EP4504036A1

EP4504036A1 - Implantable platform for imaging in-vivo

Info

Publication number: EP4504036A1
Application number: EP23727094.7A
Authority: EP
Inventors: Manuela Teresa Raimondi; Giulio Nicola CERULLO; Claudio CONCI; Alessandra NARDINI; Giuseppe CHIRICO; Margaux BOUZIN; Mario MARINI; Laura SIRONI; Maddalena COLLINI; Roberto Osellame; Rebeca MARTINEZ VAZQUEZ; Maria Farsari; Asimina Eleni KAMPOURAKI
Original assignee: Foundation for Research and Technology-Hellas; Consiglio Nazionale delle Richerche CNR; Universita degli Studi di Milano Bicocca; Politecnico di Milano
Current assignee: Foundation for Research and Technology-Hellas; Consiglio Nazionale delle Richerche CNR; Universita degli Studi di Milano Bicocca; Politecnico di Milano
Priority date: 2022-05-10
Filing date: 2023-05-09
Publication date: 2025-02-12
Also published as: WO2023218340A1; IT202200009587A1

Abstract

An implantable platform for imaging in-vivo comprising: a planar substrate (10) transparent to light; at least one three-dimensional matrix (12) fabricated on a side of said planar substrate (10), configured to host biomaterial; wherein said at least one three-dimensional matrix (12) is obtained by means of two-photon photo-polymerisation and is luminescent under infrared excitation by two-photon absorption; at least one micro-optical device (11), comprising at least one lens (20, 21, 22), fabricated by means of two-photon photo-polymerisation or nanoimprinting lithography on the other side of said planar substrate (10) placed in correspondence of said a least one three-dimensional matrix (12); wherein said implantable platform for imaging in-vivo is configured to be implantable below the skin of a laboratory animal.

Description

Implantable platform for imaging in-vivo

DESCRIPTION

The present invention refers to an implantable platform for imaging in-vivo and a method for a minimally invasive solution for the in-vivo inspection of cellular dynamics and/or tissue’s morphological changes and/or drug release.

Biomaterial, understood as a natural or synthetic material that is suitable for implantation in living organisms, in particular humans, needs a complex and standardized validation procedure aimed to demonstrate the biocompatibility of the material. In fact, medical devices proposed for worldwide distribution must necessarily follow international standards which are the basis for harmonised regulatory processes that ensure minimum standards of safety, quality, and performance of such medical devices. New biomaterials must be tested in vivo, following a procedure of subcutaneous implantation, as regulated by the ISO 10993-6 set of standards. Within this set of norms, the reaction of the implant of the biomaterial must be assessed through histopathological analysis on a four to six timepoints, at which the formation of the fibrotic capsule, signs of inflammation like presence of polymorphonuclear cells, giant cells (Langhans cells), plasma cells, neo-vascularisation and/or degradation of the implanted material must be quantified on laboratory animals. This procedure entails obviously multiple replicas of the measurements and, consequently, the sacrifice of an extremely large number of rodents

(rats, rabbits or guinea pigs), with very high costs and a large ethical burden for the society and the consumers. The optimization of the regulatory norms can be obtained by changing the current approach with which the analysis of the reaction to implants in-vivo are made.

With this purpose, several attempts have been made to use intravital microscopy to assess the immune reaction to the biomaterial implants, enabling the continuous and prolonged observation in-vivo of the material implanted at a subcutaneous level. Intravital high resolution microscopy can be better implemented in multiphoton excitation that exploits the simultaneous absorption or scattering of two or more photons by the living tissues. It can be used to quantify various factors relevant to assess the immune reaction to the implant by exploiting auto-fluorescence of cell cytoplasm and the endogenous second harmonic signal coming from collagen or higher order multi-photon processes (third harmonic, three and four photons excitationor resonant Raman scattering). By exploiting these signals, we can follow the cellular dynamics and the angiogenesis on individual animals at different time-points after the implantation, without the need to suppress or subject the recipient to invasive surgical operations of various nature, once the pre-set time window has been reached.

Specific observation chambers (known as "window chambers") have been used to observe reactions in-vivo. These chambers are very effective in allowing observations. However, they observe a situation that is drastically different from physiological ones due to a very invasive surgical implant procedure conducted in the recipient animal. Moreover, the discomfort of the recipient is not accounted for when using traditional window chambers. The recipient, in fact, beside an initial invasive surgical operation, which is ethically questionable from many points of view, undergoes severe suffering and stress for the entire duration of the implantation. For this reason, these observation chambers are also unable to provide extended temporal duration of the observation of the reaction to the implant of a biomaterial. Finally, they do not encompass any method for re- positioning at a micrometric level the field of observation at each time point. All these reasons have rendered window chambers not a suitable alternative to the directives of ISO 10993-6 for the quantification of the response of an organism to the implant of a foreign body.

To partially overcome this limitation, scaffolds with micrometric size features and sub-millimiter size were developed that are minimally invasive for the recipient animal, allows the repositioning of the field of view during different and consecutive imaging experiments, allow to quantify the density of cells recruited by the immune reaction due to the highly regular structure that is dimly fluorescence under common near infrared laser excitation, as used in intravital non-linear microscopy.

An example of an implantable medical device is disclosed in the document WO2019123227.

However, these scaffolds did not incorporate any method and device to allow effective in-vivo observation through the skin of the recipient animal. In fact, in order to ensure a correct quantification of the response of the animal to the device, this should be implanted sub- cute and imaged through it. However, skin is known to have large scattering coefficients that severely warp the phase of the light wave front, limiting the possibility to perform high resolution optical scanning imaging.

The aim of the present invention is to provide an implantable platform for imaging in-vivo that overcomes the drawback of the prior art.

Another aim is to provide an implantable platform for imaging in-vivo able to diminish at the minimum the distance between the objective lens and the observation field.

According to the present invention, the above aims and also others are achieved by an implantable platform for imaging in-vivo according to claim 1 .

Further characteristics of the invention are described in the dependent claims.

The advantages of the present solution over the solutions according to the prior art are multiple.

Diminishing at the minimum the distance between the objective lens and the observation field determines that the light penetrating the skin, the subcutaneous fat and the muscle is collimated and suffers the minimum spherical aberrations.

This goal is achieved by forming microstructures on the two sides of a planar substrate transparent to light. On one side of the planar substrate, a three-dimensional matrix is fabricated comprising a number of levels distributed in height and made according to a regular lattice design. On the opposite side of the planar substrate, a set of micro-optical devices is fabricated.

The advantages of the present solution to the inspection and quantification of the immune response of a host animal to the implant of a biomaterial, over the existing protocols based on ISO-10993 and according to prior art, are multiple. The solution proposed consists of a miniaturised device dedicated to intravital optical microscopy based on micro-optics implanted on the very same animal on which the biomaterial to be tested is implanted and allows the observation of cell dynamics and the angiogenesis on an animal for a long period of time.

The part of the device that host the biomaterial and allows the inspection of the reaction to it, is constituted by a micro-geometry with controlled porosity that also acts as guide for growth of newly formed tissue. This micro-geometry is obtained by means of two-photon photo-polymerisation in a non-cytotoxic photosensitive material called resist with photo-initiators that were proved not to be toxic for cells.

The resist, once polymerized and developed, is in turn luminescent under infrared excitation by two-photon absorption, with a spectrum peaked in the green band. Therefore, the microstructure is visible under two-photon excitation used for the imaging in-vivo and can serve as a frame of reference for multiple/repeated observations. The device will be coupled to a custom-made biomaterial shaped as a disk and will be implanted in an animal according to current standards. The implantation and observation site will be without any percutaneous access, unlike the existing window chambers commonly used up to now. The device will be colonised by the tissue of the recipient, and the micro-geometry will guide said re-growth in situ, enabling neo- vascularisation of the implanted portion. We have developed protocols for the implant of the device in mice sub-cute, either in the inner part of the hind leg or on the upper part of the back. We have observed the reaction to the implant in-vivo by means of two-photon and second harmonic scattering microscopy. Due to the micro-optics fabricated close (on the opposite side of the planar transparent substrate) to the regular micro-geometry, the device will moreover enable microscopy observations repeated over time in one and the same spatial position through the skin of the animal, thanks to the low numerical aperture laser beam. This technology hence for the first lime enables objective, continued, and prolonged quantitative analyses of the inflammatory response to the implant, by means of non-invasive real-time longitudinal observations. Thanks to this solution, it will no longer be necessary to sacrifice the animal subjected to testing at each observation time point. Finally, the device strives for a paradigm shift also in the statistical approach to the analysis of the immune reaction to biomaterial implants. In fact, the observations will be done on individual animals that will be observed for the whole implant duration at several time points. The only stress to the animal will be a mild anaesthesia to keep it still under the microscope. A stereotactic device could also help in this sense. We are then able to single out the effects due to wide variance of the biological response (biological replicates) from the intrinsic variance due to the uncertainty in the measurements and observations (technical replicates).

The advantages of the device are of economic, scientific and ethical nature. Considering the economic aspect, the device will enable reduction of development costs linked to testing of biomaterials/drugs, as well as reduction of the number of animals used for the experiment. A large number of animals implies large specific structures (animal facilities), dedicated staff, and high management costs: these are the most relevant costs for the biomaterial validation.

From an ethical standpoint, the number of animals sacrificed is consequently reduced, and likewise the suffering linked to the implantation of standard window chambers is reduced. The total absence of a percutaneous access, afforded by the device, completely eliminates the need for long, complex, and highly ethically questionable surgical operations on the recipient and also dramatically reduces the risk of infection. The scientific relevance of the device is based on the possibility offered to obtain accurate quantification of the reaction to the implant, providing counting of cells, divided by type, and of vessel growth in contact with the biomaterial. The relevance and impact of these observations can be further improved by means of

Machine Learning methods to cluster data and to quantify correlations among the various types of immune cells observed. To this purpose, the device will be coupled to the possibility to impart to the host animal batches of fluorescently labelled immune cells: different labels per different types of immune cells. The characteristics and advantages of the present invention will emerge clearly from the ensuing detailed description of a practical embodiment thereof, which is illustrated by way of non-limiting example in the attached drawings, wherein:

Figure 1 is a schematic illustration of an implantable platform for imaging in-vivo, shown in perspective, according to the present invention;

Figure 2 shows an upper portion of an implantable platform for imaging in-vivo, shown from the side, according to the present invention;

Figure 3 shows an upper portion of an implantable platform for imaging in-vivo, shown in perspective from below, according to the present invention;

Figure 4 shows an upper portion of an implantable platform for imaging in-vivo, shown from below, according to the present invention;

Figure 5 shows an upper portion of an implantable platform for imaging in-vivo, shown from above, according to the present invention;

Figure 6 shows a partial section of an upper portion of an implantable platform for imaging in-vivo, according to a first embodiment of the present invention;

Figure 7 shows a partial section of an upper portion of an implantable platform for imaging in-vivo, according to a second embodiment of the present invention;

Figure 8 shows a partial section of an upper portion of an implantable platform for imaging in-vivo, according to a third embodiment of the present invention;

Figure 9 shows a schematic illustration of an implanted medical platform inclusive of a laser source and a photon detector according to the present invention;

Figure 10 shows a schematic representation of the device coupled to a microscopy objective to be used in a optical scanning microscope on an implantable platform according to the present invention;

Figure 11 shows a porous microstructure used in an implantable platform according to the present invention;

Figure 12, 13 show examples of the images that can be obtained through the microlenses (as in the scheme of Figure 10) at high resolution on different fields of view and different spectral windows, like the green emission of cells cytoplasm autofluorescence

(Figure 12) or the red emission of nuclear dyes (Figure 13).

Referring to the attached figures, an implantable platform for in- vivo imaging, according to the present invention comprises a planar substrate 10, transparent to the visible-near infrared light: a set of micro-optical elements 11 are placed on the top side of the substrate

10, a set of one or more (four in the sketch) regular porous microstructure 12 and spacer 13 are placed on the back side of the substrate 10.

The planar substrate 10, transparent to light, can be done with glass, treated or untreated, COC polymer or Ormocomp™, or other photoresists This double face substrate 10 can be preferably combined with a second substrate 14, placed below the planar substrate 10, possibly but not limited to, a slab of biomaterial. This slab is fabricated with the biomaterial whose immunogenicity is to be tested and is coupled by gluing or bonding to the set of spacers 13 with biocompatible (acrylic) glues. The shape and size of the spacers 13 is tailored to ensure tight adhesion and to avoid perfusion of the glue to the central observation regular microporous structures 12.

In the present embodiment, the micro-optical elements 11 comprises two larger lenses 20, and two lenses arrays 21 placed on the top of the planar substrate 10 positioned to form a quadrilateral.

Depending on the size of the microlenses, their focal length is in the range 0.3-0.6 mm with an overall magnification of 200-300.

On the back side of the planar substrate 10, in correspondence of the lens 20 and 21, four microstructure 12 are placed. Peripherally to the lens 20 and 21 , the spacer 13 (or pillar) are placed.

As shown in the figures, the lens 20 are single planoconvex lens, the lens 21 are an array of seven planoconvex lenses (smaller than the lens 20) grouped together, but can be of different shapes as a single biconvex lens 22, coupled with the planar transparent substrate to form a single optical element.

The implantable platform for in-vivo imaging is implanted below the skin 24 of a laboratory animal 25 and a laser source 26 for multi- photon or for confocal microscopy can be used to get an image thereof. The optical setup is schematically composed of a scanning system that operates an angular scanning of a collimated laser source

26 on the microstructure 12 and allows the detection of fluorescence or other spectroscopic signals (non-linear scattering of various order) by means of a (preferably digital) photon detector 27.

The implantable platform is produced using the technique known as two-photon laser polymerization (2PP), or multiphoton lithography, or multiphoton writing, or direct laser writing of a resin

(SZ2080™ and its derivative). It is carried out via a laser source with a wavelength in the near infrared (NIR) Focused in a photosensitive material constituted by a pre-polymer (i.e., monomers and oligomers) and by a photo-initiator (or no photoinitiator). This phenomenon results in an overall polymerization of the local volume, causing in situ cross- linking of the monomers and of the oligomers. The likelihood of two- photon absorption has a quadratic dependence upon the intensity of the IR incident laser beam; consequently, polymerization occurs only within a volume of about 10"¹⁵ litres restricted around the locus.

Absorption of two independent and simultaneous photons causes an energy transition that cannot be obtained by absorption of a single infrared photon. Due to the very small volume on which the 2 -photon absorption occurs and by playing with the polymerization threshold determined by the concentration of the monomer and the photo- initiator, one can reach high resolution of fabrication down to about

100 nm, thus overcoming the optical diffraction limit. The microstructure 12 have the section of 0.5 mm x 0.5 mm, the height of 0.1 mm fabricated on a substrate 10 of diameter of 5 mm or 12 mm.

The microstructure 12 host the biomaterial and allows the inspection of the reaction to it, and also acts as guide for growth of newly formed tissue.

Similarly, the microlenses 20 and 21, have been fabricated by

2-photon polymerization with a random spiral scanning of the outer as shell of the lenses. The photo-polymerization of the bulk inner volume of the lenses can then be obtained by UV curing.

The bi-convex lens 22 are obtained by 2PP polymerization of a photoresist once a concave hosting niche has been obtained in the transparent substrate, possibly by laser etching, or by means of a similar method.

The microstructure 12 s obtained by means of two-photon photo-polymerisation in a non-cytotoxic photosensitive material called resist with photo-initiators that were proved in the literature not to be toxic for cells. The resist, once polymerized and developed, is in turn luminescent under infrared excitation by two-photon absorption, with a spectrum peaked in the green band. Therefore, the microstructure

12 is visible under two-photon excitation used for the imaging in-vivo and can serve as a frame of reference for multiple/repeated observations.

For two-photon polymerisation of the microstructure 12 an ytterbium femtosecond laser was used, based upon a mode-locking cavity-dumped oscillator with an emission wavelength ranging in the near infrared, or a fiber laser operating at 780 nm. The characteristic wavelength of the laser was 1030 nm, the pulse duration was approximately 300 fs, the repetition frequency was

1 MHz, the maximum pulse energy was 1 μJ, and the maximum average power was 1 W.

As photosensitive material a radical-polymerizable biocompatible hybrid resin known as SZ2080™ was in general used, constituted by a pre-polymer and by a photo-initiator known as

IRGACURE™369 (2-benzyl-2-(dimethylamino)- 1 -(4-

(morpholinylphenyl) - 1-butanone), which is not cytotoxic and is slightly fluorescent. Other possible photo-initiators are 4,4-Bis(diethylamino) benzophenone (BIS), which is more fluorescent than the lrgacure™369, and Sudan Black B, which post-treated with an ethanol solution of Sudan Black B leads to very low autofluorescence 3D structures.

SZ2080™ is an inorganic-organic hybrid, the main components of which are [3-(Methacryloyloxy) propyl] trimethoxysilane (MAPTMS),

Methacrylic acid (MAA), zirconium propoxide (ZPO) and 1 % of the

IRGACURE 369 photo-initiator. The SZ2080™ photoresist presents many advantages, such as biocompatibility, long-term stability, chemical and electrochemical inertia, good optical transmission, and good mechanical stability after polymerization, and entails a negligible distortion and reduction of the structure during development when compared to other commercially available photoresists.

For the fabrication of the microlenses 20-22, beside the use of

SZ2080™, a resin with high refraction index has been used to obtain enough dioptric power to be used when embedded in the animal tissue, whose average index of refraction is 1 .45. This second resist was obtained on a composite resin incorporating high (>45 wt%) of ultrasmall (5 nm) zirconia or titania stabilized nanoparticles into a zirconium acrylate or polymeric precursor. The micro-optics thus obtained on this resist by 2PP, can then be subject to pyrolysis (at least T = 500 Celsius) with minimal shrinkage of the volume.

Refraction index of at least Rl = 1 .9 should be obtained to have enough dioptric power of the micro-lenses to ensure good imaging results from the device.

This resin was deposited by drop-casting on coverslips, in a controlled amount: 30-40 μL for each 12-mm coverslip and 2 μL for each 5-mm coverslip, which subsequently were subjected to a step of baking at 105 Celsius for 60 min.

At this point, each sample was micro-structured via direct exposure to a focused laser beam. Slides were used, which enable translation of the sample, mounted on a sample-carrier fixed with respect to the movement system in the three spatial dimensions, thus enabling writing of complex geometries in the resin. The commands for movement were issued via G-code software, a language commonly used in numeric control machine tools.

Regarding the fabrication of the microstructure 12, in an embodiment provided by way of example, the software program was written so as to fill, first with all the columns, the circular surface set in the parameters; then, all the gratings in the various planes were formed, first in one direction and then in the other; lastly, reinforced columns (pillars) were obtained, designed to bestow greater stability upon the entire micro-grid.

Regarding the fabrication of the micro-lenses 20-22, in an embodiment by way of example, the scanning of the dome shells of the lenses was programmed in a concentric circumference fashion, starting at the largest radius and decreasing towards the centre. The starting point on each circumference was chosen randomly. This scanning method ensures good optical quality of the 2PP fabricated microlenses.

Finally, the non-irradiated portions of resin were removed by means of a specific alcohol-based developing solution (50% (v/v) 3- pentanone, 50% (v/v) iso-propyl alcohol, Sigma-Aldrich, USA). For the case of the micro-lenses (13 in figures), for which only the outer shell was 2PP fabricated, the bulk volume was then cured by exposure to

UV light (385 nm).

Each microstructure 12 in a possible embodiment, had a shape constituted by a three-dimensional matrix of thin vertical lines interlaced with various thin horizontal lines. These structures constitute a number of levels and determine a sequence of cubic pores. These cubic pores, each of which have typically a side of 50 μm, are positioned alongside each other to form a resulting square structure with a total side of 500 μm. The vertical levels are five in number, and the consequent total height was 100 μm. The vertical spacing could be varied according to the need. The horizontal spacing of 50 μm is the minimum needed to allow the cell and vessels infiltration within the atlas.

At least one truncated cone 28 was polymerised alongside the microstructure 12. The idea behind this was to provide information on the spatial positioning along the vertical co-ordinate of penetration (Z).

The truncated cone 28 is a reference device, which defines the height focused in the analysis by means of the two-photon excitation fluorescence (TPEF) microscope, or more in general the multi-photon excitation fluorescence microscope, and alternatively by means of a confocal microscope. In the implementation step, the diameters of the lower base and upper base were defined so as to obtain a linear dependence between the diameter of the cone, at a given height, and the height from the support 10 or the height from any other reference element.

Consequently, the operator has to measure the dimension

(diameter) of the cone 28 in the current focal plane to verify the height of the level observed. Another application of the reference cone is the check of the optical magnification that will change with the imaging depth due to the coupling of the microlenses with the microscope objective.

Instead of the cone 28, it is possible to use other three- dimensional figures that have at least one of their sides inclined, or in any case have a variable cross section, which enables a linear dependence, or even a non-linear dependence, to be obtained between a measurable dimension and the height of the level with respect to a reference plane.

If then the cone 28 is positioned in an asymmetrical way with respect to the microstructure 12 it is also possible to define the origin of the axes X and Y, and hence define with a single marker the three co-ordinates of the microstructure 12.

It may for example be envisaged to position the cone 28, in a unique way, alongside one side of the microstructure 12, in the proximity of a corner, and with respect to the comer positioned in a counterclockwise direction (or clockwise direction).

The cone 28 will be located close to a corner that will be the origin of the axes, where the axis Y will be set along the side, alongside which the cone 28 is positioned.

In another embodiment of the present invention, the microstructure 12may be inclusive of the truncated cone 28; i.e., instead of providing the truncated cone 28 on the support 10 in a particular position, the truncated cone 28 is provided within the grid structure of the microstructure 12 itself.

To facilitate spatial recognition of the device, and in particular to determine the axes X and Y, other reference points 30 were provided in the form of identifiable marks.

Preferably, every two columns, of the microstructure 12, there is a column having a diameter ten times larger than that of the other columns. These pillars are the strongest elementary structure in the entire device and play a supporting role for the device and are endowed with sufficient fluorescent signal under two-photon excitation that are clearly visible in two-photon excitation fluorescence (TPEF) microscopy. This is essential to facilitate the focusing operation, the repositioning of the field of view under following observations and the correction of the image aberration due to the microlenses and to the distortion of the beam caused by the biological tissue. On one horizontal layer of the atlas, preferably the top one, one fabricates also a composition of two or more thicker lines 31 , approximately of the thickness of the pillars. These lines form a unique symbol, different for each of the four or more atlas fabricated on the transparent substrate, that can be clearly detected under TPE and facilitate the identification of which, among the various Atlas fabricated on the device, is under observation during the optical measurement through the microlenses.

This is essential to facilitate the positioning of the device implanted in the animal with respect to the external optical system that exploit the microlenses.

The present invention, as shown in the figures for a particular embodiment, is comprised typically of four atlas lattices microstructure

12 fabricated symmetrically on the vertex of a square on the transparent support 10. In addition, on the same support there are denser structures spacers 13 that can have different shapes (cubic or

C-shaped, for example), having at least one side of 500 μm and a height of 100 μm, and in any case a height equal to the height of the microstructure 12, which perform a function of structural support and gluing or bonding position for the second substrate 14 to be positioned above it. The implantable platform thus obtained can be used, once implanted, for observing growth and vascularisation of the tissue in vivo via the micro-lenses. This can be obtained by scanning a near infrared femtosecond laser on the entrance pupil of each of the micro- lenses or arrays of microlenses by means of a scanning setup 26 and

27. The microlenses can be exploited as single optical elements of in parallel on an array when coupled to an optical scanning microscope

(confocal or non-linear excitation one) to provide images of cells at high resolution on different fields of view (Figures 12, 13) and different spectral windows, like the green emission of cells’ cytoplasm autofluorescence (Figure 12) or the red emission of nuclear dyes (Fig ure 13), allowing to distinguish and segment cells and different components of the cells. In order to address the individual micro- lens/atlas (MIA) of the (at least) four fabricated on a typical embodiment of the device, one can exploit a spatial light modulator

(SLM). Each quadrant of the SLM can address the individual MIA by activating only one quadrant of the SLM at a time. The alignment of external optical system onto individual MIA set is done by a random search on the SLM polar plane. To this purpose the marks will be exploited. The SLM will also allow to correct for the residual optical aberrations of the microlenses and for the tissue scattering, that may warp severely the light wave front when imaging through the skin. To this latter purpose we will exploit the high number of modes available on a SLM.

Allowing in vivo imaging through the skin, the device can be used for testing reactions to new biomaterials. With the devices set on the support, the second substrate 14 adheres to the spacers. For this purpose, it is possible to use a mono-component glue, or else a biocompatible glue that can be polymerized by UV radiation, or else a bonding process.

The main advantages of two-photon excitation fluorescence

(TPEF) lie in the depth of penetration, in the high localization of the excitation volume, and in the selectivity of the signal that depends on the choice of the fluorescent probes. This enables application of samples that are dense (to a depth of 300-1000 micrometers) and turbid. In addition to this, one should exploit the possibility offered by

SLM to correct for the tissue scattering by exploiting a concept of physical optics known as optical phase conjugation.

In addition to TPEF, one can take advantage of other non-linear excitation processes, second harmonic generation and third harmonic generation scattering, as well as three photons excitation fluorescence or stimulated Raman scattering. These techniques allow to follow the formation of collagen I, one signature of the immune response to implant of biomaterials and present also around the endothelium of vessels, and a number of proteinaceous components of the cells as well as lipid deposits in the tissue. All these techniques have the potential to allow to follow the immune response in-vivo by optical microscopy.

The images are obtained by two-photon excitation .induced by radiation having a wavelength of 800 nm to 1200 nm. The images taken at each level (different heights from the base of the microstructure 12) are captured by a computer and analysed and assembled to make up a three-dimensional image.

For each of the atlas on the device and from each level of the device itself, an image will hence be obtained, from which it is possible to uniquely identify the height, via the cone 28, from a reference plane that may be the device itself or the support 10. The use of reference systems internal to the device that encode for the position of observation renders such positioning independent of possible imperfections in calibration of the scanning system of the two-photon microscope. Moreover, the known regular geometrical structure of the device enables control of the presence of image aberration due to the micro-optics 20, 21 or 22 of the device and to the optics of the scanning system 26 and/or to the distortions of the laser beam as a result of propagation in the biological tissue.

Claims

1. An implantable platform for imaging in-vivo comprising: a planar substrate (10) transparent to light; at least one three-dimensional matrix (12) fabricated on a side of said planar substrate (10), configured to host biomaterial; wherein said at least one three-dimensional matrix (12) is obtained by means of two-photon photo-polymerisation and is luminescent under infrared excitation by two-photon absorption; at least one micro-optical element (11 ), comprising at least one lens (20, 21 , 22), fabricated on the other side of said planar substrate

(10) placed in correspondence of said a least one three-dimensional matrix (12); wherein said implantable platform for imaging in-vivo is configured to be implantable below the skin of a laboratory animal.

2. An implantable platform for in-vivo imaging according to claim

1 characterized in that said at least one micro-optical element (11 ) comprises a plurality of lenses (21 ).

3. An implantable platform for in-vivo imaging according to claim

1 characterized in that said at least one micro-optical element (11 ) comprise at least one planoconvex lens.

4. An implantable platform for in-vivo imaging according to claim

1 characterized in that said at least one micro-optical element (11 ) comprise at least one biconvex lens.

5. An implantable platform for in-vivo imaging according to claim 1 characterized by comprising a second substrate (14), placed below said planar substrate (10).

6. An implantable platform for in-vivo imaging according to claim

1 characterized in that said a least one three-dimensional matrix (12) comprises a matrix of columns interlaced to various horizontal thin lines to determine a sequence of cubic pores.

7. An implantable platform for in-vivo imaging according to claim

1 characterized in that said a least one three-dimensional matrix (12) comprises a reference solid (28) having a cross section that varies with height.

8. An implantable platform for in-vivo imaging according to claim

1 characterized by comprising a plurality of spacers (13) placed laterally to said at least one three-dimensional matrix (12) on said planar substrate (10).

9. An implantable platform for in-vivo imaging according to claim

1 characterized in that said at least one micro-optical device (11 ) is fabricated by 2 -photon polymerization.

10. Method for a minimally invasive solution for the in-vivo inspection and quantification of the immune response of a host animal to the implant of a biomaterial comprising an implantable platform for imaging in-vivo according to claim 1 and wherein the microstructure is visible under two-photon excitation used for the imaging in-vivo and can serve as a frame of reference for multiple/repeated observations.