WO2020120102A1

WO2020120102A1 - Attachment for an immersion objective

Info

Publication number: WO2020120102A1
Application number: PCT/EP2019/082199
Authority: WO
Inventors: Francesco Pampaloni; Ernst H. K. Stelzer
Original assignee: Johann Wolfgang Goethe-Universität Frankfurt am Main
Priority date: 2018-12-14
Filing date: 2019-11-22
Publication date: 2020-06-18

Abstract

Attachment (10) for an immersion objective (14) for performing an inverse immersion microscopy is provided. The attachment (10) comprises an attachment portion (12) for receiving an end portion of an immersion objective (14), wherein the attachment portion (12) defines a detecting axis (20) coinciding with the optical axis (22) of the immersion objective (14), when the immersion objective (14) is received in the attachment portion (12), and a container portion (16). The container portion (16) defines, when the immersion objective (14) is received in the attachment portion (12) and oriented in an inverse orientation, together with a front surface of the received immersion objective (14) a container (34) for retaining an immersion fluid (38), comprises a transparent wall portion (50) defining an intended illumination direction (52) being orthogonal to the transparent wall portion (50) and tilted to the detecting axis (20), and is arranged, such that when the immersion objective (14) is in the inverse orientation and received in the attachment portion (12) a sample (54) being located on the detecting axis (20) can be illuminated through the transparent wall portion (50) and through an immersion fluid (38) being retained by the container (34).

Description

Attachment for an immersion Objective

TECHNICAL FIELD

The present invention lies in the field of microscopy.

BACKGROUND

Microscopy is used in a large number of different disciplines for a large number of different applications and can be used, for example, to investigate and to image the inner structure of biological samples.

For this purpose, a sample can be prepared with a fluorescent dye specifically attaching to corresponding structures of the sample. The sample can be illuminated to excite the fluores cent dye and the resulting fluorescent light emitted by the sample can be detected with an objective, also designated as objective lens.

Microscopy arrangements can be divided into inverse setups and non-inverse setups. Differ ent from non-inverse microscopy setups where the detection objective is arranged above the sample, in inverse microscopy setups the detection objective is arranged below of the sample or beside the sample. Therefore, the space above the sample can be directly and easily acces sible without removing or moving any measurement components and can be used for chang ing measurement parameters, such as adding or removing samples or sample media, e.g. individually by pipetting or by replacing one or more complete sample plates, cuvettes or microtiter plates. Therefore, inverse microscopy setups allow for a comparatively simple und uncomplicated sample handling.

For effectively imaging the three dimensional structure of a sample the sample can be scanned with a beam in the form of sheet to sequentially excite different planes of the sample, such that for each plane the fluorescing structures can be detected without simultaneously detecting the non-fluorescing structures in other non-exited sample planes.

An example for an inverse microscopy setup being suitable for investigating biological sam ples by an illumination and detection from below through a water prism for compensating for optical aberrations is provided in the article“Open-top selective plane illumination micro scope for conventionally mounted specimens”, R. McGorty et al., Optics Express 16151, Vol. 13, No. 12, 15 June 2015. SUMMARY OF THE INVENTION

The problem to be solved by the present invention is to improve the imaging quality in a mi croscopy setup which allows for a simple und uncomplicated sample handling. This problem is solved by the independent claim. Preferred embodiments are defined in the dependent claims.

The present invention provides an attachment for an immersion objective for performing an inverse immersion microscopy, wherein the attachment comprises:

• an attachment portion for receiving an end portion of an immersion objective, wherein the attachment portion defines a detecting axis coinciding with the optical axis of the immersion objective, when the immersion objective is received in the attachment por tion, and

• a reservoir portion, which defines, when the immersion objective is received in the at tachment portion and oriented in an inverse orientation, together with

a front surface of the received immersion objective,

a reservoir for retaining an immersion fluid,

wherein said attachment further comprises an optical interface allowing for passing illu mination light along an intended illumination direction through the attachment to a sam ple being located on said detecting axis.

When an immersion objective is received in the attachment portion within an inverse mi croscopy arrangement, a fluorescent sample can be positioned on the detection axis, such that the sample can be illuminated from below or in horizontal direction though the opti cal interface to excite the fluorescent dye of the sample. A portion of the emitted fluores cent light which is emitted towards the immersion objective can propagate through the retained immersion fluid and from the retained immersion fluid directly into the immer sion objective and can be detected in the downward direction or in the horizontal direction defined by the optical axis of the detecting inverse oriented immersion objective.

Accordingly, the attachment allows using an inverse microscopy arrangement for an im mersion microscopy. Compared to non-immersion microscopy where the detected light entered from a medium with a comparatively low refractive index, such as air, into the objective, immersion microscopy increases the numerical aperture by using an immersion liquid having a comparatively high refractive index, which allows for a higher resolution and for an improved imaging quality. Therefore, the attachment according to the present invention allows combining the ad vantages of inverse microscopy with the advantages of immersion microscopy, i.e. a sim ple und uncomplicated sample handling with a high imaging quality. A scanning of a sam ple or of a plurality of samples is possible by relatively moving the objective together with the attachment and the retained immersion fluid with respect to the sample or the plurali ty of samples.

In a preferred embodiment, said optical interface is formed by a transparent wall portion of said reservoir portion, said transparent wall portion allowing for passing said illumina tion light therethrough along an intended illumination direction being orthogonal to the transparent wall portion and tilted to the detecting axis. Herein, said reservoir portion and said transparent wall portion are arranged such that, when the (immersion) objective is in the inverse orientation and received in the attachment portion, a sample being located on the detecting axis can be illuminated through the transparent wall portion and through an immersion fluid being retained by the reservoir.

In a preferred embodiment, the transparent wall portion is planar and/or at least partially transparent for light in the in the ultraviolet range, in the visible range, in the near infra red range, in the infrared range, in wavelengths ranges therein and/or for one or more wavelengths therein. At least partially transparent means that at least 30%, preferably at least 50% of the light is transmitted.

In an alternative embodiment, said optical interface is formed by a further attachment portion for receiving an end portion of a further immersion objective, wherein said first attachment portion defines said intended illumination direction coinciding with the opti cal axis of the further immersion objective when the further immersion objective is re ceived in the further attachment portion. In other words, the attachment may comprise a further attachment portion that is similar or even identical in structure with the aforemen tioned attachment portion but that serves for providing the optical interface via which the illumination light can be applied. In particular, the immersion objective and the further immersion objective may be identical as well, and each of them may be adapted for being selectively used in an illumination mode and in a detecting mode. In some embodiments, the attachment portion and/or the further attachment portion defines an inner surface being complementaiy to an outer surface of a respective immersion objective. In these embodiments, the respective attachment and the respective immersion objective can be connected with a form-fit connection and the respective immersion objective can be re ceived simply by attaching or plugging the attachment onto the immersion objective. Fur ther, the attachment and the immersion objective can be connected to be movable as a unit. Moreover, it is possible that the form-fit connection sufficiently seals the reservoir or contributes to sealing the reservoir, which is partially defined by a surface of the immer sion objective, in order to prevent a leaking of the retained immersion fluid.

The immersion fluid can be water, a gel, an oil or an aqueous solution. Hence, the attach ment can usable with immersion fluids of quite different viscosities.

In the inverse orientation, in which an immersion fluid can be retained by the reservoir, the detecting axis being defined by the attachment portion can be tilted with respect to the vertical direction of gravity by an angle a in a range o°£ a £ 90°, preferably in a range 20°£ a £ 70°, more preferably in a range 40°£ a £ 50°. Accordingly, in the inverse orien tation, when the immersion objective is received, the inlet surface or inlet window of the immersion objective can face in an upward direction having a vertical component or can face in horizontal direction.

In some embodiments the attachment portion comprises an inner surface defining a frus tum of a right circular cone and/or defining a right circular cylinder. Herein, the expres sion“right cylinder” or“right cone” indicates that the respective cylinder axis or cone axis is perpendicular to the base surface. A conventional commercially available immersion objective usually comprises an outer surface portion defining a frustum of a right circular cone and/or an outer surface portion defining a right circular cylinder. Therefore, the at tachments according to these embodiments can be adapted for the use with corresponding conventional immersion objectives. Therein, the inner surface of the attachment portion can be partially or completely complementary to a corresponding portion of the outer sur face of the end portion of the corresponding immersion objective. This corresponding por tion of the outer surface of the immersion objective does not include the front surface of the immersion objective, which contributes in defining the reservoir.

In one or more embodiments the detecting axis is tilted with respect to the intended illu mination axis by an angle b in a range 70°£ b £ iio°, preferably in a range 8o°£ b £ ioo°, more preferably in a range 8s°£ b £ 95°, in particular by 90°. An angle of 90° allows de tecting fluorescent light being emitted perpendicular to an illuminating beam and that the detected excited sample plane or line section is orthogonal the optical axis of the immer sion objective. Further, the illumination through the transparent wall portion can be or thogonal to the transparent wall portion. This allows for an imaging with high quality and low optical aberrations. However, the other above mentioned angle ranges still allow for advantageous measurements.

Preferably, in the inverse orientation the reservoir comprises an upwards, preferably ver tically upwards facing opening allowing dipping the sample, a cuvette comprising the sample and/or a micro well comprising the sample into a retained immersion fluid, such that the sample is located on the detecting axis and can be illuminated along said intended illumination direction through said optical interface, e.g. through the transparent wall portion or through the further attachment portion. Due to a surface tension of the immer sion fluid, the retained immersion fluid can be partially arranged above the reservoir. Therefore, the sample need not necessarily be located in the reservoir volume but can also be arranged above the reservoir opening, when being directly or indirectly dipped into the retained immersion fluid. In these embodiments, the illuminated detected sample can be directly in contact with the retained immersion fluid or can be disposed in a sample medi um within the cuvette or micro well, such that the sample can be detected without air be tween the sample and the inlet surface or inlet window of the immersion objective.

The sample can be a biological fluorescent sample with a diameter in a range between 0,1 pm and 1000 pm, for example.

In some embodiments the attachment is integrally formed and/or comprises or consists of a formed foil of a transparent polymer, preferably of fluorinated ethylene propylene, FEP. These attachments can be easily manufactured at low cost by using a correspondingly shaped mold to form the foil in a thermoforming process. Further, the foil material may in advantageous embodiments by itself provide the optical properties which are required for the transparent wall portion.

In these embodiments the formed foil can have a thickness of too pm or less, preferably of 50 pm or less.

In an alternative embodiment, the attachment is made from a rubberlike material, in par ticular PDMS.

In a preferred embodiment, the reservoir portion may be connected or connectable with a supply of immersion fluid allowing for a replenishment of immersion fluid during micros copy. Especially during prolonged time-lapsed imaging of multiple positions, it is advan tageous to provide for a replenishment of the immersion liquid during microscopy to en sure high-quality data throughout the whole experiment. For this purpose, in a preferred embodiment, the reservoir portion is connected or connectable with a corresponding sup ply of immersion fluid. For example, in some embodiments, the reservoir portion may comprise an inlet port for connecting a supply line for supplying the immersion fluid, al lowing for a continuous supply of immersion fluid as needed. In addition, the reservoir portion may further comprise an outlet port for communicating with a discharge line for discharging immersion fluid from said reservoir. This, in combination with the inlet port, allows not only for replenishment of the immersion fluid, but also for replacing the im mersion fluid, at least in part, during microscopy. The supply line and discharge line can be formed by rubber tubes or the like, and the supply and/or discharge can be driven us ing a suitable pump, such as a peristaltic or syringe pump. The pump is preferably elec tronically controlled to ensure a desired amount of immersion fluid in the reservoir, and/or a desired flow of immersion fluid through the reservoir.

The present invention further provides a system, comprising an immersion objective and an attachment according to one or more of the before mentioned embodiments, wherein the attachment portion is adapted for receiving an end portion of the immersion objective.

The present invention also provides an arrangement for performing an immersion micros copy, wherein the arrangement comprises the before mentioned system, wherein the end portion of the immersion objective is received in the attachment portion and wherein the system is arranged in an inverse orientation, in which the detection axis is horizontal or is tilted with respect to the vertical direction by an angle a in a range o°< a £ 90°, preferably in a range 20°£ a £ 70°, more preferably in a range 40°£ a £ 50°, in particular by an an gle of 45⁰. The arrangement further comprises an illumination source arranged for provid ing a beam along an illumination axis with a vertical upwards directed component or in horizontal direction. In the arrangement the illumination axis extends through the optical interface, for example a transparent wall portion or a further attachment portion, and is tilted with respect to the detecting axis by an angle g, preferably in the range 70°£ g £ no°, more preferably in the range 8o°£ g £ ioo°, in particular in the range 8s°£ g £ 95°.

In some embodiments of the arrangement, the illumination axis and the detecting axis intersect each other and/or the illumination axis and the detecting axis both intersect a common sample location having a diameter of 3 mm or smaller, preferably 1 mm or small er, more preferably 500 pm or smaller. Accordingly, it is not necessary that the illumina tion axis and the detecting axis are precisely adjusted to intersect each other in a single point of space having hardly any dimension, although this is preferred. The sample loca tion can define a volume within which a sample can be located and illuminated, such that the sample emits fluorescent light towards the immersion objective, which can be detected by the immersion objective.

According to some embodiments, the arrangement further comprises a cuvette for keeping a sample medium with the sample disposed therein, wherein the cuvette comprises a first planar bottom wall and a second planar bottom wall and wherein the cuvette can be ar ranged such that a sample in the cuvette can be illuminated through the first bottom wall along the illumination axis, and such that fluorescent light stemming from the illuminated sample can travel through the second bottom wall along the detecting axis towards the immersion objective. In these embodiments, the cuvette containing a sample in a sample medium can dip into the retained immersion fluid, such that the sample can be detected by an inverse immersion microscopic measurement.

In the arrangements according to the before mentioned embodiments, the normal direc tion of the first bottom wall can be tilted with respect to the normal direction of the second bottom wall by an angle g in the range 70°£ g £ no°, preferably in the range 8o°£ g £ ioo°, more preferably in the range 8s°£ g £ 95⁰, in particular by an angle of 90°. This al lows that the first and second bottom wall can be orthogonal to the illumination direction and to the detection direction, respectively, which can reduce optical aberrations.

In one or more of the before mentioned embodiments the arrangement further comprises a microtiter plate with a plurality of said cuvettes, and/or further comprises an XYZ-stage configured to position the microtiter plate or the cuvette relative to the system and the illumination source.

The illumination source can comprise an illumination objective. In some embodiments, the illumination objective may be identical with the immersion objective used for detect ing light, and hence resemble the“further immersion objective” referred to above, that may be received in the“further attachment portion”. Alternatively or additionally the il lumination source can be configured for providing a beam with a focus in the form of light sheet.

Finally, the present invention provides a method for forming an attachment according to one or more of the before mentioned embodiments, wherein the method comprises

- providing a mold being partially shaped according to an end portion of an immersion objective and having a projection for defining the volume of the reservoir,

- using the mold for shaping a transparent polymer foil in a thermoforming process ac cording to the shape of the mold, and

- removing a portion of the shaped foil, such that, when the end portion of the immer sion objective is received in the formed foil, a front surface of the received immersion objective forms an inner wall of the reservoir and the reservoir comprises on opening.

Accordingly, the method allows for a manufacturing process which can be quite simple and fast and which typically requires only a small amount of material, such that the at tachment can be produced with low costs.

A further embodiment relates to an alternative method for forming an attachment accord ing to one of the types recited above, said method comprising the following steps: - providing a negative mold comprising a base portion and at least one insert, said insert having a shape matching the shape of at least a part of an end portion of an objective that is to be received in an attachment portion of said attachment,

- placing said insert in said base portion,

- pouring molding material, in particular a rubber material into said base portion,

- allowing said molding material to solidify, in particular by curing said molding materi al, to thereby form said attachment,

- removing said at least one insert from said base portion, and

- removing said attachment from said base portion.

BRIEF DESCRIPTION OF THE DRAWINGS

Further details and advantages of the present invention will become apparent from the fol lowing description, in which preferred embodiments are described in detail with reference to the appended drawings, in which:

Fig. l shows an attachment according to an embodiment of the present invention,

Fig. 2 shows a mold and a foil for forming the attachment of figure l,

Fig. 3 shows a system according to an embodiment of the present invention compris ing the attachment figure l and a corresponding immersion objective,

Fig. 4 shows the system of figure 3 which retains an immersion fluid,

Fig. 5 shows the system of figure 3 for imaging a sample within a cuvette,

Fig. 6 shows two photographs of an arrangement according to an embodiment of the present invention comprising the system of figure 3,

Fig. 7 shows a part of an arrangement according to another embodiment of the pre sent invention comprising a microtiter plate with a plurality of cuvettes,

Fig. 8a, 8b each show a part of an arrangement according to a further embodiment of the present invention for imaging the sample on an object plate,

Fig. 9 shows a schematic perspective view of a further embodiment of an attachment of the invention comprising inlet and outlet ports for supplying/discharging immersion fluid, Fig. to shows the attachment of Fig. g as attached to an immersion objective, Fig. na shows a schematic side view of an attachment including an attachment portion in which an immersion objective for light detection is received, and including a further attachment portion in which a further objective for illumination pur poses is received,

Fig. lib shows a schematic side view of a two-sided attachment similar to that of Fig.

11a, and a further two sided attachment oriented upside down, in which a total of four objectives are received,

Fig. 12 is a perspective view of a mold for forming the attachment of Fig. 11a, and Fig. 13 is a top view of the mold of Fig. 12. Fig. 14 is a perspective view of a negative mold for forming a two sided attachment in a disassembled state.

Fig. 15 is a perspective view of the negative mold of Fig. 14 in the assembled state.

In the drawings same elements are designated with same reference numbers.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

Figure 1 shows an attachment 10 according to an embodiment of the present invention. The attachment 10 comprises an attachment portion 12 for receiving an end portion of an immer sion objective 14 (shown figure 3). The attachment 10 further comprises a reservoir portion 16.

The attachment portion 12 comprises an inner surface which is complimentary to an outer surface of the end portion of the corresponding immersion objective 14, such that the at tachment 10 can be plugged onto the end portion of the corresponding immersion objective 14 or attached to the corresponding immersion objective 14, such that an end portion of the immersion objective 14 can be received in the attachment portion 12, and such that the at tachment 10 and the immersion objective 14 can be connected by a form fit connection.

Figure 3 shows a system 18 according to an embodiment of the present invention, which comprises the attachment 10 of figure 1 and the before mentioned corresponding immersion objective 14. In the illustration of figure 3 the attachment 10 and the immersion objective 14 are not connected and the immersion objective 14 is not received by the attachment 10. The attachment portion 12 defines a detecting axis 20, which is shown in figure 1 and which coin cides with the optical axis 22 (shown in figure 3) of the immersion objective 14, when the end portion of the immersion objective 14 is received in the attachment portion 12.

The immersion objective 14 of the system 18 of figure 3 corresponds to a conventional im mersion objective having a cylindrical portion 24 in the shape of a right circular cylinder and a frustoconical portion 26 in the shape frustum of a right circular cone. Both portions 24 and 26 are axial portions. In the embodiment of figure 3 and figure 1, the complete inner surface of the attachment portion 12 is complimentary to a corresponding portion of the outer sur face of the immersion objective 14, wherein this corresponding outer surface portion com prises an outer surface part of the frustoconical portion 26 as well as an outer surface part of the cylindrical portion 24.

In other embodiments, however, which are not shown, not the complete inner surface but only a portion of the inner surface of the attachment portion 12 is complimentary to a corre sponding outer surface portion of a corresponding immersion objective.

Further, in systems according to other embodiments of the present invention, which are not shown, the immersion objective can have a different shape, for example only cylindrical, only frustoconical or another shape. Accordingly, also the shape of the inner surface of the at tachment portion, which can at least partially correspond to a complementaiy surface of the outer surface of the immersion objective, such that an end portion of the immersion objective can be received for attaching the attachment, can be different in these embodiments. An im mersion objective which is suitable for a use in combination with the present invention can be any objective being suitable for performing an immersion microscopy.

As shown in the embodiments of figure 1 and figure 3, the attachment 10 is shaped as a cover, cap or hollow sleeve, wherein the reservoir portion 16 partially extends or projects in the di rection defined by the detecting axis 20. In these embodiments, the attachment 10 consists of a portion of a formed foil defining a hollow body having a rear opening 28 for inserting the end portion of the immersion objective 14 and a leading opening 30, as shown in figure 1.

When the immersion objective 14 is received in the attachment portion 12 of the attachment 10, as shown in figure 4, the reservoir portion 16 and a front surface 32 (see Fig. 3) of the immersion objective 14 together define a reservoir 34. Because the inner volume of the reser voir 34 is partially confined by the immersion objective 14, the reservoir 34 proper is only provided in the received or connected state of the system 18 shown in figure 4, but not in the disconnected state shown in figure 3. When the immersion objective 14 is received, as shown in figure 4, the immersion objective 14 and the attachment 10 are in a relative orientation, which is a predefined except for a relative rotation about the detecting axis 20. In other words, when being received, the immersion objective 14 of figure 3 and figure 4 cannot be inserted further into the attachment 10, and the immersion objective 14 and the attachment 10 are in a predefined relative axial orientation.

The reservoir 34 defines a volume and comprises an opening 36. When the connected system 18 is in a corresponding inverse orientation, as shown in figure 4, the opening 36 faces verti cally upwards, opposite to the force of gravity. For the embodiment of figure 4, the opening 36 faces vertically upwards for an orientation in which the detecting axis 20, which coincides with the optical axis 22 of the received immersion objective 14, is tilted by an angle of 45⁰ with respect to vertical direction and in which the attachment 10 is in a corresponding rota tional position in respect to a rotation about the detecting axis 20. An inverse orientation is defined by the orientation of the detecting axis 20 and/or the optical axis 22 and is not changed to a different inverse orientation for a rotation about the detecting axis 20 and/or the optical axis 22.

In other embodiments the opening 36 faces vertically upwards in other inverse orientations, wherein the angle between the vertical direction and the detecting axis 20 is > 0° and £ 90°.

In one or more corresponding inverse orientations, the reservoir 34 can retain an immersion fluid 38, wherein the immersion fluid 38 can correspond to water, gel, oil, an aqueous solu tion or any other liquid. Due to a surface tension of the immersion fluid 38, the immersion fluid 38 may not only be contained within the reservoir volume but may also protrude above the reservoir volume and the opening 36 of the reservoir 34, as shown in figure 4. Therefore, the detecting axis 20 and the optical axis 22 can extend through a retained immersion fluid 38 without necessarily extending simultaneously through the volume of the reservoir 34, as shown in figure 4. In other embodiments, however, which are not shown, the volume of the reservoir 34 can be intersected by the detecting axis 20 and by the optical axis 22.

In the system shown in figure 4, the attachment 10, the received immersion objective 14 and the retained immersion fluid 38 can be moved together as a unit. A sufficient sealing of the reservoir 34 can be provided by a form fit between the attachment 10 and immersion objec tive 14. Alternatively or additionally, the sealing can be provided by a glue arranged between an inner wall of the attachment 10 and an outer of the wall immersion objective 14 and/or by an outer tape or elastic ring (not shown) for tightening a wall of said attachment 10 against an outer surface of the immersion objective 14.

Referring to figure 2, a method for forming the attachment 10 of figure 1 is explained. Figure 2 shows a mold 40 being partially shaped as an end portion of the immersion objective 14. The shape of the mold 40 only differs from the shape of the end portion of the immersion objective 14 by an additional projection 42 defining the shape of the reservoir portion 16 and by additional holes or vacuum channels 44. Figure 2 further shows a transparent polymer foil 46 being arranged above the mold 40. The method comprises a thermoforming process in which the foil 46 is heated, drawn over the mold 40 and drawn or pressed against the mold 40 in order to adopt the shape of the mold 40. During the thermoforming process the foil 46 may be sucked against the surface of the mold 40 by applying a vacuum through the vacuum channels 44. After the forming process a portion of the formed foil 46 is removed in order to provide the leading opening 30 of the attachment 10. The removal of the before mentioned portion of the formed foil 46 is performed by cutting along the cutting line 48 which is indi cated by a dashed line in figure 2. According to an exemplary embodiment, an FEP-foil with a thickness of 50 pm is used to form the attachment 10.

Referring to figure 1, the reservoir portion 16 of the attachment 10 comprises a transparent wall portion 50, which resembles an example of the aforementioned optical interface allow ing for passing illumination light along an illumination direction. The transparent wall por tion 50 is preferably planar and defines an intended illumination direction 52 being normal or orthogonal to the transparent wall portion 50. The“illumination direction” 52 may be characterized by a corresponding“illumination axis” also referred to herein. The intended illumination direction 52 corresponds to a preferred illumination direction for illuminating a sample through the optical interface, and in particular through the transparent wall portion 50. In case of the transparent wall portion 50, an illumination direction orthogonal thereto is preferred because optical aberrations can be minimized. However, the sample can also be illuminated through the transparent wall portion in other directions which differ from the intended illumination direction 52 and imaged with a sufficiently high quality.

In the embodiment of figure 1 the intended illumination direction 52 is orthogonal to the di rection of the detecting axis 20. In other embodiments these directions are tilted with respect to each other by other angels.

With reference to figure 5, the imaging of a sample 54 being disposed within a cuvette in a sample medium 58 is explained. The sample can correspond to a biological sample compris ing a plurality of cells and cell structures and being prepared to be at least partially transpar ent for the light of an illumination beam 60 and comprising a fluorescent dye being excitable by the light of the illumination beam 60. The cuvette 56 comprises a first bottom wall 62 be ing planar and at least partially transparent for the light of the illumination beam 60 and fur ther comprises a second bottom wall 64 being planar and at least partially transparent for the fluorescent light being emitted by the fluorescent dye in response to an excitation by the light of the illumination beam 60. The normal direction of the first bottom wall 62 is orthogonal with respect to the normal di rection of the second bottom wall 64. The sample 54 is located at the bottom of the cuvette 56 and in touch with first bottom wall 62 and with the second bottom wall 64. The cuvette 56 with the sample 54 is dipped into the retained immersion fluid 38 and arranged and oriented such that the first bottom wall 62 is parallel the transparent wall portion 50 of the attach ment 10 and such that the second bottom wall 64 and the sample 54 are intersected by the detecting axis 20 (coinciding with the optical axis 22), wherein the second bottom wall 64 is orthogonal to the detecting axis 20. The orientation of the system 18 in figure 5 is the same as in figure 4.

In figure 5 the sample 54 is illuminated and excited by the illumination beam 60, which trav els through the transparent wall portion 50, through the retained immersion fluid 38 be tween the transparent wall portion 50 and the first bottom wall 62, through the first bottom wall 62 and through the sample. Therein, the illumination beam 60 is orthogonal to the transparent wall portion 50 and orthogonal to the first bottom wall 62. In response to the excitation, fluorescent light is emitted from the sample 54 along the detecting axis 20 being orthogonal to the illumination beam 60 towards an inlet window of the immersion objective 14. In particular, this fluorescent light travels vertically through the second bottom wall 62, between the second bottom wall 62 and the window of the immersion objective 14 through the retained immersion fluid 38 and through the inlet window into the immersion objective 14-

Figure 6 shows an arrangement 66 according to an embodiment of the present invention, wherein the arrangement 66 comprises the system 18 of figure 5 in the orientation of figure 5, an illumination source comprising an illumination objective 68 defining the illumination axis for providing the illumination beam 60 along the illumination axis, two of the before men tioned cuvettes 56 containing samples and sample medium 58 and an XYZ-stage 70 for posi tioning the cuvettes 56 relative to the system 18 and to the illumination objective 68, in par ticular to the optical axis of the illumination objective 68 along which the illumination beam 60 is provided.

In figure 6a) none of the cuvettes is inserted into the immersion fluid 38 retained by the res ervoir 34 of the system 18. In figure 6b) the right one of the cuvettes 56 has been moved downwards and dipped into the retained immersion fluid 38 by the XYZ-stage 70, such that the sample can be imaged as explained before with respect to figure 5.

Preferably, the illumination beam 60 is formed as a light sheet, such that it can excite a com plete plane of the sample 54 simultaneously, wherein this excited plane is orthogonal the op tical axis of the detecting objective 14. In other embodiments, however, the illumination beam 60 has a circular cross-section, such that a complete sample plane can be excited by scanning the beam 60 in a corresponding direction.

In order to image a three-dimensional structure of the sample 56, the cuvette 56 can be moved with respect to the illumination beam 60 and the system 18, which preferably are kept in a fixed spatial relation or orientation with respect to each other, such that the sample 56 is laterally shifted with respect to the illumination beam 60 and can be scanned by the illumina tion beam 60.

In alternative embodiments, the scanning is performed by moving the system 18 and the il lumination objective 68 together as a unit with respect to the one or more of the cuvettes 56, which can be fixedly or stationarily arranged.

For the scanning of an individual sample 54, the relative movement between the system 18 and the cuvette 56 is preferably performed in the direction of the detecting axis 20, such that the sequentially imaged sample planes are pairwise parallel and such that the illumination beam 60 remains orthogonal to the first bottom wall 62 and such that the propagation direc tion of the detected fluorescent light remains orthogonal to the second bottom wall 64. This allows for a high resolution imaging with minimal optical aberrations.

Figure 7 shows a part of an arrangement according to another embodiment of the present invention which does not comprise only two cuvettes 56 but a microtiter plate 80 comprising a plurality of cuvettes 156, each having a first and a second bottom wall 62, 64 corresponding to the before mentioned first and second bottom walls 62, 64 of the cuvette 56 and having the same orientation with respect to each other. In figure 7, the microtiter plate 80 is oriented horizontally and the system 18 is oriented as explained with respect to figure 4, such that the detecting axis 20 and the optical axis 22 are tilted by 45⁰ with respect to the vertical direc tion.

In this embodiment of figure 7, the distance between adjacent cuvettes 156 is selected and adapted according to the size and dimension of the attachment 10 and the corresponding immersion objective 14. In particular, when the microtiter plate 80 and the system 18 are relatively arranged for detecting and imaging a sample 54 on the bottom of one of the cu vettes 156, as shown in figure 7, the distance between the system 18 and an adjacent cuvette 156 (third cuvette 156 from the left hand side in figure 7) is smaller than the distance between the system 18 and the cuvette 156 of the detected sample 54 (second cuvette 156 from the left hand side in figure 7) and such that the system 18 does not abut against the microtiter plate 80. This dense arrangement of the cuvettes 156 allows imaging and scanning a plurality of samples 54, each being contained in a corresponding one of the cuvettes 156, wherein the samples 54 can be measured and investigated quite efficiently with a high throughput and with a compact arrangement.

In the before mentioned embodiments inverse setups are used, in which the sample is illumi nated from below and is detected in a downward direction. When detecting in a downward direction the detected emitted fluorescent light propagates in a direction having a component pointing in the downward vertical direction of gravity. In other words, the inlet aperture of the detecting immersion objective 14 faces in an upward direction having a component point ing in the opposite direction of the downward vertical direction of gravity. When illuminating from below the illuminating light propagates in a direction having a component pointing in the opposite direction of the downward vertical direction of gravity.

However, the present invention also comprises arrangements with inverse setups according to other embodiments, in which one of the detecting axis 20 and the intended illumination axis 52 extends in horizontal direction. The other axis of the detecting axis 20 and the in tended illumination axis 52 preferably extends in vertical direction, but can also extent in other directions having a vertical component.

The inverse setups allows for a comparatively simple und uncomplicated sample handling.

Figure 8 shows an arrangement according to a further embodiment, which differs from the arrangement of figure 8 in that it comprises an object plate 82 instead of a microtiter plate 80. On the object plate 82 a sample 54 is disposed and surrounded by a sample medium (not shown in figure 8). A portion of the downwards facing surface of the object plate 82 is in touch with the retained immersion fluid 38, such that the sample can be imaged with high resolution and quality by detecting the fluorescent light, which travels through the object plate 82 and the retained immersion fluid 38 and which enters the immersion objective 14 directly from the retained immersion fluid 38.

In figure 8a) and 8b) the plane of the object plate 82 is horizontally arranged. In this embod iment a scanning of the sample 54 can be performed by tilting the illumination beam 60 to gether with the system 18, such that a different plane of the sample 54 becomes illuminated and detected. This is illustrated in figure 8, where the illumination beam 60 and the detecting axis 20 are fixedly arranged with respect to each other and are orthogonal to each other: In figure 8a) the detecting axis 20 is tilted with respect to the object plate 82 by an angle q_i. In figure 8b) the system 18 and the illumination beam 60 have been tilted together with respect to the object plate 82, such that detecting axis 20 is tilted with respect to the object plate 82 by an angle q₂ being larger than q_i and such that a different plane of the sample 54 is illumi nated and detected. Fig. 9 shows a perspective view of another embodiment of an attachment to. Fig. to shows the same attachment to as attached to an immersion objective 14. The attachment 10 has an attachment portion 12 which in this embodiment is a ring that fits tightly over a cylin drical portion of an immersion objective 14 shown in Fig. 10. The attachment 10 further has a reservoir portion 16 having a transparent wall portion 50 and two triangular wall portions 84. In in one of the triangular wall portions 84, an inlet port 86 is provided. Simi larly, in the other triangular wall portion 84, an outlet port 88 is provided. The inlet port 86 can be connected with a supply line (not shown) for providing immersion fluid to the reservoir 34 that is formed when the attachment 10 is attached to the immersion objective 14 in the way shown in Fig. 10. The outlet port 88 can be connected with a discharge line (not shown) for discharging immersion fluid from said reservoir 34.

By supplying immersion fluid through the inlet port 86, the reservoir 34 can be initially filled. In addition, during microscopy, immersion fluid can be replenished as desired without having to interrupt the microscopy procedure. This is particularly important dur ing prolonged time-lapsed imaging of multiple positions, to ensure high-quality data throughout the whole experiment.

Moreover, immersion fluid can be discharged from the discharge port 88, thereby allow ing, in combination with the inlet port, not only for replenishment of the immersion fluid, but also for replacing the immersion fluid, at least in part, during microscopy.

The supply line and discharge line can be formed by rubber tubes or the like, and the sup ply and/or discharge can be driven using a suitable pump, such as a peristaltic or syringe pump. The pump is preferably electronically controlled to ensure a desired amount of im mersion fluid in the reservoir, and/or a desired flow of immersion fluid through the reser voir.

Fig. 11a is a schematic side view of an arrangement 18 comprising an object plate 82 with a sample 54 arranged thereon as well as an alternative embodiment of an attachment 10 which is configured for receiving two immersion objectives 14, 14’. The whole arrangement shown in Fig. 11a is symmetric, in that the immersion objectives 14, 14’ are identical with each other, and that the attachment 10 has a symmetrical configuration as well. As is further seen in Fig. 11a, the attachment 10 forms a reservoir portion 16 in which an immersion fluid (not shown in Fig. 11a) is contained. The immersion fluid is in contact with both, the lower side of the object plate 82, and the outer lenses of the respective immersion objectives 14, 14’. In the arrangement shown in Fig. 11a, the immersion objective 14 shown on the right serves for de tection of light, whereas the further immersion objective 14’ shown on the left serves for the illumination. However, during microscopy, the two immersion objectives 14, 14’ can switch roles, in that the illumination is provided by means of the immersion objective 14 on the right and the detection light is received by the immersion objective 14’ on the left.

In order to receive the two immersion objectives 14, 14’, the attachment 10 comprises two attachment portions 12, 12’, each for receiving one of the immersion objectives 14, 14’. As before, one of the attachment portions 12 defines a detecting axis 20 coinciding with the opti cal axis 22 of the immersion objective 14 to be received therein. The further attachment por tion 12’ defines the intended illumination direction coinciding with the optical axis 22 of the further immersion objective 14’ to be received in the further attachment portion 12’. Note that in this regard, the further attachment portion 12’ resembles an optical interface allowing for passing illumination light along an intended illumination direction through the attachment 10 to the sample 54 being located on the detecting axis 20.’ While in Fig. 11a, a simple object plate 82 is shown, in other embodiments, a multi-well plate could be used instead. In yet fur ther embodiments, an object plate 82 can be used that has a functionalized surface with ap propriately designed regions to which the sample 54 will adhere. For example, the functional ized object plate can have regions of different hydrophilicity and hydrophobicity or the like.

Fig. 11b shows a schematic side view of a yet further arrangement 18, which is essentially built up of two arrangements of the type shown in Fig. 11a, of which one is arranged below the object plate 82 in the same (upright) configuration as shown in Fig. 11a, and the other is arranged above the object plate 82 in an upside down configuration. This also means that the reservoir 16 of the upper arrangement is upside down, such that the immersion fluid 38 has a tendency to flow out of the reservoir 16. However, due to the surface tension, the immersion fluid 38 does not completely flow out of the reservoir 16 of the upper arrangement, but forms a meniscus as is schematically shown in the figure. While not shown in Fig. 11b, and inlet port similar to the inlet port 86 shown in Fig. 9 and 10 can be provided for constantly replenishing immersion fluid 38 to compensate immersion fluid 38 that drips out of the reservoir 16 dur ing operation. A corresponding inlet port may also be provided for the reservoir 16 of the lower arrangement.

In operation, the object plate 82 may be moved with respect to the arrangement in a direction indicated by the arrow in Fig. 11b. As before, each of the objective lenses 14, 14’ can be selec tively used for illumination and for detection. By providing a total of four objective lenses 14, 14’, the imaging speed and/or quality can be increased.

While the arrangement shown in Fig. 11b includes four objective lenses 14, 14’ that are each arranged in a vertical plane, it is also conceivable to arrange the four objective lenses 14, 14’ in a horizontal plane. However, in this case, if the sample 54 is to rest on an object plate as is shown in Fig. 11a and Fig. 11b, this will call for a very narrow strip like object plate. In such a horizontal arrangement, a very narrow, strip-like multiwell plate could likewise be used.Figures 12 and 13 show a perspective view and a top view of a mold 90 for forming the attachment 10 as shown in Fig. 11a. While the shape of the attachment 10 is only schematical ly shown in Fig. 11a, from figures 12 and 13 the shape of the attachment 10 can be better dis cerned. The manufacturing process of the attachment 10 using the mold 90 shown in figures 12 and 13 is conceptually the same as the manufacturing process described with reference to Fig. 2 above, and comprises a thermoforming or vacuum forming process using a polymer foil. The details of the manufacturing process shall not be repeated here.

However, instead of forming the attachment from a sheet material, such as a foil or the like, it is also possible to produce the attachment by molding from a liquid or pliable raw material. In particularly advantageous embodiments, the attachment is made from a resilient, rubber like material, for example from silicon rubber. Rubberlike materials have certain advantages over attachments made from formed sheet materials such as polymer foils. For example, due to the resilient nature of the rubberlike material, it is particularly easy to ensure fluid tight ness of the interconnection between the attachment portion 12 and the immersion objective 14. Moreover, the resilience of the rubberlike material also allows for translating the immer sion objective 14 along its respective optical axis 22 while inserted in the attachment, to thereby provide optimum focusing or refocusing.

Fig. 14 shows an example of a 3D-printed negative mold 92 in a disassembled state for the production of a“double sided attachment”, i.e. an attachment allowing for receiving two im mersion objectives 14, 14’ in corresponding attachment portions 12, 12’. Fig. 15 shows the same negative mold 92 in an assembled state. As seen from Figs. 14 and 15, the negative mold 92 comprises a base portion 94 and two frusto-conical inserts 96. The base portion 94 has a platform 98 which forms the bottom of the reservoir of the attachment formed using the mold 92 and a trench 100 surrounding said platform 98, wherein said trench 100 is for form ing wall portions of the reservoir. In the platform 98, frusto-conical recesses 102 are formed in which the frusto-conical inserts 96 are to be inserted for acquiring the assembled state shown in Fig. 15. The frusto-conical inserts 96 define voids in the attachment formed using the mold 92, which correspond to the“attachment portions” 12, 12’ for receiving the immer sion objectives 14, 14’, respectively. The attachment can simply be formed by pouring liquid PDMS material into the mold 92 and curing the material. After the PDMS material is cured, first the frusto-conical inserts 96 are removed, and thereafter the PDMS attachment can be removed from the base portion 94 of the negative mold 92. While specific embodiments have been described in detail, it is not intended that the scope of protection is limited by the specific embodiments. The scope of protection is defined by the appended claims.

LIST OF REFERENCE SIGNS:

10 attachment

12 attachment portion

12 further attachment portion

14 immersion objective

14 further immersion objective’ i6 reservoir portion

i8 system

20 detecting axis

22 optical axis

24 cylindrical portion

26 frustoconical portion

28 rear opening

30 leading opening

32 front surface

34 reservoir

36 opening

38 immersion fluid

40 mold

42 projection

44 vacuum channels

46 foil

48 cutting line

50 transparent wall portion

52 intended illumination direction

54 sample

56, 156 cuvette

58 sample medium

60 illumination beam

62 first bottom wall

64 second bottom wall

66 arrangement

68 illumination objective

70 XYZ-stage

80 microtiter plate

82 object plate

84 triangular wall part 86 inlet port

88 outlet port

90 mold for forming attachment with two attachment portions as shown in Fig.

11a

92 negative mold for producing two sided attachment

94 base portion of mold 92

96 frusto-conical insert to mold 92

98 platform

100 trench

102 frusto-conical recess

Claims

1. Attachment (to) for an immersion objective (14) for performing an inverse immersion microscopy, wherein the attachment (10) comprises

• an attachment portion (12) for receiving an end portion of an immersion objective (14), wherein the attachment portion (12) defines a detecting axis (20) coinciding with the optical axis (22) of the immersion objective (14), when the immersion objective (14) is received in the attachment portion (12), and

• a reservoir portion (16), which defines, when the immersion objective (14) is received in the attachment portion (12) and oriented in an inverse orientation, together with a front surface of the received immersion objective (14), a reservoir (34) for retaining an immersion fluid (38), wherein said attachment further comprises an optical interface allowing for passing illumination light along an intended illumination direction through the attachment (10) to a sample (54) being located on said detecting axis (20).

2. Attachment (10) of claim 1, wherein said optical interface is formed by a transparent wall portion (50) of said reservoir portion (16), said transparent wall portion (50) allowing for passing said illumination light therethrough along an intended illumination direction (52) being orthogonal to the transparent wall portion (50) and tilted to the detecting axis (20), and wherein said container portion (16) and said transparent wall portion (50) are arranged such that, when the immersion objective (14) is in the inverse orientation and received in the attachment portion (12), a sample (54) being located on the detecting axis (20) can be illuminated through the transparent wall portion (50) and through an immersion fluid (38) being retained by the reservoir (34).

3. Attachment (10) of one of the preceding claims, wherein the transparent wall portion is planar and/or at least partially transparent for light in the in the ultraviolet range, in the visible range, in the near infrared range, in the infrared range, in wavelengths ranges therein and/or for one or more wavelengths therein.

4. Attachment (10) of claim 1, wherein said optical interface is formed by a further attachment portion (12') for receiving an end portion of a further immersion objective (14’), wherein said further attachment portion (14') defines said intended illumination direction coinciding with the optical axis (22') of the further immersion objective (14') when the further immersion objective (14') is received in the further attachment portion (12').

5. Attachment (10) of one of the preceding claims, wherein the attachment portion (12) and/or the further attachment portion (12’) defines an inner surface being complementary to an outer surface of a respective immersion objective (14, 14’)·

6. Attachment (10) of one of the preceding claims, wherein the immersion fluid (38) is water, a gel, an oil or an aqueous solution.

7. Attachment (10) of one of the preceding claims, wherein in the inverse orientation, in which an immersion fluid (38) can be retained by the reservoir (34), the detecting axis (20) defined by the attachment portion (12) is tilted with respect to the vertical direction of gravity by an angle a in a range o°£ a £ 90°, preferably in a range 20°£ a £ 70⁰, more preferably in a range 40°£ a £ 50°.

8. Attachment (10) of one of the preceding claims, wherein the attachment portion (12) and/or the further attachment portion (12’) comprises an inner surface defining a frustum of a right circular cone and/or defining a right circular cylinder.

9. Attachment (10) of one of the preceding claims, wherein the detecting axis (20) is tilted with respect to the intended illumination direction (52) by an angle b in a range 70°£ b £ iio°, preferably in a range 8o°£ b £ ioo°, more preferably in a range 8s°£ b £ 95°, in particular by 90°.

10. Attachment (10) of one of the preceding claims, wherein in the inverse orientation the reservoir (34) comprises an upwards facing opening allowing to dip the sample (54), a cuvette (56, 156) comprising the sample (54) and/or a micro well comprising the sample (54) into a retained immersion fluid (54), such that the sample (38) is located on the detecting axis (20) and can be illuminated along said intended illumination direction.

11. Attachment (10) of one of the preceding claims, wherein the sample (54) is a biological fluorescent sample with a diameter in a range between 0,1 pm and 1000 pm.

12. Attachment (to) of one of the preceding claims, wherein the attachment (to) is integrally formed and/or comprises or consists of

- a formed foil (46) of a transparent polymer, preferably of fluorinated ethylene propylene, FEP, wherein the formed foil preferably has a thickness of loopm or less, preferably of 50pm or less, or

- a rubberlike material, in particular PDMS.

13. The attachment (10) of one of the preceding claims, wherein said reservoir portion (16) is connected or connectable with a supply of immersion fluid allowing for a replenishment of immersion fluid during microscopy,

wherein said reservoir portion (16) preferably comprises an inlet port for connecting a supply line for supplying said immersion fluid (38), wherein said reservoir portion preferably further comprises an outlet port for connecting a discharge line for discharging immersion fluid from said reservoir portion (16).

14. A system (18), comprising

- an immersion objective (14) and

- an attachment (10) according to one of the preceding claims, wherein the attachment portion (12) is adapted for receiving an end portion of the immersion objective (14).

15. Arrangement (66) for performing an immersion microscopy, comprising

- a system (18) of claim 14, wherein the end portion of the immersion objective (14) is received in the attachment portion (12) and wherein the system (18) is arranged in an inverse orientation, in which the detection axis (20) is horizontal or is tilted with respect to the vertical direction by an angle a in a range o°< a £ 90⁰, preferably in a range 20°£ a £ 70°, more preferably in a range 40°£ a £ 50⁰, in particular by an angle of 45⁰, and

- an illumination source (68) arranged for providing a beam (60) along an illumination axis with a vertical upwards directed component or in horizontal direction, wherein the illumination axis extends through the optical interface and is tilted with respect to the detecting axis (20) by an angle g, preferably in the range 70°£ g £ no°, more preferably in the range 8o°£ g £ ioo°, in particular in the range 8s°£ g £ 95

16. Arrangement (66) of claim 15, wherein the illumination axis and the detecting axis (20) intersect each other and/or wherein the illumination axis and the detecting axis (20) both intersect a common sample location having a diameter of 3 mm or smaller, preferably 1 mm or smaller, more preferably 500 pm or smaller.

17. Arrangement (66) of claim 15 or 16, further comprising a cuvette (56, 156) for keeping a sample medium (58) with the sample (54) disposed therein, wherein the cuvette (56, 156) comprises a first planar bottom wall (62) and a second planar bottom wall (64) and wherein the cuvette (56, 156) can be arranged, such that

- through the first bottom wall (62) a sample (54) in the cuvette (56, 156) can be illuminated along the illumination axis, and that

- through the second bottom wall (64) fluorescent light stemming from the illuminated sample (54) can travel along the detecting axis (20) towards the immersion objective (14).

18. Arrangement (66) of claim 17, wherein the normal direction of the first bottom wall (62) is tilted with respect to the normal direction of the second bottom wall (64) by an angle g in the range 70°£ g £ no°, preferably in the range 8o°£ g £ ioo°, more preferably in the range 8s°£ g £ 95°, in particular by an angle of 90°.

19. Arrangement (66) of claim 17 or 18, further comprising a microtiter plate (80) with a plurality of said cuvettes (156), and/or

further comprising an XYZ-stage (70) configured to position the microtiter plate (80) or the cuvette (56) relative to the system (18) and the illumination source.

20. Arrangement (66) of one of claims 15 to 19, wherein the illumination source comprises an illumination objective (68) and/or wherein the illumination source is configured for providing a beam (60) with a focus in the form of light sheet.

21. Method for forming an attachment according to one claims 1 to 13, comprising - providing a mold (40) being partially shaped according to an end portion of an immersion objective (14) and having a projection (42) for defining the volume of the reservoir (34),

- using the mold (40) for shaping a transparent polymer foil (46) in a thermoforming process according to the shape of the mold (40),

- removing a portion of the shaped foil, such that, when the end portion of the immersion objective (14) is received in the formed foil, a front surface of the received immersion objective (14) forms an inner wall of the reservoir (34) and the reservoir (34) comprises on opening (36).

22. Method for forming an attachment (10) according to one of claims 1 to 13, said method comprising the following steps:

- providing a negative mold (92) comprising a base portion (94) and at least one insert (96), said insert (96) having a shape matching the shape of at least a part of an end portion of an objective (14) that is to be received in an attachment portion (12) of said attachment (10, no),

- placing said insert (94) in said base portion (94),

- allowing said molding material to solidify, in particular by curing said molding material, to thereby form said attachment (10),

- removing said at least one insert (96) from said base portion (94), and

- removing said attachment (10, no) from said base portion (94).