizes possible surface effects, for example the un- specific adsorption of proteins. While the surface area is minimized in SPIM experiments, the visibility of these surfaces in the three dimensional data sets still allows us to clearly assess whether phenomena of microtubule dynamics are associated with proximity to or contact with a surface.

SPIM technology for three-dimensional cell culture

So far relatively few data are available on how cells interact and communicate with each other in the three-dimensional context of a tissue. Cell fate in living organisms— for example polarization, growth, migration or apoptosis ( see Schmeichel KL, Bissell MJ: Modeling tissue- specific signalling and organ function in three dimensions. J Cell Sci 2003, 116:2377-2388; Even-Ram S, Yamada KM: Cell migration in 3D matrix. Curr Opin Cell Biol 2005, 17:524- 532;. Friedl P, Hegerfeldt Y, Tusch M: Collective cell migration in morphogenesis and cancer. Int JDev Biol 2004, 48:441-449; Nelson WJ: Epithelial cell polarity from the outside looking in. News Physiol Sci 2003, 18:143-146; Debnath J, Mills KR, Collins NL, Reginato MJ, Muthuswamy SK, Brugge JS: The role of apoptosis in creating and maintaining luminal space within normal and oncogene-expressing mammary acini. Cell 2002, 111 :29-40) is determined by three-dimensional and temporal information exchange between neighbouring cells as well as by cues from the microenvironment (e.g. ECM proteins or growth factors).

Ultimately, the whole physiology of healthy organisms or pathologic organisms depends on information flow and processing that is based on both biochemical and mechanical cues (Thery M, Racine V, Pepin A, Piel M, Chen Y, Sibarita J-B, Bornens M: The extracellular matrix guides the orientation of the cell division axis. Nat Cell Biol 2005, in press; Cukierman E, Pankov R, Stevens DR, Yamada KM: Taking cell-matrix adhesions to the third dimension. Science 2001, 294:1708-1712). It is believed that the life sciences are currently undergoing a paradigm shift towards the investigation of cells maintained in an environment closely mimicking the mechanical, chemical and cytological properties of real tissues (Abbott A: Biology's new dimension. Nature 2003, 424:870-872). An improved understanding of how cells react to physiological stimulations and constraints is leading to substantial scientific and technological advancements in cancer research (Friedl P, WoIfK: Tumor-cell invasion and migra- tion: diversity and escape mechanisms. Nat Rev Cancer 2003, 3:362-374, and Bissell MJ,

Radisky D: Putting tumors in context. Nat Rev Cancer 2001 , 1 :46-54), immunology (Friedl P, Gunzer M: Interaction of T cells with APCs: the serial encounter model. Trends Immunol 2001, 22:187-192 and Friedl P, den Boer AT, Gunzer M: Tuning immune responses diversity and adaptation of the immunological synapse. Nat Rev Immunol 2005, 5:532-545) and tissue engineering (RadisicM,ParkH, Shing H, Consi T, Schoen FJ, LangerR, Freed LE, Vuηjak- Novakovic G: Functional assembly of engineeredmyocardium by electrical stimulation of cardiac myocytes cultured on scaffolds. Proc Natl Acad Sci USA 2004, 101 : 18129- 18134 and Griffith LG, Naughton G: Tissue Engineering— Current Challenges and Expanding Opportunities. Science 2002, 295:1009-1014). Indeed, in three-dimensional cell cultures, the bounda- ries between in vivo and in vitro experiments tend to disappear, which has important implications, particularly for drug discovery (discussed in Schmeichel KL, Bissell MJ: Modeling tissue-specific signalling and organ function in three dimensions. J Cell Sci 2003, 116:2377- 2388). The invention comprises a basic 'SPIM-compatible' technology to investigate three- dimensional cell cultures. Several types of matrices are commercially available for three- dimensional cell cultures. The matrices are extracted either from living systems (e.g. Ma- trigel) or from synthetic systems (e.g. Puramatrix). Matrigel reproduces the mechanical and biochemical characteristics of natural ECM and consequently exerts an environmental pressure on the cells that is close to a physiological situation. For many years it has been known that MDCK cells cultured in a collagen gel or Matrigel for 7-10 days form hollow cysts, consisting of a monolayer of 50-100 polarized cells Montesano R, Schaller G, Orci L: Induction of epithelial tubular morphogenesis in vitro by fibroblast-derived soluble factors. Cell 1991, 66:697-711 and O'Brien LE, Zegers MMP, Mostov KE: Building epithelial architecture: insights from three-dimensional culture models. Nat Rev MoI Cell Biol 2002, 3 :531-537).

MDCK cells also undergo a branching tubulogenesis when exposed to hepatocyte growth factor (HGF) (reported in Montesano R, Schaller G, Orci L: Induction of epithelial tubular morphogenesis in vitro by fibroblast-derived soluble factors. Cell 1991, 66:697-711 and Lubarsky B, Krasnow MA: Tube morphogenesis: making review and shaping biological tubes. Cell 2003, 112:19-28). MDCK cells thus represent an interesting model system for investigating the morphogenesis of epithelia (O'Brien LE, Zegers MMP, Mostov KE: Building epithelial architecture: insights from three-dimensional culture models. Nat Rev MoI Cell Biol 2002, 3:531-537). In order to test both the instruments and the understanding of the underlying biology of cyst and tubules formation, MDCK was culture inside Matrigel or collagen type I matrices. We observed the structures with SPIM using the agarose chamber approach illustrated in Figure 3.

Claims

Titel: Microscope Specimen Holder

Anmelder: EMBL

Unser Zeichen: 90559WO

Claims

1. Specimen support (10) for a microscope, the specimen support (10) comprising at least one window (12) formed in a wall of the specimen support (10), wherein in use a specimen (2) is viewable with the microscope through the at least one window (12) and wherein in use the specimen (2) is embedded in a matrix (4), the matrix (4) being through the at least one window in contact with a liquid surrounding at least partially said specimen support (10).

2. Specimen support (10) according to claim 1, wherein the matrix (4) comprises at least one transparent polymer.

3. Specimen support (10) according to claim 1 or 2, wherein the matrix (4) comprises at least one biopolymer gel.

4. Specimen support (10) according to any of the preceding claims, wherein the at least one window (12) comprises transparent plastics material.

5. Specimen support (10) according to any of the preceding claims, wherein the at least one window (12) and the matrix (4) are permeable to biochemical agents. 6. Specimen support (10) according to any of the preceding claims, wherein the at least one window has a thickness of less than 20 μm

7. Specimen support (10) according to any of the preceding claims, wherein the at least one window (12) and the matrix (4) are transparent to visible light.

8. Specimen support (10) according to any of the preceding claims, wherein in use the specimen support (10) is mounted movably with respect to an objective (20) of the microscope. 9. Specimen support (10) according claim 8, further comprising a specimen support moving device for moving the specimen support in at least one direction with respect to the objective (20).

10. Specimen support (10) according to claim 9, wherein the specimen support is reversibly mounted to the specimen support moving device.

11. Specimen support (10) according to claim 9 or 10, wherein the specimen support is magnetically mountable to the specimen support unit moving device. 12. A specimen support (100) for a microscope, wherein in use a specimen (200) is viewable with the microscope, the specimen (200) being embedded in a droplet matrix (400), the droplet matrix (400) being attached to the specimen support (100) and being in contact with a liquid surrounding at least partially said specimen support (100). 13. Specimen support (100) according to claim 12, wherein the droplet matrix (400) comprises at least one transparent polymer.

14. Specimen support (100) according to claim 12 or 13, wherein the drop-shaped matrix (4) comprises at least one biopolymer gel.

15. Specimen support (100) according to any of claims 12 to 14, wherein the droplet matrix (400) is permeable to biochemical agents.

16. Specimen support (100) according to any of claims 12 to 15, wherein the droplet matrix (400) is transparent to visible light.

17. Specimen support (100) according to any of claims 12 to 16, wherein in use the specimen support (100) is mounted movably with respect to an objective (20) of the microscope.

18. Specimen support (100) according claim 17, further comprising a specimen support moving device for moving the specimen support in at least one direction with respect to the objective (20).

19. Specimen support (100) according to claim 18, wherein the specimen support is reversibly mounted to the specimen support moving^" device.

20. Specimen support (100) according to claim 18 or 19, wherein the specimen support is magnetically mountable to the specimen support unit moving device.

21. Specimen holding device for a microscope comprising:

- a sealable chamber(30) that is mountable to an objective of the microscope; and - a specimen support (10, 100) according to any of the preceding claims within the sealable chamber (30).

22. Specimen holding device according to claim 21, wherein the sealable chamber (30) is a perfusion chamber with at least one inlet and at least one outlet for a fluid.

23. Specimen holding device according to any of claims 21 or 22, wherein the sealable chamber (30) is a perfusion chamber with at least one inlet and at least one outlet for gas exchange inside the sealable chamber (30). 24. Specimen holding device according to any of claims 21 to 23, wherein the sealable chamber (30) comprises a temperature control for controlling the temperature of the fluid inside the sealable chamber (30).

25. Specimen holding device according to any of claims 21 to 24, wherein the sealable cham- ber (30) comprises a sensor for measuring the pH of the fluid inside the sealable chamber

(30).

26. Specimen holding device according to any of claims 21 to 25, wherein the sealable chamber (30) comprises a sensor for measuring the liquid level of the fluid inside the sealable chamber (30). 27. Specimen holding device according to any of the preceding claims wherein the microscope is a single plane illumination microscope (SPIM).

28. Method for viewing a specimen (2) with a microscope, the method comprising:

dipping a specimen support (100) into a solution comprising the specimen;

pulling the specimen support (100) out of the solution, wherein the specimen support

(100) is adapted to form at least one droplet of a liquid containing the specimen (2) on the specimen support (100);

arranging the at least one droplet attached to the specimen support (100) in front of an objective (20) of the microscope;

viewing the specimen (2) in the droplet with the microscope.

29. Method for viewing a specimen (2) according to claim 28, wherein arranging the at least one droplet attached to the specimen support (100) in front of an objective (20) of the microscope comprises placing the at least one droplet attached to the specimen support (100) in a sealable chamber (30), wherein the sealable chamber (30) is mountable to an objective of the microscope.

30. Method for viewing a specimen (2) according to claim 28 or 29, further comprising adding a matrix forming agent to the droplet, thereby forming a droplet matrix (400).

31. Method for viewing a specimen (2) according to any of claims 28 to 30, wherein the matrix forming agent comprises a polymer.

32. Method for viewing a specimen (2) according to any of claims 28 to 31, wherein the ma- trix forming agent comprises a bio-polymer.

33. Method for viewing a specimen (2) according to any of claims 28 to 32, wherein the microscope is a single plane illumination microscope (SPIM).