WO2024094851A1

WO2024094851A1 - Robotic micro-biopsy system and robotic micro-biopsy method

Info

Publication number: WO2024094851A1
Application number: PCT/EP2023/080674
Authority: WO
Inventors: Ali ERTÜRK; Furkan ÖZTÜRK; Harsharan Singh BHATIA; Moritz NEGWER; Mihail TODOROV; Saketh Kapoor
Original assignee: Deep Piction Gmbh
Priority date: 2022-11-03
Filing date: 2023-11-03
Publication date: 2024-05-10

Abstract

The present invention provides to a robotic micro-biopsy system and a corresponding robotic micro-biopsy method. The system provided comprises a sample holder configured and arranged for holding an object on which micro-biopsy is to be performed; a light-sheet microscope system configured and arranged for imaging the object on which micro-biopsy is to be performed; a robotic system with at least one robotic arm configured and arranged for performing or at least assisting the micro-biopsy on the object; and a biopsy needle system that is configured and arranged for micro-biopsy on the object and that comprises at least one biopsy needle to be attached to the robotic arm, optionally via a needle holder.

Description

Robotic Micro-Biopsy System and Robotic Micro-Biopsy Method

Field of the Invention

The present disclosure and invention relate to a robotic micro-biopsy system and a robotic micro-biopsy method .

Background

Scientists who use mice or whole organs to study drugs , diseases , or the spread of diseases on cellular level in a whole human organ or in a mouse body use light sheet imaging to scan the whole sample in 3D . In contrast to CT or MRI , light sheet imaging provides a cellular level resolution . This can be used, for example , to recogni ze single cell metastases on a si ze scale of about 20 pm in a whole mouse body, which would be invisible for other imaging modalities , for example , CT with a typical resolution : 0 . 5 mm) . Light sheet imaging utili zes a microscope and a planar laser source to light up only one slice of a mouse or whole organs of the sample . While the sample is moved under the laser plane , sequential slices of the sample are captured by the microscope . However, the samples need to be labelled so that speci fic cells and molecules emit fluorescence light under the laser irradiation and cleared, i . e . , made transparent so that the laser can pass through the thick tissue of the sample .

So , light sheet imaging is a capable technology for microscopic tissue imaging, in particular on basically three- dimensional samples like whole mice or whole organs . However, imaging is only one part in the complex process of drug development . In addition to imaging, it is also highly desirable to extract distinct parts of the imaged tissue for further examinations and processing . One example for illustration is developing a drug against cancer . A mouse with a metastatic cancer is treated with a new drug, but after light sheet imaging three cancer micro-metastases , the si ze few cells , are detected to be not cured around a distinct area . These micro-metastases are to be extracted, so that further analysis can be conducted to understand the reason why the drug did not cure them and to be able to improve the drug accordingly . There are methods available to isolate such small targets previously identi fied by light sheet imaging . However, such current technologies are either too tedious and take a lot of time , for example , laser capture microdissection, or the extraction si ze or accuracy are not suf ficient for the obj ective at hand, for example , in fine needle aspiration and core needle biopsy .

For instance , " laser capture microdissection" ( LCM) is the standard way for micro sample extractions from uncleared and cleared tissue . The disadvantage of LCM is that it can only work on a thin slice ( around 12 pm) of tissue . In order to do LCM, 3D large tissue samples are first cut into a signi ficant amount of extremely thin slices ( e . g . , via cryosectioning) . Said slices are then scanned again to find the target area . Afterwards these areas are cut out with a focused laser beam and ej ected from their places with an unfocused laser beam . However, when the slicing is done , the integrity of the whole 3D sample is lost . 3D information and a connection there between is no longer available and layers can no longer be analyzed .

Summary

Ob ective

It is thus an obj ective of the present invention and disclosure to provide apparatuses and methods to overcome the disadvantages currently faced in the state-of-the-art . Speci fically, it is an obj ective of the present invention and disclosure to provide systems/apparatuses and methods for accurately and ef ficiently extracting micro-scale tissue or cell samples from preferably three-dimensional intact biological specimens , for instance , whole mice or whole organs and further from cleared tissues .

This obj ective is accomplished with the systems/apparatuses and methods according to the present application . In the following, details of the present disclosure and invention will be outlined . The present invention is also defined by the appended independent claims , whereas the dependent claims describe optional features and distinct embodiments .

Advantages

The present invention and disclosure provide a robotic microbiopsy system and method that allows the extraction of small , micro-scale cell and tissue samples with high accuracy, stability, and ef ficiency .

Detailed Description

The systems/apparatuses and methods according to the present invention and disclosure relate to biopsy, speci fically microbiopsy systems and methods . The expression "biopsy" as used herein is in line of the common understanding of the skilled reader, namely describing a process that involves extraction of sample cells or tissues from biological samples for further examination . In the context of the present invention, such extraction is preferably done during fluorescent imaging from clari fied tissues which are mostly tough to penetrate in comparison to fresh tissues that are typical biopsied in clinical settings . The attribute "micro" as used herein indicates the spatial scale on which the biopsy is performed as well as the si ze of the sample to be extracted . Accordingly, the spatial scale is in the order of tens of micrometres and the si ze of the sample may be as small as a couple of cells .

The systems/apparatuses and methods according to the present invention and disclosure further relate to biopsy facilitated and/or fully conducted by robotic technology . This implies that robotic systems are involved to di f ferent degrees in the biopsy-process , i . e . , to assist and facilitate or completely perform the extraction of tissue or cell samples . The very foundation of the biopsy-proces s is preferably provided by light sheet imaging of the sample , for example , whole mice or whole organs . More speci fically, but not limiting, the context of the present invention disclosure may also be described as light sheet microscopy-guided robotic micro-biopsy on cleared biological samples such as tissues , organs and organisms .

According to the present disclosure and invention, a robotic micro-biopsy system for cleared tissue samples is provided that comprises the following components . A sample holder configured and arranged for holding an obj ect on which microbiopsy is to be performed . A light-sheet microscope system configured and arranged for imaging the obj ect on which microbiopsy is to be performed . A robotic system with at least one robotic arm configured and arranged for performing or at least assisting the micro-biopsy on the obj ect . And a biopsy needle system that is configured and arranged for micro-biopsy on the obj ect and that comprises at least one biopsy needle to be attached to the robotic arm, optionally via a needle holder .

The above defined system allows ef ficient biopsy with high accuracy in particular on complex and three-dimensional tissue samples , for example , whole mice or whole organs . Preferably, a micro-biopsy is thus enabled that goes along with imaging over the entire depth of the sample . This is in contrast to conventional biopsy-methods , where only superficial imaging possible , but no imaging into the depth of the sample from where cells or tissues are to be removed . The respective sample holder is for stably and reliably holding the respective obj ect , i . e . , the sample , such that during the biopsy-process the sample is as stationary and stable as possible so as to ensure the highest possible extraction accuracy . The sample holder can be a x-y- z moveable stage or the like . The "obj ect" mentioned herein, basically represents the sample to be examined by micro-biopsy and is thus preferably a biological sample , i . e . , tissue . Preferably, in order to improve sample handling and biopsy-accuracy, the obj ect is formed by the sample fully or partially embedded in a structural support matrix . The light sheet microscope provides light sheet imaging of the sample to identi fy regions of interest from where tissue cells are supposed to be extracted . Most importantly, using light sheet imaging allows depth-imaging of the sample all the way up to the biopsy-site from where tissue or cells are to be removed and thus also advanced biopsy-control . This is distinctively di f ferent to conventional biopsy, where only superficial imaging is available . "Light sheet imaging" or " light sheet microscopy" is used herein in the normal understanding of the skilled person, namely as the respective and established imaging technology also known as light sheet fluorescence microscopy ( LSFM) . The extraction is then facilitated or completely performed by a robotic system that is provided to controllably move the respective biopsy-equipment , for example , the respective parts of the biopsy needle system . The light sheet imaging data previously obtained but also continuously obtained during the biopsy-process is not only for identi fying the regions of interest in the first place , but also to provide useful control data for the robotic system and to guide the biopsy needle into the region of interest . It is thus preferred to provide for by the present invention and perform light sheet " live" imaging to facilitate the biopsyprocess . The terminology "cleared tissue" mentioned above relates to organic/biological tis sue that has been chemically altered so as to become essentially optically transparent using known methods , i . e . , preferably transparent in the wavelength range visible by the human eye and/or employed for light sheet imaging . Such known methods are disclosed, for example , in WO 2018 /224289 , which is incorporated herein by reference for all purposes . This enables the usage of light sheet imaging for initial imaging and respective guidance during the biopsy-process . It is understood from the above explanations of the interactions of the various system components of the robotic micro-biopsy system, that the system also comprises respective functional , for example , electric connections between the components and respective controllers that enable the various components to interact with each other and to carry out the desired functions . Evidently, the system also comprises respective sensors that provide necessary input data for the functional interaction and control of the various system components .

According to an embodiment and aspect of the system, the sample holder and/or the needle holder is obtained by designing the sample holder and/or the needle holder using finite element analysis in view of optimi zing sti f fness with respect to the forces encountered in micro-biopsy, and producing the sample holder and/or the needle holder by additive manufacturing and/or machining of a lightweight material , optionally aerospace grade aluminium, based on the results of the finite element analysis-based design .

The above detailed sample holder and/or needle holder are speci fically designed and produced with respect to the requirements faced in the micro-biopsy systems and methods according to the present invention and disclosure . The respectively optimi zed holders ensure on the one hand optimal retention of the sample , for example , a whole mouse or organ typically additionally embedded in a structural support matrix, such that during the biopsy-process the sample does not move to the extent that it corrupts the tissue extraction and its accuracy . Likewise , the needle holder is also optimi zed with respect thereto such that the biopsy-needles used can readily withstand the forces exerted upon them during the biopsy-process . This way al so , relative movements that impede the accuracy can be suppressed . In another embodiment and aspect of the system, the system for robotic micro-biopsy is so configured that image data obtained by the light-sheet microscope system is used to perform microbiopsy on the obj ect , preferably wherein image data obtained by the light-sheet microscope is used for guidance of the biopsy needle and/or control of the robotic system and/or the biopsy needle system .

As indicated above , it is not only beneficial to acquire light sheet image data for identi fying regions of interest in the first place . Additionally, light sheet image data proves very helpful when it comes to conducting the biopsy-process . That respective data can be used to control the robotic system such that it accurately advances the biopsy needle system to the desired place for extraction of the desired tissue cells .

In another embodiment and aspect of the system, the robotic system further comprises multiple robotic arms and optionally ancillary actuators configured and arranged for performing or at least assisting the micro-biopsy on the obj ect ; and/or the at least one robotic arm is a harmonic drive robotic arm, and/or the robotic system further comprises DC and/or stepper motors with a closed loop encoding system and narrow motion tolerances for driving the at least one robotic arm .

The above-mentioned details of the robotic system all contribute to the accuracy of the biopsy-process . The presence of multiple robotic arms increases the procedural flexibility and is useful for adding further functionalities . Using a harmonic drive robotic arm or the additional systems mentioned above ensures a smooth and accurate movement of the arm itsel f at the same time providing suf ficient forces for the biopsyprocess and backlash- free motion . The system according to the present invention and disclosure may also comprise several robotic systems as detailed herein for yet further flexibility and versatility . Pursuant to a further embodiment and aspect of the system, the biopsy needle system further comprises a combination of the at least one biopsy needle and a stylet , wherein the stylet is configured to be removably inserted into the biopsy needle .

In another embodiment and aspect of the system, the biopsy needle system further comprises : a single biopsy needle extraction system comprising one biopsy needle and one stylet configured to be removably inserted into the biopsy needle and an extraction system connected to the biopsy needle ; or a dual biopsy needle vacuum extraction system comprising a first biopsy needle and second biopsy needle with a smaller opening diameter than the first biopsy needle and a stylet configured to be removably inserted into the second biopsy needle and an extraction system connected to the second biopsy needle , wherein the extraction system is one of a pneumatic- or a hydraulic pressure control system or a pneumatic- or hydraulic vacuum and pumping system .

A combination of stylet and needle is advantageous in that , for example , the stylet keeps the needle free of tissue and cells during the first stages of tissue penetration and up to reaching the region of interest . Also , a sharp tip of the stylet facilitates needle propagation through tissue . Once said region is reached, the stylet can be withdrawn from the needle and the needle is then functional to extract desired tissue itsel f , either by natural tissue penetration within the hollow inner area of the needle or facilitated by section forces coming from respective systems that are mentioned above and have proven optimal for the respective purpose .

According to another embodiment and aspect of the system, the biopsy needle system is so configured that the at least one biopsy needle is as such stationary or rotates and/or vibrates so as to facilitate tissue penetration of the obj ect .

As a standard, the biopsy needles of the biopsy needle system only move in a translatory fashion during the biopsy-process . However, as needed and depending on the application scenario , the biopsy-needle itsel f can rotate around its own longitudinal axis or vibrate in various directions , preferably perpendicular to its longitudinal axis . Such additional movements can prove helpful in the biopsy-process , in particular, for tissue penetration, depending on the individual sample type .

In another embodiment and aspect of the system, cylindrical shape from the obj ect , the cell or tissue sample comprising 1 to 200000 human or animal cells , preferably comprising 1 to 10 human or animal cells , 1 to 5 human or animal cells or a single human or animal cell . Advantageously, due to its high accuracy, stability, and ef ficiency, the system is particularly suitable for the extraction of small tissue samples of preferably cylindrical shape comprising 1 to 10 human or animal cells , 1 to 5 human or animal cells or even a single human or animal cell . The present invention and the present disclosure also embrace a robotic micro-biopsy method that can be optionally performed using the robotic microbiopsy system as detailed in the present application . The respective method of the present disclosure and invention can on the one hand be seen to be a stand-alone method but can optionally also be considered to be the method carried out speci fically with the above-defined robotic micro-biopsy system/ apparatus .

The robotic micro-biopsy method comprises the following steps , preferably in the indicated order but not limited thereto . A step of obtaining an obj ect for micro-biopsy, said step comprising the sub-steps of ( c ) embedding a human or animal tissue for micro-biopsy so as to obtain the obj ect for microbiopsy and (b ) clearing of the tissue prior to embedding . A step of imaging the obj ect by l ight-sheet microscopy . And a step of extracting a cell or tissue sample via a robotic system from the obj ect , wherein the extraction is guided by image data obtained from the light-sheet microscope . Preferably, the method according to the present invention and disclosure is an ex vi vo method . Further preferably, the method according to the present invention and disclosure is not a method for treatment of the human or animal body by surgery or therapy or a diagnostic method practiced on the human or animal body .

The above step of obtaining an obj ect for micro-biopsy is basically the very first step of providing an ob ect/ sample to be analyzed, for example , a pristine mouse or organ . As mentioned previously, in order to enable the use of light sheet imaging or microscopy, the sample needs to be cleared . That means that chemical processes have to be performed in order to make the sample optical transparent - within the wavelength range visible by the human eye and/or used by the light sheet imaging and microscopy . Methods for clearing are known in the art and are disclosed, for example , in WO

2018 /224289 , which is incorporated herein by reference for all purposes . The respectively cleared sample then is to be embedded in the structural support matrix in order to be mechanically stabili zed for handling and for fixation on the sample holder . Light sheet imaging is then performed to identi fy regions of interest and further optional performed during the micro-biopsy-process facilitated or fully conducted by a robotic system . The respective method according to the present invention and disclosure thereby enables biopsy with high ef ficiency and accuracy on the micro-scale .

According to a further embodiment and aspect of the method, the step of obtaining the obj ect comprises a sub-step of ( a ) labelling, preferably fluorescent labelling, the human or animal tissue , preferably prior to sub-step (b ) , simultaneously with sub-step (b ) and/or after sub-step (b ) and prior to sub-step ( c ) . In that context , the structural support matrix or its material has preferably a refractive index that di f fers by not more than 20% , preferably by not more than 15% , more preferably by not more than 10% , still more preferably by not more than 5% and still more preferably by not more than 2 % from the refractive index of the cleared human or animal tissue . The material of the structural support matrix is preferably a resin- , polymer- , gel-based or composite material and is preferably agarose , more preferably 2 % agarose , or a polymer other than agarose , said polymer having a refractive index matching the refractive index of the cleared human or animal tissue .

Methods for measuring the refractive index are well-known in the art . The references to , and values of , refractive index mentioned herein refer to the refractive index at room temperature ( i . e . 25 ° C ) and normal atmospheric pressure ( i . e . 760 mmHg) .

A prior labeling step is beneficial for highlighting certain types of tissues and cells that will then more clearly appear on light sheet imaging and help identi fy regions of interest . Also , in the context of using light sheet imaging and microscopy, it is beneficial that the material in which the sample is embedded for fixation is not obstructing the light sheet imaging . This implies on the one hand that the material used for embedding as such does not negatively interfere with the light used for the light sheet imaging . In addition, it also means that the material used for embedding is such that it does not cause optical arti facts due to the transition between the material for embedding and the sample . For instance , harmoni zing the refractive index between the material used for embedding and that of the cleared tissue of the sample can reduce arti facts like interfacial reflections or refractions . For example , similar optical properties between the material used for embedding and that of the cleared tissue can suppress arti facts and improve the imaging quality of light sheet imaging and microscopy, which in turn are beneficial for defining regions of interest and guiding the robotic system .

In line with a further embodiment and aspect of the method, the step of extracting a cell or tissue sample further comprises penetrating the human or animal tissue with a biopsy needle in which a stylet is situated so as to extend beyond the tissue penetrating opening of the biopsy needle ; withdrawing the stylet from the biopsy needle once a tissue target region is reached; and extracting the cell or tissue sample via the biopsy needle , optionally via a suction force applied through the biopsy needle . And/or the step of extracting a cell or tissue sample further comprises penetrating the human or animal tissue with a first biopsy needle until a tissue target region is reached, wherein the first biopsy needle is optionally a biopsy needle in which a stylet is situated so as to extend beyond the tissue penetrating opening of the first biopsy needle and the stylet is withdrawn from the first biopsy needle once the tissue target region is reached; advancing a second biopsy needle within the first biopsy needle until the tissue target region is reached, wherein a stylet is situated in said second biopsy needle so as to extend beyond the tissue penetrating opening of the second biopsy needle ; withdrawing the stylet from the second biopsy needle once a tissue target region is reached; and extracting the cell or tissue sample via the second biopsy needle , optionally via a suction force applied through the biopsy needle and/or optionally via retraction of the second biopsy needle .

The additional aspects outlined in the preceding paragraph further contribute to the practicality, ef ficiency and reliability of the robotic micro-biopsy method according to the present invention disclosure .

In a further embodiment and aspect of the method, the cell or tissue sample is preferably of cylindrical shape and comprises 1 to 200000 human or animal cells , preferably 1 to 10 human or animal cells , 1 to 5 human or animal cells or a single human or animal cell . Advantageously, due to its high accuracy, stability, and ef ficiency, the method is particularly suitable for the extraction of small tissue samples of preferably cylindrical shape comprising 1 to 10 human or animal cells , 1 to 5 human or animal cells or even a single human or animal cell .

In a further embodiment and aspect of the method, the obj ect is a whole organ or a whole mouse .

In a further embodiment and aspect of the method, the cell or tissue sample is a sample of a cancer metastasis . Due to its high accuracy, stability, and ef ficiency, the method is particularly suitable for the extraction of cancer metastasi s samples , which can be small .

According to the final embodiment and aspect , the method further comprises , a step of trans ferring and storing the extracted cell or tissue sample in a sample container, optionally tubes or well plates , and optionally analyzing the cell or tissue sample .

In line with this embodiment , the expected cell or tissue sample can be suitably stored for any post-biopsy treatment or examination .

The analyzing of the cell or tissue sample may include any known analysis method such as , for example , mass spectrometry .

List of Figures

Fig . 1 shows a schematic illustration of a distinct embodiment of the robotic micro-biopsy system/ apparatus according to the present invention .

Preferred Example and Embodiment

A distinct but non-limiting embodiment of the robotic microbiopsy system/ apparatus according to the present invention and disclosure will be described with respect to Fig . 1 below . The robotic micro— biopsy system 1 in Fig . 1 comprises a sample holder 10 that is configured and arranged to stably hold an obj ect 11 on which micro-biopsy is to be performed . The obj ect is preferably a biological sample that can come in the form of any suitable biological tissue , preferably in the form of a whole mouse or a whole organ . Preferably, the obj ect is a biological tissue embedded in the structural support matrix that in turn is then retained on the sample holder 10 . The sample holder 10 needs to be configured so as to ensure that during light sheet imaging and micro-biopsy the obj ect 11 , i . e . , the sample thereon is optimally retained such that all necessary acts can be carried out on the obj ect . For example , the sample holder 10 needs to ensure that the obj ect 11 does not move unintentionally, when the biopsy- forces are exerted thereon . In addition, the sample holder 10 also needs to be so configured that it is compatible with the requirements of light sheet imaging and microscopy .

The micro-biopsy system as exempli fied in Fig . 1 also comprises a light sheet microscope system 20 that is so arranged and configured that it can produce light sheet images of the obj ect 11 held on the sample holder 10 . For example , the light sheet microscope system 20 can be situated above the sample holder 10 , preferably such that its optical axis is perpendicular to the plane of the sample holder 10 on which the obj ect 11 is held . Alternatively, the sample holder 10 may also be part of a designated sample area or table of the light sheet microscope system 20 . A non-limiting example of a suitable light sheet microscope is the "Miltenyi UltraMicroscope" .

The micro-biopsy system further comprises a robotic system 30 that itsel f at least comprises one robotic arm 31 that is configured and arranged and so controlled, for example , by respective control systems of the robotic system 30 itsel f or any other control systems of the robotic micro-biopsy system 1 that it can interact with the obj ect 11 held on the sample holder 10 . Set interaction preferably means that the robotic system 30 and in particular its at least one robotic arm 31 can facilitate or fully perform the micro-biopsy on the obj ect 11 . For that purpose , the robotic system 30 , in particular its at least one robotic arm 31 is functionally connected to the biopsy needle system 40 . Preferably, at a suitable end of the at least one robotic arm 31 , a needle holder 42 is functionally attached, whereas said needle holder 42 in turn retains a suitable biopsy needle 41 , for example , a "A-MAX Paed 18G" biopsy needle optionally used with a stylet therein . Said parts are so configured and arranged within the system that at least one robotic arm 31 can control and move the biopsy needle 41 so as to perform a micro-biopsy . Using the respective robotic system 30 ensures high dynamic accuracy and ef ficiency when it comes to conducting a micro-biopsy . Also , any suitable light sheet imaging data can be used for controlling the robotic system 30 so as to basically provide guidance for the movement of the biopsy needle 41 . One nonlimiting example of a suitable robotic system is the "MECA500" robotic arm with several harmonic drives that provide backlash- free motion .

Part of the biopsy needle system 40 are means to produce suction forces on the biopsy needle 41 for cell and tissue extraction . Non limiting examples are simply syringe for manual operation, a digitally controlled sample suction device or a microcontroller driven automated suction control device .

The system and method of the invention were also exempli fied in the following non-limiting experimental example :

Proteomics validation of molecular integrity of tissue extraction using the system and method of the invention

Next , the inventors test molecular integrity of small samples extracted from whole mouse bodies and whole human organs using MS . As sectioning/ imaging for LSM is impractical for large volume samples at scale , they developed a robotic micro-biopsy system, using biopsy needles to isolate ROI s for subsequent proteomics analysis. This included: 1) stabilization of whole mouse body for robotic extraction while imaging, 2) minimizing biopsy needle deflections during extractions, and 3) biopsy needles that can penetrate into hard cleared tissues.

Firstly, to stabilize the sample and ensure that any loose tissue is firmly placed, the inventors investigated different resins and agarose concentrations to modulate bed stiffness, compatibility with the clearing solutions and imaging. They found that a 2% agarose embedding of whole mouse bodies was advantageous for the purpose.

Next, they customized different 3D-printed mouse holder adaptors and needle holders, according to their strength and force-deflection criteria and chose the one with the least deflection .

Last, testing various needle sizes and shapes, they found that stylet needles (size of 18 gauge (G) and/or 22G) provided a good penetration and sample extraction precision without contamination of undesired tissue. They further optimized the system to work concomitantly with the light-sheet microscope. The resulting robotic micro-biopsy system allowed the extraction of small tissue regions from cleared samples while imaging, thereby enabling non-destructive and repetitive tissue isolation at scale for any downstream molecular analysis including MS.

To demonstrate the utility of the robotic micro-biopsy system, they cleared LysM-eGFP mice (expressing eGFP in mainly myeloid lineage cells) .

They co-labeled nuclei with propidium iodide (PI) to identify the ROIs for robotic micro-biopsy extraction. Imaging of the whole body allowed to spatially identify locations of all 425 LysM-eGFP⁺ cells, which were mostly found to be in bone marrow niches. They then focused on the cranium and scapula, two bones with irregular 3D structures, thus hard to study with standard 2D sections. They chose 3 ROIs from the parietal cranium region and 6 ROIs from scapula (3 from the lateral border (LysM-eGFP-) and 3 ROIs from the medial border (LysM- eGFP⁺) . The robotic micro-biopsy system extracted ROIs were then analyzed using the MS pipeline as described above.

In the cranium, they identified the shared signature of 1984 proteins in all 3 ROIs with Pearson correlation between 0.72 and 0.76. To verify the precision of robotic micro-biopsy extraction, they compared the results with proteomics of freshly isolated skull marrow cells (Kolabas, Z.I., Kuemmerle, L.B., Perneczky, R., Fdrstera, B., Buttner, M., Caliskan, O.S., Ali, M., Rong, Z . , Mai, H., Hummel, S., et al. (2021) . Multi-omics and 3D-imaging reveal bone heterogeneity and unique calvaria cells in neuroinflammation. bioRxiv 2021.12.24.473988; doi: https://doi.org/10.1101/2021.12.24.473988.) : They found -2200 shared proteins out of total 2550 identified protein groups when compared with isolated skull-proteome confirming the high precision of robotic extraction. Among others, 7 protein groups identified here (Ptprc, Vwf, Sirp, Mrc, Dagl, Fam3, Lgals) were earlier shown to be expressed both at transcript and proteome level in freshly isolated skull marrow, which further validates the extraction precision using robotic arm. In the scapula, they observed distinct signal of LysM-eGFP in medial vs. lateral border bone. PGA clearly separated the two groups as well as the ROIs itself, particularly from medial border, indicating inter-regional and intra-regional heterogeneity among these extracts. They identified 1250 proteins across conditions with high Pearson correlation coefficients (0.88- 0.98) .

They found a common signature of 764 proteins in lateral border and medial border bone, whereas 336 and 22 proteins were unique to the respective regions. Differential expression analyses showed upregulation of 10 proteins, including those related to the innate and adaptive immune system such as antigen presenting molecule H2-L (MHC classlb) , B-cell/T-cell receptor pathway related proteins and cytokine signaling proteins such as signal transducer and activator of transcription 3 (Stat3) , which are involved in biological processes of inflammatory response regulation to antigenic stimuli. Among 33 downregulated proteins were signalosome related proteins ( CopsVa ) and proteins related to actin filament network formation ( Fhodl ) . These results demonstrate that the robotic micro-biopsy system and method can be applied to whole adult mouse bodies after end-to-end imaging to investigate spatial-molecular heterogeneity and diverse biology .

These results also demonstrate that the robotic micro-biopsy system is highly accurate .

Advantageously, it was found that the robotic biopsy system o f the invention can achieve a tissue micro-biopsy with an accuracy of less than 100 pm .

Claims

1. A robotic micro-biopsy system (1) for cleared tissue samples, the system comprising: a sample holder (10) configured and arranged for holding an object (11) on which micro-biopsy is to be performed; a light-sheet microscope system (20) configured and arranged for imaging the object (11) on which micro-biopsy is to be performed; a robotic system (30) with at least one robotic arm (31) configured and arranged for performing or at least assisting the micro-biopsy on the object; and a biopsy needle system (40) that is configured and arranged for micro-biopsy on the object and that comprises at least one biopsy needle (41) to be attached to the robotic arm, optionally via a needle holder (42) .

2. The robotic micro-biopsy system according to claim 1, wherein : the sample holder and/or the needle holder is obtained by: designing the sample holder and/or the needle holder using finite element analysis in view of optimizing stiffness with respect to the forces encountered in micro-biopsy; and producing the sample holder and/or the needle holder by additive manufacturing and/or machining of a lightweight material, optionally aerospace grade aluminium, based on the results of the finite element analysis-based design.

3. The robotic micro-biopsy system according to claim 1 or 2, wherein : the system for robotic micro-biopsy is so configured that image data obtained by the light-sheet microscope system is used to perform micro-biopsy on the object not only at the surface but several millimetres in depth of the object, preferably wherein image data obtained by the light-sheet microscope is used for guidance of the biopsy needle and/or control of the robotic system and/or the biopsy needle system.

4 . The robotic micro-biopsy system according to any one of the preceding claims , wherein : the robotic system further comprises multiple robotic arms and optionally ancillary actuators configured and arranged for performing or at least assisting the micro-biopsy on the obj ect ; and/or the at least one robotic arm is a harmonic drive robotic arm, and/or the robotic system further comprises DC and/or stepper motors with a closed loop encoding system and narrow motion tolerances for driving the at least one robotic arm .

5 . The robotic micro-biopsy system according to any one of the preceding claims , wherein : the biopsy needle system further comprises : a combination of the at least one biopsy needle and a stylet , wherein the stylet is configured to be removably inserted into the biopsy needle .

6 . The robotic micro-biopsy system according to any one of the preceding claims , wherein : the biopsy needle system further comprises : a single biopsy needle extraction system comprising one biopsy needle and one stylet configured to be removably inserted into the biopsy needle and an extraction system connected to the biopsy needle ; or a dual biopsy needle vacuum extraction system comprising a first biopsy needle and second biopsy needle with a smaller opening diameter than the first biopsy needle and a stylet configured to be removably inserted into the second biopsy needle and an extraction system connected to the second biopsy needle , wherein the extraction system is one of a pneumatic- or a hydraulic pressure control system or a pneumatic- or hydraulic vacuum and pumping system .

. The robotic micro-biopsy system according to any one of the preceding claims , wherein : the biopsy needle system is so configured that the at least one biopsy needle is as such stationary or rotates and/or vibrates so as to facilitate tissue penetration of the ob j ect .

8 . The robotic micro-biopsy system according to any one of the preceding claims , wherein : the robotic micro-biopsy system is configured and arranged for extracting a cell or ti ssue sample of preferably cylindrical shape from the obj ect , the cell or tissue sample comprising 1 to 200000 human or animal cells , preferably compris ing 1 to 10 human or animal cells , 1 to 5 human or animal cells or a single human or animal cell .

9 . A robotic micro-biopsy method, optionally performed using the robotic micro-biopsy system according to any one of the preceding claims 1 to 8 , comprising, preferably in the following order : a step of obtaining an obj ect for micro-biopsy, said step comprising the sub-steps of ( c ) embedding a human or animal tissue for micro-biopsy so as to obtain the obj ect for microbiopsy and (b ) clearing of the tissue prior to embedding; a step of imaging the obj ect by light-sheet microscopy; and a step of extracting a cell or tis sue sample via a robotic system from the obj ect , wherein the extraction is guided by image data obtained from the light-sheet microscope .

10 . The robotic micro-biopsy method according to claim 9 , wherein the step of obtaining the obj ect comprises a sub-step of ( a ) labelling, preferably fluorescent labelling, the human or animal tissue , preferably prior to sub-step (b ) , simultaneously with sub-step (b ) and/or after sub-step (b ) and prior to sub-step ( c ) .

11 . The robotic micro-biopsy method according to claim 9 or 10 , wherein the sub-step of ( c ) embedding the human or animal tissue comprises embedding the tissue in a structural support matrix, the material of which is preferably a resin- , polymer- , gelbased or composite material .

12 . The robotic micro-biopsy method according to claim 11 , wherein the structural support matrix and/or its material has a refractive index that di f fers by not more than 20% , preferably by not more than 15% , more preferably by not more than 10% , still more preferably by not more than 5% and still more preferably by not more than 2 % from the refractive index of the cleared human or animal tissue .

13 . The robotic micro-biopsy method according to claim 11 or 12 , wherein the material of the structural support matrix is agarose , preferably 2 % agarose , or a polymer other than agarose , said polymer having a refractive index matching the refractive index of the cleared human or animal tissue .

14 . The robotic micro-biopsy method according to any one of the preceding claims , wherein the step of extracting a cell or tissue sample further comprises : penetrating the human or animal tissue with a biopsy needle in which a stylet is situated so as to extend beyond the tissue penetrating opening of the biopsy needle ; withdrawing the stylet from the biopsy needle once a tissue target region is reached; and extracting the cell or tissue sample via the biopsy needle , optionally via a suction force applied through the biopsy needle .

15 . The robotic micro-biopsy method according to any one of the preceding claims , wherein the step of extracting a cell or tissue sample further comprises : penetrating the human or animal tissue with a first biopsy needle until a tissue target region is reached, wherein the first biopsy needle is optionally a biopsy needle in which a stylet is situated so as to extend beyond the tissue penetrating opening of the first biopsy needle and the stylet is withdrawn from the first biopsy needle once the tissue target region is reached; advancing a second biopsy needle within the first biopsy needle until the tissue target region is reached, wherein a stylet is situated in said second biopsy needle so as to extend beyond the tissue penetrating opening of the second biopsy needle ; withdrawing the stylet from the second biopsy needle once a tissue target region is reached; and extracting the cell or tissue sample via the second biopsy needle , optionally via a suction force applied through the biopsy needle and/or optionally via retraction of the second biopsy needle .

16 . The robotic micro-biopsy method according to any one o f the preceding claims , wherein the cell or tissue sample is preferably of cylindrical shape and comprises 1 to 200000 human or animal cells , preferably 1 to 10 human or animal cells , 1 to 5 human or animal cells or a single human or animal cell , and/or the obj ect is a whole organ or a whole mouse , and or the cell or tissue sample is a sample of a cancer metastasis .

17. The robotic micro-biopsy method according to any one of the preceding claims, further comprising: a step of transferring and storing the extracted cell or tissue sample in a sample container, optionally tubes or well plates, and optionally analyzing the cell or tissue sample.