Method for optical 3D printing in a photoresist and use of a lens protection device

Abstract

A method for optical 3D printing in a photoresist, comprising the steps of: a) providing a lens and a lens protective device, comprising • a transparent film and • a mounting device made of a flexible material, wherein the film and mounting device are integrally joined by welding or fusing, b) releasably attaching the lens protective device to the lens by tensioning the film, and c) curing a photoresist using the lens, wherein the photoresist is separated from the lens by the film of the lens protective device.

Description

The invention relates to a method for optical 3D printing in a photoresist and to the use of an objective protection device with film for protecting objectives in optical printing by means of photoresists (multiphoton lithography) and in other optical methods.Multiphoton polymerization has already been demonstrated in the 1990s to produce 3D nano- or microstructures. Based thereon, the method of "multiphoton lithography", also known as "Fedosecond direct laser writing" (fDLW), resulted.The necessary microscope objectives are characterized by two characteristics: magnification and numerical aperture (NA). Objectives with NA in the range 0.1-1.4 typically have a working distance (i.e. distance between objective and the focal point where the image or object is formed) of between a few millimeters to about 210 μm. For greatly enlarging applications or the pressure of particularly small structures, the use of an immersion medium is essential.The principle of immersion to increase the optical resolution, for example in microscopy, is known. An immersion medium is brought into contact with the objective and the object or object carrier to be examined. Refractive index differences at material boundaries are thereby reduced and thus undesired reflections are lowered. In the case of water, living cells or tissues can also be examined, which would otherwise dry out.It is known to use glass panes (e.g. object slides, coverslips) for the separation between objective and photoresist. In the immersion mode (FIG. 1, right), the photoresist is applied on the side facing away from the objective, so that the photoresist itself does not come directly into contact with the objective. Three-dimensional objects can be printed in this manner.In dip-in mode (as shown on the left in FIG. 1 ), the photoresist (between objective and object carrier) itself serves as an immersion medium. The disadvantage is that a time-consuming cleaning of the objective is necessary. The risk is likewise disadvantageous that seals or the like can be attacked by the immersion medium or cleaning chemicals within the objective.DE 10 2018 221 670 A1 discloses a membrane holder with membrane which is introduced between the objective and an object at a distance from the objective so that no disturbing forces are transmitted to the objective. No attachment to the objective takes place. The diaphragm is moved relative to the objective.WO 2015 / 084737 A 2 discloses a protective element for microscope objectives with glass cover glass or transparent plastic part. Glass is disadvantageous because, as a planar plate, it is not capable of imaging the rounded shapes of the objectives and thus includes the objective. The glass cover occupies a large amount of space, the plastic disclosed is too thick. Both have the result that either structures of as small a size cannot be printed because of the space requirement or the objective collides with the printed object.In JP 2006235208 A, a lens cover that covers the surface of an objective lens used for object observation, particularly a lens cover made of a resin film that eliminates the influence on imaging performance of a water immersion objective lens and enables cells and the like to be observed in a sterilized state, is described. The disadvantage of the lens cover is not suitable for immersion in aggressive photoresists.The document US 2007 / 0024982 A1 discloses an imaging system of a microlithographically projected exposure apparatus which makes it possible to protect the outer optical surfaces from soiling. In an imaging system with a projection objective, which serves to project an image of a mask adjustable in an object plane onto a light-sensitive layer adjustable in an image plane, a membrane substantially transparent for an operating wavelength of the projection objective is arranged to an outer optical surface of the projection objective in such a way that a space is formed between this optical surface and the membrane, which space is configured to receive a liquid or gaseous medium. The diaphragm is attached to movable rollers by which it is tensioned. These moving parts adversely affect the cost and susceptibility to errors of the imaging system.JP 2006343670 A relates to an objective protection device having an optically transparent cover element for covering at least the edge surface of an objective and a fastening means for fastening the cover element to the objective in order to reduce the cleaning work on the objective and to protect the objective from the adhesion of bacteria or dirt and from the action of an external force when cleaning is carried out. The objective protection device is disadvantageously not to be used for use in optical 3D printing processes and for immersion in aggressive photoresists.It is an object of the invention to provide a method for optical 3D printing in a photoresist using a protective device for objectives which is easy to clean, disposable and thus also sterile and cost-effective to produce. The protective device should have a low intrinsic weight and have a low complex structure. The protection device is intended to reduce the working distance of the objective only minimally, but also its production resolution, to protect the objective from aggressive photoresists or cleaning agents, and to prevent cross-contamination of the objective and substrate.The invention relates to the use of an objective protection device comprising• a transparent film and• a fastening device made of a flexible material, the film and fastening device being integrally connected to one another by means of welding or fusion, for protecting an objective during optical 3D printing in a photoresist.In embodiments, the protection device is used for detachable attachment to an objective. The lens is covered by the re-detachable fastening, i.e. the lens is directly connected to the film when fastened. Nowhere else (than on the objective) is a fastening of the protective device necessary.The distance between the film and the objective remains constant after assembly (i.e. the detachable fastening according to the invention). This means that according to the invention the film is always in contact with the objective during optical printing or optical visualization or manipulation. The transparent film is flexible.The invention also relates to a method for optical 3D printing in a photoresist, having the steps: a) providing an objective and an objective protection device, comprising• a transparent film and• a fastening device made of a flexible material, wherein the film and the fastening device are integrally connected to one another by welding or melting together, b) releasable fastening of the objective protection device to the objective while tensioning the film, and c) curing of a photoresist by means of the objective, wherein the photoresist is separated from the objective by the film of the objective protection device.The tensioning of the film in step b) takes place in such a way that the film is tensioned, either because it has already been tensioned beforehand by the fastening device itself or by a tensioning aid, or because it is tensioned only by contacting with the objective, because in the latter, too, the fastening device serves for tensioning.In embodiments of use, the releasable attachment is effected while tensioning the film.In embodiments, the method is designed as a method for optically visualizing or manipulating particles, structures, bacteria, cells or cell composites in a medium, comprising the steps mentioned above.Embodiments of the invention also include an integrated lens protection device that is particularly well suited for use and method according to the invention. This is an objective protection device for protecting objectives, comprising• a transparent film, anda fastening device, wherein the film and the fastening device are connected to one another in an individable manner.It is particularly well suited for single use. This is because it is formed in one piece, so that it is easier to produce in mass production and easier to dispose of with a handle. It is referred to below as an "integrated objective protection device", wherein all preferred embodiments of the invention are also possible for this (optimized) integrated objective protection device.The fixing device of the invention serves for fixing the film to the objective lens.In embodiments of use, the fastening device is designed as a clamping device.The objective protection device is sterile in embodiments of the method and use.Embodiments of the invention comprise a sterile kit comprising said objective protection device and a sterile outer packaging. This outer packaging could be, for example, a plastic bag which is sterile in the interior.In embodiments, the film is a polymer film, in particular one made of polyethylene (PE). In one embodiment, the film is so thin that objects can be printed side by side without the objective colliding with the previously printed object during the printing in the secondary position.In embodiments, the transparent film according to the invention is 1 mm-0.05 mm thick, preferably 0.2 mm-0.05 mm, particularly preferably <160 μm, or even <100 μm, better still 10±3 μm, in particular <10 μm, i.e. as thick as keep-fresh film (~10 μm) or thinner.In embodiments, the film according to the invention consists of a material selected from polyethylene (in particular mylar, i.e. biaxially oriented polyester BO-PET), polyamide, fluorinated polymers and mixtures thereof, and particular preference is given to FEP (fluoroethylenepropylene). The film is thus advantageously suitable for particularly aggressive photoresists or media. It is also advantageous to use particularly flexible material for the film, for example polydimethylsiloxane (PDMS).The fastening device according to the invention is made of a flexible material in embodiments.In one embodiment, it has a ring shape, preferably it is an O-ring.In one embodiment of the invention, the fastening device is detachably fastened to the objective by means of threads, clamps, magnets and / or screws. Expediently, a counterpart can be located on the objective for this purpose. In the method, step b) would then be a detachable fastening of the objective protection device to the objective by means of such threads, clamps, magnets and / or screws.In one embodiment of the invention, in the lens protector, the film is made of a material selected from polyethylene, polyamide, fluorinated polymers and mixtures thereof, and the fixing device is made of a flexible material and is annularly constructed.In one embodiment, the transparent film and the fastening device are connected to one another in an indivisibly, i.e. the objective protection device is one-piece / one-piece. This can be done, for example, by means of adhesive / glue or welding / fusion (see FIGS. 6, 7 ). The advantage of this embodiment is that a one-piece article is produced, which is favorable for mass production and then for use as disposable article and disposal. Then, an external diaphragm holder is no longer necessary.In one embodiment of the invention, the objective protection device is used with the aim• optical printing, and / or• The Visual Visualization or Visual Manipulation of Particles, Structures, Bacteria, Cells or Cell Composites.Optical printing is 3D printing or also single / multiphoton / fluorescence imaging. The optical printing can take place with electromagnetic radiation, in particular with laser, preferably with a wavelength of 1030±30 nm, 780 nm±30 nm or 515±30 nm.In particular in the visual visualization or manipulation of biological substrates such as bacteria, cells or cell composites, the sterility which is possible with the invention is advantageous. This sterility is also advantageous in particular in the 3D printing of structures for colonization with abovementioned biological substrates.Optical manipulation also includes the principle of optical forceps (optical trapping). The optical trap is a photonic device for manipulating, i.e. holding and moving, minute objects.In one embodiment, in the case of optical printing, the method additionally comprises the step of subsequently partially curing (structuring) a photoresist, which is separated from the objective by the film, by means of the objective.In one embodiment of the invention, the film and / or the fastening device is sterile. In particular, the objective protection device is preferably sterile. In the method, this relates in particular already to the provided objective protection device in step a). It is expediently packaged in sterile fashion.Advantages:The invention permits easy cleaning of the objective. This is because, for example in the case of optical printing, the film protects the photoresist from non-sterile conditions on the objective, but at the same time without adversely affecting the writing resolution. Even in the case of visualization or manipulation of bacteria, for example, these would be protected by the film from immortal conditions of the objective.In this regard, the objective protection device can also be sterilized.The lens protector is disposable because it is easily mass-produced and thus suitable as a disposable product for the above-mentioned uses.It has a low own weight compared to lens protection devices with glass, which does not make the highly accurate dynamic positioning of the lens (for example when changing the lens under locking positions) difficult due to high weight.The objective protection device in the use and method according to the invention has low technical complexity, it is a daily article, is cost-effective and less susceptible to errors.The film can image the usual lateral slopes of the objectives (conical region), which would not be possible with a straight glass pane. A non-planar glass object, on the other hand, would be extremely complicated to produce.The attachment of the film by the objective protection device to the objective in a detachable manner simplifies the change of the objective during printing or microscopical operations. A "followed" objective turret is made possible. No disturbing contacts are necessary during the change of objective, the precision thereby being increased. Advantageously, a lateral voxel diameter of less than 150 nm is achieved.It is also possible to mount or mount the protective device before the actual lens installation and outside the printer or microscope.There is no limitation as to the height of the object to be printed. All advantages of the dip-in mode from DE102011012484A1 are retained, only the working distance is reduced by the distance between the lens termination and the film side facing away (i.e. by the small thickness of such a film).In the invention, the film protects the objective from aggressive liquids, such as acidic photoresists or cleaning agents.The invention can be used for any desired objectives or is also suitable for retrofitting existing objectives.Preferred EmbodimentsIn a preferred embodiment of the method, it has the additional step: a2) moistening the film and / or the objective with an immersion medium.Steps a2) and b) can be interchanged, but the order a2) b is preferred).The immersion medium can be, for example, water, oil or air; the immersion medium is preferably selected from water and oil and / or other immersion liquids whose refractive index is adapted to the objective used.Logically, it is such that an immersion medium is usefully applied on the side facing the objective when it is applied to the film, so that it is in contact with the film and objective and no air gap forms. In this preferred embodiment, the detachable fastening in step b) must also be carried out in such a way that the contact of the film with the objective is finally established via the immersion medium.In a preferred embodiment of the method according to the invention, the refractive index of the film or of the immersion liquid differs only by <0.1 from that of the photoresist or of the medium. Photoresist or medium is located on the film side which faces away from the objective. The difference is preferably <0.01, particularly preferably <0.001.In preferred embodiments of the method, the film is made of a material selected from polyethylene, polyamide, fluorinated polymers and mixtures thereof.In preferred embodiments of use, the film and fastening device are sterile.In preferred embodiments of use, the film is made of a material selected from polyethylene, polyamide, fluorinated polymers and mixtures thereof, wherein the fastening device is annularly constructed from a flexible material. FIG. 1 shows two methods from the prior art (left: dip-in mode, right: immersion mode). Figures 2a and 2b show applications / results of the invention with different photoresists. Fig. 3 shows an embodiment of the invention. FIG. 4 shows an embodiment of the invention in dip-in mode (left: objective without a protruding mounting edge, right: objective with a protruding mounting edge and a defined gap). FIG. 5 shows an embodiment with a film made of polyethylene, an O-ring as clamping device and Insolen / deionized water as immersion medium. FIG. 6 shows an embodiment with an inseparably (integrally) connected film and fastening device. FIG. 7 shows an embodiment of the objective protection device according to the invention with a film and fastening device (left and center) joined in an indivisibly with the aid of adhesive and in a detachable fastening to an objective (right).The subject matter of the invention is illustrated by the main claims. Preferred embodiments are set forth as subordinate claims.All the various embodiments can be combined with one another in any desired manner. Preferred embodiments for steps of the process according to the invention can also be used for the plant according to the invention.Exemplary Embodiments1. Gap between the film and the writing objectiveProjecting mounting edges on the objectives create a distance between the film and the objective (see FIG. 4 on the right). This was determined for the objectives used (by means of confocal microscopy) and was between 0 and 40 μm. This gap is filled with an immersion medium.Further data are shown in Table 1. Table 1: Experimental Pressure Settings Using the Invention. Table 1: Experimental Pressure Settings Using the Invention.40x oil1,4Alwayssol 518F0,19 ±0.05 μmIP-DipStacked line structure (dx=dy / dz) 500 nm / 990 nm25x oil0,8The reaction. H 2 O24,4 ±0.4μmIP-DipStacked line structure (dx=dy / dz) 1 μm / 2.12 μm25x water0,8The reaction. H 2 O24,4 ±0.4μmGM10 Methacrylated GelatinMouse Cell Framework25x oil0,8Alwayssol 518F24,4 ±0.4μmEpoxy Lacquer580 μm high40x water1,2The reaction. H 2 O36,5 ±1.2 μmSilver nitrateLine structure 200 μm Throughm.*... The addition to the magnification indicates the immersion medium for which the objective is designed. The immersion medium used in the experiment is given in column 3.**... Gap between the film and the objective lens2. Sensitivity to Film and Gap - Resolution AchievedFilm and gap sensitivity for different numerical apertures (NA) was analyzed. The Strehl ratio as a measure for the optical quality of the construction (here: objective, immersion medium and polyethylene film with a thickness of 10 μm) is 0.8 both for the high resolution variant and for the low resolution variant. Thicker films and higher NA reduced the resolution markedly.The poorly matched low resolution variant of NA=0.8 oil objective with water as immersion medium reached a Strehl ratio of 0.981.All in all, thinner films and smaller immersion gaps achieved the best results. That is, shorter beam paths are less sensitive to high refractive index discrepancies. Higher NAs require better refractive index adjustments for experimentally established gaps and film thicknesses.The kitchen foil used was inexpensive (~10 μm) which was clamped and fastened on the objective by means of an O-ring. A drop of immersion medium was previously placed on the objective. Excess immersion medium can escape at the sides.The resolutions achieved (lateral and axial) were compared with the known dip-in method without a protective device. The voxel dimension was determined by scanning electron microscopy.The high resolution variant (40x, 1.4 NA) reached a lateral voxel diameter of <150 nm.The low resolution variant (25x, 0.8 NA, water as immersion medium) reached voxel diameters comparable to conventional dip-in methods.3. Comparison of Different PhotoresistsThe invention was carried out using methacrylate-based standard resists from a 3D printer manufacturer, but also using aqueous protein biotinte, black epoxy-based photoresist, or aqueous silver ink, as described below.Figure 2a shows on the left (A) a bright field microscope image during the production of a 3D framework from aqueous protein photoresist which contains 25% by weight of GM10 (a gelatin derivative with a high degree of methacrylication) and 2% by weight of LAP (lithium phenyl 2,4,6-trimethylbenzoylphosphinate) as photoinitiator in phosphate buffer. The ease of removal of the film reduces the risk of biological contaminants and allows the sterile conditions (in the photoresist) to be maintained without difficulty.On the right (B), FIG. 2 ashows the microscope image of the 580 μm-high tower of black photoresist (DELO Katiobond OM VE 114929 from DELO Industrial Adhesives). It was thus possible to print objects which extend far beyond the working distance of the writing lens. This is because this working distance is only 210 μm for the objective, and 25x 0.8NA objective for 170 μm cover glass thickness.FIG. 2B shows (C) the electron micrograph of a metallic line structure printed from aqueous silver reagent (AgNO 3, NH 3, trisodium citrate initiator Na 3 C 6 H 5 O 7).4. Comparative ExamplesComparative examples with deposition of a metal film on unprotected object in conventional dip-in methods resulted in irrevocable destruction of the objective.Reference numerals denote reference numerals1 Objective 2 Film 3 Fastening device 4 Photoresist or medium 5 Immersion medium 6 Object holder 7 Protruding mounting edge

Claims

Method for optical 3D printing in a photoresist, having the steps: a) providing an objective and an objective protection device, comprising • a transparent film and • a fastening device made of a flexible material, wherein the film and fastening device are integrally connected to one another by means of welding or melting together, b) releasably fastening the objective protection device to the objective while tensioning the film, and c) curing a photoresist by means of the objective, wherein the photoresist is separated from the objective by the film of the objective protection device.Method according to claim 1, with the additional step a2) moistening the foil and / or the objective with an immersion medium.Method according to either of Claims 1 and 2, wherein the refractive index of the film or of the immersion liquid differs only by <0.1 from that of the photoresist or of the medium.The method of any one of claims 1 to 3, wherein the film is made of a material selected from polyethylene, polyamide, fluorinated polymers, and mixtures thereof.Use of an objective protection device, comprising • a transparent film and • a fastening device made of a flexible material, wherein the film and fastening device are integrally connected to one another by means of welding or fusion, for protecting an objective in optical 3D printing in a photoresist.Use according to claim 5, wherein the film and the fastening device are sterile.Use according to any one of claims 5 or 6, wherein the film is made of a material selected from polyethylene, polyamide, fluorinated polymers and mixtures thereof, and wherein the fastening device is annularly constructed from a flexible material.

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