Biological sample processing with a microfluidic cartridge

Abstract

Biological sample processing system (1) comprising a microfluidic cartridge (4), an imaging device such as a microscope (13), a sample processing unit (2) including a microfluidic cartridge and sample support holder (6a, 6b) for holding the microfluidic cartridge (4) against a sample support (3), and a control system (9). The microfluidic cartridge comprises a substrate (10), a fluid flow network (22) formed in the substrate (10), a reaction chamber (20) formed within the substrate and configured to be sealingly closed against the sample support (3), and a viewing window formed in the substrate (10) configured to allow optical access to the reaction chamber (20) for an imaging device (13) of the biological sample processing system.

Description

[0001] P2979PC02

[0002] BIOLOGICAL SAMPLE PROCESSING WITH A MICROFLUIDIC CARTRIDGE

[0003] The present invention relates to a biological sample processing system with a microfluidic cartridge for analyzing samples fixed on a support, using an imaging system including a microscope.

[0004] Samples may include whole tissue samples, surgical biopsies or needle biopsies of tissue types, blood samples or cell smears. The samples may also be of microbial nature such as bacteria, or samples of living tissue such as tissue cultures.

[0005] Spatial biology techniques allow the study of the complex cellular interplays in healthy and pathological tissues. By preserving the spatial information and allowing analyses at single-cell resolution, a better understanding of the cellular composition of histological samples has been made possible. Among these, RNA fluorescence in situ hybridization (FISH) technologies are shown to enable a precise profiling of key cellular players, while uncovering their spatial distribution and interactions. Thanks to the detection of key messenger RNA (mRNA) molecules, they also made possible the identification of cells responsible for the production of secreting molecules, which are essential to comprehend signaling networks and cell activation statuses.

[0006] Manual RNA FISH protocols are long and complex protocols prone to multiple errors, in particular:

[0007] • Multiple consecutive reagent incubation and wash steps are necessary, increasing the chance of human errors while performing the assays,

[0008] • RNAs are highly unstable and sensitive molecules, prone to degradation in case of longer manual handling and increased risk of RNase contamination,

[0009] • Multiple off-line imaging steps are required in between hybridization steps, making the whole protocol more complex and exposing tissues to increased risk of degradation by the multiple addition and removal of glass coverslips required for imaging steps,

[0010] • The increased complexity and total duration of the protocol significantly impact on reproducibility of the assays when repeating the analysis on different days and / or large sample cohorts.

[0011] The automation of RNA FISH protocols would thus represent a significant advantage both in terms of performance and of the increase adoptability of the technology, by improving its efficiency, reproducibility, and user-friendliness. WO2017 / 137402 and WO2019 / 012005 describe devices and methods for in situ imaging of a biological tissue sample immobilized on a sample support subjected to multiplexed cycle staining that enables imaging of various molecular targets through multi-molecular read-outs on the same sample. A microfluidic chip interfaces with the sample support and forms a microfluidic chamber surrounding the tissue sample to channel liquid flow over the tissue sample. Multi-cycle multiplexing as described for instance in WO2017 / 137402 and WO2019 / 012005 involves the elution of the target antibody or the inactivation of labeling molecules after each staining and imaging cycle. In many bio-chemical assays the biochemical reagents (DNA, antibodies, etc.) and the target sample (cells, tissues, etc.) need incubation inside the microfluidic chamber for durations that may exceed tens of minutes (for instance 30 minutes per staining and imaging cycle) which may impact imaging accuracy and repeatability due to variations in temperature and pressure, in particular for very small particles such as RNA targets at the single molecule level.

[0012] Thus, the automation of RNA FISH protocols consists of multiple challenges:

[0013] • Temperature oscillations: the different steps of probe hybridization, signal detection, signal removal, wash, and imaging included in the RNA FISH protocol must be done at a specific temperature to ensure maximum sensitivity and specificity of the assay.

[0014] • Long incubations: the RNA FISH protocol requires long incubations at temperatures higher than room temperature to allow sufficient probe hybridization for the detection of key RNA molecules.

[0015] • Single molecule detection: RNA FISH protocols allow the detection of RNA targets at a single molecule level. Although a valuable biological information, this makes RNA detection challenging and generally harder than in situ protein detection, since in mammalian cells a protein to mRNA ratio of about 104 is estimated. For certain key functional genes, only a few RNA molecules per cell are present at any time. Therefore, to have enough sensitivity it is necessary to detect enough RNA molecules in the selected tissue focus plane.

[0016] • Dotted detection: the RNA signal is detected as dotted, where each dot corresponds to a specific RNA molecule. This makes RNA signal more sensitive to imaging issues due to easily recognizable dot shape alterations, with respect to more simple protein detection.

[0017] • Long imaging time: in order to be able to detect efficiently single RNA molecules with a hybridization-based amplification system, the exposure times required during image acquisition become relatively long, in particular when scanning a large tissue imaging area.

[0018] In applications for in-situ imaging of a biological sample immobilized on a slide as described in the aforementioned publications WO 2017 / 137402 and WO 2019 / 012005, the microfluidic cartridge has a microfluidic chamber with a low height, typically less than 250 pm and a surface area greater than 100 mm2. The automation of the RNA FISH assay in a platform comprising automated probe hybridization, signal detection, and wash steps through such a microfluidic cartridge, and microscopic imaging through the transparent viewing window of the cartridge has shown unsatisfactory results because of the following encountered issues linked to each of the previously described challenges:

[0019] • Temperature-related focus drifts: continuous temperature oscillations among different steps and in particular between hybridization (generally done at 37-40 °C for higher specificity) and imaging steps (generally done at room temperature for maximum stability), induce mechanical changes to the microfluidic chip and the mechanical unit that controls the proper clamping of the microfluidic chip to the histological slide, which cause a drift of the sample during imaging. Since the tissue is detected by an autofocus system at the beginning of the imaging step, those changes induce a temperature-related focus drift, causing the sample to appear increasingly out of focus throughout the imaging process.

[0020] • Pressure-related focus drifts: long incubation times at temperatures higher than room temperature reguire application of pressure in the reaction chamber to ensure the prevention of bubble formation. Long incubations with applied pressure before an imaging step induce pressure variations during the following imaging step, therefore resulting in pressure-related focus drifts, significantly impairing imaging guality.

[0021] • Low sensitivity due to generally increased pressure and resulting bad focus plane selection: RNA single molecule detection in combination with the low number of RNA copies per cell, imply that the selection of a suboptimal focus on the histological sample could result in a dramatic reduction of RNA detectability and data guality collection. Some microscope parameters, such as the objective collar ring correction, are fixed parameters that cannot be changed throughout a protocol, and they significantly affect the autofocus plane selection and image guality. These parameters are determined when the reaction chamber is in normal condition and the imaging window in the microfluidic chip is flat. Due to a general change of pressure during the automated RNA FISH protocol (directly caused by incubation steps with applied pressure and indirectly by temperature changes within the closed chamber during imaging), the reaction chamber changes its height therefore inducing a change in the interface between the objective and the sample. This, in turn, results in a worsen autofocusing capacity and generally lower imaging guality, which determines a reduced detection of guantifiable RNA signal.

[0022] • Signal loss due to microfluidic chip curvature and RNA signal distortion: RNA dotted shape is recognized by image analysis software for RNA signal guantifications. Change of pressure in the reaction chamber leading to curvature of the imaging window can result in a loss of focus in specific region of the image and in a signal distortion particularly evident on RNA circular dots in specific regions of each region of interest (ROIs). This leads to undetectable signal with a strong negative impact on assay sensitivity.

[0023] • Regional out of focus regions due to assay parallelization among multiple staining units: increased imaging time due to low RNA signal intensity also increases in parallel the risk of having out of focus tile acguisition if another protocol is run in parallel on the same device. Pressure oscillations in the clamping system of the staining unit due to activation of pressure-linked elements in parallel (e.g. valve opening and closing) can induce loss of focus and lack of signal detectability. Having long imaging times significantly increases the risk and occurrence of such issue.

[0024] It is an object of this invention to provide a biological sample processing system for imaging and analyzing tissues samples with a microfluidic chip, that allows efficient, economical and reliable staining and high resolution imaging, in particular that allows accurate imaging with subcellular resolution such as for RNA FISH assays.

[0025] It is advantageous to provide a biological sample processing system that enables rapid and efficient imaging of tissue samples, especially for sequential multiplex processing of the biological sample with a sequence of reagents.

[0026] It is advantageous to provide a biological sample processing system that preserves the integrity of the tissue sample with minimum damage or undesired alterations, especially for sequential multiplex processing of the biological sample with a sequence of reagents.

[0027] It is advantageous to provide a biological sample processing system that enables automated analysis and that is cost-effective to operate and maintain.

[0028] Objects of the invention have been achieved by providing a biological sample processing system according to the independent claim(s). Dependent claims set forth various advantageous embodiments.

[0029] Disclosed herein is a biological sample processing system comprising a microfluidic cartridge, an imaging device such as a microscope, a sample processing unit including a microfluidic cartridge and sample support holder for holding the microfluidic cartridge against a sample support, and a control system. The microfluidic cartridge comprises a substrate, a fluid flow network formed in the substrate, and a reaction chamber formed within the substrate and configured to be sealingly closed against the sample support. A viewing window formed in the substrate is configured to allow optical access to the reaction chamber for the imaging device of the biological sample processing system.

[0030] According to a first aspect of the invention, the biological sample processing system further comprises a pressure release device connected to the fluid flow network, the pressure release device controlled by the control system and operable to adjust the pressure in the reaction chamber to substantially atmospheric pressure Patm .

[0031] In an advantageous embodiment, the biological sample processing further comprises a waste depot connected to an outlet line of the fluid flow network via a waste line, the outlet line connected to an outlet of the reaction chamber, the pressure release device comprising a pressure release line connected via a valve arrangement to the outlet line, the valve arrangement configured to switch the connection of the outlet line from the waste line to the pressure release line.

[0032] In an advantageous embodiment, the pressure release line is connected downstream of the valve arrangement to a waste container at atmospheric pressure.

[0033] In an advantageous embodiment, the pressure release line is connected downstream of the valve arrangement to a pressure release control system.

[0034] In an advantageous embodiment, the pressure release control system is a passive control system comprising a chamber with an elastically variable volume, for instance comprising a membrane or a spring mounted piston.

[0035] In an advantageous embodiment, the pressure control system comprises a pressure release valve.

[0036] In an advantageous embodiment, the control system is configured to actuate the pressure release device after a staining cycle and prior to an image capture cycle.

[0037] According to a second aspect of the invention, the biological sample processing system further comprises a pneumatic circuit, and a pneumatic clamping pressure mechanism supplied with pneumatic pressure from the pneumatic circuit configured to apply pressure on the microfluidic cartridge and sample support holder to press the sample support against the microfluidic cartridge. The pneumatic circuit comprises a primary pressure regulator and a clamping pressure regulator configured to step down the pressure regulated by the primary pressure regulator and isolate the pneumatic circuit connected downstream of the clamping pressure regulator from the pneumatic circuit connected upstream of the clamping pressure regulator, the pneumatic clamping pressure mechanism being connected downstream of the clamping pressure regulator.

[0038] In an advantageous embodiment, the pneumatic clamping pressure mechanism comprises a piston that presses the sample support holder towards the microfluidic cartridge holder.

[0039] In an advantageous embodiment, the biological sample processing system comprises a plurality of said sample handling devices all fed by the pneumatic circuit. In this embodiment, the biological sample processing system may comprise a plurality of clamping pressure regulators.

[0040] In an advantageous embodiment, each of said plurality of pneumatic clamping pressure mechanisms is connected upstream to only one of each of said plurality of clamping pressure regulators.

[0041] In an advantageous embodiment, the pneumatic clamping pressure mechanism comprises a switching mechanism including a valve, for instance a solenoid valve, to actuate the piston into the clamped position.

[0042] In an advantageous embodiment, the pneumatic clamping pressure mechanism comprises a return spring to return the piston to an unclamped position when the sample support holder and microfluidic cartridge holder are moved to the open position for loading or removal of a microfluidic cartridge and sample support.

[0043] According to a third aspect of the invention, the control system comprises an image capture and processing module configured to control the imaging device and compose an image of a tissue sample, said image composed of a plurality of individual image tiles captured sequentially over an image capture procedure time and stitched together, wherein the image capture and processing module is further configured to generate, during said image capture procedure, a plurality of autofocus maps of the tissue sample image.

[0044] In an advantageous embodiment, at least one of the plurality of autofocus maps is a reference autofocus map generated at a beginning of the image capture procedure time, the reference autofocus map formed of a grid of a predefined number N of focus points distributed over a surface of an image to be captured, at least one or more of the plurality of autofocus maps generated subsequently to the reference autofocus map each comprising a reduced number N1 of focus points (N1 < N) corresponding to a lower resolution autofocus map than that of the reference autofocus map.

[0045] In an advantageous embodiment, individual autofocus maps of the plurality of autofocus maps are generated in alternance with the capture of one or more image tiles.

[0046] In an advantageous embodiment, the biological sample processing system is configured to execute a multiplexing process comprising a plurality of cycles, each cycle with said reagent staining procedure and said image capture procedure, the image capture procedure executed after the reagent staining procedure, wherein in each said cycle said plurality of autofocus maps are generated.

[0047] According to a fourth aspect of the invention, the control system comprises an image capture and processing module configured to control the imaging device and compose an image of a tissue sample, said image composed of a plurality of individual image tiles captured sequentially over an image capture cycle time and stitched together, wherein the image capture and processing module is further configured to generate a plurality of stacked image tile slices, the stacked image tile slices being combined into a single image tile using maximum intensity projection (MIP) configured such that a pixel with a highest light intensity across the stack is retained at each pixel location.

[0048] In an advantageous embodiment, a distance step between slices of said plurality of stacked image tile slices is in a range of 50% to 120% of a depth of field of the imaging device, more preferably in a range of 80% to 110% of the depth of field of the imaging device.

[0049] In an advantageous embodiment, the reaction chamber and viewing window have a surface area greater than 100mm2 and a height of the reaction chamber between an inner surface of the viewing window and a surface of the sample support is less than 250pm.

[0050] In an advantageous embodiment, the biological sample processing system further comprises a plurality of reagent sources connectable to the fluid flow network of the microfluidic cartridge, the biological sample processing system configured for multiplex cycling of reagents through the reaction chamber.

[0051] Further objects and advantageous features of the invention will be apparent from the claims, from the detailed description, and annexed drawings, in which: Figure 1a is a schematic simplified diagram of a biological sample processing system according to an embodiment of the invention;

[0052] Figure 1b is a schematic simplified diagram of a piston clamping system of a biological sample processing system according to an embodiment of the invention;

[0053] Figure 1c is a schematic simplified diagram of a chamber pressure adjustment system of a biological sample processing system according to an embodiment of the invention;

[0054] Figures 2a and 2b are illustrations of a cartridge holder and microfluidic cartridge of a biological sample processing system according to an embodiment of the invention;

[0055] Figure 2c is a cross-section illustration of the cartridge holder and microfluidic cartridge of a biological sample processing system according to an embodiment of the invention;

[0056] Figures 3a and 3b are top and bottom perspective views of a microfluidic cartridge of a biological sample processing system according to an embodiment of the invention;

[0057] Figure 3c is a cross-sectional view through line lllc-lllc of figure 3b;

[0058] Figure 3d is a perspective exploded view of the microfluidic cartridge of figure 3b;

[0059] Figure 4a is a schematic enlarged representation of a section of the microfluidic cartridge chamber and a tissue sample mounted on a sample support illustrating a situation where the pressure differential between an inside of the chamber and an outside (the atmosphere) is 0;

[0060] Figure 4b is a view similar to figure 4a illustrating a deformation of the viewing window due to a positive pressure differential and figure 4c is a view similar to figure 4a showing a deformation of the viewing window due to a negative pressure differential;

[0061] Figure 5 is a flow chart illustrating steps in a staining and imaging method of operating a biological sample processing system according to an embodiment of the invention;

[0062] Figure 6a is a schematic diagram illustrated a sequence of generation of autofocus maps and imaging steps over time for the capture of an image of a tissue sample in a staining cycle according to an embodiment of the invention;

[0063] Figure 6b is a 3D plot of an example of a reference autofocus map obtained prior to an image acquisition step of the whole tissue sample;

[0064] Figure 6c is a 3D plot illustrating the points of a plurality of autofocus maps acquired during an imaging step;

[0065] Figure 7 illustrates three images of a portion of a tissue sample, the images formed by a stack of images (termed herein slices) captured at different focusing heights, whereby the image on the left is formed of a stack of five image slices separated by one micron depth to cover a height of four microns in total, the middle image illustrating a stack of three image slices covering four microns in height, the slices separated by two microns steps, and the image on the right formed by a stack of two image slices separated by a distance of four microns. Referring to the figures, in particular 1 , 2a and 2b, a biological sample processing system according to embodiments of the invention comprises a control unit 9, an imaging device such as a microscope 13, a sample handling device 2, and a microfluidic cartridge 4 mounted on the sample handling device 2. The biological sample processing system 1 is for analyzing biological tissue samples 5 fixed to a sample support 5.

[0066] The sample support 3 may be in form of a conventional microscope slide, for instance made of glass and having typical dimensions of 3 x 2 cm surface area and about 1 mm thickness. Such microscope slides are widely used for fixing tissue samples for placement under a microscope objective to analyze the samples manually or by an automated imaging system. Other supports, whether conventional or not, may however also be used for fixing a tissue sample for analysis with an imaging system according to embodiments of the invention.

[0067] Various tissue samples may be analysed, a non-exhaustive list of typical tissue samples for analysis by a biological sample processing system according to embodiments of the invention including for instance whole tissue samples, surgical biopsies or needle biopsies of different tissue types, cell pellets, blood samples or cell smears from different species. The samples may also be of microbial nature.

[0068] The imaging device comprises an image processing system connected to the microscope 13 with associated electronic circuit and software for capturing and processing images viewed through the microscope. Imaging systems for capture, processing and storage of images are perse well known and do not need to be further described herein.

[0069] The biological sample processing system may further comprise a reagent storage and delivery module 7 controlled by the control unit 9 for supplying reagents, buffer solutions, and washing solutions to the microfluidic cartridge 4, in particular for flow through a reaction chamber 20 formed between the microfluidic cartridge 4 clamped against the sample support 3, for analysis of the sample 5.

[0070] The biological sample processing system may according to embodiments of the invention may comprise a plurality of sample handling devices on a moveable handling platform as described in WO2021 / 058782 which is incorporated herein by reference. Each sample handling device 2 comprises a microfluidic cartridge holder 6b, a sample support holder 6a, and a coupling therebetween to allow movement of the cartridge holder relative to the tissue support holder for mounting and dismounting of the sample support 3. In the illustrated embodiment, the microfluidic cartridge holder is provided in a form of a lid rotatably coupled via a hinge forming the coupling to a base forming the tissue support holder.

[0071] Within the scope of the invention, it may however also be envisaged to have the microfluidic cartridge holder as the base and the tissue slide holder as the lid moveably mounted to the base. This configuration can for example be used in combination with inverted microscopy.

[0072] The microfluidic cartridge comprises a viewing window 16 to allow image capture of the sample 5 under observation.

[0073] The biological sample processing system comprises a pressure mechanism that applies pressure on the microfluidic cartridge 4 against the tissue support 3. The pressure ensures that a seal 24 arranged between a substrate 10 of the microfluidic cartridge 4 and the tissue support 3 is hermetically closed to withstand a pressure in the reaction chamber 20 during injection of reagent and other fluids in the reaction chamber. The pressure applied by the pressure actuator ensures that the maximum pressure attained in the reaction chamber does not cause the seal 24 to leak.

[0074] The biological sample processing system further comprises a reagent storage and delivery module 7 for directing the flow of reagents and other fluids from the reagents source 7a to the fluid flow network 22 of the microfluidic cartridge 4. The reagent storage and delivery module 7 thus comprises inlet couplings for reagent conduits such as reagent tubes for the inlet and outlet of reagents, and an interface surrounded by a sealing element that couples to the fluid flow network 22 on the microfluidic cartridge 4.

[0075] The microfluidic cartridge according to the embodiment of the invention comprises a substrate 10, a fluid flow network 22 formed within the substrate 10, a seal 24, and a viewing window 16. The fluid flow network comprises inlet channel 22i for coupling to the reagent storage and delivery module 7 in the base of the sample handling device, and outlet channels 22o for outflow of reagents from the reaction chamber 20. The fluid flow network is configured to provide a substantially uniform flow of reagents through the reaction chamber 20, intended to ensure substantially advective transport of reagents into the biological sample 5 fixed on the tissue support 3.

[0076] The seal 24 is mounted in a groove formed into a mounting face 12 of the substrate 10 that surrounds the reaction chamber 20, the reaction chamber 20 being formed between the tissue support 3 and the viewing window 16 enclosed by the seal 24 sandwiched between the substrate 10 and tissue support 3.

[0077] A sticky layer 15 may be bonded on an outer side of the substrate to facilitate mounting of the microfluidic cartridge to the cartridge holder 6b prior to closing of the cartridge holder 6b to the tissue support holder 6a.

[0078] The height of the reaction chamber 20, between the sample support 3 and viewing window 24, may typically be in a range of 50pm to 250pm, whereas the surface area of the viewing window is typically more than 100mm2, up to for example 600mm2. A length L of the reaction chamber 20 in the direction of flow of the reagents therethrough may typically be in a range of 10 -25mm or more.

[0079] The viewing window 16 comprises a transparent or semi-transparent cover that may be made of glass, sapphire, or a transparent polymer. The thickness of the viewing window 16 is typically in a range of 100pm to 1500pm. The transparent cover may be separately formed from the substrate 10 and assembled thereto by adhesive bonding 19, by welding, or by overmolding with a material of the substrate 10. The substrate 10 may advantageously be formed of a molded polymer, for instance an injection molded polymer such as COP, COC, PC, PSU and PEEK that may be transparent or opaque.

[0080] The transparent viewing window 16 of the microfluidic cartridge 4 covers a significant portion of the microfluidic chamber 20 and thus has a relatively large surface area, whereas the thickness of transparent viewing window is typically in the order of the chamber height as mentioned above, such that operating pressure differential Pchamber - Patm between the fluid inside of the chamber atmosphere and the atmosphere outside of the chamber, can deform the viewing window 16 in a non-negligeable manner.

[0081] As illustrated in figure 4b, a positive pressure differential in the chamber relative to the atmosphere causes the viewing window to deflect outwardly to increase the chamber height H, namely the height between an inner (bottom) side 25 of the viewing window and an inner (upper) side 29 of the sample support 3 on which the tissue sample 5 is positioned, whereas a negative pressure differential causes the viewing window 16 to deflect towards the sample support 3 as illustrated in figure 4b. In view of the very low chamber height H and the relatively large surface area viewing window with a relatively low thickness, the deformation of the viewing window 16 may have a significant effect on the focus distance of the microscope objective relative to the tissue sample for capture of an in-focus image over the surface area of the viewing window 16.

[0082] The variation of the focus distance as a function of the horizontal X-Y position of the microscope objective, which defines the position of the image tile to be captured, may be obtained by an autofocus scan of the tissue sample in an initial step prior to image capture to generate a reference autofocus map as illustrated in figure 6b, which may be stored in a memory of the control system 9 for the subsequent imaging step. A change in pressure however affects the focusing distance, such that the reference autofocus map depends on the internal pressure in the reaction chamber 20.

[0083] According to an aspect of the invention, the biological sample processing system 1 comprises a pressure release device 8 connected to the fluid flow network 22 connected to the control system 9 operable to adjust the pressure in the reaction chamber 20 to substantially atmospheric pressure Patm or to a pressure in a range between atmospheric pressure Patm and atmospheric pressure Patmplus a small positive control pressure PCOntro of up to 250mbar. The pressure release device 8 may be actuated prior, or just prior, to an image capture cycle, which is performed after completion of the staining cycle.

[0084] The reaction chamber pressure after actuation of the pressure release device is advantageously kept in a pressure range between ambient pressure and 250mbar above atmospheric pressure and with a pressure variation over time not higher than +-50mbar / hour and at a chamber temperature maintained in between 10°C to 40°C. More specifically the reaction chamber release pressure is preferably kept in a range between 25 to 100mbar above atmospheric pressure with a pressure variation over time not higher than +- 25mbar in between two multiple autofocus map during the imaging cycle or between imaging cycles. The release pressure in the reaction chamber can be controlled by a separate line connecting the reaction chamber to a pressure control system.

[0085] In the illustrated embodiment, the pressure release device comprises a pressure release line 30 connected via a valve arrangement V having one or more valves, in turn connected to the outlet 18o, 17o of the reaction chamber 20.

[0086] In one embodiment, the pressure release line 30 is connected for instance to a waste container at atmospheric pressure and the valve arrangement V switches the connection of the outlet line 17o of the fluid flow network from a waste line 31 , that feeds into waste containers of the waste depot 28, to the pressure release line 30. The waste line 31 may be at a pressure setting that is configured to obtain a desired pressure above atmospheric pressure within the reaction chamber 20 during the staining cycle. Once this staining cycle is completed, the pressure in the reaction chamber may be released down to atmospheric pressure such that the viewing window 16 is in its undeformed state.

[0087] In a variant, the pressure release line 30 may be connected to a pressure release control system 32 at a controlled positive release pressure (Patm+ Pcontni), and the valve arrangement V switches the connection of the outlet line 17o of the fluid flow network from a waste line 31 , that feeds into waste containers of the waste depot 28, to the pressure release line 30 connected to the pressure release control system 32.

[0088] Advantageously, the controlled small positive pressure during the pressure release operation allows to reduce bubble formation during the pressure release operation, while limiting the pressure in the reaction chamber such that there is only a small or negligeable deformation of the viewing window that does not adversely affect the imaging process.

[0089] In embodiments, the pressure release control system 32 may be a passive control system. In an embodiment, the passive control system comprises a variable volume chamber with an elastic membrane or a spring mounted piston configured to allow expansion of the chamber to limit the pressure in the chamber so that it does not exceed the desired maximum release pressure (Patm+ Pcontni). In another embodiment, the pressure control system may comprise a chamber with a pressure release valve set to open at the desired maximum release pressure (Patm + Pcontro), for instance Patm plus 250mbar, such that the maximum pressure in the release chamber does not exceed the maximum desired release pressure (Patm+ Pcontni).

[0090] In other embodiments, the pressure control system may also include an active control system, for instance including a pressure sensor and a pump to adjust the pressure in the pressure release line 30 to a desired release pressure (Patm+ Pcontni).

[0091] According to another aspect of the invention, the biological sample processing system 1 comprises a pneumatic clamping pressure mechanism 14 that applies pressure on the sample support 3 against the microfluidic cartridge 4 to sealingly close the sample support 3 against the microfluidic cartridge 4. The pressure applied by the pneumatic clamping pressure mechanism 14 is generated by a pneumatic circuit 27, the pneumatic clamping pressure mechanism 14 comprising for instance a piston 25 that presses the sample support holder 6a towards the microfluidic cartridge holder 6b. The pneumatic circuit 27 is however utilized in the biological sample processing system to drive other components, for instance there may be a plurality of sample handling devices to mounted in the biological sample processing system 1 , all fed by the pneumatic circuit 27. Operation of other devices using the pneumatic circuit 27 causes fluctuation in the pressure of the pneumatic circuit 27 that may affect the pressure of the piston 25 and thus the clamping force of the sample support holder 6a against the microfluidic cartridge holder 6b. The pneumatic circuit comprises a primary pressure regulator 1 to ensure a stable pressure but this nevertheless cannot entirely smoothen out the pressure variations due to operation of other devices using the pneumatic circuit 27.

[0092] According to an aspect of the invention, the pneumatic circuit 27 further comprises a clamping pressure regulator P2 that steps down the pressure regulated by the primary pressure regulator P1 and isolates the pneumatic circuit connected downstream of the clamping pressure regulator P2 from the rest of the pneumatic circuit 27. The pneumatic clamping pressure mechanism 14 is connected downstream of the clamping pressure regulator P2 such that the piston 25 is isolated from the rest of the pneumatic circuit 27 thus ensuring a stable desired pressure of the sample support 3 against the microfluidic cartridge 4.

[0093] In order to operate the piston 25, the pneumatic clamping pressure mechanism 14 may comprise a switching mechanism 26 such as a valve, for instance a solenoid valve, to actuate the piston into the clamped position. A return spring may be integrated in the pneumatic clamping pressure mechanism 14 to return the piston 25 to an unclamped position when the sample support holder 6a and microfluidic cartridge holder 6b are moved to the open position for loading or removal of a microfluidic cartridge and sample support.

[0094] More generally, since an image capture cycle can take a relatively long time, for instance 1-3 hours, focus drift over time may be due to various factors such as:

[0095] - the varying deformation of the viewing window as a function of varying pressure, or

[0096] - the distance between the microscope and the tissue can drift due to thermal dilatation variations of components supporting the microscope 13 or the microfluidic cartridge 4 and tissue sample support 3, or

[0097] - the distance between the microscope and the tissue sample can drift due to the tissue sample position as a function of clamping pressure between the tissue sample support 3 and the microfluidic cartridge 4.

[0098] According to an aspect of the invention, a solution to the problem of focus drift over an image capture cycle of a whole tissue sample, which for instance may correspond to over 60% of the surface area of the viewing window 16, includes the generation of multiple autofocus maps of the tissue sample image over the image capture cycle time, as schematically illustrated in figures 5, 6a and 6c.

[0099] An autofocus map defines the optimal focus position for an objective, at selected points of a predefined 2D grid, for imaging an object within the predefined 2D grid. The autofocus map thus defines 3D coordinates, i.e. X-Y-Z positions, for an objective to have an optimal focus for imaging the object at each of the selected points of the predefined grid.

[0100] The multiple autofocus maps may be acquired in alternance with the imaging process compensating for the drift of focus over time. The amount, the time duration and the distribution of the autofocus maps may be controlled by software, for instance an autofocus mapping module installed in the control system 9. Imaging of tissue samples may occur multiple times during a multiplexing protocol, in particular during multiple cycles of the protocol as illustrated in figure 5. Cycles of the multiplexing protocol may include staining and imaging procedures, the imaging procedure (or step) following the staining procedure (or step). Multiple autofocus maps are acquired during an imaging procedure of each cycle of the multiplexing protocol that includes an imaging procedure, as illustrated in figure 5

[0101] Typically, the focus drift will be faster at the beginning of the image capture cycle, to the extent that its effect can be significant within the duration of a single reference (full-grid) autofocus map. To mitigate this, a sub-selection of the full grid may be acquired right after the reference map which is in an order of magnitude faster than the generation of the reference autofocus map, ensuring that the image tiles acquired at the beginning of the image capture cycle are as sharp as possible. A sub-selection may for instance comprise the acquisition of a reduced number of focus points, corresponding to a lower resolution compared to the reference autofocus map, such as one in two, or one in three or one in four points of the initial reference acquisition.

[0102] The reference (full-grid) autofocus map covers every point of a predefined 2D grid of X„x Yn= N points over the tissue sample. Integers Xnand Ynmay typically be each in a range of 5 to 20, preferably in a range of 8 to 15, for instance in a range of 10 to 14. Thus, the predefined 2D full grid may have a number of points N in a range of 25 to 400, preferably in a range of 64 to 225, for instance in a range of 100 to 196, depending on the selected predefined resolution of the reference full grid autofocus map. At each point, the autofocus mapping module determines if tissue sample is visible in the field-of-view of the current position, and subsequently the optimal focus position is determined. If no tissue sample is present at the current position, the objective 13 moves to the next point of the predefined 2D grid. Once every point is visited, outliers are removed from the grid and a 3D model of the autofocus map is fitted on the remaining points (i.e. without the outliers). This 3D model of the autofocus map determines the X-Y-Z position of the objective 13 during the acquisition of each of the image tiles.

[0103] For subsequent autofocus maps generated in the imaging procedure of one cycle of the multiplexing protocol, a sub-selection of the initial full grid reference autofocus map grid, i.e. with a reduced number of points N1, is used. After the focus has been acquired on a point of the sub-selection grid, the autofocus mapping module finds a point for which the focus has not been yet acquired and which is furthest away from all previously acquired focus points, and then moves the objective to this point and finds the focus. For each new point, the autofocus mapping module determines if the model is sufficiently insensitive to noise in the measurements. Once this is the case, an updated 3D model of the autofocus map is once again fitted on the acquired focus points.

[0104] By way of example, an initial full grid reference autofocus map may take minutes to complete, for instance in a range of 3-5 minutes, whereas a reduced autofocus map with lower resolution may for instance take 1 min.

[0105] According to another aspect of the invention, for the accurate image capture of very small features such as fluorescent RNA dots, stacks of image slices are acquired, which are combined into a single image using maximum intensity projection (MIP). Because the fluorescent RNA dots are much smaller structures than the fluorescent probes typically seen in proteomics, a sharper focus is necessary to discern them. The signal of an out of focus dot is spread over a larger area of the image sensor than an in-focus dot, and the amplitude of an out of focus dot signal can often be lower than the noise or the background. In a situation where the tissue sample has an intrinsic thickness that is greater than the depth of field of a microscope objective, for instance 2-3 times the depth of field of the objective, there is no single position of the objective that guarantees that all very small features such as fluorescent RNA dots are in focus.

[0106] To address this issue, according to another aspect of the invention, a plurality of image slices, forming a stack of image slices, are acquired, the stack of image slices being combined into a single image using maximum intensity projection (MIP). For each pixel location in the stack of image slices, the MIP method selects the pixel with the highest intensity across the stack. Since in-focus dots have a higher light intensity signal than out of focus dots, if their position is within the height range of the image stack, the in-focus dots will be retained and present in the final image.

[0107] The largest distance step between slices and the minimum height range of the stack to minimize imaging time will depend on the typical tissue sample thickness and the depth of field of the microscope objective. Preferably, the largest distance step between slices is about equal to or less than the depth of field of the microscope objective. To minimize imaging time, the distance step between slices is preferably in a range of 50% to 120% of the depth of field of the microscope objective, more preferably in a range of 80% to 110% of the depth of field of the microscope objective.

[0108] In the images of the example of figure 7, taken by a microscope objective with a depth of field of about 2 urn, a stack of three image slices separated by steps of 2 urn (centre image) reveals almost the same quality image as a stack of five image slices separated by steps of 1 urn (left image), whereas a stack of two image slices separated by steps of 4 urn (right image) reveals a loss of information on luminescent RNA dots. Thus, in this example a stack of image slices with an image slice separation distance corresponding to the depth of field of the microscope objective would be optimal to minimize image generation time and obtain a good quality image presenting the fluorescent RNA dots.

[0109] List of references used biological sample processing system 1 sample handling device 2 microfluidic cartridge holder 6b viewing opening sample support holder 6a inlet conduit 17i outlet conduit 17o microfluidic cartridge 4 substrate 10 mounting face 12

[0110] Inlet 18i

[0111] Outlet 18o sticky layer 15 adhesive 19 fluid flow network 22 inlet channels 22i outlet channels 22o viewing window 16 inner side 25 reaction chamber 20 top side 25 (= inner side of the viewing window) bottom side 29 (=top side of the sample support) seal 24 pressure release device 8 valve arrangement V pressure release line 30 pressure release control system 32 reagent storage and delivery module 7 reagent sources 7a pump P waste depot 28 waste container(s) waste line 31 control system 9 reagent pump and valve control 23 negative pressure pulse control 21 image capture and processing system 19 autofocus mapping module user interface 11 imaging device (e.g. microscope) 13 pneumatic circuit 27 primary pressure regulator P1 clamping pressure regulator P2 pneumatic clamping pressure mechanism 14 piston 25 switching mechanism 26 valve, e.g. solenoid valve sample support 3 top side 29 tissue sample 5

Claims

CLAIMS1 . Biological sample processing system (1 ) comprising a microfluidic cartridge (4), an imaging device such as a microscope (13), a sample processing unit (2) including a microfluidic cartridge and sample support holder (6a, 6b) for holding the microfluidic cartridge (4) against a sample support (3), and a control system (9), the microfluidic cartridge comprising a substrate (10), a fluid flow network (22) formed in the substrate (10), a reaction chamber (20) formed within the substrate and configured to be sealingly closed against the sample support (3), a viewing window formed in the substrate (10) configured to allow optical access to the reaction chamber (20) for the imaging device (13) of the biological sample processing system, the control system comprising an image capture and processing module configured to control the imaging device and compose an image of a tissue sample, said image composed of a plurality of individual image tiles captured sequentially over an image capture procedure time and stitched together, characterized in that the image capture and processing module is further configured to generate during said image capture procedure a plurality of autofocus maps of the tissue sample image, wherein at least one of the plurality of autofocus maps is a reference autofocus map generated at a beginning of the image capture procedure time, the reference autofocus map formed of a grid of a predefined number N of focus points distributed over a surface of an image to be captured, at least one or more of the plurality of autofocus maps generated subsequently to the reference autofocus map each comprising a reduced number N1 of focus points (N1 < N) corresponding to a lower resolution autofocus map than that of the reference autofocus map.

2. The biological sample processing system according to the preceding claim wherein the image capture and processing module is configured to generate individual autofocus maps of the plurality of autofocus maps in alternance with the capture of one or more image tiles.

3. The biological sample processing system according to any preceding claim wherein the biological sample processing system is configured to execute a multiplexing process comprising a plurality of cycles, each cycle with said reagent staining procedure and said image capture procedure, the image capture procedure executed after the reagent staining procedure, wherein in each said cycle said plurality of autofocus maps are generated.

4. The biological sample processing system according to any preceding claim wherein the image capture and processing module is further configured to generate a plurality of stacked image tile slices, the stacked image tile slices being combined into a single imagetile using maximum intensity projection (MIP) configured such that a pixel with a highest light intensity across the stack is retained at each pixel location.

5. The biological sample processing system according to the preceding claim wherein a distance step between slices of said plurality of stacked image tile slices is in a range of 50% to 120% of a depth of field of the imaging device.

6. The biological sample processing system according to claim 4 wherein the distance step between slices of said plurality of stacked image tile slices is in a range of 80% to 110% of the depth of field of the imaging device.

7. The biological sample processing system according to any preceding claim wherein the reaction chamber and viewing window have a surface area greater than 100mm2 and a height of the reaction chamber between an inner surface (25) of the viewing window (24) and a surface of the sample support (3) is less than 250pm.

8. Biological sample processing system (1 ) comprising a microfluidic cartridge (4), an imaging device such as a microscope (13), a sample processing unit (2) including a microfluidic cartridge and sample support holder (6a, 6b) for holding the microfluidic cartridge (4) against a sample support (3), and a control system (9), the microfluidic cartridge comprising a substrate (10), a fluid flow network (22) formed in the substrate (10), a reaction chamber (20) formed within the substrate and configured to be sealingly closed against the sample support (3), a viewing window formed in the substrate (10) configured to allow optical access to the reaction chamber (20) for the imaging device (13) of the biological sample processing system, the control system comprising an image capture and processing module configured to control the imaging device and compose an image of a tissue sample, said image composed of a plurality of individual image tiles captured sequentially over an image capture procedure time and stitched together, characterized in that the image capture and processing module is further configured to generate during said image capture procedure a plurality of autofocus maps of the tissue sample image, wherein the biological sample processing system is configured to execute a multiplexing process comprising a plurality of cycles, each cycle with said reagent staining procedure and said image capture procedure, the image capture procedure executed after the reagent staining procedure, wherein in each said cycle said plurality of autofocus maps are generated.

9. The biological sample processing system according to the preceding claim wherein at least one of the plurality of autofocus maps is a reference autofocus map generated at a beginning of the image capture procedure time, the reference autofocus map formed of a gridof a predefined number N of focus points distributed over a surface of an image to be captured, at least one or more of the plurality of autofocus maps generated subsequently to the reference autofocus map each comprising a reduced number N1 of focus points (N1 < N) corresponding to a lower resolution autofocus map than that of the reference autofocus map.

10. The biological sample processing system according to any preceding claim 8-9 wherein the image capture and processing module is configured to generate individual autofocus maps of the plurality of autofocus maps in alternance with the capture of one or more image tiles.

11. The biological sample processing system according to any preceding claim 8-10 wherein the image capture and processing module is further configured to generate a plurality of stacked image tile slices, the stacked image tile slices being combined into a single image tile using maximum intensity projection (MIP) configured such that a pixel with a highest light intensity across the stack is retained at each pixel location.

12. The biological sample processing system according to the preceding claim 8-11 wherein a distance step between slices of said plurality of stacked image tile slices is in a range of 50% to 120% of a depth of field of the imaging device, more preferably in a range of 80% to 110% of the depth of field of the imaging device.

13. The biological sample processing system according to any preceding claim 8-12 wherein the reaction chamber and viewing window have a surface area greater than 100mm2 and a height of the reaction chamber between an inner surface (25) of the viewing window (24) and a surface of the sample support (3) is less than 250pm.

14. Biological sample processing system (1) comprising a microfluidic cartridge (4), an imaging device such as a microscope (13), a sample processing unit (2) including a microfluidic cartridge and sample support holder (6a, 6b) for holding the microfluidic cartridge (4) against a sample support (3), and a control system (9), the microfluidic cartridge comprising a substrate (10), a fluid flow network (22) formed in the substrate (10), a reaction chamber (20) formed within the substrate and configured to be sealingly closed against the sample support (3), a viewing window formed in the substrate (10) configured to allow optical access to the reaction chamber (20) for the imaging device (13) of the biological sample processing system, the control system comprising an image capture and processing module configured to control the imaging device and compose an image of a tissue sample, said image composed of a plurality of individual image tiles captured sequentially over an image capture cycle time and stitched together, characterized in that the image capture and processing module is furtherconfigured to generate a plurality of stacked image tile slices, the stacked image tile slices being combined into a single image tile using maximum intensity projection (MIP) configured such that a pixel with a highest light intensity across the stack is retained at each pixel location.

15. The biological sample processing system according to the preceding claim wherein a distance step between slices of said plurality of stacked image tile slices is in a range of 50% to 120% of a depth of field of the imaging device.

16. The biological sample processing system according to claim 14 wherein the distance step between slices of said plurality of stacked image tile slices is in a range of 80% to 110% of the depth of field of the imaging device.

17. The biological sample processing system according to any preceding claim 14-16 wherein the image capture and processing module is further configured to generate during said image capture procedure a plurality of autofocus maps of the tissue sample image.

18. The biological sample processing system according to the preceding claim wherein at least one of the plurality of autofocus maps is a reference autofocus map generated at a beginning of the image capture procedure time, the reference autofocus map formed of a grid of a predefined number N of focus points distributed over a surface of an image to be captured, at least one or more of the plurality of autofocus maps generated subsequently to the reference autofocus map each comprising a reduced number N1 of focus points (N1 < N) corresponding to a lower resolution autofocus map than that of the reference autofocus map.

19. The biological sample processing system according to the preceding claim wherein individual autofocus maps of the plurality of autofocus maps are generated in alternance with the capture of one or more image tiles.

20. The biological sample processing system according to any preceding claim 14-18 in combination with claim 17 wherein the biological sample processing system is configured to execute a multiplexing process comprising a plurality of cycles, each cycle with said reagent staining procedure and said image capture procedure, the image capture procedure executed after the reagent staining procedure, wherein in each said cycle said plurality of autofocus maps are generated.

21. The biological sample processing system according to any preceding claim wherein the biological sample processing system comprises a pressure release device (8) connected to the fluid flow network, the pressure release device controlled by the controlsystem and operable to adjust the pressure in the reaction chamber (20) to substantially atmospheric pressure Patm, or to a pressure in a range from atmospheric pressure Patm to atmospheric pressure Patmplus 250mbar.

22. The biological sample processing system according to the preceding claim further comprising a waste depot (28) connected to an outlet line (17o) of the fluid flow network via a waste line (31), the outlet line connected to an outlet (18o) of the reaction chamber, the pressure release device comprising a pressure release line (30) connected via a valve arrangement (V) to the outlet line, the valve arrangement configured to switch the connection of the outlet line from the waste line (31) to the pressure release line (30).

23. The biological sample processing system according to the preceding claim wherein the pressure release line is connected downstream of the valve arrangement to a waste container at atmospheric pressure or to a pressure release control system (32).

24. The biological sample processing system according to the preceding claim wherein the pressure release control system (32) is a passive control system comprising a chamber with an elastically variable volume, for instance comprising a membrane or a spring mounted piston.

25. The biological sample processing system according to either of the two directly preceding claims wherein the pressure control system comprises a pressure release valve26. The biological sample processing system according to any preceding claim 21-25 wherein the control system is configured to actuate the pressure release device after a staining cycle and prior to an image capture cycle.

27. The biological sample processing system according to any preceding claim comprising a pneumatic circuit (27), and a pneumatic clamping pressure mechanism (14) supplied with pneumatic pressure from the pneumatic circuit configured to apply pressure on the microfluidic cartridge and sample support holder (6a, 6b) to press the sample support (3) against the microfluidic cartridge (4), wherein the pneumatic circuit comprises a primary pressure regulator (P1) and a clamping pressure regulator (P2) configured to step down the pressure regulated by the primary pressure regulator and isolate the pneumatic circuit connected downstream of the clamping pressure regulator (P2) from other portions of the pneumatic circuit, the pneumatic clamping pressure mechanism being connected downstream of the clamping pressure regulator (P2).

28. The biological sample processing system according to the preceding claim wherein the pneumatic clamping pressure mechanism comprises a piston (25) that presses the sample support holder (6a) towards the microfluidic cartridge holder (6b).

29. The biological sample processing system according to any preceding claim 27-28 comprising a plurality of the sample handling devices all fed by the pneumatic circuit (27).

30. The biological sample processing system according to the preceding claim comprising a plurality of clamping pressure regulators, wherein each of said plurality of pneumatic clamping pressure mechanisms is connected upstream to only one of each of said plurality of clamping pressure regulators.

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